Meuren LM, Coelho SVA, de Arruda LB. Evaluation of DENV-Induced Endothelial Cell Permeability by Measurements of Transendothelial Electrical Resistance (TEER) and Extravasation of Proteins and Virus. Methods Mol Biol. 2022;2409:207-222.

DOI: 10.1007/978-1-0716-1879-0_14 

This chapter will discuss reliable and relatively easy and fast strategies to evaluate the integrity of endothelial cell monolayers when infected by dengue virus (DENV). Human brain microvascular endothelial cells (HBMEC) were exploited here as general model of vessel wall core, but it may also be used as an in vitro simplified model of blood brain barrier (BBB). The integrity of endothelial cells monolayer can be inferred using a transwell culture system by: (1) measuring transendothelial electrical resistance (TEER) using a Voltohmmeter; (2) analyzing the monolayer permeability to fluorescent-conjugated proteins and fluorimetric assay; (3) investigating virus extravasation by quantitative RT-PCR and plaque conventional assay. The rational to use those strategies is that vascular alterations are often observed during dengue infection, being associated to disease severity. The vasculature core consists of a barrier of endothelial cells, which are tightly adhered by the expression of adhesion molecules and tight junctions. This structure must be preserved in order to control the flux of cells and metabolites from the circulation to the tissues and to maintain vascular homeostasis. Therefore, experimental assays that allow evaluation of endothelial integrity can be useful platforms to further understand disease pathogenesis and screen pharmaceutical interventions to control vascular disturbance.

Keywords: Endothelial cells, Blood brain barrier, Transendothelial electrical resistance, Endothelial permeability, Transwell

da Silva Prata RB, Mendes MA, Soares VC, França-Costa J, Sales AM, Duppré NC, de Matos Borges V, da Silva TP, Bozza PT, Bozza MT, Sarno EN, Moraes MO, Sperandio da Silva GM, Pinheiro RO. Arginase 1 is a marker of protection against illness in contacts of leprosy patients. Sci Rep. 2022 May 12;12(1):7850.

DOI: 10.1038/s41598-022-11944-9 

Leprosy household contacts are generally more prone to develop the disease compared to the general population. Previous studies have demonstrated that genes related to the alternative activation (M2) profile in macrophages are associated with the increased bacillary load in multibacillary leprosy patients (MB), and that contacts of MB patients have a higher risk of contracting the disease. In addition, positive serological responses to PGL-1 or LID-1 are associated with a higher risk of disease. We performed a 5-year follow-up of contacts of leprosy patients and evaluated the pattern of gene and protein expression in cells from contacts that developed leprosy during this period. Leprosy household contacts had decreased soluble CD163 and heme oxygenase 1 (HO-1) serum levels when compared with healthy donors and leprosy patients. In contrast, arginase 1 activities were higher in contacts when compared with both healthy donors and leprosy patients. Of the contacts, 33 developed leprosy during the follow-up. Gene expression analysis revealed reduced ARG1 expression in these contacts when compared with contacts that did not develop disease. Arginase activity was a good predictive marker of protection in contacts (sensitivity: 90.0%, specificity: 96.77%) and the association with serology for anti-PGL-1 and anti-LID-1 increased the sensitivity to 100%. Altogether, the data presented here demonstrate a positive role of arginase against leprosy and suggest that the evaluation of arginase activity should be incorporated into leprosy control programs in order to aid in the decision of which contacts should receive chemoprophylaxis.

Kiarely Souza E, Pereira-Dutra FS, Rajão MA, Ferraro-Moreira F, Goltara-Gomes TC, Cunha-Fernandes T, Santos JDC, Prestes EB, Andrade WA, Zamboni DS, Bozza MT, Bozza PT. Lipid droplet accumulation occurs early following Salmonella infection and contributes to intracellular bacterial survival and replication. Mol Microbiol. 2022 Feb;117(2):293-306.

DOI: 10.1111/mmi.14844

Salmonellosis is a public health problem caused by Salmonella sp., a highly adapted facultative intracellular pathogen. After internalization, Salmonella sp. Manipulates several host processes, mainly through the activation of the type III secretion system (T3SS), including modification of host lipid metabolism and lipid droplet (LD) accumulation. LDs are dynamic and complex lipid-rich organelles involved in several cellular processes. The present study investigated the mechanism involved in LD biogenesis in Salmonella-infected macrophages and its role in bacterial pathogenicity. Here, we reported that S. Typhimurium induced a rapid time-dependent increase of LD formation in macrophages. The LD biogenesis was demonstrated to depend on Salmonella's viability and SPI1-related T3SS activity, with the participation of Toll-Like Receptor (TLR) signaling. We also observed that LD accumulation occurs through TLR2-dependent signaling and is counter-regulated by TLR4. Last, the pharmacologic modulation of LD formation by inhibiting diacylglycerol O-acyltransferase 1 (DGAT1) and cytosolic phospholipase A2 (cPLA2) significantly reduced the intracellular bacterial proliferation and impaired the prostaglandin E2 (PGE2) synthesis. Collectively, our data suggest the role of LDs on S. typhimurium intracellular survival and replication in macrophages. This data set provides new perspectives for future investigations about LDs in host–pathogen interaction.

Keywords: PGE2; Salmonella Typhimurium; TLRs; lipid droplets; lipid metabolism

Costa-da-Silva AC, Nascimento DO, Ferreira JRM, Guimarães-Pinto K, Freire-de-Lima L, Morrot A, Decote-Ricardo D, Filardy AA, Freire-de-Lima CG. Immune Responses in Leishmaniasis: An Overview. Trop Med Infect Dis. 2022 Mar 31;7(4):54.

DOI: 10.3390/tropicalmed7040054

Leishmaniasis is a parasitic, widespread, and neglected disease that affects more than 90 countries in the world. More than 20 Leishmania species cause different forms of leishmaniasis that range in severity from cutaneous lesions to systemic infection. The diversity of leishmaniasis forms is due to the species of parasite, vector, environmental and social factors, genetic background, nutritional status, as well as immunocompetence of the host. Here, we discuss the role of the immune system, its molecules, and responses in the establishment, development, and outcome of Leishmaniasis, focusing on innate immune cells and Leishmania major interactions.

Keywords: immunology; immunomodulation; immunoparasitology; infection; leishmaniasis.

Prat-Luri B, Neal C, Passelli K, Ganga E, Amore J, Firmino-Cruz L, Petrova TV, Müller AJ, Tacchini-Cottier F. The C5a-C5aR1 complement axis is essential for neutrophil recruitment to draining lymph nodes via high endothelial venules in cutaneous leishmaniasis. Cell Rep. 2022 May 3;39(5):110777.

DOI: 10.1016/j.celrep.2022.110777

Neutrophils are specialized innate immune cells known for their ability to fight pathogens. However, the mechanisms of neutrophil trafficking to lymph nodes are not fully clear. Using a murine model of dermal infection with Leishmania parasites, we observe a transient neutrophil influx in draining lymph nodes despite sustained recruitment to the infection site. Cell-tracking experiments, together with intravital two-photon microscopy, indicate that neutrophil recruitment to draining lymph nodes occurs minimally through lymphatics from the infected dermis, but mostly through blood vessels via high endothelial venules. Mechanistically, neutrophils do not respond to IL-1β or macrophage-derived molecules. Instead, they are guided by the C5a-C5aR1 axis, using L-selectin and integrins, to extravasate into the draining lymph node parenchyma. We also report that C5, the C5a precursor, is locally produced in the draining lymph node by lymphatic endothelial cells. Our data establish and detail organ-specific mechanisms of neutrophil trafficking.

Keywords: neutrophils, migration, lymph node, lymphatics, blood vessels, complement, Leishmania, cutaneous leishmaniasis, swarming.

Incluir o Abstract

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Barroso MV, Gropillo I, Detoni MAA, Thompson-Souza GA, Muniz VS, Vasconcelos CRI, Figueiredo RT, Melo RCN, Neves JS. Structural and Signaling Events Driving Aspergillus fumigatus-Induced Human Eosinophil Extracellular Trap Release. Front Microbiol. 2021 Feb 18;12:633696.

DOI: 10.3389/fmicb.2021.633696

Eosinophils are granulocytes classically involved in allergic diseases and in the host immune responses to helminths, fungi, bacteria and viruses. The release of extracellular DNA traps by leukocytes is an important mechanism of the innate immune response to pathogens in various infectious conditions, including fungal infections. Aspergillus fumigatus is an opportunistic fungus responsible for allergic bronchopulmonary aspergillosis (ABPA), a pulmonary disease marked by prominent eosinophilic inflammation. Previously, we demonstrated that isolated human eosinophils release extracellular DNA traps (eosinophil extracellular traps; EETs) when stimulated by A. fumigatus in vitro. This release occurs through a lytic non-oxidative mechanism that involves CD11b and Syk tyrosine kinase. In this work, we unraveled different intracellular mechanisms that drive the release of extracellular DNA traps by A. fumigatus-stimulated eosinophils. Ultrastructurally, we originally observed that A. fumigatus-stimulated eosinophils present typical signs of extracellular DNA trap cell death (ETosis) with the nuclei losing both their shape (delobulation) and the euchromatin/heterochromatin distinction, followed by rupture of the nuclear envelope and EETs release. We also found that by targeting class I PI3K, and more specifically PI3Kδ, the release of extracellular DNA traps induced by A. fumigatus is inhibited. We also demonstrated that A. fumigatus-induced EETs release depends on the Src family, Akt, calcium and p38 MAPK signaling pathways in a process in which fungal viability is dispensable. Interestingly, we showed that A. fumigatus-induced EETs release occurs in a mechanism independent of PAD4 histone citrullination. These findings may contribute to a better understanding of the mechanisms that underlie EETs release in response to A. fumigatus, which may lead to better knowledge of ABPA pathophysiology and treatment.

Keywords: A. fumigatus; ABPA; allergic bronchopulmonary aspergillosis; eosinophils; extracellular DNA traps.

Oliveira-Maciel D, Dos-Santos JS, Oliveira-Silva G, Mello MF, da Fonseca-Martins AM, Carneiro MPD, Ramos TD, Firmino-Cruz L, Gomes DCO, Rossi-Bergmann B, de Matos Guedes HL. MPLA and AddaVax® Adjuvants Fail to Promote Intramuscular LaAg Vaccine Protectiveness against Experimental Cutaneous Leishmaniasis. Microorganisms. 2021 Jun 11;9(6):1272.

DOI: 10.3390/microorganisms9061272

There is so far no vaccine approved for human leishmaniasis, mainly because of the lack of appropriate adjuvants. This study aimed to evaluate in mice the capacity of a mixture of monophosphoryl lipid A (MPLA) and AddaVax^® adjuvants in enhancing the efficacy of a Leishvacin^®-like vaccine comprised of Leishmania amazonensis whole antigens (LaAg). For that, mice were immunized with LaAg plus MPLA/AddaVax^® by the intramuscular route (i.m.) prior to challenge with 2 × 10⁵ and 2 × 10⁶ living parasites. Immunization with LaAg alone reduced the lesion growth of the 2 × 10⁵-challenged mice only in the peak of infection, but that was not accompanied by reduced parasite load, and thus not considered protective. Mice given a 2 × 10⁶ -challenge were not protected by LaAg. The association of LaAg with MPLA/AddaVax^® was able to enhance the cutaneous hypersensitivity response compared with LaAg alone. Despite this, there was no difference in proliferative cell response to antigen ex vivo. Moreover, regardless of the parasite challenge, association of LaAg with MPL/AddaVax^® did not significantly enhance protection in comparison with LaAg alone. This work demonstrated that MPL/AddaVax^® is not effective in improving the efficacy of i.m. LaAg vaccine against cutaneous leishmaniasis.

Keywords: Leishmania amazonensis; LaAg vaccine; adjuvants; MPLA; AddaVax^®; intramuscular; immunization; C57BL/6

Ferreira AC, Soares VC, de Azevedo-Quintanilha IG, Dias SDSG, Fintelman-Rodrigues N, Sacramento CQ, Mattos M, de Freitas CS, Temerozo JR, Teixeira L, Damaceno Hottz E, Barreto EA, Pão CRR, Palhinha L, Miranda M, Bou-Habib DC, Bozza FA, Bozza PT, Souza TML. SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes. Cell Death Discov. 2021 Mar 1;7(1):43.

DOI: 10.1038/s41420-021-00428-w

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with 2019 coronavirus disease (COVID-19). Here, we show that SARS-CoV-2 engages inflammasome and triggers pyroptosis in human monocytes, experimentally infected, and from patients under intensive care. Pyroptosis associated with caspase-1 activation, IL-1ß production, gasdermin D cleavage, and enhanced pro-inflammatory cytokine levels in human primary monocytes. At least in part, our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe COVID-19.

Silva RCMC, Panis C, Pires BRB. Lessons from transmissible cancers for immunotherapy and transplant. Immunol Med. 2021 Dec 28:1-16.

DOI: 10.1080/25785826.2021.2018783

The emergence of horizontal transmission of cancer between vertebrates is an issue that interests scientists and medical society. Transmission requires: (i) a mechanism by which cancer cells can transfer to another organism and (ii) a repressed immune response on the part of the recipient. Transmissible tumors are unique models to comprehend the responses and mechanisms mediated by the major histocompatibility complex (MHC), which can be transposed for transplant biology. Here, we discuss the mechanisms involved in immune-mediated tissue rejection, making a parallel with transmissible cancers. We also discuss cellular and molecular mechanisms involved in cancer immunotherapy and anti-rejection therapies.

Keywords: Transmissible cancer, MHC, transplant, immunotherapy

Vasconcellos LRC, Martimiano L, Dantas DP, Fonseca FM, Mata-Santos H, Travassos L, Mendez-Otero R, Bozza MT, Pimentel-Coelho PM. Intracerebral Injection of Heme Induces Lipid Peroxidation, Neuroinflammation, and Sensorimotor Deficits. Stroke. 2021 May;52(5):1788-1797.

DOI: 10.1161/STROKEAHA.120.031911

Background and purpose: Heme is a red blood cell component released in the brain parenchyma following intracerebral hemorrhage. However, the study of the pathophysiological mechanisms triggered by heme in the brain is hampered by the lack of well-established in vivo models of intracerebral heme injection. This study aims to optimize and characterize a protocol of intrastriatal heme injection in mice, with a focus on the induction of lipid peroxidation, neuroinflammation and, ultimately, sensorimotor deficits. We also evaluated the involvement of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome sensor, in the behavior deficits induced by heme in this model.

Methods: Mice were injected with heme in the striatum for the evaluation of neuroinflammation and brain damage through histological and biochemical techniques. Immunoblot was used to evaluate the expression of proteins involved in heme/iron metabolism and antioxidant responses and the activation of the MAPK (mitogen-activated protein kinase) signaling pathway. For the assessment of neurological function, we followed-up heme-injected mice for 2 weeks using the rotarod, elevated body swing, and cylinder tests. Mice injected with the vehicle (sham), or autologous blood were used as controls.

Results: Heme induced lipid peroxidation and inflammation in the brain. Moreover, heme increased the expression of HO-1 (heme oxygenase-1), ferritin, p62, and superoxide dismutase 2, and activated the MAPK signaling pathway promoting pro-IL (interleukin)-1β production and its cleavage to the active form. Heme-injected mice exhibited signs of brain damage and reactive astrogliosis around the injection site. Behavior deficits were observed after heme or autologous blood injection in comparison to sham-operated controls. In addition, behavior deficits and IL-1β production were reduced in Nlrp3 knockout mice in comparison to wild-type mice.

Conclusions: Our results show that intracerebral heme injection induces neuroinflammation, and neurological deficits, in an NLRP3-dependent manner, suggesting that this is a feasible model to evaluate the role of heme in neurological.

Keywords: behavior; blood cells; cerebral hemorrhage; heme; inflammation.

Silva RCMC, Tan L, Rodrigues DA, Prestes EB, Gomes CP, Gama AM, Oliveira PL, Paiva CN, Manoury B, Bozza MT. Chloroquine inhibits pro-inflammatory effects of heme on macrophages and invivo. Free Radic Biol Med. 2021 Sep;173:104-116.

DOI: 10.1016/j.freeradbiomed.2021.07.028

Background: Chloroquine has been used successfully to treat Malaria, including by chloroquine-resistant Plasmodium sp., indicating that it has effects on disease itself. Since heme has inflammatory effects and contributes to the pathogenesis of hemolytic diseases, we hypothesize that the anti-inflammatory effect of chloroquine is partially due to its inhibitory effect on heme-induced macrophage activation and on inflammatory tissue damage.

Methods: Bone marrow derived macrophages (BMDMs) were incubated with chloroquine before stimulation with heme, in different conditions, to evaluate cytokines secretion, ROS production, mitogen activated protein kinases (MAPK) or spleen tyrosine kinase (Syk) activation, alone or combined with LPS. The effects of chloroquine upon heme inflammation were also evaluated in vivo, through simultaneous i.p. injection of LPS and heme, intratracheal instillation of Poly-IC followed by heme injection, and in a rhabdomyolysis model.

Results: Chloroquine inhibited TNF secretion, mitochondrial ROS production, MAPK, and Syk activation induced by heme. Inhibition of TNF production could be mimicked by zinc ionophore quercetin, but not by primaquine, a chloroquine analog with low affinity for heme. IL-6 and IL-1β secretions induced by heme in the presence of PRRs agonists were inhibited by chloroquine, but not by calcium chelator BAPTA or inhibitor of endosomal acidification concamycin B. Chloroquine also protected mice from heme inflammatory effects in vivo, inhibiting lethal synergism with PRR agonists, lung pathology caused by heme injection after intratracheal instillation of Poly-IC, and delaying death after rhabdomyolisis.

Keywords: Chloroquine; Cytokines; Heme; Inflammation; ROS

Moura Silva H, Kitoko JZ, Queiroz CP, Kroehling L, Matheis F, Yang KL, Reis BS, Ren-Fielding C, Littman DR, Bozza MT, Mucida D, Lafaille JJ. c-MAF-dependent perivascular macrophages regulate diet-induced metabolic syndrome. Sci Immunol. 2021 Oct;6(64):eabg7506.

DOI: 10.1126/sciimmunol.abg7506

Macrophages are an essential part of tissue development and physiology. Perivascular macrophages have been described in tissues and appear to play a role in development and disease processes, although it remains unclear what the key features of these cells are. Here, we identify a subpopulation of perivascular macrophages in several organs, characterized by their dependence on the transcription factor c-MAF and displaying nonconventional macrophage markers including LYVE1, folate receptor 2, and CD38. Conditional deletion of c-MAF in macrophage lineages caused ablation of perivascular macrophages in the brain and altered muscularis macrophages program in the intestine. In the white adipose tissue (WAT), c-MAF–deficient perivascular macrophages displayed an altered gene expression profile, which was linked to an increased vascular branching. Upon feeding high-fat diet (HFD), mice with c-MAF–deficient macrophages showed improved metabolic parameters compared with wild-type mice, including less weight gain, greater glucose tolerance, and reduced inflammatory cell profile in WAT. These results define c-MAF as a central regulator of the perivascular macrophage transcriptional program in vivo and reveal an important role for this tissue-resident macrophage population in the regulation of metabolic syndrome.

Jennings-Almeida B, Castelpoggi JP, Ramos-Junior ES, Ferreira EO, Domingues RMCP, Echevarria-Lima J, Coutinho-Silva R, Moreira-Souza ACA, Mariño E, Mackay CR, Zamboni DS, Bellio M, Scharfstein J, Lobo LA, Oliveira AC. Dietary Fiber Drives IL-1β-Dependent Peritonitis Induced by Bacteroides fragilis via Activation of the NLRP3 Inflammasome. J Immunol. 2021 May 15;206(10):2441-2452.

DOI: 10.4049/jimmunol.2000078

Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1β contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1β secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1β–dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis, activates canonical pathway of NLRP3 promoting IL-1β secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1β production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1β production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1β–dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R–deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1β secretion outside the gut.

Viana AS, Nunes Botelho AM, Moustafa AM, Boge CLK, Pires Ferreira AL, da Silva Carvalho MC, Guimarães MA, Costa BSS, de Mattos MC, Maciel SP, Echevarria-Lima J, Narechania A, O'Brien K, Ryan C, Gerber JS, Carvalho BTF, Figueiredo AMS, Planet PJ. Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Associated with Bacteremia and Monocyte Evasion, Rio de Janeiro, Brazil. Emerg Infect Dis. 2021;27(11):2825-2835.

DOI: 10.3201/eid2711.210097

We typed 600 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 51 hospitals in the Rio de Janeiro, Brazil, metropolitan area during 2014–2017. We found that multiple new clonal complex (CC) 5 sequence types had replaced previously dominant MRSA lineages in hospitals. Whole-genome analysis of 208 isolates revealed an emerging sublineage of multidrug-resistant MRSA, sequence type 105, staphylococcal cassette chromosome mec II, spa t002, which we designated the Rio de Janeiro (RdJ) clone. Using molecular clock analysis, we hypothesized that this lineage began to expand in the Rio de Janeiro metropolitan area in 2009. Multivariate analysis supported an association between bloodstream infections and the CC5 lineage that includes the RdJ clone. Compared with other closely related isolates, representative isolates of the RdJ clone more effectively evaded immune function related to monocytic cells, as evidenced by decreased phagocytosis rate and increased numbers of viable unphagocytosed (free) bacteria after in vitro exposure to monocytes.

Keywords: Brazil; MRSA; MRSA and other staphylococci; Rio de Janeiro; Staphylococcus aureus; antimicrobial resistance; bacteremia; bacteria; bacterial infection; bloodstream infections; drug resistance; methicillin-resistant Staphylococcus aureus; molecular epidemiology; monocyte evasion; monocytes; multidrug-resistance; phagocytosis; phylogenetics.

Silva JBNF, Calcia TBB, Silva CP, Guilherme RF, Almeida-Souza F, Lemos FS, Calabrese KS, Caruso-Neves C, Neves JS, Benjamim CF. ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice. Int J Mol Sci. 2021 Oct 27;22(21):11634.

DOI: 10.3390/ijms222111634

Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor β (TGFβ), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1β (IL-1β) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.

Keywords: sepsis; renal tubulointerstitial injury; resolvin; ATRvD1; inflammation; kidney 

Guimaraes-Costa AB, Shannon JP, Waclawiak I, Oliveira J, Meneses C, de Castro W, Wen X, Brzostowski J, Serafim TD, Andersen JF, Hickman HD, Kamhawi S, Valenzuela JG, Oliveira F. A sand fly salivary protein acts as a neutrophil chemoattractant. Nat Commun. 2021 May 28;12(1):3213.

DOI: 10.1038/s41467-021-23002-5

Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

Borges PA, Waclawiak I, Georgii JL, Fraga-Junior VDS, Barros JF, Lemos FS, Russo-Abrahão T, Saraiva EM, Takiya CM, Coutinho-Silva R, Penido C, Mermelstein C, Meyer-Fernandes JR, Canto FB, Neves JS, Melo PA, Canetti C, Benjamim CF. Adenosine Diphosphate Improves Wound Healing in Diabetic Mice Through P2Y12 Receptor Activation. Front Immunol. 2021 Mar 22;12:651740.

