RESUMO
Production of nitric oxide (NO) by LPS-activated macrophages is due to a complex cellular signaling initiated by TLR4 that leads to the transcription of IFN-ß, which activates IRF-1 and STAT-1, as well as to the activation of NF-κB, required for iNOS transcription. High concentrations of LPS can also be uptaken by scavenger receptors (SRs), which, in concert with TLR4, leads to inflammatory responses. The mechanisms by which TLR4 and SRs interact, and the pathways activated by this interaction in macrophages are not elucidated. Therefore, our main goal was to evaluate the role of SRs, particularly SR-A, in LPS-stimulated macrophages for NO production. We first showed that, surprisingly, LPS can induce the expression of iNOS and the production of NO in TLR4-/- mice, provided exogenous IFN-ß is supplied. These results indicate that LPS stimulate receptors other than TLR4. The inhibition of SR-A using DSS or neutralizing antibody to SR-AI showed that SR-A is essential for the expression of iNOS and NO production in stimulation of TLR4 by LPS. The restoration of the ability to express iNOS and produce NO by addition of rIFN-ß to inhibited SR-A cells indicated that the role of SR-AI in LPS-induced NO production is to provide IFN-ß, probably by mediating the internalization of LPS/TLR4, and the differential inhibition by DSS and neutralizing antibody to SR-AI suggested that other SRs are also involved. Our results reinforce that TLR4 and SR-A act in concert in LPS activation and demonstrated that, for the production of NO, it does mainly by synthesizing IRF-3 and also by activating the TRIF/IRF-3 pathway for IFN-ß production, essential for LPS-mediated transcription of iNOS. Consequently STAT-1 is activated, and IRF-1 is expressed, which together with NF-κB from TLR4/MyD88/TIRAP, induce iNOS synthesis and NO production. SUMMARY SENTENCE: TLR4 and SRs act in concert activating IRF-3 to transcribe IFN-ß and activate STAT-1 to produce NO by LPS-activated macrophages.
Assuntos
NF-kappa B , Óxido Nítrico , Camundongos , Animais , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Receptor 4 Toll-Like/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , Receptores Depuradores/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
BACKGROUND: The continuous proliferation of intestinal stem cells followed by their tightly regulated differentiation to epithelial cells is essential for the maintenance of the gut epithelial barrier and its functions. How these processes are tuned by diet and gut microbiome is an important, but poorly understood question. Dietary soluble fibers, such as inulin, are known for their ability to impact the gut bacterial community and gut epithelium, and their consumption has been usually associated with health improvement in mice and humans. In this study, we tested the hypothesis that inulin consumption modifies the composition of colonic bacteria and this impacts intestinal stem cells functions, thus affecting the epithelial structure. METHODS: Mice were fed with a diet containing 5% of the insoluble fiber cellulose or the same diet enriched with an additional 10% of inulin. Using a combination of histochemistry, host cell transcriptomics, 16S microbiome analysis, germ-free, gnotobiotic, and genetically modified mouse models, we analyzed the impact of inulin intake on the colonic epithelium, intestinal bacteria, and the local immune compartment. RESULTS: We show that the consumption of inulin diet alters the colon epithelium by increasing the proliferation of intestinal stem cells, leading to deeper crypts and longer colons. This effect was dependent on the inulin-altered gut microbiota, as no modulations were observed in animals deprived of microbiota, nor in mice fed cellulose-enriched diets. We also describe the pivotal role of γδ T lymphocytes and IL-22 in this microenvironment, as the inulin diet failed to induce epithelium remodeling in mice lacking this T cell population or cytokine, highlighting their importance in the diet-microbiota-epithelium-immune system crosstalk. CONCLUSION: This study indicates that the intake of inulin affects the activity of intestinal stem cells and drives a homeostatic remodeling of the colon epithelium, an effect that requires the gut microbiota, γδ T cells, and the presence of IL-22. Our study indicates complex cross kingdom and cross cell type interactions involved in the adaptation of the colon epithelium to the luminal environment in steady state. Video Abstract.
