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Structural and Signaling Events Driving Aspergillus fumigatus-Induced Human Eosinophil Extracellular Trap Release
Detalhes
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
Abstract
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.
MPLA and AddaVax ® Adjuvants Fail to Promote Intramuscular LaAg Vaccine Protectiveness against Experimental Cutaneous Leishmaniasis
Detalhes
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.
Abstract
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
SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes
Detalhes
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.
Abstract
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.
Lessons from transmissible cancers for immunotherapy and transplant
Detalhes
Silva RCMC, Panis C, Pires BRB. Lessons from transmissible cancers for immunotherapy and transplant. Immunol Med. 2021 Dec 28:1-16.
Abstract
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
Intracerebral Injection of Heme Induces Lipid Peroxidation, Neuroinflammation, and Sensorimotor Deficits
Detalhes
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.
Abstract
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.
Chloroquine inhibits pro-inflammatory effects of heme on macrophages and invivo
Detalhes
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.
Abstract
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
c-MAF-dependent perivascular macrophages regulate diet-induced metabolic syndrome
Detalhes
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.
Abstract
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.
Dietary Fiber Drives IL-1β-Dependent Peritonitis Induced by Bacteroides fragilis via Activation of the NLRP3 Inflammasome
Detalhes
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.
Abstract
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.
Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Associated with Bacteremia and Monocyte Evasion, Rio de Janeiro, Brazil
Detalhes
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.
Abstract
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.
ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice
Detalhes
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.
Abstract
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
A sand fly salivary protein acts as a neutrophil chemoattractant
Detalhes
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.
Abstract
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.
Adenosine Diphosphate Improves Wound Healing in Diabetic Mice Through P2Y 12 Receptor Activation
Detalhes
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.
Abstract
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.
Leishmania Parasites Drive PD-L1 Expression in Mice and Human Neutrophils With Suppressor Capacity
Detalhes
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.
Abstract
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.
Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Advantages of Stain-Free Technology
Detalhes
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
Abstract
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.
Skeletal Muscle Is an Early Site of Zika Virus Replication and Injury, Which Impairs Myogenesis
Detalhes
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.
Abstract
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.
ZIKV Disrupts Placental Ultrastructure and Drug Transporter Expression in Mice
Detalhes
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
Abstract
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 (5×107 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.
o 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
Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors
Detalhes
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
Abstract
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
Yellow fever vaccine protects mice against Zika virus infection
Detalhes
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
Abstract
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.
X-linked immunodeficient (XID) mice exhibit high susceptibility to Cryptococcus gattii infection
Detalhes
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
Abstract
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.
The role of Toll-like receptor 9 in a murine model of Cryptococcus gattii infection
Detalhes
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
Abstract
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.
The influence of the gut microbiota on the development of food allergy in children
Detalhes
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
Abstract
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.
The immunodominant antibody response to Zika virus NS1 protein is characterized by cross-reactivity to self
Detalhes
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
Abstract
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., 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.
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Universidade Federal do Rio de Janeiro Centro de Ciências da Saúde – Bloco D (sala D1-029) Cidade Universitária – Ilha do Fundão
CEP 21.941-590 – Rio de Janeiro – RJ – Telefone: (21) 3938-6748 – E-mail: pos_imuno@micro.ufrj.br
Universidade Federal do Rio de Janeiro
Centro de Ciências da Saúde – Bloco D (sala D1-029) Cidade Universitária – Ilha do Fundão
CEP 21.941-590 – Rio de Janeiro – RJ
Telefone: (21) 3938-6748
E-mail: pos_imuno@micro.ufrj.br