DOI: 10.3389/fimmu.2021.651740

Chronic wounds are a public health problem worldwide, especially those related to diabetes. Besides being an enormous burden to patients, it challenges wound care professionals and causes a great financial cost to health system. Considering the absence of effective treatments for chronic wounds, our aim was to better understand the pathophysiology of tissue repair in diabetes in order to find alternative strategies to accelerate wound healing. Nucleotides have been described as extracellular signaling molecules in different inflammatory processes, including tissue repair. Adenosine-5'-diphosphate (ADP) plays important roles in vascular and cellular response and is immediately released after tissue injury, mainly from platelets. However, despite the well described effect on platelet aggregation during inflammation and injury, little is known about the role of ADP on the multiple steps of tissue repair, particularly in skin wounds. Therefore, we used the full-thickness excisional wound model to evaluate the effect of local ADP application in wounds of diabetic mice. ADP accelerated cutaneous wound healing, improved new tissue formation, and increased both collagen deposition and transforming growth factor-β (TGF-β) production in the wound. These effects were mediated by P2Y12 receptor activation since they were inhibited by Clopidogrel (Clop) treatment, a P2Y12 receptor antagonist. Furthermore, P2Y1 receptor antagonist also blocked ADP-induced wound closure until day 7, suggesting its involvement early in repair process. Interestingly, ADP treatment increased the expression of P2Y12 and P2Y1 receptors in the wound. In parallel, ADP reduced reactive oxygen species (ROS) formation and tumor necrosis factor-α (TNF-α) levels, while increased IL-13 levels in the skin. Also, ADP increased the counts of neutrophils, eosinophils, mast cells, and gamma delta (γδ) T cells (Vγ4+ and Vγ5+ cells subtypes of γδ+ T cells), although reduced regulatory T (Tregs) cells in the lesion. In accordance, ADP increased fibroblast proliferation and migration, myofibroblast differentiation, and keratinocyte proliferation. In conclusion, we provide strong evidence that ADP acts as a pro-resolution mediator in diabetes-associated skin wounds and is a promising intervention target for this worldwide problem.

Keywords: P2Y12 recepor; adenosine diphosphate (ADP); diabetes; inflammation; mice; purinergic signaling; skin; wound healing.

da Fonseca-Martins AM, de Souza Lima-Gomes P, Antunes MM, de Moura RG, Covre LP, Calôba C, Rocha VG, Pereira RM, Menezes GB, Gomes DCO, Saraiva EM, de Matos Guedes HL. Leishmania Parasites Drive PD-L1 Expression in Mice and Human Neutrophils With Suppressor Capacity. Front Immunol. 2021 Jun 15;12:598943.

DOI: 10.3389/fimmu.2021.598943

Neutrophils play an important role in the outcome of leishmaniasis, contributing either to exacerbating or controlling the progression of infection, a dual effect whose underlying mechanisms are not clear. We recently reported that CD4+ and CD8+ T cells, and dendritic cells of Leishmania amazonensis-infected mice present high expression of PD-1 and PD-L1, respectively. Given that the PD-1/PD-L1 interaction may promote cellular dysfunction, and that neutrophils could interact with T cells during infection, we investigated here the levels of PD-L1 in neutrophils exposed to Leishmania parasites. We found that both, promastigotes and amastigotes of L. amazonensis induced the expression of PD-L1 in the human and murine neutrophils that internalized these parasites in vitro. PD-L1-expressing neutrophils were also observed in the ear lesions and the draining lymph nodes of L. amazonensis-infected mice, assessed through cell cytometry and intravital microscopy. Moreover, expression of PD-L1 progressively increased in neutrophils from ear lesions as the disease evolved to the chronic phase. Co-culture of infected neutrophils with in vitro activated CD8+ T cells inhibits IFN-γ production by a mechanism dependent on PD-1 and PD-L1. Importantly, we demonstrated that in vitro infection of human neutrophils by L braziliensis induced PD-L1+ expression and also PD-L1+ neutrophils were detected in the lesions of patients with cutaneous leishmaniasis. Taken together, these findings suggest that the Leishmania parasite increases the expression of PD-L1 in neutrophils with suppressor capacity, which could favor the parasite survival through impairing the immune response.

Keywords: Leishmania; PD-L1; human cutaneous leishmaniasis; murine leishmaniasis; neutrophils; skin; supression.

Neris RLS, Dobles AMC, Gomes AV. Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Advantages of Stain-Free Technology. Methods Mol Biol. 2021;2261:443-456.

DOI: 10.1007/978-1-0716-1186-9_28

Western blotting is one of the most used techniques in research laboratories. It is popular because it is an easy way of semiquantifying protein amounts in different samples. In Western blotting, the most commonly used method for controlling the differences in the amount of protein loaded is to independently quantify housekeeping proteins (typically actin, GAPDH or tubulin). Another less commonly used method is total protein normalization using stains, such as Ponceau S or Coomassie Brilliant Blue, which stains all the proteins on the blots. A less commonly used but powerful total protein staining technique is stain-free normalization. The stain-free technology is able to detect total protein in a large linear dynamic range and has the advantage of allowing protein detection on the gel before transblotting. This chapter discusses the theory, advantages, and method used to do total protein quantification using stain-free gels for normalization of Western blots.

Keywords: Immunoblotting, Loading control, Stain-free technology, Total protein normalization, Western blotting

Gavino-Leopoldino D, Figueiredo CM, da Silva MOL, Barcellos LG, Neris RLS, Pinto LDM, Araújo SMB, Ladislau L, Benjamim CF, Da Poian AT, Clarke JR, Figueiredo CP, Assunção-Miranda I. Skeletal Muscle Is an Early Site of Zika Virus Replication and Injury, Which Impairs Myogenesis. J Virol. 2021 Oct 27;95(22):e0090421

DOI: 10.1128/JVI.00904-21

Zika virus (ZIKV) infection became a worldwide concern due to its correlation with the development of microcephaly and other neurological disorders. ZIKV neurotropism is well characterized, but the role of peripheral viral amplification to brain infection remains unknown. Here, we found that ZIKV replicates in human primary skeletal muscle myoblasts, impairing its differentiation into myotubes but not interfering with the integrity of the already-formed muscle fibers. Using mouse models, we showed ZIKV tropism to muscle tissue either during embryogenesis after maternal transmission or when infection occurred after birth. Interestingly, ZIKV replication in the mouse skeletal muscle started immediately after ZIKV inoculation, preceding viral RNA detection in the brain and causing no disruption to the integrity of the blood brain barrier, and remained active for more than 2 weeks, whereas replication in the spleen and liver were not sustained over time. In addition, ZIKV infection of the skeletal muscle induces necrotic lesions, inflammation, and fiber atrophy. We also found a reduction in the expression of regulatory myogenic factors that are essential for muscle repair after injury. Taken together, our results indicate that the skeletal muscle is an early site of viral amplification and lesion that may result in late consequences in muscle development after ZIKV infection.

IMPORTANCE Zika Virus (ZIKV) neurotropism and its deleterious effects on central nervous system have been well characterized. However, investigations of the initial replication sites for the establishment of infection and viral spread to neural tissues remain underexplored. A complete description of the range of ZIKV-induced lesions and others factors that can influence the severity of the disease is necessary to prevent ZIKV’s deleterious effects. ZIKV has been shown to access the central nervous system without significantly affecting blood-brain barrier permeability. Here, we demonstrated that skeletal muscle is an earlier site of ZIKV replication, contributing to the increase of peripheral ZIKV load. ZIKV replication in muscle promotes necrotic lesions and inflammation and also impairs myogenesis. Overall, our findings showed that skeletal muscle is involved in pathogenesis and opens new fields in the investigation of the long-term consequences of early infection.

Keywords: Zika virus replication; muscle inflammation; myogenesis; pathogenesis; skeletal muscle; viral dissemination.

Andrade CBV, Monteiro VRS, Coelho SVA, Gomes HR, Sousa RPC, Nascimento VMO, Bloise FF, Matthews SG, Bloise E, Arruda LB, Ortiga-Carvalho TM. ZIKV Disrupts Placental Ultrastructure and Drug Transporter Expression in Mice. Front Immunol. 2021 May 21;12:680246.

DOI: 10.3389/fimmu.2021.680246

Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome.

Keywords: ABCA1; ABCG1; P-glycoprotein (P-gp) breast cancer resistance protein (BCRP); ZIKV; chemokine 2; cytokine; placenta; ultrastructure.

Juliano MA, Costa SM, Alves AMB, Cordeiro MT, Marques ETA, Scharfstein J, Arruda LB. Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors. Pharmaceuticals (Basel). 2021 Jan 12;14(1):56.

DOI: 10.3390/ph14010056

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.

Keywords: dengue; bradykinin; endothelial cells; kallikrein-kinin system; contact pathway; bradykinin receptor B2

Vicente Santos AC, Guedes-da-Silva FH, Dumard CH, Ferreira VNS, da Costa IPS, Machado RA, Barros-Aragão FGQ, Neris RLS, Dos-Santos JS, Assunção-Miranda I, Figueiredo CP, Dias AA, Gomes AMO, de Matos Guedes HL, Oliveira AC, Silva JL. Yellow fever vaccine protects mice against Zika virus infection. PLoS Negl Trop Dis. 2021 Nov 4;15(11):e0009907. 

DOI: 10.1371/journal.pntd.0009907

Zika virus (ZIKV) emerged as an important infectious disease agent in Brazil in 2016. Infection usually leads to mild symptoms, but severe congenital neurological disorders and Guillain-Barré syndrome have been reported following ZIKV exposure. Creating an effective vaccine against ZIKV is a public health priority. We describe the protective effect of an already licensed attenuated yellow fever vaccine (YFV, 17DD) in type-I interferon receptor knockout mice (A129) and immunocompetent BALB/c and SV-129 (A129 background) mice infected with ZIKV. YFV vaccination provided protection against ZIKV, with decreased mortality in A129 mice, a reduction in the cerebral viral load in all mice, and weight loss prevention in BALB/c mice. The A129 mice that were challenged two and three weeks after the first dose of the vaccine were fully protected, whereas partial protection was observed five weeks after vaccination. In all cases, the YFV vaccine provoked a substantial decrease in the cerebral viral load. YFV immunization also prevented hippocampal synapse loss and microgliosis in ZIKV-infected mice. Our vaccine model is T cell-dependent, with AG129 mice being unable to tolerate immunization (vaccination is lethal in this mouse model), indicating the importance of IFN-γ in immunogenicity. To confirm the role of T cells, we immunized nude mice that we demonstrated to be very susceptible to infection. Immunization with YFV and challenge 7 days after booster did not protect nude mice in terms of weight loss and showed partial protection in the survival curve. When we evaluated the humoral response, the vaccine elicited significant antibody titers against ZIKV; however, it showed no neutralizing activity in vitro and in vivo. The data indicate that a cell-mediated response promotes protection against cerebral infection, which is crucial to vaccine protection, and it appears to not necessarily require a humoral response. This protective effect can also be attributed to innate factors, but more studies are needed to strengthen this hypothesis. Our findings open the way to using an available and inexpensive vaccine for large-scale immunization in the event of a ZIKV outbreak.

Diniz-Lima I, da Rosa PR, da Silva-Junior EB, Guimarães-de-Oliveira JC, de Freitas EO, de Oliveira Nascimento D, Morrot A, Nimrichter L, Previato JO, Mendonça-Previato L, Freire-de-Lima L, Decote-Ricardo D, Freire-de-Lima CG. X-linked immunodeficient (XID) mice exhibit high susceptibility to Cryptococcus gattii infection. Sci Rep. 2021 Sep 15;11(1):18397.

DOI: : 10.1038/s41598-021-97041-9

Cryptococcosis is an opportunistic disease caused by the fungus Cryptococcus neoformans and Cryptococcus gattii. It starts as a pulmonary infection that can spread to other organs, such as the brain, leading to the most serious occurrence of the disease, meningoencephalitis. The humoral response has already been described in limiting the progression of cryptococcosis where the B-1 cell seems to be responsible for producing natural IgM antibodies, crucial for combating fungal infections. The role of the B-1 cell in C. neoformans infection has been initially described, however the role of the humoral response of B-1 cells has not yet been evaluated during C. gattii infections. In the present study we tried to unravel this issue using XID mice, a murine model deficient in the Btk protein which compromises the development of B-1 lymphocytes. We use the XID mice compared to BALB/c mice that are sufficient for the B-1 population during C. gattii infection. Our model of chronic lung infection revealed that XID mice, unlike the sufficient group of B-1, had early mortality with significant weight loss, in addition to reduced levels of IgM and IgG specific to GXM isolated from the capsule of C. neoformans. In addition to this, we observed an increased fungal load in the blood and in the brain. We described an increase in the capsular size of C. gattii and the predominant presence of cytokines with a Th2 profile was also observed in these animals. Thus, the present study strongly points to a higher susceptibility of the XID mouse to C. gattii, which suggests that the presence of B-1 cells and anti-GXM antibodies is fundamental during the control of infection by C. gattii.

da Silva-Junior EB, Firmino-Cruz L, Guimarães-de-Oliveira JC, De-Medeiros JVR, de Oliveira Nascimento D, Freire-de-Lima M, de Brito-Gitirana L, Morrot A, Previato JO, Mendonça-Previato L, Decote-Ricardo D, de Matos Guedes HL, Freire-de-Lima CG. The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection. Sci Rep. 2021 Jan 14;11(1):1407.

DOI: 10.1038/s41598-021-80959-5

Toll-like receptor 9 (TLR9) is crucial to the host immune response against fungi, such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, but its importance in Cryptococcus gattii infection is unknown. Our study aimed to understand the role of TLR9 during the course of experimental C. gattii infection in vivo, considering that the cryptococcal DNA interaction with the receptor could contribute to host immunity even in an extremely susceptible model. We inoculated C57BL/6 (WT) and TLR9 knock-out (TLR9-/-) mice intratracheally with 104 C. gattii yeast cells. TLR9-/- mice had a higher mortality rate compared to WT mice and more yeast cells that had abnormal size, known as titan cells, in the lungs. TLR9-/- mice also had a greater number of CFUs in the spleen and brain than WT mice, in addition to having lower levels of IFN-γ and IL-17 in the lung. With these markers of aggressive cryptococcosis, we can state that TLR9-/- mice are more susceptible to C. gattii, probably due to a mechanism associated with the decrease of a Th1 and Th17-type immune response that promotes the formation of titan cells in the lungs. Therefore, our results indicate the participation of TLR9 in murine resistance to C. gattii infection.

SOUSA, S. M. dos S.; BANDEIRA, D. R.; QUINTANILHA , L. F. da C.; BALDAÇARA, R. P. de C.; CAVALCANTE, C. P. A.; SILVA, J. B. N. F. The influence of the gut microbiota on the development of food allergy in children . Research, Society and Development, [S. l.], v. 10, n. 14, p. e293101422156, 2021.

DOI: 10.33448/rsd-v10i14.22156

The gut microbiota, which is formed mainly early in life, plays a crucial role in the formation of the immune system and in food tolerance. Dysbiosis, which means an overgrowth of pathobiont bacteria, creates imbalance and inflammation of the gut microbiota, affecting its function. Because of this, there was interest in evaluating the influence of the microbiota on the development of food allergies in children, analyzing the interference of dysbiosis on the immune system, by the method of integrative review in the last 10 years, since there is an increase in the prevalence of food allergies in children in the last decade. This research is characterized as an integrative literature review, in which articles published in the last 10 years, selected through the inclusion criteria, were analyzed. It is based on the theme "The influence of the microbiota on the development of food allergy in children". Literature published in English, Portuguese and Spanish was sought as the study priority. The intestinal microbiota is directly related to the immune system, and malfunction of one is followed by imbalance of the other. Thus, the composition of a comprehensive and functional gut microbiota contributes to a more resilient immune system and can lead to a greater tolerance to food allergies. However, we conclude that dysbiosis is more conducive to the onset of allergies. Despite the genetic contribution, it has the contribution of environmental factors and hygienic factors that favor the onset of food allergy.

Keywords:  microbiota; dysbiosis; food hypersensitivity.; Dysbiosis; Food hypersensitivity.

Cavazzoni CB, Bozza VBT, Lucas TCV, Conde L, Maia B, Mesin L, Schiepers A, Ersching J, Neris RLS, Conde JN, Coelho DR, Lima TM, Alvim RGF, Castilho LR, de Paula Neto HA, Mohana-Borges R, Assunção-Miranda I, Nobrega A, Victora GD, Vale AM. The immunodominant antibody response to Zika virus NS1 protein is characterized by cross-reactivity to self. J Exp Med. 2021 Sep 6;218(9):e20210580. 

DOI: 10.1084/jem.20210580

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations.

Rigoni TS, Vellozo NS, Cabral-Piccin M, Fabiano-Coelho L, Lopes UG, Filardy AA, DosReis GA, Lopes MF. RANK Ligand Helps Immunity to Leishmania major by Skewing M2-Like Into M1 Macrophages. Front Immunol. 2020 May 12;11:886.

DOI: https://doi.org/10.3389/fimmu.2020.00886

Macrophages host Leishmania major infection, which causes cutaneous Leishmaniasis in humans. In the murine model, resistance to infection depends on the host immunity mediated by CD4 T-cell cytokines and macrophages. In association to other stimuli, the Th1 cytokine IFN-γ induces NO-mediated microbial killing by M1/classically-activated macrophages. By contrast, the Th2 cytokine IL-4 promotes M2/alternatively activated macrophages, which express arginase-1 and shelter infection. Other cytokines, such as RANKL, might also participate in the crosstalk between T cells and macrophages to restrict parasite infection. RANKL and its receptor RANK are known to play an essential role in bone remodeling, by inducing osteoclatogenesis. It has also been shown that RANKL stimulates antigen-presenting cells, such as DCs and macrophages, to enhance T cell responses. Here we investigated how RANKL directly modulates the effector macrophage phenotypes and immunity to L. major parasites. We found that inflammatory peritoneal macrophages from B6 mice express RANK and M2 features, such as CD301 (MGL) and CD206 (mannose receptor). Nonetheless, treatment with RANKL or IFN-γ induced macrophage differentiation into more mature F40/80hi macrophages able to produce IL-12 and TNF-α. In parallel, macrophages treated with RANKL, IFN-γ, or RANKL along with IFN-γ progressively downregulated the expression of the M2 hallmarks MGL, arginase-1, and CCL17. Moreover, a synergism between IFN-γ and RANKL enhanced inducible NO synthase (iNOS) expression and NO production by macrophages. These results are consistent with the idea that RANKL helps IFN-γ to induce a M2-like to M1 phenotype shift. Accordingly, concomitant treatment with RANKL and IFN-γ promoted macrophage-mediated immunity to L. major, by inducing NO and ROS-dependent parasite killing. Furthermore, by cooperating with IFN-γ, endogenous RANKL engages CD4 T-cell help toward L. major-infected macrophages to upregulate M1 and Th1 cytokine responses. Therefore, RANKL, in combination with IFN-γ, is a potential local therapeutic tool to improve immune responses in Leishmaniasis, by skewing M2-like into effector M1 macrophages.

Bozza MT, Lintomen L, Kitoko JZ, Paiva CN, Olsen PC. The Role of MIF on Eosinophil Biology and Eosinophilic Inflammation. Clin Rev Allergy Immunol. 2020 Feb;58(1):15-24.

DOI: https://doi.10.1007/s12016-019-08726-z

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that participates in innate and adaptive immune responses. MIF contributes to the resistance against infection agents, but also to the cellular and tissue damage in infectious, autoimmune, and allergic diseases. In the past years, several studies demonstrated a critical role for MIF in the pathogenesis of type-2-mediated inflammation, including allergy and helminth infection. Atopic patients have increased MIF amounts in affected tissues, mainly produced by immune cells such as macrophages, Th2 cells, and eosinophils. Increased MIF mRNA and protein are found in activated Th2 cells, while eosinophils stock pre-formed MIF protein and secrete high amounts of MIF upon stimulation. In mouse models of allergic asthma, the lack of MIF causes an almost complete abrogation of the cardinal signs of the disease including mucus secretion, eosinophilic inflammation, and airway hyper-responsiveness. Additionally, blocking the expression of MIF in animal models leads to significant reduction of pathological signs of eosinophilic inflammation such as rhinitis, atopic dermatitis, eosinophilic esophagitis and helminth infection. A number of studies indicate that MIF is important in the effector phase of type-2 immune responses, while its contribution to Th2 differentiation and IgE production is not consensual. MIF has been found to intervene in different aspects of eosinophil physiology including differentiation, survival, activation, and migration. CD4+ T cells and eosinophils express CD74 and CXCR4, receptors able to signal upon MIF binding. Blockage of these receptors with neutralizing antibodies or small molecule antagonists also succeeds in reducing the signals of inflammation in experimental allergic models. Together, these studies demonstrate an important contribution of MIF on eosinophil biology and in the pathogenesis of allergic diseases and helminth infection.

Castro LL, Kitoko JZ, Xisto DG, Olsen PC, Guedes HLM, Morales MM, Lopes-Pacheco M, Cruz FF, Rocco PRM. Multiple doses of adipose tissue-derived mesenchymal stromal cells induce immunosuppression in experimental asthma. Stem Cells Transl Med. 2020 Feb;9(2):250-260.

DOI: https://doi.org/10.1002/sctm.19-0120

In experimental house dust mite (HDM)-induced allergic asthma, therapeutic administration of a single dose of adipose tissue-derived mesenchymal stromal cells (MSCs) ameliorates lung inflammation but is unable to reverse remodeling. We hypothesized that multiple doses of MSCs might exert better therapeutic effects by reducing lung inflammation and remodeling but might also result in immunosuppressive effects in experimental asthma. HDM was administered intranasally in C57BL/6 mice. After the last HDM challenge, mice received two or three doses of MSCs (105 cells per day) or saline intravenously. An additional cohort of mice received dexamethasone as a positive control for immunosuppression. Two and three doses of MSCs reduced lung inflammation, levels of interleukin (IL)-4, IL-13, and eotaxin; total leukocyte, CD4+ T-cell, and eosinophil counts in bronchoalveolar lavage fluid; and total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes. Two and three doses of MSCs also reduced collagen fiber content and transforming growth factor-β levels in lung tissue; however, the three-dose regimen was more effective, and reduced these parameters to control levels, while also decreasing α-actin content in lung tissue. Two and three doses of MSCs improved lung mechanics. Dexamethasone, two and three doses of MSCs similarly increased galectin levels, but only the three-dose regimen increased CD39 levels in the thymus. Dexamethasone and the three-dose, but not the two-dose regimen, also increased levels of programmed death receptor-1 and IL-10, while reducing CD4+CD8low cell percentage in the thymus. In conclusion, multiple doses of MSCs reduced lung inflammation and remodeling while causing immunosuppression in HDM-induced allergic asthma.

Corrêa R, Silva LFF, Ribeiro DJS, Almeida RDN, Santos IO, Corrêa LH, de Sant'Ana LP, Assunção LS, Bozza PT, Magalhães KG. Lysophosphatidylcholine Induces NLRP3 Inflammasome-Mediated Foam Cell Formation and Pyroptosis in Human Monocytes and Endothelial Cells. Front Immunol. 2020 Jan 9;10:2927.

DOI: https://doi.org/10.3389/fimmu.2019.02927

Foam cells are specialized lipid-loaded macrophages derived from monocytes and are a key pathological feature of atherosclerotic lesions. Lysophosphatidylcholine (LPC) is a major lipid component of the plasma membrane with a broad spectrum of proinflammatory activities and plays a key role in atherosclerosis. However, the role of LPC in lipid droplet (LD) biogenesis and the modulation of inflammasome activation is still poorly understood. In the present study, we investigated whether LPC can induce foam cell formation through an analysis of LD biogenesis and determined whether the cell signaling involved in this process is mediated by the inflammasome activation pathway in human endothelial cells and monocytes. Our results showed that LPC induced foam cell formation in both types of cells by increasing LD biogenesis via a NLRP3 inflammasome-dependent pathway. Furthermore, LPC induced pyroptosis in both cells and the activation of the inflammasome with IL-1β secretion, which was dependent on potassium efflux and lysosomal damage in human monocytes. The present study described the IL-1β secretion and foam cell formation triggered by LPC via an inflammasome-mediated pathway in human monocytes and endothelial cells. Our results will help improve our understanding of the relationships among LPC, LD biogenesis, and NLRP3 inflammasome activation in the pathogenesis of atherosclerosis.

da Mota JB, Echevarria-Lima J, Kyle-Cezar F, Melo M, Bellio M, Scharfstein J, Oliveira AC. IL-18R signaling is required for γδ T cell response and confers resistance to Trypanosoma cruzi infection. J Leukoc Biol. 2020 Oct;108(4):1239-1251. doi: 10.1002/JLB.4MA0420-568R.