Assuntos
Microbioma Gastrointestinal , Inulina , Humanos , Animais , Camundongos , Inulina/farmacologia , Dieta , Fibras na Dieta , Celulose , Epitélio , Comunicação CelularRESUMO
External and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.
Assuntos
Interleucina-10 , Interleucina-17 , Células Cultivadas , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Transdução de Sinais , Células Th17RESUMO
The mechanisms involved in the development of resistance to infection/reinfection by Schistosoma mansoni still arouse great interest and controversy. Some authors demonstrate that resistance to infection is attributed to a mixed Th1 and Th2 response and resistance to reinfection after repeated treatments through mechanisms associated with the Th2 response. Through flow cytometry, the phenotypic characterization of B and T lymphocytes in individuals residing in endemic areas with low parasite loads over 10 years was evaluated for the first time in humans. In this study, individuals with low parasite loads for Schistosoma mansoni had a higher proportion of Th1 and Th2 cells. In addition, lymphocytes from these individuals showed a higher degree of expression of costimulatory molecules CD28 and CTLA-4 and regulatory molecules FoxP3 and IL-10, when compared to individuals with high parasite loads. Our data indicate that the control of the parasite load of S. mansoni must be associated with a Th1, Th2, and regulatory response, and that further studies are needed to elucidate the possibility of mechanisms associated with the hyporesponsiveness of lymphocytes from individuals with high parasite loads.
Assuntos
Esquistossomose mansoni , Animais , Linfócitos B , Humanos , Contagem de Linfócitos , Schistosoma mansoni , Esquistossomose mansoni/parasitologia , Células Th2RESUMO
Schistosomiasis is a parasitic disease that affects about 166 million people around the world. It is estimated that 5%-10% of individuals with schistosomiasis develop severe forms of the disease, which are characterized by pulmonary hypertension, ascites, periportal fibrosis, and other significant complications. The chronic phase of the disease is associated with a Th2 type immune response, but evidence also suggests there are roles for Th1 and Th17 in the development of severe disease. The aim of this study was to evaluate the CD4+ T lymphocyte profile of patients with different degrees of periportal fibrosis secondary to schistosomiasis. These individuals had been treated for schistosomiasis, but since they live in a S. mansoni endemic area, they are at risk of reinfection. They were evaluated in relation to the degree of periportal fibrosis and classified into three groups: without fibrosis or with incipient fibrosis (WF/IFNE), n=12, possible periportal fibrosis/periportal fibrosis, n=13, and advanced periportal fibrosis/advanced periportal fibrosis with portal hypertension, n=4. We observed in the group without fibrosis a balance between the low expression of Th2 cytokines and high expression of T reg cells. As has already been described in the literature, we found an increase of the Th2 cytokines IL-4, IL-5, and IL-13 in the group with periportal fibrosis. In addition, this group showed higher expression of IL-17 and IL-10 but lower IL-10/IL-13 ratio than patients in the WF/IFNE group. Cells from individuals who present any level of fibrosis expressed more TGF-ß compared to the WF/IFNE group and a positive correlation with left lobe enlargement and portal vein wall thickness. There was a negative correlation between IL-17 and the thickness of the portal vein wall, but more studies are necessary in order to explore the possible protective role of this cytokine. Despite the fibrosis group having presented a higher expression of pro-fibrotic molecules compared to WF/IFNE patients, it seems there is a regulation through IL-10 and T reg cells that is able to maintain the low morbidity of this group.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Fibrose/etiologia , Fibrose/metabolismo , Schistosoma/imunologia , Esquistossomose/complicações , Esquistossomose/parasitologia , Animais , Biomarcadores , Citocinas/metabolismo , Suscetibilidade a Doenças , Feminino , Fibrose/patologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and animals. Currently available live attenuated vaccines against brucellosis still have drawbacks. Therefore, subunit vaccines, produced using epitope-based antigens, have the advantage of being safe, cost-effective and efficacious. Here, we identified B. abortus small RNAs expressed during early infection with bone marrow-derived macrophages (BMDMs) and an apolipoprotein N-acyltransferase (Int) was identified as the putative target of the greatest expressed small RNA. Decreased expression of Int was observed during BMDM infection and the protein sequence was evaluated to rationally select a putative immunogenic epitope by immunoinformatic, which was explored as a vaccinal candidate. C57BL/6 mice were immunized and challenged with B. abortus, showing lower recovery in the number of viable bacteria in the liver, spleen, and axillary lymph node and greater production of IgG and fractions when compared to non-vaccinated mice. The vaccinated and infected mice showed the increased expression of TNF-α, IFN-γ, and IL-6 following expression of the anti-inflammatory genes IL-10 and TGF-ß in the liver, justifying the reduction in the number and size of the observed granulomas. BMDMs stimulated with splenocyte supernatants from vaccinated and infected mice increase the CD86+ marker, as well as expressing greater amounts of iNOS and the consequent increase in NO production, suggesting an increase in the phagocytic and microbicidal capacity of these cells to eliminate the bacteria.
Assuntos
Zoonoses Bacterianas/prevenção & controle , Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Brucelose/prevenção & controle , Aciltransferases/genética , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Zoonoses Bacterianas/imunologia , Zoonoses Bacterianas/microbiologia , Vacina contra Brucelose/administração & dosagem , Vacina contra Brucelose/genética , Brucella abortus/genética , Brucelose/imunologia , Brucelose/microbiologia , Simulação por Computador , Modelos Animais de Doenças , Mapeamento de Epitopos/métodos , Humanos , Imunogenicidade da Vacina , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Cultura Primária de Células , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Schistosomiasis is a debilitating parasitic disease that affects more than 200 million people worldwide and causes approximately 280,000 deaths per year. Inside the definitive host, eggs released by Schistosoma mansoni lodge in the intestine and especially in the liver where they induce a granulomatous inflammatory process, which can lead to fibrosis. The molecular mechanisms initiating or promoting hepatic granuloma formation remain poorly understood. Inflammasome activation has been described as an important pathway to induce pathology mediated by NLRP3 receptor. Recently, other components of the inflammasome pathway, such as NLRP6, have been related to liver diseases and fibrotic processes. Nevertheless, the contribution of these components in schistosomiasis-associated pathology is still unknown. In the present study, using dendritic cells, we demonstrated that NLRP6 sensor is important for IL-1ß production and caspase-1 activation in response to soluble egg antigens (SEA). Furthermore, the lack of NLRP6 has been shown to significantly reduce periovular inflammation, collagen deposition in hepatic granulomas and mRNA levels of α-SMA and IL-13. Livers of Nlrp6-/- mice showed reduced levels of CXCL1/KC, CCL2, CCL3, IL-5, and IL-10 as well as Myeloperoxidase (MPO) and Eosinophilic Peroxidase (EPO) enzymatic activity. Consistently, the frequency of macrophage and neutrophil populations were lower in the liver of NLRP6 knockout mice, after 6 weeks of infection. Finally, it was further demonstrated that the onset of hepatic granuloma and collagen deposition were also compromised in Caspase-1-/- , IL-1R-/- and Gsdmd-/- mice. Our findings suggest that the NLRP6 inflammasome is an important component for schistosomiasis-associated pathology.
Assuntos
Fígado/imunologia , Fígado/patologia , Receptores de Superfície Celular/metabolismo , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Animais , Antígenos de Helmintos/metabolismo , Antígenos de Helmintos/farmacologia , Caspase 1/genética , Caspase 1/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Fibrose , Técnicas de Inativação de Genes , Granuloma/imunologia , Granuloma/metabolismo , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Hepatopatias/imunologia , Hepatopatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Receptores de Superfície Celular/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Esquistossomose mansoni/parasitologiaRESUMO
Antibiotic-induced dysbiosis is a key predisposing factor for Clostridium difficile infections (CDIs), which cause intestinal disease ranging from mild diarrhea to pseudomembranous colitis. Here, we examined the impact of a microbiota-derived metabolite, short-chain fatty acid acetate, on an acute mouse model of CDI. We found that administration of acetate is remarkably beneficial in ameliorating disease. Mechanistically, we show that acetate enhances innate immune responses by acting on both neutrophils and ILC3s through its cognate receptor free fatty acid receptor 2 (FFAR2). In neutrophils, acetate-FFAR2 signaling accelerates their recruitment to the inflammatory sites, facilitates inflammasome activation, and promotes the release of IL-1ß; in ILC3s, acetate-FFAR2 augments expression of the IL-1 receptor, which boosts IL-22 secretion in response to IL-1ß. We conclude that microbiota-derived acetate promotes host innate responses to C. difficile through coordinate action on neutrophils and ILC3s.