DOI: https://doi.org/10.1002/jlb.4ma0420-568r

IFN-γ-producing γδ T cells have been suggested to play an important role in protection against infection with Trypanosoma cruzi. However, little is known about the mechanisms leading to functional differentiation of this T cell subset in this model. In the current work, we investigated the possibility that the IL-18/MyD88 pathway is central for the generation of effector γδ T cells, playing a role for resistance against infection. We found that splenic γδ+CD3+ cells were rapidly expanded (10–14 days post infection), which was accompanied by an early γδ T cell infiltration into the heart. In the following days, intracardiac parasitism was reduced, the protective immunity being accompanied by decreased γδ T cells tissue infiltration. As predicted, there was a drastic reduction of γδ T cells in Myd88- and Il18r1-deficient mice, both transgenic strains displaying a susceptible phenotype with increased intracardiac parasitism. In vivo and in vitro assays confirmed that IL-18R deficiency hampered γδ T cell proliferation. Further characterization revealed that T. cruzi infection up-regulates IL-18R expression in WT γδ+ T cell population whereas Il18r1−/− mice showed impaired generation of cytotoxic GzB+ and IFN-γ-producing γδ T cells. Consistently, in vitro cytotoxicity assay confirmed that cytolytic function was impaired in Il18r1-deficient γδ T cells. As a proof of concept, adoptive transfer of WT γδ T cells rescues Il18r1-deficient mice from susceptibility, reducing parasitemia and abrogating the mortality. Collectively, our findings implicate the IL-18R-MyD88 signaling in the mechanisms underlying generation of immunoprotective γδ T cells response in experimental Trypanosoma cruzi infection.

Dias SSG, Soares VC, Ferreira AC, Sacramento CQ, Fintelman-Rodrigues N, Temerozo JR, Teixeira L, Nunes da Silva MA, Barreto E, Mattos M, de Freitas CS, Azevedo-Quintanilha IG, Manso PPA, Miranda MD, Siqueira MM, Hottz ED, Pão CRR, Bou-Habib DC, Barreto-Vieira DF, Bozza FA, Souza TML, Bozza PT. Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators. PLoS Pathog. 2020 Dec 16;16(12):e1009127.

DOI: https://doi.org/10.1371/journal.ppat.1009127

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

Duque TLA, Siqueira MS, Travassos LH, Moreira OC, Bozza PT, Melo RCN, Henriques-Pons A, Menna-Barreto RFS. The induction of host cell autophagy triggers defense mechanisms against Trypanosoma cruzi infection in vitro. Eur J Cell Biol. 2020 Jan;99(1):151060.

DOI: https://doi.org/10.1016/j.ejcb.2019.151060

Trypanosoma cruzi causes Chagas disease, a neglected illness that affects millions of people worldwide, especially in Latin America. The balance between biochemical pathways triggered by the parasite and host cells response will ultimately define the progression of a life-threatening disease, justifying the efforts to understand cellular mechanisms for infection restrain. In this interaction, parasite and host cells are affected by different physiological responses as autophagy modulation, which could be under intense cellular stress, such as nutrient deprivation, hormone depletion, or infection. Autophagy is a constitutive pathway that leads to degradation of macromolecules and cellular structures and may induce cell death. In Trypanosoma cruzi infection, the relevance of host autophagy is controversial regarding in vitro parasite intracellular life cycle. In the present study, we evaluated host cell autophagy during T. cruzi infection in phagocytic and non-professional phagocytic cells. We described that the presence of the parasite increased the number of LC3 puncta, a marker for autophagy, in cardiac cells and peritoneal macrophages in vitro. The induction of host autophagy decreased infection in macrophages in early and late time-periods. We suggest that starved phagocytic cells reduced internalization, also confirmed by inert particles and dead trypomastigotes. Whereas, in cardiac cells, starvation-induced autophagy decreased lipid droplets and infection in later time-point, by reducing parasite differentiation/proliferation. In ATG5 knockout MEF cells, we confirmed our hypothesis of autophagy machinery activation during parasite internalization, increasing infection. Our data suggest that host autophagy downregulates T. cruzi infection through impairing parasite intracellular life cycle, reducing the infection in primary culture cells.

Ferreira, Marina & Gonçalves, Diego & Medeiros, Elisa & Peralta, José Mauro & Guimaraes, Allan. (2021). “Feast-Fit-Fist-Feat”: Overview of Free-living Amoeba Interactions with Fungi and Virulence as a Foundation for Success in Battle. Current Tropical Medicine Reports. 8.

DOI: https://link.springer.com/article/10.1007/s40475-020-00220-3

Free-living amoebae (FLAs) are ubiquitous and can co-habit similar niches and interact with fungi. Herein, we discuss theories on FLAs and the origin, evolution, and conservation of fungal virulence, proposing the “feast-fit-fist-feat” hypothesis that covers the knowledge on FLA-fungi interactions, and could be extended during evolutionarily host escalation. Overall, by bridling this selective pressure, fungi might return to environment and by serendipity, infect superior hosts. The selected traits might grant the fungus with an enhanced capacity to cause damage, or virulence. The fungal virulence factors that might be expressed during infection to amoeba and that grant a fungal benefit during infection to mammals are discussed. However, how they are induced during infection of FLAs is still an open field. Here we discuss also the “Trojan Horse” role of FLAs and the importance of co-infections and disease outcome.

Fintelman-Rodrigues N, Sacramento CQ, Ribeiro Lima C, Souza da Silva F, Ferreira AC, Mattos M, de Freitas CS, Cardoso Soares V, da Silva Gomes Dias S, Temerozo JR, Miranda MD, Matos AR, Bozza FA, Carels N, Alves CR, Siqueira MM, Bozza PT, Souza TML. Atazanavir, Alone or in Combination with Ritonavir, Inhibits SARS-CoV-2 Replication and Proinflammatory Cytokine Production. Antimicrob Agents Chemother. 2020 Sep 21;64(10):e00825-20.

DOI: https://doi.org/10.1128/aac.00825-20

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.

Firmino-Cruz L, Decote-Ricardo D, Gomes DCO, Morrot A, Freire-de-Lima CG, de Matos Guedes HL. How to B(e)-1 Important Cell During Leishmania Infection. Front Cell Infect Microbiol. 2020 Jan 14;9:424.

DOI: https://doi.org/10.3389/fcimb.2019.00424

B-1 cells are an innate-like population of B lymphocytes that are subdivided into B-1a and B-1b distinguished by the presence or absence of CD5, respectively. B-1 cells can act as regulatory B cells, are able to present antigen and produce IL-10. Leishmaniasis in humans is a complex of diseases caused by parasites of the genus Leishmania. More than 20 species can infect humans, with each species causing the development of different immunological responses in the host. Susceptibility is usually related to the production of anti-inflammatory cytokines while the production of Th1 cytokines is indicative of resistance. However, few studies have attempted to evaluate the role of B-1 cells during either the in vivo infection or in vitro interaction with Leishmania parasites. In vivo studies were performed using XID mice model, BALB/Xid mice have a mutation in the Bruton's tyrosine kinase, which is an important enzyme for developing B-1 and maturing B-2 lymphocytes leading to the presence of immature B-2 cells. Here, we compile these studies and assess the influence of B-1 cells on disease progression with different Leishmania species.

Firmino-Cruz L, Ramos TD, da Fonseca-Martins AM, Oliveira-Maciel D, Oliveira-Silva G, Dos Santos JS, Cavazzoni C, Morrot A, Gomes DCO, Vale AM, Decoté-Ricardo D, Freire-de-Lima CG, de Matos Guedes HL. B-1 lymphocytes are able to produce IL-10, but is not pathogenic during Leishmania (Leishmania) amazonensis infection. Immunobiology. 2020 Jan;225(1):151857.

DOI: 10.1016/j.imbio.2019.10.006

Over the years research has found an association between B lymphocytes and pathogenesis during Leishmania sp. infections. Recently we demonstrated that B-2 lymphocytes are the main producers of IL-10 during L. amazonensis infection, and that the disease severity in BALB/c mice was attributed to these IL-10-producing B-2 lymphocytes. Here, we aim to understand the role of peritoneal B-1 lymphocytes in the pathogenesis of L. amazonensis infection. We found that infection resulted in a decrease in the number of B-1a lymphocytes and increase in B-1b lymphocytes in the peritoneal cavity of WT BALB/c mice but not in B lymphocyte deficient mice (BALB/Xid) mice. In vitro interaction between B-1 lymphocytes and L. amazonensis showed that the amastigote form of the parasite was able to induce higher levels of IL-10 in B-1 lymphocytes derived from infected BALB/c mice than the promastigote. Moreover, B-1 lymphocytes derived from infected mice produced more IL-10 than B-1 lymphocytes derived from naïve mice under amastigote interaction. However, the repopulation of BALB/Xid mice with B-1 lymphocytes from WT BALB/c mice did not affect the lesion development. Together, these results suggest that although B-1 lymphocytes are able to produce IL-10 during in vitro interaction with L. amazonensis, they are not directly related to pathogenesis in vivo.

Gonçalves DS, Rodriguez de La Noval C, Ferreira MDS, Honorato L, Araújo GRS, Frases S, Pizzini CV, Nosanchuk JD, Cordero RJB, Rodrigues ML, Peralta JM, Nimrichter L, Guimarães AJ. Histoplasma capsulatum Glycans From Distinct Genotypes Share Structural and Serological Similarities to Cryptococcus neoformans Glucuronoxylomannan. Front Cell Infect Microbiol. 2021 Jan 8;10:565571.

DOI: https://doi.org/10.3389/fcimb.2020.565571

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans (Cn), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides, and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo. In this work, we characterized the ability of Hc to produce cellular-attached (C-gly-Hc) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly-Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly-Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly-Hc and E-gly-Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly-Hc and E-gly-Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly-Hc and E-gly-Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella. These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis.

Linhares-Lacerda L, Temerozo JR, Ribeiro-Alves M, Azevedo EP, Mojoli A, Nascimento MTC, Silva-Oliveira G, Savino W, Foguel D, Bou-Habib DC, Saraiva EM. Neutrophil extracellular trap-enriched supernatants carry microRNAs able to modulate TNF-α production by macrophages. Sci Rep. 2020 Feb 17;10(1):2715.

DOI: https://doi.org/10.1038/s41598-020-59486-2

Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.

Marques da Fonseca L, Jacques da Silva LR, Santos Dos Reis J, Rodrigues da Costa Santos MA, de Sousa Chaves V, Monteiro da Costa K, Sa-Diniz JN, Freire de Lima CG, Morrot A, Nunes Franklim T, de Alcântara-Pinto DC, Freire de Lima ME, Previato JO, Mendonça-Previato L, Freire-de-Lima L. Piperine Inhibits TGF-β Signaling Pathways and Disrupts EMT-Related Events in Human Lung Adenocarcinoma Cells. Medicines (Basel). 2020 Apr 8;7(4):19.

DOI: https://doi.org/10.3390/medicines7040019

Piperine, an amide extracted from the Piper spices, exhibits strong anti-tumor properties. However, its effect on the epithelial–mesenchymal transition (EMT) process has never been investigated. Herein, we evaluate the toxic effect of piperine on lung adenocarcinoma (A549), breast adenocarcinoma (MDA-MB-231) and hepatocellular carcinoma (HepG2) cell lines, as well as its ability to inhibit EMT-related events induced by TGF-β1 treatment. Methods: The cell viability was investigated by MTT assay. Protein expression was evaluated by Western blot. Gene expression was monitored by real-time PCR. Zymography assay was employed to detect metalloproteinase (MMP) activity in conditioned media. Cell motility was assessed by the wound-healing and phagokinetic gold sol assays. Results: The results revealed that piperine was cytotoxic in concentrations over 100 µM, showing IC50 values for HepG2, MDA-MB-231 and A549 cell lines of 214, 238 and 198 µM, respectively. In order to investigate whether piperine would reverse the TGF-β1 induced-EMT, the A549 cell line was pretreated with sublethal concentrations of the natural amide followed by the addition of TGF-β1. Besides disrupting EMT-related events, piperine also inhibited both ERK 1/2 and SMAD 2 phosphorylation.

Oliveira IA, Allonso D, Fernandes TVA, Lucena DMS, Ventura GT, Dias WB, Mohana-Borges RS, Pascutti PG, Todeschini AR. Enzymatic and structural properties of human glutamine:fructose-6-phosphate amidotransferase 2 (hGFAT2). J Biol Chem. 2021 Jan-Jun;296:100180. 33303629.

DOI: https://doi.org/10.1074/jbc.ra120.015189

Glycoconjugates play a central role in several cellular processes, and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in Escherichia coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi–bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics, and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.

Ramos TD, Silva JD, da Fonseca-Martins AM, da Silveira Pratti JE, Firmino-Cruz L, Maciel-Oliveira D, Dos-Santos JS, Tenorio JIN, de Araujo AF, Freire-de-Lima CG, Diaz BL, Cruz FF, Rocco PRM, de Matos Guedes HL. Combined therapy with adipose tissue-derived mesenchymal stromal cells and meglumine antimoniate controls lesion development and parasite load in murine cutaneous leishmaniasis caused by Leishmania amazonensis. Stem Cell Res Ther. 2020 Aug 31;11(1):374.

DOI: https://doi.org/10.1186/s13287-020-01889-z

Leishmaniasis is a neglected disease caused by Leishmania spp. One of its characteristics is an imbalance of host immune responses to foster parasite survival. In this setting, mesenchymal stromal cells (MSCs) may be a viable therapeutic alternative, given their well-established immunomodulatory potential. In this study, we compared the effects of therapy with bone marrow (BM)- and adipose tissue (AD)-derived MSCs in leishmaniasis caused by Leishmania amazonensis in C57BL/6 mice. After determining the most effective MSC source, we then combined these cells with meglumine antimoniate (a pentavalent antimonial commonly used for the treatment of leishmaniasis) to treat the infected mice.

Rodrigues DAS, Prestes EB, Gama AMS, Silva LS, Pinheiro AAS, Ribeiro JMC, Campos RMP, Pimentel-Coelho PM, De Souza HS, Dicko A, Duffy PE, Fried M, Francischetti IMB, Saraiva EM, Paula-Neto HA, Bozza MT. CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes. PLoS Pathog. 2020 Aug 14;16(8):e1008230.

DOI: https://doi.org/10.1371/journal.ppat.1008230

Neutrophil extracellular traps (NETs) evolved as a unique effector mechanism contributing to resistance against infection that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that patients suffering from severe malaria had increased amounts of circulating DNA and increased neutrophil elastase (NE) levels in plasma. We used cultured erythrocytes and isolated human neutrophils to show that Plasmodium-infected red blood cells release macrophage migration inhibitory factor (MIF), which in turn caused NET formation by neutrophils in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4). NET production was dependent on histone citrullination by peptidyl arginine deiminase-4 (PAD4) and independent of reactive oxygen species (ROS), myeloperoxidase (MPO) or NE. In vitro, NETs functioned to restrain parasite dissemination in a mechanism dependent on MPO and NE activities. Finally, C57/B6 mice infected with P. berghei ANKA, a well-established model of cerebral malaria, presented high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NETs in resistance against Plasmodium infection.

Rodrigues Mantuano N, Stanczak MA, Oliveira IA, Kirchhammer N, Filardy AA, Monaco G, Santos RC, Fonseca AC, Fontes M, Bastos CS Jr, Dias WB, Zippelius A, Todeschini AR, Läubli H. Hyperglycemia Enhances Cancer Immune Evasion by Inducing Alternative Macrophage Polarization through Increased O-GlcNAcylation. Cancer Immunol Res. 2020 Oct;8(10):1262-1272.

DOI: https://doi.org/10.1158/2326-6066.cir-19-0904

Diabetes mellitus (DM) significantly increases the risk for cancer and cancer progression. Hyperglycemia is the defining characteristic of DM and tightly correlates with a poor prognosis in patients with cancer. The hexosamine biosynthetic pathway (HBP) is emerging as a pivotal cascade linking high glucose, tumor progression, and impaired immune function. Here we show that enhanced glucose flow through the HBP drives cancer progression and immune evasion by increasing O-GlcNAcylation in tumor-associated macrophages (TAM). Increased O-GlcNAc skewed macrophage polarization to a M2-like phenotype supporting tumor progression. Finally, we found an upregulation of M2 markers on TAMs in DM2 patients with colorectal cancer compared with nondiabetic normoglycemic patients. Our results provide evidence for a new and targetable mechanism of cancer immune evasion in patients with hyperglycemia, advocating for strict control of hyperglycemia in patients with cancer.

Rodriguez-de la Noval C, Ruiz Mendoza S, de Souza Gonçalves D, da Silva Ferreira M, Honorato L, Peralta JM, Nimrichter L, Guimarães AJ. Protective Efficacy of Lectin-Fc(IgG) Fusion Proteins In Vitro and in a Pulmonary Aspergillosis In Vivo Model. J Fungi (Basel). 2020 Oct 27;6(4):250.

DOI: https://doi.org/10.3390/jof6040250

Aspergillosis cases by Aspergillus fumigatus have increased, along with fungal resistance to antifungals, urging the development of new therapies. Passive immunization targeting common fungal antigens, such as chitin and β-glucans, are promising and would eliminate the need of species-level diagnosis, thereby expediting the therapeutic intervention. However, these polysaccharides are poorly immunogenic. To overcome this drawback, we developed the lectin-Fc(IgG) fusion proteins, Dectin1-Fc(IgG2a), Dectin1-Fc(IgG2b) and wheat germ agglutinin (WGA)-Fc(IgG2a), based on their affinity to β-1,3-glucan and chitooligomers, respectively. The WGA-Fc(IgG2a) previously demonstrated antifungal activity against Histoplasma capsulatum, Cryptococcus neoformans and Candida albicans. In the present work, we evaluated the antifungal properties of these lectin-Fc(s) against A. fumigatus. Lectin-Fc(IgG)(s) bound in a dose-dependent manner to germinating conidia and this binding increased upon conidia germination. Both lectin-Fc(IgG)(s) displayed in vitro antifungal effects, such as inhibition of conidia germination, a reduced length of germ tubes and a diminished biofilm formation. Lectin-Fc(IgG)(s) also enhanced complement deposition on conidia and macrophage effector functions, such as increased phagocytosis and killing of fungi. Finally, administration of the Dectin-1-Fc(IgG2b) and WGA-Fc(IgG2a) protected mice infected with A. fumigatus, with a 20% survival and a doubled life-span of the infected mice, which was correlated to a fungal burden reduction in lungs and brains of treated animals. These results confirm the potential of lectin-Fc(IgGs)(s) as a broad-spectrum antifungal therapeutic. 

Silva de Castro I, Gordon SN, Liu J, Bissa M, McKinnon K, Trinh HV, Doster MN, Schifanella L, Liyanage NP, Cao J, Cheng O, Foulds K, Roederer M, Koup RA, Shen X, Tomaras GD, Venzon DJ, Forthal DN, Fouts T, Montefiori DC, Tartaglia J, Rao M, Ostrowski M, Franchini G, Vaccari M. Expression of CD40L by the ALVAC-Simian Immunodeficiency Virus Vector Abrogates T Cell Responses in Macaques. J Virol. 2020 Feb 28;94(6):e01933-19.

DOI: https://doi.org/10.1128/jvi.01933-19

Immunization with recombinant ALVAC/gp120 alum vaccine provided modest protection from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) acquisition in humans and macaques. Vaccine-mediated protection was associated with the elicitation of IgG against the envelope V2 loop and of envelope-specific CD4+ T cell responses. We hypothesized that the simultaneous expression of the costimulatory molecule CD40L (CD154) by the ALVAC-HIV vector could increase both protective humoral and cellular responses. We engineered an ALVAC-SIV coexpressing CD40L with SIVmac251 (ALVAC-SIV/CD40L) gag, pol, and env genes. We compared its immunogenicity in macaques with that of a canonical ALVAC-SIV, with both given as a vector-prime/gp120 in alum boost strategy. The ALVAC-SIV/CD40L was superior to the ALVAC-SIV regimen in inducing binding and tier 1 neutralizing antibodies against the gp120. The increase in humoral responses was associated with the expression of the membrane-bound form of the CD40L by CD4+ T cells in lymph nodes. Unexpectedly, the ALVAC-SIV/CD40L vector had a blunting effect on CD4+ Th1 helper responses and instead favored the induction of myeloid-derived suppressor cells, the immune-suppressive interleukin-10 (IL-10) cytokine, and the down-modulatory tryptophan catabolism. Ultimately, this strategy failed to protect macaques from SIV acquisition. Taken together, these results underlie the importance of balanced vaccine-induced activating versus suppressive immune responses in affording protection from HIV.

Silva JC, Rodrigues NC, Thompson-Souza GA, Muniz VS, Neves JS, Figueiredo RT. Mac-1 triggers neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone citrullination. J Leukoc Biol. 2020 Jan;107(1):69-83.

DOI: https://doi.org/10.1002/jlb.4a0119-009rr

Aspergillus fumigatus (A. fumigatus) is an environmental fungus and a human pathogen. Neutrophils are critical effector cells during the fungal infections, and neutropenia is a risk factor for the development of pulmonary aspergillosis. Neutrophil extracellular traps (NETs) are released by neutrophils in response to A. fumigatus and inhibit the conidial germination. In this work, we observed that the receptors TLR2, TLR4, and Dectin-1 were dispensable for the A. fumigatus induced NET release. In contrast CD11b/CD18 was critical for the NET release in response to A. fumigatus conidia, and this required the CD11b I-domain-mediated recognition, whereas the blockade of the CD11b lectin domain did not affect the A. fumigatus induced NET release. A. fumigatus induced NET release relied on the activity of spleen tyrosine kinase (Syk), Src family kinase(s), and class IA PI3 kinase δ. Although A. fumigatus promoted histone citrullination, this process was dispensable for the NET release in response to A. fumigatus conidia. The A. fumigatus induced NET release required the reactive oxygen species generation by the NOX2 complex, in a downstream pathway requiring CD11b/CD18, Src kinase family activity, Syk and PI3K class IA δ. Our findings thus reveal the signaling pathways involved in the formation of NETs in response to A. fumigatus.

Silva RCMC, Travassos LH, Paiva CN, Bozza MT. Heme oxygenase-1 in protozoan infections: A tale of resistance and disease tolerance. PLoS Pathog. 2020 Jul 21;16(7):e1008599.

DOI: https://doi.org/10.1371/journal.ppat.1008599

Heme oxygenase (HO-1) mediates the enzymatic cleavage of heme, a molecule with proinflammatory and prooxidant properties. HO-1 activity deeply impacts host capacity to tolerate infection through reduction of tissue damage or affecting resistance, the ability of the host to control pathogen loads. In this Review, we will discuss the contribution of HO-1 in different and complex protozoan infections, such as malaria, leishmaniasis, Chagas disease, and toxoplasmosis. The complexity of these infections and the pleiotropic effects of HO-1 constitute an interesting area of study and an opportunity for drug development.

Sousa-Pereira D, Oliveira TS, Paiva RO, Chaves OA, Netto-Ferreira JC, Echevarria-Lima J, Echevarria A. Synthetic (E)-3-Phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium Chloride Derivatives as Promising Chemotherapy Agents on Cell Lines Infected with HTLV-1. Molecules. 2020 May 29;25(11):2537. 