Assuntos
Acetatos/imunologia , Clostridioides difficile/imunologia , Infecções por Clostridium/imunologia , Enterocolite Pseudomembranosa/imunologia , Imunidade Inata/imunologia , Neutrófilos/imunologia , Receptores Acoplados a Proteínas G/imunologia , Animais , Inflamassomos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologiaRESUMO
Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19 (a prototypical lipoprotein of B. abortus) was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/imunologia , Monócitos/imunologia , RNA Bacteriano/imunologia , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella abortus/genética , Brucella abortus/imunologia , Brucella abortus/fisiologia , Brucelose/imunologia , Brucelose/microbiologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulação para Baixo/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , RNA Bacteriano/genética , Células THP-1RESUMO
NLRP3 inflammasome is a protein complex crucial to caspase-1 activation and IL-1ß and IL-18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus-induced IL-1ß secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7-dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL-1ß secretion in infected murine macrophages. Additionally, our results suggest that B. abortus-induced IL-1ß secretion depends on a P2X7-independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.
Assuntos
Brucella abortus/metabolismo , Brucelose/imunologia , Inflamassomos/metabolismo , Macrófagos/imunologia , Óxido Nítrico/metabolismo , Animais , Imunidade Inata , Interleucina-1beta/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2X7/genéticaRESUMO
In human brucellosis, the liver is frequently affected. Brucella abortus triggers a profibrotic response on hepatic stellate cells (HSCs) characterized by inhibition of MMP-9 with concomitant collagen deposition and TGF-ß1 secretion through type 4 secretion system (T4SS). Taking into account that it has been reported that the inflammasome is necessary to induce a fibrotic phenotype in HSC, we hypothesized that Brucella infection might create a microenvironment that would promote inflammasome activation with concomitant profibrogenic phenotype in HSCs. B. abortus infection induces IL-1ß secretion in HSCs in a T4SS-dependent manner. The expression of caspase-1 (Casp-1), absent in melanoma 2 (AIM2), Nod-like receptor (NLR) containing a pyrin domain 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC) was increased in B. abortus-infected HSC. When infection experiments were performed in the presence of glyburide, a compound that inhibits NLRP3 inflammasome, or A151, a specific AIM2 inhibitor, the secretion of IL-1ß was significantly inhibited with respect to uninfected controls. The role of inflammasome activation in the induction of a fibrogenic phenotype in HSCs was determined by performing B. abortus infection experiments in the presence of the inhibitors Ac-YVAD-cmk and glyburide. Both inhibitors were able to reverse the effect of B. abortus infection on the fibrotic phenotype in HSCs. Finally, the role of inflammasome in fibrosis was corroborated in vivo by the reduction of fibrotic patches in liver from B. abortus-infected ASC, NLRP, AIM2, and cCasp-1/11 knock-out (KO) mice with respect to infected wild-type mice.
Assuntos
Brucella abortus/fisiologia , Brucelose/imunologia , Células Estreladas do Fígado/imunologia , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Animais , Brucella abortus/genética , Brucelose/genética , Brucelose/microbiologia , Caspase 1/genética , Caspase 1/imunologia , Fibrose/genética , Fibrose/imunologia , Fibrose/microbiologia , Células Estreladas do Fígado/microbiologia , Humanos , Inflamassomos/genética , Interleucina-1beta/genética , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologiaRESUMO
Brucella spp. infection is frequently acquired through contaminated aerosols. The role of interleukin-1 beta (IL-1ß) in the early pulmonary response to respiratory Brucella infection is unknown. As shown here, IL-1ß levels in lung homogenates and bronchoalveolar lavage fluid (BALF) of mice intratracheally inoculated with B. abortus were increased at 3 and 7 days p.i. At 7 days p.i., pulmonary CFU numbers were higher in IL-1 receptor (IL-1R) knockout (KO) mice than in wild type (WT) mice. At different times p.i. CFU in lungs and BALF were higher in mice lacking some inflammasome components (caspase-1, AIM2, NLRP3) than in WT mice. At 2 days p.i. pulmonary levels of IL-1ß and CXCL1 (neutrophils chemoattractant) were lower in caspase-1/11 KO mice. At day 3 p.i., neutrophils counts in BALF were lower in caspase-1/11 KO mice than in WT mice. During in vitro infections, IL-1ß secretion was lower in alveolar macrophages from caspase-1/11, NLRP3 or AIM2 KO mice than in WT controls. Similarly, IL-1ß production by B. abortus-infected alveolar epithelial cells was reduced by pretreatment with a specific caspase-1 inhibitor. This study shows that IL-1R, probably through IL-1ß action, and the NLRP3 and AIM2 inflammasomes are involved in pulmonary innate immune protective mechanisms against respiratory B. abortus infection.
Assuntos
Brucella abortus/imunologia , Brucelose/imunologia , Inflamassomos/metabolismo , Pulmão/imunologia , Receptores de Interleucina-1/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Brucella abortus/patogenicidade , Caspase 1/metabolismo , Caspases/genética , Caspases/metabolismo , Caspases Iniciadoras , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Imunidade Inata , Inflamassomos/farmacologia , Interleucina-1beta/metabolismo , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Substâncias Protetoras/farmacologia , Serpinas/metabolismo , Proteínas Virais/metabolismoRESUMO
The helminth Schistosoma mansoni is one of main causes of human schistosomiasis, a health and economic concern in some of the world's poorest countries. Current treatment regimens can lead to serious side effects and are not suitable for breastfeeding mothers. As such, efforts have been undertaken to develop a vaccine to prevent infection. Of these, Sm29 is a promising candidate that has been associated with resistance to infection/reinfection in humans and mice. Its ability to induce resistance to reinfection has also been recently demonstrated using a vaccine formulation containing Freund's adjuvant. However, Freund's adjuvant is unsuitable for use in human vaccines. We therefore evaluated the ability of Sm29 to induce protection against S. mansoni reinfection when formulated with either alum or MPLA as an adjuvant, both approved for human use. Our data demonstrate that, in contrast to Sm29 with MPLA, Sm29 with alum reduced parasite burden after reinfection compared to a control. We next investigated whether the immune response was involved in creating the differences between the protective (Sm29Alum) and non-protective (Sm29MPLA) vaccine formulations. We observed that both formulations induced a similar mixed-profile immune response, however, the Sm29 with alum formulation raised the levels of antibodies against Sm29. This suggests that there is an association between a reduction in worm burden and parasite-specific antibodies. In summary, our data show that Sm29 with an alum adjuvant can successfully protect against S. mansoni reinfection in mice, indicating a potentially effective vaccine formulation that could be applied in humans.
Assuntos
Antígenos de Helmintos/imunologia , Imunidade Humoral , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Compostos de Alúmen/administração & dosagem , Compostos de Alúmen/química , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/química , Resistência à Doença , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , VacinaçãoRESUMO
Hepatocytes may rupture after a drug overdose, and their intracellular contents act as damage-associated molecular patterns (DAMPs) that lead to additional leukocyte infiltration, amplifying the original injury. Necrosis-derived DNA can be recognized as a DAMP, activating liver non-parenchymal cells (NPCs). We hypothesized that NPCs react to DNA by releasing interferon (IFN)-1, which amplifies acetaminophen (APAP)-triggered liver necrosis. We orally overdosed different knockout mouse strains to investigate the pathways involved in DNA-mediated amplification of APAP-induced necrosis. Mice were imaged under intravital confocal microscopy to estimate injury progression, and hepatocytes and liver NPCs were differentially isolated for gene expression assays. Flow cytometry (FACS) using a fluorescent reporter mouse estimated the interferon-beta production by liver leukocytes under different injury conditions. We also treated mice with DNase to investigate the role of necrosis DNA signaling in IFN-1 production. Hepatocytes released a large amount of DNA after APAP overdose, which was not primarily sensed by these cells. However, liver NPCs promptly sensed such environmental disturbances and activated several DNA sensing pathways. Liver NPCs synthesized and released IFN-1, which was associated with concomitant hepatocyte necrosis. Ablation of IFN-1 recognition in interferon α/ß receptor (IFNAR-/-) mice delayed APAP-mediated liver necrosis and dampened IFN-1 sensing pathways. We demonstrated a novel loop involving DNA recognition by hepatic NPCs and additional IFN-1 mediated hepatocyte death.