DOI: https://doi.org/10.3390/molecules25112537

Synthesis of four compounds belonging to mesoionic class, (E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives (5a–d) and their biological evaluation against MT2 and C92 cell lines infected with human T-cell lymphotropic virus type-1 (HTLV-1), which causes adult T-cell leukemia/lymphoma (ATLL), and non-infected cell lines (Jurkat) are reported. The compounds were obtained by convergent synthesis under microwave irradiation and the cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Results showed IC₅₀ values of all compounds in the range of 1.51–7.70 μM in HTLV-1-infected and non-infected cells. Furthermore, it was observed that 5b could induce necrosis after 24 h for Jurkat and MT2 cell lines. The experimental (fluorimetric method) and theoretical (molecular docking) results suggested that the mechanism of action for 5b could be related to its capacity to intercalate into DNA. Moreover, the preliminary pharmacokinetic profile of the studied compounds (5a–d) was obtained through human serum albumin (HSA) binding affinity using multiple spectroscopic techniques (circular dichroism, steady-state and time-resolved fluorescence), zeta potential and molecular docking calculations. The interaction HSA:5a–d is spontaneous and moderate (K_a ~ 10⁴ M⁻¹) via a ground-state association, without significantly perturbing both the secondary and surface structures of the albumin in the subdomain IIA (site I), indicating feasible biodistribution in the human bloodstream

Thompson-Souza GA, Santos GMP, Silva JC, Muniz VS, Braga YAV, Figueiredo RT, Melo RCN, Santos ALS, Pinto MR, Neves JS. Histoplasma capsulatum-induced extracellular DNA trap release in human neutrophils. Cell Microbiol. 2020 Jul;22(7):e13195.

DOI: https://doi.org/10.1111/cmi.13195

Neutrophils are leukocytes that are capable of eliminating both intra- and extracellular pathogens by mechanisms such as phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). Histoplasma capsulatum var. capsulatum (H. capsulatum) is a dimorphic fungus with a global distribution that causes histoplasmosis, a disease that is endemic in different geographic areas and is spreading worldwide. The release of NETs has been described as an important host defense mechanism against different fungi; however, there are no reports demonstrating that this process is implicated in neutrophil response to H. capsulatum infection. Therefore, the aim of this work is to investigate whether isolated human neutrophils release NETs in response to H. capsulatum and the potential mechanisms involved, as well as delineate the NETs antifungal activity. Using both confocal fluorescence and scanning electron microscopy techniques, we determined that NETs are released in vitro in response to H. capsulatum via an oxidative mechanism that is downstream of activation of the Syk and Src kinase pathways and is also dependent on CD18. NETs released in response to H. capsulatum yeasts involve the loss of neutrophil viability and are associated with elastase and citrullinated histones, however also can occur in a PAD4 histone citrullination independent pathway. This NETs also presented fungicidal activity against H. capsulatum yeasts. Our findings may contribute to the understanding of how neutrophils recognize and respond as immune effector cells to H. capsulatum, which may lead to better knowledge of histoplasmosis pathophysiology and treatment.

Vargas G, Honorato L, Guimarães AJ, Rodrigues ML, Reis FCG, Vale AM, Ray A, Nosanchuk JD, Nimrichter L. Protective effect of fungal extracellular vesicles against murine candidiasis. Cell Microbiol. 2020 Oct;22(10):e13238.

DOI: https://doi.org/10.1111/cmi.13238

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFβ, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, −20 or −80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.

Arcanjo A, Logullo J, Menezes CCB, de Souza Carvalho Giangiarulo TC, Dos Reis MC, de Castro GMM, da Silva Fontes Y, Todeschini AR, Freire-de-Lima L, Decoté-Ricardo D, Ferreira-Pereira A, Freire-de-Lima CG, Barroso SPC, Takiya C, Conceição-Silva F, Savino W, Morrot A. The emerging role of neutrophil extracellular traps in severe acute respiratory syndrome coronavirus 2 (COVID-19). Sci Rep. 2020 Nov 12;10(1):19630.

DOI: https://doi.org/10.1038/s41598-020-76781-0

The novel coronavirus SARS-CoV-2 causes COVID-19, a highly pathogenic viral infection threatening millions. The majority of the individuals infected are asymptomatic or mildly symptomatic showing typical clinical signs of common cold. However, approximately 20% of the patients can progress to acute respiratory distress syndrome (ARDS), evolving to death in about 5% of cases. Recently, angiotensin-converting enzyme 2 (ACE2) has been shown to be a functional receptor for virus entry into host target cells. The upregulation of ACE2 in patients with comorbidities may represent a propensity for increased viral load and spreading of infection to extrapulmonary tissues. This systemic infection is associated with higher neutrophil to lymphocyte ratio in infected tissues and high levels of pro-inflammatory cytokines leading to an extensive microthrombus formation with multiorgan failure. Herein we investigated whether SARS-CoV-2 can stimulate extracellular neutrophils traps (NETs) in a process called NETosis. We demonstrated for the first time that SARS-CoV-2 in fact is able to activate NETosis in human neutrophils. Our findings indicated that this process is associated with increased levels of intracellular Reactive Oxygen Species (ROS) in neutrophils. The ROS-NET pathway plays a role in thrombosis formation and our study suggest the importance of this target for therapy approaches against disease.

Albuquerque SO, Barros TG, Dias LRS, Lima CHDS, Azevedo PHRA, Flores-Junior LAP, Dos Santos EG, Loponte HF, Pinheiro S, Dias WB, Muri EMF, Todeschini AR. Biological evaluation and molecular modeling of peptidomimetic compounds as inhibitors for O-GlcNAc transferase (OGT). Eur J Pharm Sci. 2020 Nov 1;154:105510.

DOI: https://doi.org/10.1016/j.ejps.2020.105510

The vital enzyme O-linked β-N-acetylglucosamine transferase (OGT) catalyzes the O-GlcNAcylation of intracellular proteins coupling the metabolic status to cellular signaling and transcription pathways. Aberrant levels of O-GlcNAc and OGT have been linked to metabolic diseases as cancer and diabetes. Here, a new series of peptidomimetic OGT inhibitors was identified highlighting the compound LQMed 330, which presented better IC50 compared to the most potent inhibitors found in the literature. Molecular modeling study of selected inhibitors into the OGT binding site provided insight into the behavior by which these compounds interact with the enzyme. The results obtained in this study provided new perspectives on the design and synthesis of highly specific OGT inhibitors.

Gonçalves DS, Ferreira MDS, Guimarães AJ. Extracellular Vesicles from the Protozoa Acanthamoeba castellanii: Their Role in Pathogenesis, Environmental Adaptation and Potential Applications. Bioengineering (Basel). 2019 Feb 1;6(1):13.

DOI: 10.3390/bioengineering6010013

Extracellular vesicles (EVs) are membranous compartments of distinct cellular origin and biogenesis, displaying different sizes and include exosomes, microvesicles, and apoptotic bodies. The EVs have been described in almost every living organism, from simple unicellular to higher evolutionary scale multicellular organisms, such as mammals. Several functions have been attributed to these structures, including roles in energy acquisition, cell-to-cell communication, gene expression modulation and pathogenesis. In this review, we described several aspects of the recently characterized EVs of the protozoa Acanthamoeba castellanii, a free-living amoeba (FLA) of emerging epidemiological importance, and compare their features to other parasites’ EVs. These A. castellanii EVs are comprised of small microvesicles and exosomes and carry a wide range of molecules involved in many biological processes like cell signaling, carbohydrate metabolism and proteolytic activity, such as kinases, glucanases, and proteases, respectively. Several biomedical applications of these EVs have been proposed lately, including their use in vaccination, biofuel production, and the pharmaceutical industry, such as platforms for drug delivery.

Abreu SC, Xisto DG, de Oliveira TB, Blanco NG, de Castro LL, Kitoko JZ, Olsen PC, Lopes-Pacheco M, Morales MM, Weiss DJ, Rocco PRM. Serum from Asthmatic Mice Potentiates the Therapeutic Effects of Mesenchymal Stromal Cells in Experimental Allergic Asthma. Stem Cells Transl Med. 2019 Mar;8(3):301-312.

DOI: 10.1002/sctm.18-0056

Asthma is a chronic inflammatory disease characterized by airway inflammation and remodeling, which can lead to progressive decline of lung function. Although mesenchymal stromal cells (MSCs) have shown beneficial immunomodulatory properties in preclinical models of allergic asthma, effects on airway remodeling have been limited. Mounting evidence suggests that prior exposure of MSCs to specific inflammatory stimuli or environments can enhance their immunomodulatory properties. Therefore, we investigated whether stimulating MSCs with bronchoalveolar lavage fluid (BALF) or serum from asthmatic mice could potentiate their therapeutic properties in experimental asthma. In a house dust mite (HDM) extract asthma model in mice, unstimulated, asthmatic BALF-stimulated, or asthmatic serum-stimulated MSCs were administered intratracheally 24 hours after the final HDM challenge. Lung mechanics and histology; BALF protein, cellularity, and biomarker levels; and lymph-node and bone marrow cellularity were assessed. Compared with unstimulated or BALF-stimulated MSCs, serum-stimulated MSCs further reduced BALF levels of interleukin (IL)-4, IL-13, and eotaxin, total and differential cellularity in BALF, bone marrow and lymph nodes, and collagen fiber content, while increasing BALF IL-10 levels and improving lung function. Serum stimulation led to higher MSC apoptosis, expression of various mediators (transforming growth factor-β, interferon-γ, IL-10, tumor necrosis factor-α-stimulated gene 6 protein, indoleamine 2,3-dioxygenase-1, and IL-1 receptor antagonist), and polarization of macrophages to M2 phenotype. In conclusion, asthmatic serum may be a novel strategy to potentiate therapeutic effects of MSCs in experimental asthma, leading to further reductions in both inflammation and remodeling than can be achieved with unstimulated MSCs.

Alisson-Silva F, Mantuano NR, Lopes AL, Vasconcelos-Dos-Santos A, Vale AM, Costa MM, Cannon JL, Oliveira AC, Todeschini AR. CD43 sialoglycoprotein modulates cardiac inflammation and murine susceptibility to Trypanosoma cruzi infection. Sci Rep. 2019 Jun 13;9(1):8628.

DOI: 10.1038/s41598-019-45138-7

CD43 (leukosialin) is a large sialoglycoprotein abundantly expressed on the surface of most cells from the hematopoietic lineage. CD43 is directly involved in the contact between cells participating in a series of events such as signaling, adherence and host parasite interactions. In this study we examined the role of CD43 in the immune response against Trypanosoma cruzi, the protozoan parasite that causes Chagas’ disease, a potential life-threatening illness endemic in 21 Latin American countries according to the WHO. The acute stage of infection is marked by intense parasitemia and cardiac tissue parasitism, resulting in the recruitment of inflammatory cells and acute damage to the heart tissue. We show here that CD43−/− mice were more resistant to infection due to increased cytotoxicity of antigen specific CD8+ T cells and reduced inflammatory infiltration in the cardiac tissue, both contributing to lower cardiomyocyte damage. In addition, we demonstrate that the induction of acute myocarditis involves the engagement of CD43 cytoplasmic tripeptide sequence KRR to ezrin-radixin-moiesin cytoskeletal proteins. Together, our results show the participation of CD43 in different events involved in the pathogenesis of T. cruzi infection, contributing to a better overall understanding of the mechanisms underlying the pathogenesis of acute chagasic cardiomyopathy.

Barros JF, Waclawiak I, Pecli C, Borges PA, Georgii JL, Ramos-Junior ES, Canetti C, Courau T, Klatzmann D, Kunkel SL, Penido C, Canto FB, Benjamim CF. Role of Chemokine Receptor CCR4 and Regulatory T Cells in Wound Healing of Diabetic Mice. J Invest Dermatol. 2019 May;139(5):1161-1170. 

DOI: 10.1016/j.jid.2018.10.039

Wound healing is a well-coordinated process that involves inflammatory mediators and cellular responses; however, if any disturbances are present during this process, tissue repair is impaired. Chronic wounds are one of the serious long-term complications associated with diabetes mellitus. The chemokine receptor CCR4 and its respective ligands, CCL17 and CCL22, are involved in regulatory T cell recruitment and activation in inflamed skin; however, the role of regulatory T cells in wounds is still not clear. Our aim was to investigate the role of CCR4 and regulatory T cells in cutaneous wound healing in diabetic mice. Alloxan-induced diabetic wild- type mice (diabetic) developed wounds that were difficult to heal, differently from CCR4–/– diabetic mice (CCR4–/– diabetic), and also from anti-CCL17/22 or anti-CD25–injected diabetic mice that presented with accelerated wound healing and fewer regulatory T cells in the wound bed. Consequently, CCR4–/– diabetic mice also presented with alteration on T cells population in the wound and draining lymph nodes; on day 14, these mice also displayed an increase of collagen fiber deposition. Still, cytokine levels were decreased in the wounds of CCR4–/– diabetic mice on day 2. Our data suggest that the receptor CCR4 and regulatory T cells negatively affect wound healing in diabetic mice.

Barroso MV, Graça-Reis A, Cattani-Cavalieri I, Gitirana LB, Valenca SS, Lanzetti M. Mate tea reduces high fat diet-induced liver and metabolic disorders in mice. Biomed Pharmacother. 2019 Jan;109:1547-1555. 

DOI: 10.1016/j.biopha.2018.11.007

High-fat diet (HFD)-induced obesity is a worldwide health problem and can cause lipid accumulation in the liver. We evaluated the hepatoprotective effect of mate tea treatment in mice submitted to an HFD. C57BL/6 mice were fed an HFD for 13 weeks with and without mate tea. A separate group of mice was treated with fenofibrate as a positive control (a regular drug for lipid disorders). Histological analyses, glucose tolerance tests (GTT), and quantification of mediators related to lipid peroxidation, oxidative stress and blood biomarkers for lipid profile were performed. The weight of animals and major organs related to hepatic steatosis was determined, and proinflammatory cytokines and the participation of the Nrf2 pathway and adiponectin were evaluated. Mate tea prevented the accumulation of lipid droplets in hepatocytes as well as weight gain in animals submitted to the HFD. Mate tea treatment also prevented increases in the liver weight, heart weight and amount of visceral and subcutaneous white adipose tissue. Mate tea was able to prevent the deregulation of glucose uptake, as evaluated by GTT, and improved the indicators of oxidative stress, such as nitrite levels, catalase activity, and oxidative damage, as evaluated by protein carbonylation and the MDA levels. Mate tea had an anti-inflammatory effect, preventing the increase of IL-1β and KC and upregulating the expression of Nrf2. Mate tea prevented insulin increase and HDL cholesterol decrease but did not affect total cholesterol or triglycerides levels. Treatment also prevented adiponectin increase. Mate tea may be a good resource to reduce hepatic steatosis in the future since it has anti-diabetic, anti-inflammatory and antioxidant effects, which prevent the accumulation of fat in the liver.

Bezerra IPDS, Oliveira-Silva G, Braga DSFS, de Mello MF, Pratti JES, Pereira JC, da Fonseca-Martins AM, Firmino-Cruz L, Maciel-Oliveira D, Ramos TD, Vale AM, Gomes DCO, Rossi-Bergmann B, de Matos Guedes HL. Dietary Vitamin D3 Deficiency Increases Resistance to Leishmania (Leishmania) amazonensis Infection in Mice. Front Cell Infect Microbiol. 2019 Apr 9;9:88. 

DOI: 10.3389/fcimb.2019.00088

The leishmaniases are a group of diseases caused by Leishmania parasites, which have different clinical manifestations. Leishmania (Leishmania) amazonensis is endemic in South America and causes cutaneous leishmaniasis (CL), which can evolve into a diffuse form, characterized by an anergic immune response. Since the leishmaniases mainly affect poor populations, it is important to understand the involvement of immunonutrition, how the immune system is modulated by dietary nutrients and the effect this has on Leishmania infection. Vitamin D3 (VitD) is an immunonutrient obtained from diet or endogenously synthesized, which suppresses Th1 and Th17 responses by favoring T helper (Th) 2 and regulatory T cell (Treg) generation. Based on these findings, this study aims to evaluate dietary VitD influence on L. (L.) amazonensis experimental infection in C57BL/6 and BALB/c mice. Thus, C57BL/6 and BALB/c VitD deficient (VDD) mice were generated through dietary VitD restriction 45 days prior to infection. Both strains of VDD mice showed a more controlled lesion development compared to mice on a regular diet (Ctrl). There were no differences in serum levels of anti-Leishmania IgG1 and IgG2a, but there was a decrease in IgE levels in BALB/c VDD mice. Although CD4+ T cell number was not changed, the CD4+ IFN-y+ T cell population was increased in both absolute number and percentage in C57BL/6 and BALB/c VDD mice compared to Ctrl mice. There was also no difference in IL-4 and IL-17 production, however, there was reduction of IL-10 production in VDD mice. Together, our data indicate that VitD contributes to murine cutaneous leishmaniasis susceptibility and that the Th1 cell population may be related to the resistance of VDD mice to L. (L.) amazonensis infection.

Cavalcanti De Albuquerque R, Granato A, Silva Castro I, Carvalho Torres R, Santos Souza F, Lima MA, Celestino Bezerra Leite AC, de Melo Espíndola O, Echevarria-Lima J. Phenotypic and functional changes in gamma delta T lymphocytes from HTLV-1 carriers. J Leukoc Biol. 2019 Sep;106(3):607-618. 

DOI: 10.1002/JLB.MA1118-467R

Human T-cell lymphotropic virus type-1 (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic inflammatory disease that leads to gradual loss of motor movement as a result of the death of spinal cord cells through immune mediated mechanisms. The risk to develop HAM/TSP disease positively correlates with the magnitude of HTLV-1 proviral load. Gamma-delta T lymphocytes have been recognized as important players in a variety of infectious diseases. Therefore, we have investigated interactions between HTLV-1 infection and γδ T lymphocytes during HAM/TSP. Similar frequencies of total γδ T lymphocytes and their Vγ9δ2+ and Vγ9δ2neg subpopulations were observed in HAM/TSP patients. However, T lymphocytes obtained from HTLV-1 carriers displayed significantly higher rates of spontaneous proliferation and NKp30 expression when compared to cells from uninfected donors. In addition, an important decrease in the frequency of granzyme B+ γδ T lymphocytes (approximately 50%) was observed in HAM/TSP patients. Higher proportion of IFN-γ+ γδ T lymphocytes was found in HTLV-1-infected patients, which positively correlated with the HTLV-1 proviral load in peripheral blood mononuclear cells. Collectively, our data indicates that HTLV-1 infection leads to phenotypic and functional changes in the population of γδ T lymphocyte population, suggesting that HTLV-1 infection modulates functions associated to these cells, which might be involved in controlling the infection or in the development of HTLV-1-associated diseases.

Chaves OA, de Castro IS, Goulart CM, Bellieny MSS, Netto-Ferreira JC, Echevarria-Lima J, Echevarria A. In vitro and in vivo cytotoxic activity and human serum albumin interaction for a methoxy-styryl-thiosemicarbazone. Invest New Drugs. 2019 Oct;37(5):994-1005.

DOI: 10.1007/s10637-018-00722-y

Thiosemicarbazone is a class of compounds with potential applications in medicine, presenting high capacity to inhibit the growth of cancer cells as well as low toxicity. Because of high interest in anticancer studies involving thiosemicarbazones as new chemotherapeutic agents, a synthetic thiosemicarbazone derivative, 4-N-(2′-methoxy-styryl)-thiosemicarbazone (MTSC) was evaluated in vivo against Ehrlich carcinoma in an animal model. In vivo results demonstrated that MTSC treatment induced the survival of mice and altered significantly the body weight of the surviving mice 12 days after tumor inoculation. Treatment with 30 mg/kg of MTSC exhibited effective cytotoxic activity with T/C values of 150.49% (1 dose) and 278% (2 doses). Its interaction with human serum albumin (HSA), which plays a crucial role in the biodistribution of a wide variety of ligands, was investigated by multiple spectroscopic techniques at 296 K, 303 K, and 310 K, as well as by theoretical calculations. The interaction between HSA and MTSC occurs via ground-state association in the subdomain IIA (Sudlow’s site I). The binding is moderate (Ka ≈ 104 M–1), spontaneous, entropically, and enthalpically driven. Molecular docking results suggested hydrogen bonding and hydrophobic interactions as the main binding forces. Overall, the interaction HSA:MTSC could provide therapeutic benefits, improving its cytotoxic efficacy and tolerability.

da Fonseca-Martins AM, Ramos TD, Pratti JES, Firmino-Cruz L, Gomes DCO, Soong L, Saraiva EM, de Matos Guedes HL. Immunotherapy using anti-PD-1 and anti-PD-L1 in Leishmania amazonensis-infected BALB/c mice reduce parasite load. Sci Rep. 2019 Dec 30;9(1):20275. 

DOI: 10.1038/s41598-019-56336-8

Leishmaniasis is a neglected disease, for which current treatment presents numerous issues. Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The roles of the programmed death-1 (PD-1) receptor on lymphocytes and its ligand (PD-L1) on antigen-presenting cells have been well studied in tumor and other infection models; but little is known about their roles in non-healing cutaneous leishmaniasis. In this study, we observed that L. amazonensis induced PD-1 expression on both CD4+ and CD8+ T cells and PD-L1 on dendritic cells on BALB/c mice. We tested the therapeutic potential of anti-PD-1 and anti-PD-L1 monoclonal antibodies (MoAbs) against a non-healing L. amazonensis infection in BALB/c mice, and that anti-PD-1 and anti-PD-L1 treatment significantly increased IFN-γ-producing CD4+ and CD8+ T cells, respectively. Compared with infection controls, mice treated with anti-PD-1 and anti-PD-L1, but not anti-PD-L2, displayed bigger lesions with significantly lower parasite loads. Treatment did not affect anti-Leishmania antibody (IgM, IgG, IgG1 and IgG2a) or IL-10 production, but anti-PD-1 treatment reduced both IL-4 and TGF-β production. Together, our results highlight the therapeutic potential of an anti-PD-1-based treatment in promoting the reinvigoration of T cells for the control of parasite burden.

da Rocha RFDB, LaRocque-de-Freitas IF, Arcanjo AF, Logullo J, Nunes MP, Freire-de-Lima CG, Decote-Ricardo D. B-1 Cells May Drive Macrophages Susceptibility to Trypanosoma cruzi Infection. Front Microbiol. 2019 Jul 9;10:1598. 

DOI: 10.3389/fmicb.2019.01598

B-1 cells can directly and indirectly influence the immune response. These cells are known to be excellent producers of natural antibodies and can secrete a variety of immunomodulatory molecules. They are also able to differentiate into B-1 cell-derived phagocytes (B-1CDP). B-1 cells can modulate macrophages to become less effective, and B-1CDP cells are more susceptible in infection models. In this work, we investigated the microbicidal ability of these cells in Trypanosoma cruzi infection in vitro. The results show that macrophages from BALB/c mice are more susceptible to infection than macrophages from XID mice. The resistance observed in macrophages from XID mice was abolished in the presence of B-1 cells, and this event seems to be associated with IL-10 production by B-1 cells, which may have contributed to the decrease of NO production. Additionally, B-1CDP cells were more permissive to intracellular T. cruzi infection than peritoneal macrophages. These findings strongly suggest that B-1 cells and B-1CDP cells have a potential role in the persistence of the parasite in host cells.

de Oliveira GP, Kitoko JZ, de Souza Lima-Gomes P, Rochael NC, de Araújo CC, Lugon PN, Dos Santos HL, Martins EGL, Ornellas FM, de Oliveira HD, Morales MM, Olsen PC, Galina A, Silva PL, Saraiva EM, Pelosi P, Rocco PRM. Glutamine Therapy Reduces Inflammation and Extracellular Trap Release in Experimental Acute Respiratory Distress Syndrome of Pulmonary Origin. Nutrients. 2019 Apr 12;11(4):831.