RESUMO
The immune response induced by Schistosma mansoni antigens is able to prevent immune-mediated diseases. Conversely, the inflammatory response in cutaneous leishmaniasis (CL), although responsible for controlling the infection, is also associated with the pathogenesis of disease. The aim of this study was to evaluate the potential of the S. mansoni Sm29 antigen to change certain aspects of the profiles of monocyte derived dendritic cells (MoDCs) and lymphocytes from subjects with CL in vitro. Expression of surface molecules and intracellular cytokines in the MoDCs and lymphocytes as well as the proliferation of Leishmania braziliensis were evaluated by flow cytometry. Levels of cytokines were evaluated in culture supernatants by ELISA. It was observed that stimulation by rSm29 increased the frequency of expression of CD83, CD80, CD86, and IL-10R in MoDCs compared to non-stimulated cultures. Additionally rSm29 decreased the frequency CD4+ and CD8+ T cells expressing CD28 and increased the frequency of CD4+CD25hi and CD4+CTLA-4+ T lymphocytes. Addition of rSm29 to cultures increased IL-10 levels and decreased levels of IL-12p40 and IFN-γ, while not altering TNF levels compared to non-stimulated cultures. This study showed that rSm29 induced a regulatory profile in MoDCs and lymphocytes and thereby regulated the exaggerated inflammation observed in CL. Considering that there are few therapeutic options for leishmaniasis, the use of rSm29 may be an alternative to current treatment and may be an important strategy to reduce the healing time of lesions in patients with CL.
Assuntos
Antígenos de Helmintos/imunologia , Células Dendríticas/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/microbiologia , Linfócitos/imunologia , Schistosoma mansoni/imunologia , Animais , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/metabolismo , Suscetibilidade a Doenças , Feminino , Humanos , Leishmaniose Cutânea/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Linfócitos/metabolismo , Masculino , Fenótipo , Proteínas Recombinantes/imunologiaRESUMO
Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR) pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP). Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and avoid the immunological surveillance of cytotoxic CD8+ T cells.
Assuntos
Brucelose/imunologia , Receptores ErbB/imunologia , Evasão da Resposta Imune/imunologia , Monócitos/imunologia , RNA Bacteriano/imunologia , Receptor 8 Toll-Like/imunologia , Animais , Brucella abortus/imunologia , Apresentação Cruzada/imunologia , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Monócitos/microbiologia , Transdução de Sinais/imunologiaRESUMO
Heat shock proteins (Hsps) are highly expressed at all sites of inflammation. As they are ubiquitous and immunodominant antigens, these molecules represent good candidates for the therapeutic use of oral tolerance in autoimmune and chronic inflammatory diseases. Evidences from human and animal studies indicate that inflammatory bowel disease (IBD) results from uncontrolled inflammatory responses to intestinal microbiota. Hsps are immunodominant proteins expressed by several immune cells and by commensal bacteria. Using an IBD mouse model, we showed that oral pretreatment with genetically modified Lactococcus lactis that produces and releases Mycobacterium Hsp65, completely prevented DSS-induced colitis in C57BL/6 mice. Protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; increased IL-10 production in colonic tissue; and expansion of CD4+Foxp3+ and CD4+LAP+ regulatory T cells in spleen and mesenteric lymph nodes. This effect was dependent on IL-10 and toll-like receptor 2. Thus, this approach may open alternative options for long-term management of IBD.
RESUMO
Asthma is a chronic disease characterized by airway inflammation, obstruction and hyperresponsiveness. Severe asthma affects a small proportion of subjects but results in most of the morbidity, costs and mortality associated with the disease. Studies have suggested that Schistosoma mansoni infection reduces the severity of asthma and prevent atopy. OBJECTIVE: We evaluated the ability of S. mansoni antigens, Sm29 and Sm29TSP-2 to modulate lymphocyte activation status in response to the allergen of the mite Dermatophagoides pteronyssinus (Der p1) in cell cultures of individuals with asthma. METHODS: Thirty four patients were enrolled in this study: seventeen patients with severe asthma (SA group), seventeen patients with mild asthma (MA group) and six controls with no asthma. Peripheral blood mononuclear cells (PBMC) were obtained and stimulated with Sm29 and Sm29TSP-2 in the presence or absence of Der p1. The expression of surface markers and cytokines on lymphocytes was evaluated by flow cytometry and the levels of IL-10 in the culture supernatant were determined by ELISA. RESULTS: The addition of Sm29 and Sm29TSP-2 antigens to PBMC cultures from both groups of subjects with asthma stimulated with Der p1 reduced the frequency of CD4+CD25low cells whereas and increased frequency of CD4+CD25high population was observed compared to unstimulated cultures. Moreover, cultures stimulated with Sm29TSP-2 showed a reduction in the frequency of T cells expressing CD69, IFN-γ, TNF and TGF-ß in the MA group and an increase in the frequency of CD4+TSLPR+ T cells in the SA group. The addition of Sm29 to the cultures reduced the frequency of CD4+CD69+ and CD4+IL-5+ T cells in all asthmatic groups, and reduced the frequency of CD4+ T cells expressing IL-13 in the MA group. The cultures stimulated with Sm29 and Sm29TSP-2 showed an increase in the level of IL-10 in the supernatants. CONCLUSION: These results suggest that the addition of Sm29 and Sm29TSP-2 to the cells cultures from subjects with asthma reduced cell activation markers and altered the cytokine production pattern in a way that can potentialy control the inflammatory response associated with asthma.
Assuntos
Antígenos de Helmintos/sangue , Asma/sangue , Citocinas/sangue , Leucócitos Mononucleares/parasitologia , Schistosoma mansoni/imunologia , Adulto , Animais , Asma/parasitologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-10/sangue , Interleucina-13/sangue , Interleucina-5/sangue , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta/metabolismoRESUMO
Schistosomiasis is an important parasitic disease worldwide that affects more than 207 million people in 76 countries and causes approximately 250,000 deaths per year. The best long-term strategy to control schistosomiasis is through immunization combined with drug treatment. Due to the ability of DNA vaccines to generate humoral and cellular immune responses, such vaccines are considered a promising approach against schistosomiasis. Sm29 and tetraspanin-2 (Sm-TSP2) are two proteins that are located in the S. mansoni tegument of adult worms and schistosomula and induce high levels of protection through recombinant protein immunization. In this study, we transfected BHK-21 cells with plasmids encoding Sm29, Sm-TSP2 or a chimera containing both genes. Using RT-PCR analysis and western blot, we confirmed that the DNA vaccine constructs were transcribed and translated, respectively, in BHK-21 cells. After immunization of mice, we evaluated the reduction in worm burden. We observed worm burden reductions of 17-22%, 22%, 31-32% and 24-32% in animals immunized with the pUMVC3/Sm29, pUMVC3/SmTSP-2, pUMVC3/Chimera and pUMVC3/Sm29 + pUMVC3/SmTSP-2 plasmids, respectively. We evaluated the humoral response elicited by DNA vaccines, and animals immunized with pUMVC3/Sm29 and pUMVC3/Sm29 + pUMVC3/SmTSP-2 showed higher titers of anti-Sm29 antibodies. The cytokine profile produced by the spleen cells of immunized mice was then evaluated. We observed higher production of Th1 cytokines, such as TNF-α and IFN-γ, in vaccinated mice and no significant production of IL-4 and IL-5. The DNA vaccines tested in this study showed the ability to generate a protective immune response against schistosomiasis, probably through the production of Th1 cytokines. However, future strategies aiming to optimize the protective response induced by a chimeric DNA construct need to be developed.