DOI: 10.3390/nu11040831

The innate immune response plays an important role in the pathophysiology of acute respiratory distress syndrome (ARDS). Glutamine (Gln) decreases lung inflammation in experimental ARDS, but its impact on the formation of extracellular traps (ETs) in the lung is unknown. In a mouse model of endotoxin-induced pulmonary ARDS, the effects of Gln treatment on leukocyte counts and ET content in bronchoalveolar lavage fluid (BALF), inflammatory profile in lung tissue, and lung morphofunction were evaluated in vivo. Furthermore, ET formation, reactive oxygen species (ROS) production, glutathione peroxidase (GPx), and glutathione reductase (GR) activities were tested in vitro. Our in vivo results demonstrated that Gln treatment reduced ET release (as indicated by cell-free-DNA content and myeloperoxidase activity), decreased lung inflammation (reductions in interferon-γ and increases in interleukin-10 levels), and improved lung morpho-function (decreased static lung elastance and alveolar collapse) in comparison with ARDS animals treated with saline. Moreover, Gln reduced ET and ROS formation in BALF cells stimulated with lipopolysaccharide in vitro, but it did not alter GPx or GR activity. In this model of endotoxin-induced pulmonary ARDS, treatment with Gln reduced pulmonary functional and morphological impairment, inflammation, and ET release in the lung.

Dos-Santos JS, Firmino-Cruz L, Ramos TD, da Fonseca-Martins AM, Oliveira-Maciel D, De-Medeiros JVR, Chaves SP, Gomes DCO, de Matos Guedes HL. Characterization of Sv129 Mice as a Susceptible Model to Leishmania amazonensis. Front Med (Lausanne). 2019 May 29;6:100.

DOI: 10.3389/fmed.2019.00100

Leishmaniasis is a complex of neglected diseases caused by parasites of the genus Leishmania, such as Leishmania (Leishmania) amazonensis, the ethiologic agent of diffuse cutaneous leishmaniasis in Brazil. In this work, we investigated a new experimental model of infection for L. amazonensis: the Sv129 mouse. First, we subcutaneously infected Sv129 mice with 2 × 105 or 2 × 106 L. amazonensis parasites of the Josefa strain. A progressive lesion developed for both inoculation doses, showing that Sv129 mice are susceptible, independent of parasite dose. We next investigated the mechanisms associated with the pathogenesis of infection. We did not observe an increase of frequency of interferon-gamma (IFN- γ)-producing CD4+ and CD8+ T cells, a phenotype similar to that seen in BALB/c mice. There was an increased of frequency and number of IL-17-producing γδ (gamma-delta) T cells in infected Sv129 mice compared to naïve SV129 and an increased frequency of this population compared to infected BALB/c mice. In addition, Sv129 mice presented high levels of both IgG1 and IgG2a, suggesting a mixed Th1 and Th2 response with a skew toward IgG1 production based on IgG1/IgG2a ratio. Susceptibility of the Sv129 mice was further confirmed with the use of another strain of L. amazonensis, LTB0016. In this work, we characterized the Sv129 mice as a new model of susceptibility to Leishmania amazonensis infection, during infection there was controlled IFN-γ production by CD4+ or CD8+ T cells and induced IL-17 production by γδ T cells.

Figueiredo CM, Neris RLDS, Gavino-Leopoldino D, da Silva MOL, Almeida JS, Dos-Santos JS, Figueiredo CP, Bellio M, Bozza MT, Assunção-Miranda I. Mayaro Virus Replication Restriction and Induction of Muscular Inflammation in Mice Are Dependent on Age, Type-I Interferon Response, and Adaptive Immunity. Front Microbiol. 2019 Oct 1;10:2246. 

DOI: 10.3389/fmicb.2019.02246

Mayaro virus (MAYV) is an emergent arbovirus first described in forest regions of the American continent, with recent and increasing notification of urban area circulation. Similar to Chikungunya (CHIKV) and other arthritogenic Alphavirus, MAYV-induced disease shows a high prevalence of persistent arthralgia, and myalgia. Despite this, knowledge regarding pathogenesis and characteristics of host immune response of MAYV infections are still limited. Here, using different ages of wild-type (WT), adult Type I Interferon receptor deficient (IFNAR–/–), and adult recombination activation gene-1 deficient (RAG–/–) mice, we have investigated the dependence of age, innate and adaptive immunity for the control of MAYV replication, tissue damage, and inflammation in mice. We have found that MAYV induces clinical signal and replicates in young WT mice, which gain the ability to restrict MAYV replication with aging. In addition, we observed that mice age and type I interferon response are related to restriction of MAYV infection and muscular inflammation in mice. Moreover, MAYV continues to replicate persistently in RAG–/– mice, being detected at blood and tissues 40 days post infection, indicating that adaptive immunity is essential to MAYV clearance. Despite chronic replication, infected adult RAG–/– mice did not develop an apparent signal of muscle damage in early and late infection. On the other hand, MAYV infection in young WT and adult IFNAR-/- mice triggers an increase in the expression of pro-inflammatory mediators, such as TNF, IL-6, KC, IL-1β, MCP-1, and RANTES, in muscle tissue, and decreases TGF-β expression, that were not significantly modulated in adult WT and RAG–/– mice. Taken together, our data demonstrated that age, innate and adaptive immunity are important to restrict MAYV replication and that adaptive immunity is also involved in MAYV-induced tissue damage. These results contribute to the comprehension of MAYV pathogenesis, and describe translational mice models for further studies of MAYV infection, vaccine tests, and therapeutic strategies against this virus.

Figueiredo CP, Barros-Aragão FGQ, Neris RLS, Frost PS, Soares C, Souza INO, Zeidler JD, Zamberlan DC, de Sousa VL, Souza AS, Guimarães ALA, Bellio M, Marcondes de Souza J, Alves-Leon SV, Neves GA, Paula-Neto HA, Castro NG, De Felice FG, Assunção-Miranda I, Clarke JR, Da Poian AT, Ferreira ST. Zika virus replicates in adult human brain tissue and impairs synapses and memory in mice. Nat Commun. 2019 Sep 5;10(1):3890.

DOI: 10.1038/s41467-019-11866-7

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.

Freitas-Mesquita AL, Dick CF, Dos-Santos ALA, Nascimento MTC, Rochael NC, Saraiva EM, Meyer-Fernandes JR. Cloning, expression and purification of 3'-nucleotidase/nuclease, an enzyme responsible for the Leishmania escape from neutrophil extracellular traps. Mol Biochem Parasitol. 2019 Apr;229:6-14.

DOI: 10.1016/j.molbiopara.2019.02.004

Leishmaniasis is one of the most significant of the neglected tropical diseases, with 350 million people in 98 countries worldwide living at risk of developing one of the many forms of the disease. During the transmission of the parasite from its vector to the vertebrate host, neutrophils are rapidly recruited to the site of the sandfly bite. Using different strategies, neutrophils can often kill a large number of parasites. However, some parasites can resist neutrophil-killing mechanisms and survive until macrophage arrival at the infection site. One of the strategies for neutrophil-mediated killing is the production of neutrophil extracellular traps (NETs). Because of its ecto-localized nuclease activity, the enzyme 3’-nucleotidase/nuclease (3’NT/NU), present in different Leishmania species, was recently identified as part of a possible parasite escape mechanism from NET-mediated death. Previous studies showed that 3’NT/NU also plays an important role in the establishment of Leishmania infection by generating extracellular adenosine that favors the parasite and macrophage interaction. This study aims to deepen the knowledge about 3’NT/NU, mainly with respect to its nuclease activity that is little studied in the current literature. For this, we cloned, expressed and purified the recombinant La3'NT/NU and have confirmed its contribution to the parasite escape from NET-mediated killing.

Gomes PS, Tanghe S, Gallego-Delgado J, Conde L, Freire-de-Lima L, Lima AC, Freire-de-Lima CG, Lima Junior JDC, Moreira O, Totino P, Rodriguez A, Todeschini AR, Morrot A. Targeting the Hexosamine Biosynthetic Pathway Prevents Plasmodium Developmental Cycle and Disease Pathology in Vertebrate Host. Front Microbiol. 2019 Feb 28;10:305. 

DOI: 10.3389/fmicb.2019.00305

Cerebral malaria (CM) is a clinical syndrome involving irreversible and lethal signs of brain injury associated to infection by parasites of the genus Plasmodium. The pathogenesis of CM derives from infection-induced proinflammatory cytokines associated with cytoadherence of parasitized red blood cells to brain microvasculature. Glycoconjugates are very abundant in the surface of Plasmodium spp., and are critical mediators of parasite virulence in host–pathogen interactions. Herein, we show that 6-Diazo-5-oxo-L-norleucine (DON) therapeutically used for blocking hexosamine biosynthetic pathway leads to recovery in experimental murine cerebral malaria. DON-induced protection was associated with decreased parasitism, which severely reduced Plasmodium transmission to mosquitoes. These findings point to a potential use of DON in combination therapies against malaria.

Gonçalves DS, Ferreira MDS, Gomes KX, Rodríguez-de La Noval C, Liedke SC, da Costa GCV, Albuquerque P, Cortines JR, Saramago Peralta RH, Peralta JM, Casadevall A, Guimarães AJ. Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose-binding proteins. Cell Microbiol. 2019 Oct;21(10):e13066. 

DOI: 10.1111/cmi.13066

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac–fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.

Gonçalves DS, Ferreira MDS, Guimarães AJ. Extracellular Vesicles from the Protozoa Acanthamoeba castellanii: Their Role in Pathogenesis, Environmental Adaptation and Potential Applications. Bioengineering (Basel). 2019 Feb 1;6(1):13.

DOI: 10.3390/bioengineering6010013

Extracellular vesicles (EVs) are membranous compartments of distinct cellular origin and biogenesis, displaying different sizes and include exosomes, microvesicles, and apoptotic bodies. The EVs have been described in almost every living organism, from simple unicellular to higher evolutionary scale multicellular organisms, such as mammals. Several functions have been attributed to these structures, including roles in energy acquisition, cell-to-cell communication, gene expression modulation and pathogenesis. In this review, we described several aspects of the recently characterized EVs of the protozoa Acanthamoeba castellanii, a free-living amoeba (FLA) of emerging epidemiological importance, and compare their features to other parasites’ EVs. These A. castellanii EVs are comprised of small microvesicles and exosomes and carry a wide range of molecules involved in many biological processes like cell signaling, carbohydrate metabolism and proteolytic activity, such as kinases, glucanases, and proteases, respectively. Several biomedical applications of these EVs have been proposed lately, including their use in vaccination, biofuel production, and the pharmaceutical industry, such as platforms for drug delivery.

Insuela DBR, Azevedo CT, Coutinho DS, Magalhães NS, Ferrero MR, Ferreira TPT, Cascabulho CM, Henriques-Pons A, Olsen PC, Diaz BL, Silva PMR, Cordeiro RSB, Martins MA, Carvalho VF. Glucagon reduces airway hyperreactivity, inflammation, and remodeling induced by ovalbumin. Sci Rep. 2019 Apr 24;9(1):6478. 

DOI: 10.1038/s41598-019-42981-6

Glucagon has been shown to be beneficial as a treatment for bronchospasm in asthmatics. Here, we investigate if glucagon would prevent airway hyperreactivity (AHR), lung inflammation, and remodeling in a murine model of asthma. Glucagon (10 and 100 µg/Kg, i.n.) significantly prevented AHR and eosinophilia in BAL and peribronchiolar region induced by ovalbumin (OVA) challenge, while only the dose of 100 µg/Kg of glucagon inhibited subepithelial fibrosis and T lymphocytes accumulation in BAL and lung. The inhibitory action of glucagon occurred in parallel with reduction of OVA-induced generation of IL-4, IL-5, IL-13, TNF-α, eotaxin-1/CCL11, and eotaxin-2/CCL24 but not MDC/CCL22 and TARC/CCL17. The inhibitory effect of glucagon (100 µg/Kg, i.n.) on OVA-induced AHR and collagen deposition was reversed by pre-treatment with indomethacin (10 mg/Kg, i.p.). Glucagon increased intracellular cAMP levels and inhibits anti-CD3 plus anti-CD28-induced proliferation and production of IL-2, IL-4, IL-10, and TNF- α from TCD4+ cells in vitro. These findings suggest that glucagon reduces crucial features of asthma, including AHR, lung inflammation, and remodeling, in a mechanism probably associated with inhibition of eosinophils accumulation and TCD4+ cell proliferation and function. Glucagon should be further investigated as an option for asthma therapy.

 Kennedy-Feitosa E, Cattani-Cavalieri I, Barroso MV, Romana-Souza B, Brito-Gitirana L, Valenca SS. Eucalyptol promotes lung repair in mice following cigarette smoke-induced emphysema. Phytomedicine. 2019 Mar 1;55:70-79.

DOI: 10.1016/j.phymed.2018.08.012 

Male mice (C57BL/6) were divided into the following groups: control (sham-exposed), cigarette smoke (CS) (mice exposed to 12 cigarettes a day for 60 days), CS + 1 mg/ml (CS mice treated with 1 mg/ml eucalyptol for 60 days), and CS + 10 mg/ml (CS mice treated with 10 mg/ml eucalyptol for 60 days). Mice in the CS and control groups received vehicle for 60 days. Eucalyptol (or the vehicle) was administered via inhalation (15 min/daily). Mice were sacrificed 24 h after the completion of the 120-day experimental procedure.

Luz NF, DeSouza-Vieira T, De Castro W, Vivarini AC, Pereira L, França RR, Silveira-Mattos PS, Costa DL, Teixeira C, Meneses C, Boaventura VS, de Oliveira CI, Lopes UG, Aronson N, Andrade BB, Brodskyn CI, Valenzuela JG, Kamhawi S, Borges VM. Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites. Front Immunol. 2018 Nov 28;9:2779.  

DOI: 10.3389/fimmu.2018.02779

Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24–48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections.

Magalhães KG, Luna-Gomes T, Mesquita-Santos F, Corrêa R, Assunção LS, Atella GC, Weller PF, Bandeira-Melo C, Bozza PT. Schistosomal Lipids Activate Human Eosinophils via Toll-Like Receptor 2 and PGD2 Receptors: 15-LO Role in Cytokine Secretion. Front Immunol. 2019 Jan 25;9:3161. 

DOI: 10.3389/fimmu.2018.03161

Parasite-derived lipids may play important roles in host-pathogen interactions and immune evasion mechanisms. Remarkable accumulation of eosinophils is a characteristic feature of inflammation associated with parasitic disease, especially caused by helminthes. Infiltrating eosinophils are implicated in the pathogenesis of helminth infection by virtue of their capacity to release an array of tissue-damaging and immunoregulatory mediators. However, the mechanisms involved in the activation of human eosinophils by parasite-derived molecules are not clear. Here we investigated the effects and mechanisms of schistosomal lipids-induced activation of human eosinophils. Our results showed that stimulation of human eosinophils in vitro with total lipid extracts from adult worms of S. mansoni induced direct activation of human eosinophils, eliciting lipid droplet biogenesis, synthesis of leukotriene (LT) C4 and eoxin (EX) C4 (14,15 LTC4) and secretion of eosinophil pre-formed TGFβ. We demonstrated that main eosinophil activating components within S. mansoni lipid extract are schistosomal-derived lysophosphatidylcholine (LPC) and prostaglandin (PG)D2. Moreover, TLR2 is up-regulated in human eosinophils upon stimulation with schistosomal lipids and pre-treatment with anti-TLR2 inhibited both schistosomal lipids- and LPC-, but not PGD2-, induced lipid droplet biogenesis and EXC4 synthesis within eosinophils, indicating that TLR2 mediates LPC-driven human eosinophil activation. By employing PGD2 receptor antagonists, we demonstrated that DP1 receptors are also involved in various parameters of human eosinophil activation induced by schistosomal lipids, but not by schistosomal LPC. In addition, schistosomal lipids and their active components PGD2 and LPC, triggered 15-LO dependent production of EXC4 and secretion of TGFβ. Taken together, our results showed that schistosomal lipids contain at least two components—LPC and PGD2—that are capable of direct activation of human eosinophils acting on distinct eosinophil-expressed receptors, noticeably TLR2 as well as DP1, trigger human eosinophil activation characterized by production/secretion of pro-inflammatory and immunoregulatory mediators.

Mustafá YM, Meuren LM, Coelho SVA, de Arruda LB. Pathways Exploited by Flaviviruses to Counteract the Blood-Brain Barrier and Invade the Central Nervous System. Front Microbiol. 2019 Mar 28;10:525.

DOI: 10.3389/fmicb.2019.00525

Human infection by different flaviviruses may cause severe neurologic syndromes, through pathogenic mechanisms that are still largely unknown. Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), yellow fever virus (YFV), dengue virus (DENV), and tick-borne encephalitis virus (TBEV) are believed to reach the central nervous system by a hematogenous route, upon crossing the blood-brain barrier. Although the disruption of BBB during flavivirus infection has been largely evidenced in experimental models, the relevance of BBB breakdown for virus entering the brain was not completely elucidated. In vitro models of BBB had demonstrated that these viruses replicated in brain microvascular endothelial cells (BMECs), which induced downregulation of tight junction proteins and increased the permeability of the barrier. Other reports demonstrated that infection of BMECs allowed the basolateral release of infectious particles, without a remarkable cytopathic effect, what might be sufficient for virus invasion. Virus replication and activation of other cells associated to the BBB, mostly astrocytes and microglia, were also reported to affect the endothelial barrier permeability. This event might occur simultaneously or after BMECs infection, being a secondary effect leading to BBB disruption. Importantly, activation of BMECs, astrocytes, and microglia by flaviviruses was associated to the expression and secretion of inflammatory mediators, which are believed to recruit leukocytes to the CNS. The leukocyte infiltrate could further mediate viral invasion through a Trojan horse mechanism and might contribute to BBB breakdown and to neurological alterations. This review discussed the previous studies regarding in vitro and in vivo models of JEV, WNV, ZIKV, YFV, DENV, and TBEV infection and addressed the pathways for BBB overcome and invasion of the CNS described for each virus infection, aiming to increment the knowledge and stimulate further discussion about the role of BBB in the neuropathogenesis of flavivirus infection.

Pratti JES, da Fonseca Martins AM, da Silva JP, Ramos TD, Pereira JC, Firmino-Cruz L, Oliveira-Maciel D, Vieira TSS, Lacerda LL, Vale AM, Freire-de-Lima CG, Gomes DCO, Saraiva EM, Rossi-Bergmann B, de Matos Guedes HL. The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy. PLoS Negl Trop Dis. 2019 Feb 25;13(2):e0007146. 

DOI: 10.1371/journal.pntd.0007146

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL.

Reis FCG, Borges BS, Jozefowicz LJ, Sena BAG, Garcia AWA, Medeiros LC, Martins ST, Honorato L, Schrank A, Vainstein MH, Kmetzsch L, Nimrichter L, Alves LR, Staats CC, Rodrigues ML. A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in Cryptococcus gattii. mSphere. 2019 Mar 20;4(2):e00080-19. 

DOI: 10.1128/mSphere.00080-19

Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release.

Souza-Moreira L, Soares VC, Dias SDSG, Bozza PT. Adipose-derived Mesenchymal Stromal Cells Modulate Lipid Metabolism and Lipid Droplet Biogenesis via AKT/mTOR -PPARγ Signalling in Macrophages. Sci Rep. 2019 Dec 0;9(1):20304.

DOI: 10.1038/s41598-019-56835-8

Mesenchymal stromal cells (MSCs) are a potential therapy for many chronic inflammatory diseases due to their regenerative, immunologic and anti-inflammatory properties. The two-way dialogue between MSCs and macrophages is crucial to tissue regeneration and repair. Previous research demonstrated that murine adipose-derived MSC conditioned medium (ASCcm) reprograms macrophages to an M2-like phenotype which protects from experimental colitis and sepsis. Here, our focus was to determine the molecular mechanism of lipid droplet biogenesis in macrophages re-educated using ASCcm. Adipose-derived MSC conditioned medium promotes phosphorylation of AKT/mTOR pathway proteins in macrophages. Furthermore, increased expression of PPARγ, lipid droplet biogenesis and PGE2 synthesis were observed in M2-like phenotype macrophages (high expression of arginase 1 and elevated IL-10). Treatment with mTOR inhibitor rapamycin or PPARγ inhibitor GW9662 suppressed lipid droplets and PGE2 secretion. However, these inhibitors had no effect on arginase-1 expression. Rapamycin, but not GW9662, inhibit IL-10 secretion. In conclusion, we demonstrate major effects of ASCcm to reprogram macrophage immunometabolism through mTOR and PPARγ dependent and independent pathways.

Vaccari M, Fourati S, Brown DR, Silva de Castro I, Bissa M, Schifanella L, Doster MN, Foulds KE, Roederer M, Koup RA, Sui Y, Berzofsky JA, Sekaly RP, Franchini G. Myeloid Cell Crosstalk Regulates the Efficacy of the DNA/ALVAC/gp120 HIV Vaccine Candidate. Front Immunol. 2019 May 14;10:1072. 

DOI: 10.3389/fimmu.2019.01072

Vaccination with DNA-SIV + ALVAC-SIV + gp120 alum results in inflammasome activation, high levels of IL-1β production, emergency myelopoiesis, and the egress of CXCR4+ CD14+ pre-monocytes from bone marrow. Previously we have shown that this vaccine-induced innate monocyte memory is associated with decreased risk of SIVmac251 acquisition. Because IL-1β also promotes the propagation of monocyte-derived suppressor (M-MDSC)-like cells, here we extended our analysis to this negative regulator subset, characterizing its levels and functions in macaques. Interestingly, we found that DNA prime engages M-MDSC-like cells and their levels are positively associated with the frequency of CD14+ classical monocytes, and negatively with the levels of CD16+ monocytes, correlates of decreased and increased risk of SIV acquisition, respectively. Accordingly, M-MDSC frequency, arginase activity, and NO were all associated with decrease of CD8 T cells responses and worse vaccination outcome. DNA vaccination thus induces innate immunity by engaging three subsets of myeloid cells, M-MDSCs, CD14+ innate monocyte memory, and CD16+ monocytes all playing different role in protection. The full characterization of the immunological space created by myeloid cell crosstalk will likely provide clues to improve the efficacy of HIV vaccine candidates.

Vasconcelos FM, Silva HLA, Poso SMV, Barroso MV, Lanzetti M, Rocha RS, Graça JS, Esmerino EA, Freitas MQ, Silva MC, Raices RSL, Granato D, Pimentel TC, Sant'Ana AS, Cruz AG, Valença SS. Probiotic Prato cheese attenuates cigarette smoke-induced injuries in mice. Food Res Int. 2019 Sep;123:697-703.

DOI: 10.1016/j.foodres.2019.06.001

The efficacy of probiotic Prato cheese against the inflammatory and oxidative damage in mice organs induced by cigarette smoke exposure was investigated. Forty C57BL/6 male mice were assigned to four groups: (CS) exposed to cigarette smoke and fed regular chow; (CS + C) exposed to cigarette smoke and fed daily conventional cheese ad libitum; (CS + PC) exposed to cigarette smoke and fed daily probiotic (Lactobacillus casei-01) cheese ad libitum; and a control group (C) exposed to ambient smoke-free air and fed regular chow. Bronchoalveolar lavage (BAL), blood, gut and liver homogenates were used for biochemical assays. The (CS + PC) group exhibited fewer BAL leukocytes, reactive oxygen species (ROS), and BAL and gut lipid peroxidation than the (CS) and (CS + C) groups, which had findings similar to the (C) group. Probiotic cheese consumption did not change the red blood cell count, but lower lactate dehydrogenase (LDH) levels in plasma, inducible nitric oxide synthase (iNOS) and peroxynitrite expression were observed compared to the (CS) and (CS + C) groups, with findings similar to the (C) group. These results suggest that probiotic Prato cheese consumption reduced oxidative stress in the lungs, gut, and liver.

Wolff T, Berrueta LA, Valente LMM, Barboza RS, Neris RLS, Guimarães-Andrade IP, Assunção-Miranda I, Nascimento AC, Gomes M, Gallo B, Iriondo C. Comprehensive characterisation of polyphenols in leaves and stems of three anti-dengue virus type-2 active Brazilian Faramea species (Rubiaceae) by HPLC-DAD-ESI-MS/MS. Phytochem Anal. 2019 Jan;30(1):62-72.

DOI: 10.1002/pca.2790

The methanol (MeOH) leaf extracts of the species Faramea bahiensis, F. hyacinthina and F. truncata (Rubiaceae) have previously shown in vitro non-cytotoxic and anti-dengue virus serotype 2 (DENV2) activities in human hepatocarcinoma cell lineage (HepG2). Chemical studies have led to the isolation of major flavonoids, but quite complex fractions of phenolic compounds still remain.

Nascimento DO, Vieira-de-Abreu A, Arcanjo AF, Bozza PT, Zimmerman GA, Castro-Faria-Neto HC. Integrin αDβ2 (CD11d/CD18) Modulates Leukocyte Accumulation, Pathogen Clearance, and Pyroptosis in Experimental Salmonella Typhimurium Infection. Front Immunol. 2018 May 24;9:1128.

DOI: 10.3389/fimmu.2018.01128

β2 integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin αDβ2 in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of αDβ2 results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in αDβ2 had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of αDβ2 in bacterial infection.

Nem de Oliveira Souza I, Frost PS, França JV, Nascimento-Viana JB, Neris RLS, Freitas L, Pinheiro DJLL, Nogueira CO, Neves G, Chimelli L, De Felice FG, Cavalheiro ÉA, Ferreira ST, Assunção-Miranda I, Figueiredo CP, Da Poian AT, Clarke JR. Acute and chronic neurological consequences of early-life Zika virus infection in mice. Sci Transl Med. 2018 Jun 6;10(444):eaar2749. 

DOI: 10.1126/scitranslmed.aar2749

Although congenital Zika virus (ZIKV) exposure has been associated with microcephaly and other neurodevelopmental disorders, long-term consequences of perinatal infection are largely unknown. We evaluated short- and long-term neuropathological and behavioral consequences of neonatal ZIKV infection in mice. ZIKV showed brain tropism, causing postnatal-onset microcephaly and several behavioral deficits in adulthood. During the acute phase of infection, mice developed frequent seizures, which were reduced by tumor necrosis factor–α (TNF-α) inhibition. During adulthood, ZIKV replication persisted in neonatally infected mice, and the animals showed increased susceptibility to chemically induced seizures, neurodegeneration, and brain calcifications. Altogether, the results show that neonatal ZIKV infection has long-term neuropathological and behavioral complications in mice and suggest that early inhibition of TNF-α–mediated neuroinflammation might be an effective therapeutic strategy to prevent the development of chronic neurological abnormalities.

Neris RLS, Figueiredo CM, Higa LM, Araujo DF, Carvalho CAM, Verçoza BRF, Silva MOL, Carneiro FA, Tanuri A, Gomes AMO, Bozza MT, Da Poian AT, Cruz-Oliveira C, Assunção-Miranda I. Co-protoporphyrin IX and Sn-protoporphyrin IX inactivate Zika, Chikungunya and other arboviruses by targeting the viral envelope. Sci Rep. 2018 Jun 28;8(1):9805. 

DOI: 10.1038/s41598-018-27855-7

The global situation of diseases transmitted by arthropod-borne viruses such as Dengue (DENV), Yellow Fever (YFV), Chikungunya (CHIKV) and Zika (ZIKV) viruses is alarming and treatment of human infection by these arboviruses faces several challenges. The discovery of broad-spectrum antiviral molecules, able to inactivate different groups of viruses, is an interesting approach. The viral envelope is a common structure among arboviruses, being a potential target for antivirals. Porphyrins are amphipathic molecules able to interact with membranes and absorb light, being widely used in photodynamic therapy. Previously, we showed that heme, Co-protoporphyrin IX (CoPPIX) and Sn-protoporphyrin IX (SnPPIX) directly inactivate DENV and YFV infectious particles. Here we demonstrate that the antiviral activity of these porphyrins can be broadened to CHIKV, ZIKV, Mayaro virus, Sindbis virus and Vesicular Stomatitis virus. Porphyrin treatment causes viral envelope protein loss, affecting viral morphology, adsorption and entry into target cells. Also, light-stimulation enhanced the SnPPIX activity against all tested arboviruses. In summary, CoPPIX and SnPPIX were shown to be efficient broad-spectrum compounds to inactivate medically and veterinary important viruses.

Nico D, Conde L, Rivera-Correa JL, Vasconcelos-Dos-Santos A, Mesentier-Louro L, Freire-de-Lima L, Arruda MB, Freire-de-Lima CG, Ferreira ODC Jr, Lopes Moreira ME, Zin AA, Vasconcelos ZFM, Otero RM, Palatnik-de-Sousa CB, Tanuri A, Todeschini AR, Savino W, Rodriguez A, Morrot A. Prevalence of IgG Autoantibodies against GD3 Ganglioside in Acute Zika Virus Infection. Front Med (Lausanne). 2018 Mar 9;5:25.

DOI: 10.3389/fmed.2018.00025

Zika virus (ZIKV) disease has become a global health emergency with devastating effects on public health. Recent evidences implicate the virus as an emergent neuropathological agent promoting serious pathologies of the human nervous system, that include destructive and malformation consequences such as development of ocular and fetal brain lesions, microcephaly in neonates, and Guillain–Barré syndrome (GBS) in adults. These neurological disorders of both central and peripheral nervous systems are thought to be associated to the neurotropic properties of the virus that has ability to infect neural stem cells as well as peripheral neurons, a hallmark of its pathogenicity. The presence of autoantibodies against gangliosides plays a pivotal role in the etiogenesis of GBS and a variety of neurological disorders. Gangliosides are a class of galactose-containing cerebrosides mainly expressed in nervous system tissues playing a critical role in the physiology of neural cells and neurogenesis. Herein, our findings indicate that patients at acute phase of ZIKV infection without any neurological signs show increased levels of IgG autoantibody against GD3 gangliosides, a class of glycolipid found to be highly expressed in neural stem cell acting in the maintenance of their self-renewal cellular capacity. It is possible that a pathological threshold of these antibodies is only acquired in secondary or subsequent infections. In the light of these evidences, we propose that the target of GD3 by autoimmune responses may possibly has an effect in the neuropathy and neurogenesis disorder seen during ZIKV infection.

Nunes CF, Nogueira JS, Vianna PHO, Ciambarella BT, Rodrigues PM, Miranda KR, Lobo LA, Domingues RMCP, Busch M, Atella GC, Vale AM, Bellio M, Nóbrega A, Canto FB, Fucs R. Probiotic treatment during neonatal age provides optimal protection against experimental asthma through the modulation of microbiota and T cells. Int Immunol. 2018 Apr 3;30(4):155-169.

DOI: 10.1093/intimm/dxy011

The incidence of allergic diseases, which increased to epidemic proportions in developed countries over the last few decades, has been correlated with altered gut microbiota colonization. Although probiotics may play a critical role in the restoration of gut homeostasis, their efficiency in the control of allergy is controversial. Here, we aimed to investigate the effects of probiotic treatment initiated at neonatal or adult ages on the suppression of experimental ovalbumin (OVA)-induced asthma. Neonatal or adult mice were orally treated with probiotic bacteria and subjected to OVA-induced allergy. Asthma-like symptoms, microbiota composition and frequencies of the total CD4+ T lymphocytes and CD4+Foxp3+ regulatory T (Treg) cells were evaluated in both groups. Probiotic administration to neonates, but not to adults, was necessary and sufficient for the absolute prevention of experimental allergen-induced sensitization. The neonatally acquired tolerance, transferrable to probiotic-untreated adult recipients by splenic cells from tolerant donors, was associated with modulation of gut bacterial composition, augmented levels of cecum butyrate and selective accumulation of Treg cells in the airways. Our findings reveal that a cross-talk between a healthy microbiota and qualitative features inherent to neonatal T cells, especially in the Treg cell subset, might support the beneficial effect of perinatal exposure to probiotic bacteria on the development of long-term tolerance to allergens.

Poggio HA, Antunes MA, Rocha NN, Kitoko JZ, Morales MM, Olsen PC, Lopes-Pacheco M, Cruz FF, Rocco PRM. Impact of one versus two doses of mesenchymal stromal cells on lung and cardiovascular repair in experimental emphysema. Stem Cell Res Ther. 2018 Nov 8;9(1):296. 

DOI: 10.1186/s13287-018-1043-6

A single administration of mesenchymal stromal cells (MSCs) has been shown to reduce lung inflammation in experimental elastase-induced emphysema; however, effects were limited in terms of lung-tissue repair and cardiac function improvement. We hypothesized that two doses of MSCs could induce further lung and cardiovascular repair by mitigating inflammation and remodeling in a model of emphysema induced by multiple elastase instillations. We aimed to comparatively investigate the effects of one versus two doses of MSCs, administered 1 week apart, in a murine model of elastase-induced emphysema.

Pinheiro TM, Mota MTO, Watanabe ASA, Biselli-Périco JM, Drumond BP, Ribeiro MR, Vedovello D, Araújo JP Jr, Pimenta PFP, Chaves BA, Silva MMCD, Batista ICA, Papa MP, Meuren LM, Lucas CGO, Matassoli FL, Gil LHVG, Bozzi A, Calzavara-Silva CE, Arruda LB, Souza DDG, Teixeira MM, Vasilakis N, Nogueira ML. Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil. PLoS Negl Trop Dis. 2018 May 29;12(5):e0006525. 

DOI: 10.1371/journal.pntd.0006525

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto.

Posso SV, Quesnot N, Moraes JA, Brito-Gitirana L, Kennedy-Feitosa E, Barroso MV, Porto LC, Lanzetti M, Valença SS. AT-RVD1 repairs mouse lung after cigarette smoke-induced emphysema via downregulation of oxidative stress by NRF2/KEAP1 pathway. Int Immunopharmacol. 2018 Mar;56:330-338.

DOI: 10.1016/j.intimp.2018.01.045

Long-term exposure to cigarette smoke (CS) results in alveolar parenchyma destruction due to chronic inflammatory response and the imbalance between oxidants and antioxidants, and proteases and antiproteases. Emphysema is the main symptom of chronic obstructive pulmonary disease. Current treatment focuses on relieving respiratory symptoms, and inflammation resolution failure is an important pathophysiological element of the disease. Specialized pro-resolving mediators (SPMs) synthesized endogenously during resolution processes demonstrated beneficial effects in murine models of airway inflammation. Here, we aimed to test the SPM AT-RvD1 in a murine model of CS-induced emphysema. AT-RvD1 restored elastic fibers and lung morphology, with reduction in MMP-3, neutrophils, and myeloperoxidase activity and increases in macrophages and IL-10 levels. AT-RvD1 also decreased levels of oxidative stress markers and ROS via upregulation of the Nrf2/Keap1 pathway. Therefore, we suggest that AT-RvD1 causes pro-resolutive action in our murine model of CS-induced emphysema by upregulation of the Nrf2/Keap1 pathway.

Siqueira MDS, Ribeiro RM, Travassos LH. Autophagy and Its Interaction With Intracellular Bacterial Pathogens. Front Immunol. 2018 May 23;9:935.

DOI: 10.3389/fimmu.2018.00935

Cellular responses to stress can be defined by the overwhelming number of changes that cells go through upon contact with and stressful conditions such as infection and modifications in nutritional status. One of the main cellular responses to stress is autophagy. Much progress has been made in the understanding of the mechanisms involved in the induction of autophagy during infection by intracellular bacteria. This review aims to discuss recent findings on the role of autophagy as a cellular response to intracellular bacterial pathogens such as, Streptococcus pyogenes, Mycobacterium tuberculosis, Shigella flexneri, Salmonella typhimurium, Listeria monocytogenes, and Legionella pneumophila, how the autophagic machinery senses these bacteria directly or indirectly (through the detection of bacteria-induced nutritional stress), and how some of these bacterial pathogens manage to escape from autophagy.

Vasconcellos LR, Siqueira MS, Moraes R, Carneiro LA, Bozza MT, Travassos LH. Heme Oxygenase-1 and Autophagy Linked for Cytoprotection. Curr Pharm Des. 2018;24(20):2311-2316. 

DOI: 10.2174/1381612824666180727100909

Heme-oxygenase (HO) catalyzes the main enzymatic step of heme degradation and generates anti-inflammatory end products with protective roles in physiological and pathological situations. The importance of HO in pathological conditions is evidenced by its pharmacological inhibition or genetic blockage in different models of stress such as infection, inflammation and oxidative stress. Under these situations, another well-known protective process triggered is autophagy. Autophagy is a homeostatic process that eliminates defective cytosolic components and organelles, allowing cells and tissues to recover through recycling of functional blocks for anabolic reactions. Recently, studies have demonstrated a link between HO activity and autophagy activation.

Abreu SC, Lopes-Pacheco M, da Silva AL, Xisto DG, de Oliveira TB, Kitoko JZ, de Castro LL, Amorim NR, Martins V, Silva LHA, Gonçalves-de-Albuquerque CF, de Castro Faria-Neto HC, Olsen PC, Weiss DJ, Morales MM, Diaz BL, Rocco PRM. Eicosapentaenoic Acid Enhances the Effects of Mesenchymal Stromal Cell Therapy in Experimental Allergic Asthma. Front Immunol. 2018 May 24;9:1147.

DOI: 10.3389/fimmu.2018.01147

Asthma is characterized by chronic lung inflammation and airway hyperresponsiveness. Despite recent advances in the understanding of its pathophysiology, asthma remains a major public health problem and, at present, there are no effective interventions capable of reversing airway remodeling. Mesenchymal stromal cell (MSC)-based therapy mitigates lung inflammation in experimental allergic asthma; however, its ability to reduce airway remodeling is limited. We aimed to investigate whether pre-treatment with eicosapentaenoic acid (EPA) potentiates the therapeutic properties of MSCs in experimental allergic asthma. Seventy-two C57BL/6 mice were used. House dust mite (HDM) extract was intranasally administered to induce severe allergic asthma in mice. Unstimulated or EPA-stimulated MSCs were administered intratracheally 24 h after final HDM challenge. Lung mechanics, histology, protein levels of biomarkers, and cellularity in bronchoalveolar lavage fluid (BALF), thymus, lymph nodes, and bone marrow were analyzed. Furthermore, the effects of EPA on lipid body formation and secretion of resolvin-D1 (RvD1), prostaglandin E2 (PGE2), interleukin (IL)-10, and transforming growth factor (TGF)-β1 by MSCs were evaluated in vitro. EPA-stimulated MSCs, compared to unstimulated MSCs, yielded greater therapeutic effects by further reducing bronchoconstriction, alveolar collapse, total cell counts (in BALF, bone marrow, and lymph nodes), and collagen fiber content in airways, while increasing IL-10 levels in BALF and M2 macrophage counts in lungs. In conclusion, EPA potentiated MSC-based therapy in experimental allergic asthma, leading to increased secretion of pro-resolution and anti-inflammatory mediators (RvD1, PGE2, IL-10, and TGF-β), modulation of macrophages toward an anti-inflammatory phenotype, and reduction in the remodeling process. Taken together, these modifications may explain the greater improvement in lung mechanics obtained. This may be a promising novel strategy to potentiate MSCs effects.

Banerjee I, Behl B, Mendonca M, Shrivastava G, Russo AJ, Menoret A, Ghosh A, Vella AT, Vanaja SK, Sarkar SN, Fitzgerald KA, Rathinam VAK. Gasdermin D Restrains Type I Interferon Response to Cytosolic DNA by Disrupting Ionic Homeostasis. Immunity. 2018 Sep 18;49(3):413-426.e5.

DOI: 10.1016/j.immuni.2018.07.006

Inflammasome complexes trigger the enzymatic activity of caspase-1, which activates interleukin-1β (IL-1β) and IL-18 (Martinon et al., 2002). Caspase-1 also targets gasdermin D, a pore-forming protein (Kayagaki et al., 2015, Shi et al., 2015). The pore-forming activity of gasdermin D resides in its N-terminal domain and is inhibited by its C-terminal domain. Caspase-1 cleaves gasdermin D at the linker region between these two domains, liberating the N-terminal domain, which migrates to the plasma membrane-forming pores with an inner diameter of 10–15 nm (Aglietti et al., 2016, Ding et al., 2016, Liu et al., 2016, Sborgi et al., 2016). An important consequence of gasdermin D activation is a lytic form of cell death called pyroptosis (He et al., 2015, Kayagaki et al., 2015, Shi et al., 2015). However, new evidence points out that gasdermin D executes additional functions independent of cell death; gasdermin D pores mediate the release of IL-1β and IL-18 without inducing cell death in response to certain ligands (Evavold et al., 2018). Additionally, after cytosolic LPS sensing by caspase-11, gasdermin D activates the NLRP3 inflammasome by inducing potassium (K+) efflux (Kayagaki et al., 2015, Rühl and Broz, 2015, Schmid-Burgk et al., 2015). Central to these distinct functions of gasdermin D is its membrane pore-forming activity. However, whether gasdermin D executes any additional immune functions is largely unknown.
A key surveillance mechanism in the cytosol, in addition to inflammasomes, is the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) pathway. cGAS is a sensor for cytosolic DNA, and the binding of DNA by cGAS triggers its nucleotidyl transferase activity leading to the synthesis of cGAMP from ATP and GTP. cGAMP stimulates the transcription of type I interferon genes via the STING adaptor-TBK1 kinase-IRF3 transcription factor axis (Burdette et al., 2011, Wu et al., 2013). The type I interferon response elicited by cGAS plays important roles in host defense (Schneider et al., 2014). However, it is increasingly appreciated that the sustained production of type I interferons at high amounts is detrimental to the host, particularly during infections with intracellular bacteria such as Francisella novicida, Mycobacterium tuberculosis, and Listeria monocytogenes (Auerbuch et al., 2004, Henry et al., 2010, Mayer-Barber et al., 2014, McNab et al., 2015, Storek et al., 2015). Therefore, the magnitude and duration of cGAS-driven type I interferon responses should be kept in check. However, how the host restrains cGAS signaling during infections is poorly defined.
Here, we demonstrate that gasdermin D activated by the Aim2 inflammasome complex suppresses cytosolic DNA-induced production of type I interferons in macrophages. Consistent with this finding, mice lacking gasdermin D displayed enhanced IFN-β response to F. novicida infection. Pyroptosis and the extracellular release of IL-1 cytokines are dispensable for the inhibition of IFN-β by gasdermin D. Mechanistically, gasdermin D-induced membrane pores leaked intracellular potassium (K+) ions, and this K+ efflux in turn impaired type I interferon responses to cytosolic DNA and F. novicida. Gasdermin D-K+ efflux axis targeted cGAS to reduce cGAMP synthesis and thus IFN-β production. In summary, this study uncovers a previously unrecognized key role for gasdermin D in restraining cytosolic DNA-elicited interferon responses. Collectively, an emerging theme from the findings of this work and the recent studies (Evavold et al., 2018, Kayagaki et al., 2015) is that the fundamental pore-forming ability of gasdermin D and the consequent ionic fluxes confer gasdermin D additional biological functions independent of the terminal cell lytic event.

Barboza RS, Valente LMM, Wolff T, Assunção-Miranda I, Neris RLS, Guimarães-Andrade IP, Gomes M. Antiviral Activity of Faramea hyacinthina and Faramea truncata Leaves on Dengue Virus Type-2 and Their Major Compounds. Chem Biodivers. 2018 Feb;15(2).

DOI: 10.1002/cbdv.201700393

The defatted fractions of the Faramea hyacinthina and F. truncata (Rubiaceae) leaf MeOH extracts showed in vitro non-cytotoxic and anti-dengue virus serotype 2 (DENV2) activity in human hepatocarcinoma cell lineage (HepG2). Submitting these fractions to the developed RP-SPE method allowed isolating the antiviral flavanone (2S)-isosakuranetin-7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (1) from both species and yielded less active sub-fractions. The new diastereoisomeric epimer pair (2S) + (2R) of 5,3′,5′-trihydroxyflavanone-7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (2a/2b) from F. hyacinthina; the known narigenin-7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (3) from both species; rutin (4) and quercetin-4′-β-d-O-glucopyranosyl-3-O-rutinoside (5) from F. hyacinthina, and kaempferol-3-O-rutinoside (6), erythroxyloside A (7) and asperuloside (8) from F. truncata have been isolated from these sub-fractions. Compounds 4 – 8 are reported for the first time in Faramea spp.

de Mattos Barbosa MG, de Andrade Silva BJ, Assis TQ, da Silva Prata RB, Ferreira H, Andrade PR, da Paixão de Oliveira JA, Sperandio da Silva GM, da Costa Nery JA, Sarno EN, Pinheiro RO. Autophagy Impairment Is Associated With Increased Inflammasome Activation and Reversal Reaction Development in Multibacillary Leprosy. Front Immunol. 2018 Jun 4;9:1223.

DOI: 10.3389/fimmu.2018.01223

Leprosy reactions are responsible for incapacities in leprosy and represent the major cause of permanent neuropathy. The identification of biomarkers able to identify patients more prone to develop reaction could contribute to adequate clinical management and the prevention of disability. Reversal reaction may occur in unstable borderline patients and also in lepromatous patients. To identify biomarker signature profiles related with the reversal reaction onset, multibacillary patients were recruited and classified accordingly the occurrence or not of reversal reaction during or after multidrugtherapy. Analysis of skin lesion cells at diagnosis of multibacillary leprosy demonstrated that in the group that developed reaction (T1R) in the future there was a downregulation of autophagy associated with the overexpression of TLR2 and MLST8. The autophagy impairment in T1R group was associated with increased expression of NLRP3, caspase-1 (p10) and IL-1β production. In addition, analysis of IL-1β production in serum from multibacillary patients demonstrated that patients who developed reversal reaction have significantly increased concentrations of IL-1β at diagnosis, suggesting that the pattern of innate immune responses could predict the reactional episode outcome. In vitro analysis demonstrated that the blockade of autophagy with 3-methyladenine (3-MA) in Mycobacterium leprae-stimulated human primary monocytes increased the assembly of NLRP3 specks assembly, and it was associated with an increase of IL-1β and IL-6 production. Together, our data suggest an important role for autophagy in multibacillary leprosy patients to avoid exacerbated inflammasome activation and the onset of reversal reaction.

Echevarria-Lima J, de Abreu Pereira D, de Oliveira TS, de Melo Espíndola O, Lima MA, Celestino Leite AC, Sandim V, Rodrigues Nascimento C, E Kalume D, B Zingali R. Protein Profile of Blood Monocytes is Altered in HTLV-1 Infected Patients: Implications for HAM/TSP Disease. Sci Rep. 2018 Sep 25;8(1):14354.

DOI: 10.1038/s41598-018-32324-2

Human T-cell lymphotropic virus type-1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The endothelial breakdown and migration of leukocytes, including monocytes, to the spinal cord are involved in HAM/TSP development. Monocytes from HTLV-1-infected individuals exhibit important functional differences when compared to cells from uninfected donors. Using proteomic shot gun strategy, performed by nanoACQUITY-UPLC system, we analyzed monocytes isolated from peripheral blood of asymptomatic carriers (AC), HAM/TSP and uninfected individuals. 534 proteins were identified among which 376 were quantified by ExpressionE software. Our study revealed a panel of changes in protein expression linked to HTLV-1 infection. Upregulation of heat shock proteins and downregulation of canonical histone expression were observed in monocytes from HTLV-1-infected patients. Moreover, expression of cytoskeleton proteins was increased in monocytes from HTLV-1-infected patients, mainly in those from HAM/TSP, which was confirmed by flow cytometry and fluorescence microscopy. Importantly, functional assays demonstrated that monocytes from HAM/TSP patients present higher ability for adhesion and transmigration thought endothelium than those from AC and uninfected individuals. The major changes on monocyte protein profile were detected in HAM/TSP patients, suggesting that these alterations exert a relevant role in the establishment of HAM/TSP.

Filardy AA, Guimarães-Pinto K, Nunes MP, Zukeram K, Fliess L, Pereira L, Oliveira Nascimento D, Conde L, Morrot A. Human Kinetoplastid Protozoan Infections: Where Are We Going Next? Front Immunol. 2018 Jul 25;9:1493.

DOI: 10.3389/fimmu.2018.01493

Kinetoplastida trypanosomatidae microorganisms are protozoan parasites exhibiting a developmental stage in the gut of insect vectors and tissues of vertebrate hosts. During the vertebrate infective stages, these parasites alter the differential expression of virulence genes, modifying their biological and antigenic properties in order to subvert the host protective immune responses and establish a persistent infection. One of the hallmarks of kinetoplastid parasites is their evasion mechanisms from host immunity, leading to disease chronification. The diseases caused by kinetoplastid parasites are neglected by the global expenditures in research and development, affecting millions of individuals in the low and middle-income countries located mainly in the tropical and subtropical regions. However, investments made by public and private initiatives have over the past decade leveraged important lines of intervention that if well-integrated to health care programs will likely accelerate disease control initiatives. This review summarizes recent advances in public health care principles, including new drug discoveries and their rational use with chemotherapeutic vaccines, and the implementation of control efforts to spatially mapping the kinetoplastid infections through monitoring of infected individuals in epidemic areas. These approaches should bring us the means to track genetic variation of parasites and drug resistance, integrating this knowledge into effective stewardship programs to prevent vector-borne kinetoplastid infections in areas at risk of disease spreading.

Firmino-Cruz L, Ramos TD, da Fonseca-Martins AM, Maciel-Oliveira D, Oliveira-Silva G, Pratti JES, Cavazzoni C, Chaves SP, Oliveira Gomes DC, Morrot A, Freire-de-Lima L, Vale AM, Freire-de-Lima CG, Decote-Ricardo D, de Matos Guedes HL. Immunomodulating role of IL-10-producing B cells in Leishmania amazonensis infection. Cell Immunol. 2018 Dec;334:20-30. 

DOI: 10.1016/j.cellimm.2018.08.014

This work aims to study the immunomodulation of B lymphocytes during L. amazonensis infection. We demonstrated in this study that follicular B cells from draining lymph nodes of infected wild type BALB/c mice are the major source of IL-10 during infection. We infected BALB/Xid mice that developed smaller lesions in comparison with the control, but the parasite load obtained from the infected tissues was similar in both groups. We observed a reduction in the number of follicular B cells from BALB/Xid mice in relation to WT mice and, consequently, lower levels of IgM, IgG, IgG1, IgG2a and IgG2b in the serum of BALB/Xid when compared with wild type mice. BALB/Xid mice also presented lower levels of IL-10 in the infected footpad, draining lymph nodes and in the spleen when compared with WT infected tissues. We did not detect differences in the number of IL-10 producing CD4+ and CD8+ T cells between WT and BALB/Xid mice; however, a strong reduction of IL-10 producing follicular B cells was noted in BALB/Xid mice. When analyzed together, our data indicate that B cells are related with lesion pathogenesis through the production of antibodies and IL-10.

Gomes-Neto JF, Sartorius R, Canto FB, Almeida TS, Dias AA, Barbosa CD, Melo GA, Oliveira AC, Aguiar PN, Machado CR, de Matos Guedes HL, Santiago MF, Nóbrega A, De Berardinis P, Bellio M. Vaccination With Recombinant Filamentous fd Phages Against Parasite Infection Requires TLR9 Expression. Front Immunol. 2018 May 29;9:1173. 

DOI: 10.3389/fimmu.2018.01173

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9−/− mice were vaccinated with rfd phages expressing the OVA257–264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8+ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9−/− animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8+ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism.

Gonçalves DS, Ferreira MDS, Liedke SC, Gomes KX, de Oliveira GA, Leão PEL, Cesar GV, Seabra SH, Cortines JR, Casadevall A, Nimrichter L, Domont GB, Junqueira MR, Peralta JM, Guimaraes AJ. Extracellular vesicles and vesicle-free secretome of the protozoa Acanthamoeba castellanii under homeostasis and nutritional stress and their damaging potential to host cells. Virulence. 2018 Dec 31;9(1):818-836.

DOI: 10.1080/21505594.2018.1451184

Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.

Guimarães-Pinto K, Nascimento DO, Corrêa-Ferreira A, Morrot A, Freire-de-Lima CG, Lopes MF, DosReis GA, Filardy AA. Trypanosoma cruzi Infection Induces Cellular Stress Response and Senescence-Like Phenotype in Murine Fibroblasts. Front Immunol. 2018 Jul 9;9:1569. 

DOI: 10.3389/fimmu.2018.01569

Trypanosoma cruzi infects and replicates within a wide variety of immune and non-immune cells. Here, we investigated early cellular responses induced in NIH-3T3 fibroblasts upon infection with trypomastigote forms of T. cruzi. We show that fibroblasts were susceptible to T. cruzi infection and started to release trypomastigotes to the culture medium after 4 days of infection. Also, we found that T. cruzi infection reduced the number of fibroblasts in 3-day cell cultures, by altering fibroblast proliferation. Infected fibroblasts displayed distinctive phenotypic alterations, including enlarged and flattened morphology with a nuclei accumulation of senescence-associated heterochromatin foci. In addition, infection induced an overexpression of the enzyme senescence-associated β-galactosidase (SA-β-gal), an activation marker of the cellular senescence program, as well as the production of cytokines and chemokines involved with the senescence-associated secretory phenotype (SASP) such as IL-6, TNF-α, IL-1β, and MCP-1. Infected fibroblasts released increased amounts of stress-associated factors nitric oxide (NO) and reactive oxygen species (ROS), and the treatment with antioxidants deferoxamine (DFO) and N-acetylcysteine reduced ROS generation, secretion of SASP-related cytokine IL-6, SA-β-gal activity, and parasite load by infected fibroblasts. Taken together, our data suggest that T. cruzi infection triggers a rapid cellular stress response followed by induction of a senescent-like phenotype in NIH-3T3 fibroblasts, enabling them to act as reservoirs of parasites during the early stages of the Chagas disease.

Kitoko JZ, de Castro LL, Nascimento AP, Abreu SC, Cruz FF, Arantes AC, Xisto DG, Martins MA, Morales MM, Rocco PRM, Olsen PC. Therapeutic administration of bone marrow-derived mesenchymal stromal cells reduces airway inflammation without up-regulating Tregs in experimental asthma. Clin Exp Allergy. 2018 Feb;48(2):205-216.  

DOI: 10.1111/cea.13048

Prophylactic administration of mesenchymal stromal cells (MSCs) derived from adipose (AD-MSC) and bone marrow tissue (BM-MSC) in ovalbumin-induced asthma hinders inflammation in a Treg-dependent manner. It is uncertain whether MSCs act through Tregs when inflammation is already established in asthma induced by a clinically relevant allergen.

Linhares-Lacerda L, Granato A, Gomes-Neto JF, Conde L, Freire-de-Lima L, de Freitas EO, Freire-de-Lima CG, Coutinho Barroso SP, Jorge de Alcântara Guerra R, Pedrosa RC, Savino W, Morrot A. Circulating Plasma MicroRNA-208a as Potential Biomarker of Chronic Indeterminate Phase of Chagas Disease. Front Microbiol. 2018 Mar 6;9:269.

DOI: 10.3389/fmicb.2018.00269

Chagas cardiomyopathy is the most severe clinical manifestation of chronic Chagas disease. The disease affects most of the Latin American countries, being considered one of the leading causes of morbidity and death in the continent. The pathogenesis of Chagas cardiomyopathy is very complex, with mechanisms involving parasite-dependent cytopathy, immune-mediated myocardial damage and neurogenic disturbances. These pathological changes eventually result in cardiac myocyte hypertrophy, arrhythmias, congestive heart failure and stroke during chronic infection phase. Herein, we show that miR-208a, a microRNA that is a key factor in promoting cardiovascular dysfunction during cardiac hypertrophy processes of heart failure, has its circulating levels increased during chronic indeterminate phase when compared to cardiac (CARD) clinical forms in patients with Chagas disease. In contrast, we have not found altered serum levels of miR-34a, a microRNA known to promote pro-apoptotic role in myocardial infarction during degenerative process of cardiac injuries thus indicating intrinsic differences in the nature of the mechanisms underlying the heart failure triggered by Trypanosoma cruzi infection. Our findings support that the chronic indeterminate phase is a progressive phase involved in the genesis of chagasic cardiopathy and point out the use of plasma levels of miR-208a as candidate biomarker in risk-prediction score for the clinical prognosis of Chagas disease.

Lixa C, Mujo A, de Magalhães MTQ, Almeida FCL, Lima LMTR, Pinheiro AS. Oligomeric transition and dynamics of RNA binding by the HuR RRM1 domain in solution. J Biomol NMR. 2018 Dec;72(3-4):179-192.

DOI: 10.1007/s10858-018-0217-y

Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization–ion mobility spectrometry–mass spectrometry (ESI–IMS–MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1. Our solution structure reveals a canonical RRM fold containing a 19-residue, intrinsically disordered N-terminal extension, which is not involved in RNA binding. NMR titration results confirm the primary RNA-binding site to the two central β-strands, β1 and β3, for a cyclooxygenase 2 (Cox2) ARE I-derived, 7-nucleotide RNA ligand. We show by 15N relaxation that, in addition to the N- and C-termini, the β2–β3 loop undergoes fast backbone dynamics (ps–ns) both in the free and RNA-bound state, indicating that no structural ordering happens upon RNA interaction. ESI–IMS–MS reveals that HuR RRM1 dimerizes, however dimer population represents a minority. Dimerization occurs via the α-helical surface, which is oppositely orientated to the RNA-binding β-sheet. By using a DNA analog of the Cox2 ARE I, we show that DNA binding stabilizes HuR RRM1 monomer and shifts the monomer–dimer equilibrium toward the monomeric species. Altogether, our results deepen the current understanding of the mechanism of RNA recognition employed by HuR.

Lucas CGO, Kitoko JZ, Ferreira FM, Suzart VG, Papa MP, Coelho SVA, Cavazzoni CB, Paula-Neto HA, Olsen PC, Iwasaki A, Pereira RM, Pimentel-Coelho PM, Vale AM, de Arruda LB, Bozza MT. Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection. Nat Commun. 2018 Aug 7;9(1):3136. 

DOI: 10.1038/s41467-018-05519-4 

Protective adaptive immunity to Zika virus (ZIKV) has been mainly attributed to cytotoxic CD8+ T cells and neutralizing antibodies, while the participation of CD4+ T cells in resistance has remained largely uncharacterized. Here, we show a neutralizing antibody response, dependent on CD4+ T cells and IFNγ signaling, which we detected during the first week of infection and is associated with reduced viral load in the brain, prevention of rapid disease onset and survival. We demonstrate participation of these components in the resistance to ZIKV during primary infection and in murine adoptive transfer models of heterologous ZIKV infection in a background of IFNR deficiency. The protective effect of adoptively transferred CD4+ T cells requires IFNγ signaling, CD8+ T cells and B lymphocytes in recipient mice. Together, this indicates the importance of CD4+ T cell responses in future vaccine design for ZIKV.

Martin-Martin I, Chagas AC, Guimaraes-Costa AB, Amo L, Oliveira F, Moore IN, DeSouza-Vieira TS, Sanchez EE, Suntravat M, Valenzuela JG, Ribeiro JMC, Calvo E. Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection. PLoS Pathog. 2018 May 3;14(5):e1007006. 

DOI: 10.1371/journal.ppat.1007006

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis—Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine.

Melo AC, Cattani-Cavalieri I, Barroso MV, Quesnot N, Gitirana LB, Lanzetti M, Valença SS. Atorvastatin dose-dependently promotes mouse lung repair after emphysema induced by elastase. Biomed Pharmacother. 2018 Jun;102:160-168.

DOI: 10.1016/j.biopha.2018.03.067

Emphysema results in a proteinase – antiproteinase imbalance, inflammation and oxidative stress. Our objective was to investigate whether atorvastatin could repair mouse lungs after elastase-induced emphysema. Vehicle (50 μL) or porcine pancreatic elastase (PPE) was administered on day 1, 3, 5 and 7 at 0.6 U intranasally. Male mice were divided into a control group (sham), PPE 32d (sacrificed 24 h after 32 days), PPE 64d (sacrificed 24 h after 64 days), and atorvastatin 1, 5 and 20 mg treated from day 33 until day 64 and sacrificed 24 h later (A1 mg, A5 mg and A20 mg, respectively). Treatment with atorvastatin was performed via inhalation for 10 min once a day. We observed that emphysema at day 32 was similar to emphysema at day 64. The mean airspace chord length (Lm) indicated a recovery of pulmonary morphology in groups A5 mg and A20 mg, as well as recovery of collagen and elastic fibers in comparison to the PPE group. Bronchoalveolar lavage fluid (BALF) leukocytes were reduced in all atorvastatin-treated groups. However, tissue macrophages were reduced only in the A20 mg group compared with the PPE group, while tissue neutrophils were reduced in the A5 mg and A20 mg groups. The redox balance was restored mainly in the A20 mg group compared with the PPE group. Finally, atorvastatin at doses of 5 and 20 mg reduced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and matrix metalloproteinase-12 (MMP-12) compared with the PPE group. In conclusion, atorvastatin was able to induce lung tissue repair in emphysematous mice.

Morrot A, da Fonseca LM, Salustiano EJ, Gentile LB, Conde L, Filardy AA, Franklim TN, da Costa KM, Freire-de-Lima CG, Freire-de-Lima L. Metabolic Symbiosis and Immunomodulation: How Tumor Cell-Derived Lactate May Disturb Innate and Adaptive Immune Responses. Front Oncol. 2018 Mar 23;8:81. 

DOI: 10.3389/fonc.2018.00081

The tumor microenvironment (TME) is composed by cellular and non-cellular components. Examples include the following: (i) bone marrow-derived inflammatory cells, (ii) fibroblasts, (iii) blood vessels, (iv) immune cells, and (v) extracellular matrix components. In most cases, this combination of components may result in an inhospitable environment, in which a significant retrenchment in nutrients and oxygen considerably disturbs cell metabolism. Cancer cells are characterized by an enhanced uptake and utilization of glucose, a phenomenon described by Otto Warburg over 90 years ago. One of the main products of this reprogrammed cell metabolism is lactate. “Lactagenic” or lactate-producing cancer cells are characterized by their immunomodulatory properties, since lactate, the end product of the aerobic glycolysis, besides acting as an inducer of cellular signaling phenomena to influence cellular fate, might also play a role as an immunosuppressive metabolite. Over the last 10 years, it has been well accepted that in the TME, the lactate secreted by transformed cells is able to compromise the function and/or assembly of an effective immune response against tumors. Herein, we will discuss recent advances regarding the deleterious effect of high concentrations of lactate on the tumor-infiltrating immune cells, which might characterize an innovative way of understanding the tumor-immune privilege.

Muniz VS, Silva JC, Braga YAV, Melo RCN, Ueki S, Takeda M, Hebisawa A, Asano K, Figueiredo RT, Neves JS. Eosinophils release extracellular DNA traps in response to Aspergillus fumigatus. J Allergy Clin Immunol. 2018 Feb;141(2):571-585.e7. 

DOI: 10.1016/j.jaci.2017.07.048

Eosinophils mediate the immune response in different infectious conditions. The release of extracellular DNA traps (ETs) by leukocytes has been described as an innate immune response mechanism that is relevant in many disorders including fungal diseases. Different stimuli induce the release of human eosinophil ETs (EETs). Aspergillus fumigatus is an opportunistic fungus that may cause eosinophilic allergic bronchopulmonary aspergillosis (ABPA). It has been reported that eosinophils are important to the clearance of A fumigatus in infected mice lungs. However, the immunological mechanisms that underlie the molecular interactions between A fumigatus and eosinophils are poorly understood.

DeSouza-Vieira T, Chan FK. Bacterial pathogenesis: Pathogenic bacteria attack RHIM. Nat Microbiol. 2017 Mar 28;2:17042.

DOI:  10.1038/nmicrobiol.2017.42

Attaching and effacing enteropathogenic Escherichia coli causes gastrointestinal inflammation and diarrhoea. In this issue of Nature Microbiology, Pearson and colleagues find that this pathology involves bacterial cleavage of a class of host cell death signal adaptors that encode a unique protein interaction motif called the RHIM.

Abreu SC, Antunes MA, Xisto DG, Cruz FF, Branco VC, Bandeira E, Zola Kitoko J, de Araújo AF, Dellatorre-Texeira L, Olsen PC, Weiss DJ, Diaz BL, Morales MM, Rocco PRM. Bone Marrow, Adipose, and Lung Tissue-Derived Murine Mesenchymal Stromal Cells Release Different Mediators and Differentially Affect Airway and Lung Parenchyma in Experimental Asthma. Stem Cells Transl Med. 2017 Jun;6(6):1557-1567.

DOI: 10.1002/sctm.16-0398

Mesenchymal stromal cells (MSCs) from different sources have differential effects on lung injury. To compare the effects of murine MSCs from bone marrow (BM), adipose tissue (AD), and lung tissue (LUNG) on inflammatory and remodeling processes in experimental allergic asthma, female C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) or saline (C). Twenty-four hours after the last challenge, mice received either saline (50 µl, SAL), BM-MSCs, AD-MSCs, or LUNG-MSCs (105 cells per mouse in 50 µl total volume) intratracheally. At 1 week, BM-MSCs produced significantly greater reductions in resistive and viscoelastic pressures, bronchoconstriction index, collagen fiber content in lung parenchyma (but not airways), eosinophil infiltration, and levels of interleukin (IL)-4, IL-13, transforming growth factor (TGF)-β, and vascular endothelial growth factor (VEGF) in lung homogenates compared to AD-MSCs and LUNG-MSCs. Only BM-MSCs increased IL-10 and interferon (IFN)-γ in lung tissue. In parallel in vitro experiments, BM-MSCs increased M2 macrophage polarization, whereas AD-MSCs and LUNG-MSCs had higher baseline levels of IL-4, insulin-like growth factor (IGF), and VEGF secretion. Exposure of MSCs to serum specimens obtained from asthmatic mice promoted reductions in secretion of these mediators, particularly in BM-MSCs. Intratracheally administered BM-MSCs, AD-MSCs, and LUNG-MSCs were differentially effective at reducing airway inflammation and remodeling and improving lung function in the current model of allergic asthma. In conclusion, intratracheal administration of MSCs from BM, AD, and LUNG were differentially effective at reducing airway inflammation and remodeling and improving lung function comparably reduced inflammation and fibrogenesis in this asthma model. However, altered lung mechanics and lung remodeling responded better to BM-MSCs than to AD-MSCs or LUNG-MSCs. Moreover, each type of MSC was differentially affected in a surrogate in vitro model of the in vivo lung environment.

Arcanjo AF, Nico D, de Castro GMM, da Silva Fontes Y, Saltarelli P, Decote-Ricardo D, Nunes MP, Ferreira-Pereira A, Palatnik-de-Sousa CB, Freire-de-Lima CG, Morrot A. Dependency of B-1 Cells in the Maintenance of Splenic Interleukin-10 Producing Cells and Impairment of Macrophage Resistance in Visceral Leishmaniasis. Front Microbiol. 2017 Jun 2;8:978. 

DOI: 10.3389/fmicb.2017.00978

Visceral leishmaniasis is a neglected disease caused by Leishmania protozoa parasites transmitted by infected sand fly vectors. This disease represents the second in mortality among tropical infections and is associated to a profound immunosuppression state of the host. The hallmark of this infection-induced host immunodeviation is the characteristic high levels of the regulatory interleukin-10 (IL-10) cytokine. In the present study, we investigated the role of B-1 cells in the maintenance of splenic IL-10 levels that could interfere with resistance to parasite infection. Using an experimental murine infection model with Leishmania (L.) infantum chagasi we demonstrated an improved resistance of B-1 deficient BALB/XID mice to infection. BALB/XID mice developed a reduced splenomegaly with diminished splenic parasite burden and lower levels of IL-10 secretion of purified splenocytes at 30 days post-infection, as compared to BALB/c wild-type control mice. Interestingly, we found that resident peritoneal macrophages isolated from BALB/XID mice were more effective to control the parasite load in comparison to cells isolated from BALB/c wild-type mice. Our findings point to a role of B-1 cells in the host susceptibility to visceral leishmaniasis.

Babdor J, Descamps D, Adiko AC, Tohmé M, Maschalidi S, Evnouchidou I, Vasconcellos LR, De Luca M, Mauvais FX, Garfa-Traore M, Brinkmann MM, Chignard M, Manoury B, Saveanu L. IRAP+ endosomes restrict TLR9 activation and signaling. Nat Immunol. 2017 May;18(5):509-518.  

DOI: 10.1038/ni.3711

The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.

Coelho SVA, Neris RLS, Papa MP, Schnellrath LC, Meuren LM, Tschoeke DA, Leomil L, Verçoza BRF, Miranda M, Thompson FL, Da Poian AT, Souza TML, Carneiro FA, Damaso CR, Assunção-Miranda I, de Arruda LB. Development of standard methods for Zika virus propagation, titration, and purification. J Virol Methods. 2017 Aug;246:65-74.

DOI: 10.1016/j.jviromet.2017.04.011

The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7 days post infection in C6/36 cells (7 × 107 and 2 × 108 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5 × 107 PFU/ml at 72 hpi), whereas ZIKV-BR titers reached 108 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research.

de Castro LL, Xisto DG, Kitoko JZ, Cruz FF, Olsen PC, Redondo PAG, Ferreira TPT, Weiss DJ, Martins MA, Morales MM, Rocco PRM. Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma. Stem Cell Res Ther. 2017 Jun 24;8(1):151.  

DOI: 10.1186/s13287-017-0600-8

Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma.

DeSouza-Vieira T, Chan FK. Bacterial pathogenesis: Pathogenic bacteria attack RHIM. Nat Microbiol. 2017 Mar 28;2:17042.

DOI: 10.1038/nmicrobiol.2017.42

Attaching and effacing enteropathogenic Escherichia coli causes gastrointestinal inflammation and diarrhoea. In this issue of Nature Microbiology, Pearson and colleagues find that this pathology involves bacterial cleavage of a class of host cell death signal adaptors that encode a unique protein interaction motif called the RHIM.

Freire-de-Lima L, Nardy AFFR, Ramos-Junior ES, Conde L, Santos Lemos J, da Fonseca LM, Lima JE, Maiolino A, Morrot A. Multiple Myeloma Cells Express Key Immunoregulatory Cytokines and Modulate the Monocyte Migratory Response. Front Med (Lausanne). 2017 Jun 27;4:92. 

DOI: 10.3389/fmed.2017.00092

Multiple myeloma (MM) is a plasma cell disorder that still remains incurable. The immune dysfunction of the host is a striking characteristic of MM, leading to tumor growth and reducing the survival rate of patients. Monocytes are precursors of conventional dendritic cells (DCs), a major player in the immunity mechanisms driving protective T cell responses against tumor. Herein, we report that human MM RPMI 8226 cell line shows a pronounced chemoattractant activity for monocytes and also expresses enhanced levels of the leukocyte chemotactic cytokines CXCL12, CCL5, MIP-1β, and CXCL10 in association with elevated levels of both key immunoregulatory interleukins such as IL-4 and IL-10. This cytokine profile was observed together with reduced expression of IFN-γ by MM RPMI 8226 cell line, a determinant interleukin involved in the acquisition of cellular-mediated protective responses against tumor cells. We further demonstrate that MM RPMI 8226 cell line expresses elevated levels of soluble form of the intercellular adhesion molecule-1 known to inhibit antitumoral T cell responses. This attractive modulation of immune responses by MM cells might provide a means to impair early antitumor responses during the establishment of cytokine-mediated immunosuppressive tumor niche.

Guimarães-Costa AB, Rochael NC, Oliveira F, Echevarria-Lima J, Saraiva EM. Neutrophil Extracellular Traps Reprogram IL-4/GM-CSF-Induced Monocyte Differentiation to Anti-inflammatory Macrophages. Front Immunol. 2017 May 17;8:523. 

DOI: 10.3389/fimmu.2017.00523

Monocyte-derived dendritic cells (mo-DCs) are essential for the development of a Th1 protective immune response against Leishmania parasites. It is well known that IL-4 and GM-CSF drive differentiation of human monocytes to dendritic cells (DCs). Here, we investigate if neutrophil extracellular traps (NETs) disrupt this process. NETs-enriched supernatants, generated after human neutrophil activation by Leishmania promastigotes, were added to monocytes and differentiation monitored by expression of molecules associated with macrophage and DCs phenotypes, cytokine production, and parasite killing. We found that NETs addition to IL-4/GM-CSF-treated monocytes prevented then to fully differentiate into DCs. No effect was observed if NETs were treated with DNase or by filtering the traps. Moreover, NETs closely interact with monocytes and downregulate the expression of the IL-4 receptor, which in turn disrupts fully differentiation of monocytes into DCs. Neutrophil elastase inhibition rescues the monocytes to DCs differentiation. Monocytes cultured with IL-4/GM-CSF and NETs differentiated into macrophages, as observed by the increased expression of CD68, CD32, and CD163, and decreased expression of CD80. Moreover, NET addition to IL-4/GM-CSF-treated monocytes rendered these cells less efficient to kill Leishmania parasites. Altogether, our results show that NETs interfere with IL-4/GM-CSF driven differentiation, reprogramming the generation of mo-DCs to an anti-inflammatory macrophage.

Mendes MA, de Carvalho DS, Amadeu TP, Silva BJA, Prata RBDS, da Silva CO, Ferreira H, Hacker MA, Nery JAC, Pinheiro RO, Sampaio EP, Sarno EN, Schmitz V. Elevated Pentraxin-3 Concentrations in Patients With Leprosy: Potential Biomarker of Erythema Nodosum Leprosum. J Infect Dis. 2017 Dec 19;216(12):1635-1643.

DOI: 10.1093/infdis/jix267

Leprosy, the leading infectious cause of disability worldwide, remains a major public health challenge in the most severely affected countries despite the sharp decline in new cases in recent years. The search for biomarkers is essential to achieve a better understanding of the molecular and cellular mechanisms underlying the disease.

Mendonça PHB, da Rocha RFDB, Moraes JBB, LaRocque-de-Freitas IF, Logullo J, Morrot A, Nunes MP, Freire-de-Lima CG, Decote-Ricardo D. Canine Macrophage DH82 Cell Line As a Model to Study Susceptibility to Trypanosoma cruzi Infection. Front Immunol. 2017 May 31;8:604.

DOI: 10.3389/fimmu.2017.00604

Trypanosoma cruzi is an obligatory intracellular protozoan parasite, and it is the etiological agent of Chagas’ disease that is endemic in the Americas. In addition to humans, a wide spectrum of mammals can be infected by T. cruzi, including dogs. Dogs develop acute and chronic disease, similar to human infection. T. cruzi can infect almost all cell types and after cell invasion, the metacyclics trypomastigotes localize in the cytoplasm, where they transform into amastigotes, the replicative form of T. cruzi in mammals. After amastigote multiplication and differentiation, parasites lyse host cells and spread through the body by blood circulation. In this work, we evaluated the in vitro ability of T. cruzi to infect a canine macrophage cell line DH82 compared with RAW264.7, a murine tissue culture macrophage. Our results have shown that the T. cruzi is able to infect, replicate and differentiate in DH82 cell line. We observed that following treatment with LPS and IFN-γ DH82 cells were more resistant to infection and that resistance was not related reactive oxygen species production in our system. In this study, we also found that DH82 cells became more susceptible to T. cruzi infection when cocultured with apoptotic cells. The analysis of cytokine production has showed elevated levels of the TGF-β, IL-10, and TNF-α produced by T. cruzi-infected canine macrophages. Additionally, we demonstrated a reduced expression of the MHC class II and CD80 by infected DH82 cell line.

Nascimento CR, Andrade D, Carvalho-Pinto CE, Serra RR, Vellasco L, Brasil G, Ramos-Junior ES, da Mota JB, Almeida LN, Andrade MV, Correia Soeiro MN, Juliano L, Alvarenga PH, Oliveira AC, Sicuro FL, de Carvalho ACC, Svensjö E, Scharfstein J. Mast Cell Coupling to the Kallikrein-Kinin System Fuels Intracardiac Parasitism and Worsens Heart Pathology in Experimental Chagas Disease. Front Immunol. 2017 Aug 2;8:840.

DOI: 10.3389/fimmu.2017.00840

During the course of Chagas disease, infectious forms of Trypanosoma cruzi are occasionally liberated from parasitized heart cells. Studies performed with tissue culture trypomastigotes (TCTs, Dm28c strain) demonstrated that these parasites evoke neutrophil/CXCR2-dependent microvascular leakage by activating innate sentinel cells via toll-like receptor 2 (TLR2). Upon plasma extravasation, proteolytically derived kinins and C5a stimulate immunoprotective Th1 responses via cross-talk between bradykinin B2 receptors (B2Rs) and C5aR. Awareness that TCTs invade cardiovascular cells in vitro via interdependent activation of B2R and endothelin receptors [endothelin A receptor (ETAR)/endothelin B receptor (ETBR)] led us to hypothesize that T. cruzi might reciprocally benefit from the formation of infection-associated edema via activation of kallikrein–kinin system (KKS). Using intravital microscopy, here we first examined the functional interplay between mast cells (MCs) and the KKS by topically exposing the hamster cheek pouch (HCP) tissues to dextran sulfate (DXS), a potent “contact” activator of the KKS. Surprisingly, although DXS was inert for at least 30 min, a subtle MC-driven leakage resulted in factor XII (FXII)-dependent activation of the KKS, which then amplified inflammation via generation of bradykinin (BK). Guided by this mechanistic insight, we next exposed TCTs to “leaky” HCP—forged by low dose histamine application—and found that the proinflammatory phenotype of TCTs was boosted by BK generated via the MC/KKS pathway. Measurements of footpad edema in MC-deficient mice linked TCT-evoked inflammation to MC degranulation (upstream) and FXII-mediated generation of BK (downstream). We then inoculated TCTs intracardiacally in mice and found a striking decrease of parasite DNA (quantitative polymerase chain reaction; 3 d.p.i.) in the heart of MC-deficient mutant mice. Moreover, the intracardiac parasite load was significantly reduced in WT mice pretreated with (i) cromoglycate (MC stabilizer) (ii) infestin-4, a specific inhibitor of FXIIa (iii) HOE-140 (specific antagonist of B2R), and (iv) bosentan, a non-selective antagonist of ETAR/ETBR. Notably, histopathology of heart tissues from mice pretreated with these G protein-coupled receptors blockers revealed that myocarditis and heart fibrosis (30 d.p.i.) was markedly and redundantly attenuated. Collectively, our study suggests that inflammatory edema propagated via activation of the MC/KKS pathway fuels intracardiac parasitism by generating infection-stimulatory peptides (BK and endothelins) in the edematous heart tissues.

Nesi RT, Barroso MV, Souza Muniz V, de Arantes AC, Martins MA, Brito Gitirana L, Neves JS, Benjamim CF, Lanzetti M, Valenca SS. Pharmacological modulation of reactive oxygen species (ROS) improves the airway hyperresponsiveness by shifting the Th1 response in allergic inflammation induced by ovalbumin. Free Radic Res. 2017 Jul-Aug;51(7-8):708-722. 

DOI: 10.1080/10715762.2017.1364377

Asthma is an allergic inflammation driven by the Th2 immune response with release of cytokines such as IL-4 and IL-13, which contribute to the airflow limitations and airway hyperresponsiveness (AHR). The involvement of oxidative stress in this process is well-established, but the specific role of the superoxide anion and nitric oxide in asthma are poorly understood. Thus, the aim of this study was to investigate the mechanisms underlying the superoxide anion/nitric oxide production and detoxification in a murine asthma model. BALB/c male mice were sensitised and challenged with ovalbumin (OVA). Pretreatments with either apocynin (14 mg/kg) or allopurinol (25 mg/kg) (superoxide anion synthesis inhibitors), aminoguanidine (50 mg/kg) (nitric oxide synthesis inhibitor) or diethyldithiocarbamate (100 mg/kg) (superoxide dismutase inhibitor) were performed 1 h before the challenge. Our data showed that apocynin and allopurinol ameliorated AHR and reduced eosinophil peroxidase, as well as IL-4 and IL-13 levels. Apocynin also abrogated leukocyte peribronchiolar infiltrate and increased IL-1β secretion. Aminoguanidine preserved lung function and shifted the Th2 to the Th1 response with a reduction of IL-4 and IL-13 and increase in IL-1β production. Diethyldithiocarbamate prevented neither allergen-induced AHR nor eosinophil peroxidase (EPO) generation. All treatments protected against oxidative damage observed by a reduction in TBARS levels. Taken together, these results suggest that AHR in an asthma model can be avoided by the down-regulation of superoxide anion and nitric oxide synthesis in a mechanism that is independent of a redox response. This down-regulation is also associated with a transition in the typical immunological Th2 response toward the Th1 profile.

Papa MP, Meuren LM, Coelho SVA, Lucas CGO, Mustafá YM, Lemos Matassoli F, Silveira PP, Frost PS, Pezzuto P, Ribeiro MR, Tanuri A, Nogueira ML, Campanati L, Bozza MT, Paula Neto HA, Pimentel-Coelho PM, Figueiredo CP, de Aguiar RS, de Arruda LB. Zika Virus Infects, Activates, and Crosses Brain Microvascular Endothelial Cells, without Barrier Disruption. Front Microbiol. 2017 Dec 22;8:2557. 

DOI: 10.3389/fmicb.2017.02557

Zika virus (ZIKV) has been associated to central nervous system (CNS) harm, and virus was detected in the brain and cerebrospinal fluids of microcephaly and meningoencephalitis cases. However, the mechanism by which the virus reaches the CNS is unclear. Here, we addressed the effects of ZIKV replication in human brain microvascular endothelial cells (HBMECs), as an in vitro model of blood brain barrier (BBB), and evaluated virus extravasation and BBB integrity in an in vivo mouse experimental model. HBMECs were productively infected by African and Brazilian ZIKV strains (ZIKVMR766 and ZIKVPE243), which induce increased production of type I and type III IFN, inflammatory cytokines and chemokines. Infection with ZIKVMR766 promoted earlier cellular death, in comparison to ZIKVPE243, but infection with either strain did not result in enhanced endothelial permeability. Despite the maintenance of endothelial integrity, infectious virus particles crossed the monolayer by endocytosis/exocytosis-dependent replication pathway or by transcytosis. Remarkably, both viruses' strains infected IFNAR deficient mice, with high viral load being detected in the brains, without BBB disruption, which was only detected at later time points after infection. These data suggest that ZIKV infects and activates endothelial cells, and might reach the CNS through basolateral release, transcytosis or transinfection processes. These findings further improve the current knowledge regarding ZIKV dissemination pathways.

Pereira JC, Ramos TD, Silva JD, de Mello MF, Pratti JES, da Fonseca-Martins AM, Firmino-Cruz L, Kitoko JZ, Chaves SP, Gomes DCO, Diaz BL, Rocco PRM, de Matos Guedes HL. Effects of Bone Marrow Mesenchymal Stromal Cell Therapy in Experimental Cutaneous Leishmaniasis in BALB/c Mice Induced by Leishmania amazonensis. Front Immunol. 2017 Aug 10;8:893. 

DOI: 10.3389/fimmu.2017.00893

Cutaneous leishmaniasis remains both a public health and a therapeutic challenge. To date, no ideal therapy for cutaneous leishmaniasis has been identified, and no universally accepted therapeutic regimen and approved vaccines are available. Due to the mesenchymal stromal cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders, including infectious, inflammatory, and allergic diseases. We evaluated the potential effects of bone marrow MSC therapy in a murine model of cutaneous leishmaniasis. In vitro, coculture of infected macrophages with MSC increased parasite load on macrophages in comparison with controls (macrophages without MSCs). In vivo, BALB/c mice were infected with 2 × 106 Leishmania amazonensis (Josefa strain) promastigotes in the footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.), i.e., in the same site of infection, or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of phosphate-buffered saline by i.l. or i.v. routes. The lesion progression was assessed by its thickness measured by pachymetry. Forty-two days after infection, animals were euthanized and parasite burden in the footpad and in the draining lymph nodes was quantified by the limiting dilution assay (LDA), and spleen cells were phenotyped by flow cytometry. No significant difference was observed in lesion progression, regardless of the MSC route of administration. However, animals treated with i.v. MSCs presented a significant increase in parasite load in comparison with controls. On the other hand, no harmful effect due to MSCs i.l. administered was observed. The spleen cellular profile analysis showed an increase of IL-10 producing T CD4+ and TCD8+ cells in the spleen only in mice treated with i.v. MSC. The excessive production of IL-10 could be associated with the disease-aggravating effects of MSC therapy when intravenously administered. As a conclusion, in the current murine model of L. amazonensis-induced cutaneous disease, MSCs did not control the damage of cutaneous disease and, depending on the administration route, it could result in deleterious effects.

Pinho-Ribeiro V, Melo AC, Kennedy-Feitosa E, Graca-Reis A, Barroso MV, Cattani-Cavalieri I, Carvalho GMC, Zin WA, Porto LC, Gitirana LB, Lanzetti M, Valença SS. Atorvastatin and Simvastatin Promoted Mouse Lung Repair After Cigarette Smoke-Induced Emphysema. Inflammation. 2017 Jun;40(3):965-979.

DOI: 10.1007/s10753-017-0541-5

Cigarette smoke (CS) induces pulmonary emphysema by inflammation, oxidative stress, and metalloproteinase (MMP) activation. Pharmacological research studies have not focused on tissue repair after the establishment of emphysema but have instead focused on inflammatory stimulation. The aim of our study was to analyze the effects of atorvastatin and simvastatin on mouse lung repair after emphysema caused by CS. Male mice (C57BL/6, n = 45) were divided into the following groups: control (sham-exposed), CSr (mice exposed to 12 cigarettes a day for 60 days and then treated for another 60 days with the vehicle), CSr+A (CSr mice treated with atorvastatin for 60 days), and CSr+S (CSr mice treated with simvastatin for 60 days). The treatment with atorvastatin and simvastatin was administered via inhalation (15 min with 1 mg/mL once a day). Mice were sacrificed 24 h after the completion of the 120-day experimental procedure. We performed biochemical, morphological, and physiological analyses. We observed decreased levels of leukocytes and cytokines in statin-treated mice, accompanied by a reduction in oxidative stress markers. We also observed a morphological improvement confirmed by a mean linear intercept counting in statin-treated mice. Finally, statins also ameliorated lung function. We conclude that inhaled atorvastatin and simvastatin improved lung repair after cigarette smoke-induced emphysema in mice.

Thompson-Souza GA, Gropillo I, Neves JS. Cysteinyl Leukotrienes in Eosinophil Biology: Functional Roles and Therapeutic Perspectives in Eosinophilic Disorders. Front Med (Lausanne). 2017 Jul 18;4:106. 

DOI: 10.3389/fmed.2017.00106

Cysteinyl leukotrienes (cysLTs), LTC4, and its extracellular metabolites, LTD4 and LTE4, have varied and multiple roles in mediating eosinophilic disorders including host defense against parasitic helminthes and allergic inflammation, especially in the lung and in asthma. CysLTs are known to act through at least 2 receptors termed cysLT1 receptor (CysLT1R) and cysLT2 receptor (CysLT2R). Eosinophils contain a dominant population of cytoplasmic crystalloid granules that store various preformed proteins. Human eosinophils are sources of cysLTs and are known to express the two known cysLTs receptors (CysLTRs). CysLTs can have varied functions on eosinophils, ranging from intracrine regulators of secretion of granule-derived proteins to paracrine/autocrine roles in eosinophil chemotaxis, differentiation, and survival. Lately, it has been recognized the expression of CysLTRs in the membranes of eosinophil granules. Moreover, cysLTs have been shown to evoke secretion from isolated cell-free eosinophil granules operating through their receptors expressed on granule membranes. In this work, we review the functional roles of cysLTs in eosinophil biology. We review cysLTs biosynthesis, their receptors, and argue the intracrine and paracrine/autocrine responses induced by cysLTs in eosinophils and in isolated free extracellular eosinophil granules. We also examine and speculate on the therapeutic relevance of targeting CysLTRs in the treatment of eosinophilic disorders.

Travassos LH, Vasconcellos LR, Bozza MT, Carneiro LA. Heme and iron induce protein aggregation. Autophagy. 2017 Mar 4;13(3):625-626.

DOI: 10.1080/15548627.2016.1271515

Heme is an essential molecule expressed in many tissues where it plays key roles as the prosthetic group of several proteins involved in vital physiological and metabolic processes such as gas and electron transport. Structurally, heme is a tetrapyrrole ring containing an atom of iron (Fe) in its center. When released into the extracellular milieu, heme exerts several deleterious effects, which make it an important player in infectious and noninfectious hemolytic diseases where large amounts of free heme are observed such as malaria, dengue fever, β-thalassemia, sickle cell disease and ischemia-reperfusion. Our recent work has uncovered an unappreciated cellular response triggered by heme or Fe, one of its degradation products, on macrophages, which is the formation of protein aggregates known as aggresome-like induced structres (ALIS). This response was shown to be fully dependent on ROS production and the activation of the transcription factor NFE2L2/NRF2. In addition, we have demonstrated that heme degradation by HMOX1/HO-1 (heme oxygenase 1) is required and that Fe is essential for the formation of ALIS, as heme analogs lacking the central atom of Fe are not able to induce these structures. ALIS formation is also observed in vivo, in a model of phenylhydrazine (PHZ)-induced hemolysis, indicating that it is an integral part of the host response to excessive free heme and that it may play a role in cellular homeostasis.

Vellozo NS, Pereira-Marques ST, Cabral-Piccin MP, Filardy AA, Ribeiro-Gomes FL, Rigoni TS, DosReis GA, Lopes MF. All-Trans Retinoic Acid Promotes an M1- to M2-Phenotype Shift and Inhibits Macrophage-Mediated Immunity to Leishmania major. Front Immunol. 2017 Nov 17;8:1560.

DOI: 10.3389/fimmu.2017.01560

As key cells, able to host and kill Leishmania parasites, inflammatory monocytes/macrophages are potential vaccine and therapeutic targets to improve immune responses in Leishmaniasis. Macrophage phenotypes range from M1, which express NO-mediated microbial killing, to M2 macrophages that might help infection. Resistance to Leishmaniasis depends on Leishmania species, mouse strain, and both innate and adaptive immunity. C57BL/6 (B6) mice are resistant and control infection, whereas Leishmania parasites thrive in BALB/c mice, which are susceptible to develop cutaneous lesions in the course of infection with Leishmania major, but not upon infection with Leishmania braziliensis. Here, we investigated whether a deficit in early maturation of inflammatory monocytes into macrophages in BALB/c mice underlies increased susceptibility to L. major versus L. braziliensis parasites. We show that, after infection with L. braziliensis, monocytes are recruited to peritoneum, differentiate into macrophages, and develop an M1 phenotype able to produce proinflammatory cytokines in both B6 and BALB/c mice. Nonetheless, more mature macrophages from B6 mice expressed inducible NO synthase (iNOS) and higher NO production in response to L. braziliensis parasites, whereas BALB/c mice developed macrophages expressing an incomplete M1 phenotype. By contrast, monocytes recruited upon L. major infection gave rise to immature macrophages that failed to induce an M1 response in BALB/c mice. Overall, these results are consistent with the idea that resistance to Leishmania infection correlates with improved maturation of macrophages in a mouse-strain and Leishmania-species dependent manner. All-trans retinoic acid (ATRA) has been proposed as a therapy to differentiate immature myeloid cells into macrophages and help immunity to tumors. To prompt monocyte to macrophage maturation upon L. major infection, we treated B6 and BALB/c mice with ATRA. Unexpectedly, treatment with ATRA reduced proinflammatory cytokines, iNOS expression, and parasite killing by macrophages. Moreover, ATRA promoted an M1 to M2 transition in bone marrow-derived macrophages from both strains. Therefore, ATRA uncouples macrophage maturation and development of M1 phenotype and downmodulates macrophage-mediated immunity to L. major parasites. Cautions should be taken for the therapeutic use of ATRA, by considering direct effects on innate immunity to intracellular pathogens.

Tipo de produção: Técnica
Subtipo de produção: Patente

Tipo de produção: Técnica
Subtipo de produção: Patente

Tipo de produção: Bibliográfica
Subtipo de produção: Livro

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.ejphar.2016.03.056

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.cub.2016.06.025

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1111/bph.13430

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.bbrc.2016.07.043

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fonc.2016.00054

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1186/s13071-016-1540-3

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1007/s11302-016-9544-1

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.18632/oncotarget.11253

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.2217/fmb-2016-0090

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1038/cti.2016.32

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fimmu.2016.00296

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pone.0162926

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fimmu.2016.00153

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1002/jbio.201600061

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3390/md14090163

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.bbi.2016.11.006

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1007/s00011-016-0937-y

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.bbalip.2016.11.006

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fmicb.2016.00348

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fmicb.2016.00367

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.intimp.2015.11.019

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.4049/jimmunol.1502492

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.ppat.1005947

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.pep.2016.01.006

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.coi.2015.11.006

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1007/s12551-015-0190-6

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.toxicon.2016.01.066

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1073/pnas.1608928113

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.21037/tcr.2016.11.30

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1128/msphere.00099-16

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pntd.0005034

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.2147/COPD.S93958

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1080/14786419.2015.1081196

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.etap.2016.03.004

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pone.0153607

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1017/s0031182016002237

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.parint.2016.01.001

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1189/jlb.4A0615-261RR

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.2174/1570159X14666151230110058

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fmicb.2016.00698

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1038/ncomms13344

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1189/jlb.3MA0915-418R

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.21037/atm.2016.09.44

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pntd.0004848

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pone.0160433

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.21037/atm.2016.05.38

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1097/ALN.0000000000000919

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.vetpar.2016.04.033

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1042/BSR20160107

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1017/S0031182015001584

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.vaccine.2016.04.018

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fmicb.2016.01233

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1186/s12936-016-1447-7

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1111/jam.13038

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pntd.0004298

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fmicb.2016.01617

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pone.0151252

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.ibmb.2015.08.007

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3892/ol.2016.4593

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1586/14787210.2016.1160779

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1080/15548627.2015.1100356

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.4049/jimmunol.1502023

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fmicb.2016.01034

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.toxicon.2016.05.007

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pone.0156037

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1017/s0007114516000027

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1249/mss.0000000000000907

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1093/nar/gkw725

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fmicb.2016.00704

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.pupt.2016.09.004

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1038/srep34666

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1074/jbc.m116.729236

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.acthis.2015.12.010

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1186/s13071-016-1822-9

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.nut.2016.10.019

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1111/odi.12551

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.resp.2016.04.002

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1017/S003118201600127X

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1096/fj.201500059

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1371/journal.pone.0147005

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1523/JNEUROSCI.1269-16.2016

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1002/iid3.132

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1038/srep32619

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1189/jlb.3a1015-480r

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1007/s12035-016-0307-3

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1016/j.micinf.2015.12.006

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fphys.2016.00151

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1038/cddis.2016.135

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1128/msphere.00085-15

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1042/bsr20160061

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fimmu.2016.00237

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.7554/elife.12318

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fimmu.2016.00184

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.1097/shk.0000000000000520

Tipo de produção: Bibliográfica
Subtipo de produção: Artigo em periódico
Nome do Detalhamento: DOI
Valor do Detalhamento: 10.3389/fphys.2016.00475