RESUMO
Recognition of bacterial pathogens by the mammalian host relies on the induction of early innate immune responses initiated by the activation of pattern-recognition receptors (PRRs) upon sensing of their cognate microbe-associated-patterns (MAMPs). Successful pathogens have evolved to intercept PRR activation and signaling at multiple steps. The molecular dissection of the underlying mechanisms revealed many of the basic mechanisms used by the immune system. Here we provide an overview of the different strategies used by bacterial pathogens and commensals to subvert and reprogram PPR-mediated innate immune responses. A particular attention is given to recent discoveries highlighting novel molecular details of the host inflammatory response in mammalian cells and current advances in our understanding of the interaction of commensals with PRR-mediated responses.
Assuntos
Bactérias/imunologia , Infecções Bacterianas/imunologia , Imunidade Inata , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais , Simbiose , Animais , Autofagia , Bactérias/classificação , Infecções Bacterianas/microbiologia , Fenômenos Fisiológicos Bacterianos , Membrana Celular , HumanosRESUMO
Lectins are proteins with a well-defined carbohydrate recognition domain. Many microbial proteins such as bacterial toxins possess lectin or lectin-like binding domains to interact with cell membranes that are decorated with glycan recognition motifs. We report a straightforward way to prepare monodisperse and biocompatible polyethylene glycol microgels, which carry glycan motifs for specific binding to lectins. The sugar-functionalized colloids exhibit a wide mesh size and a highly accessible volume. The microgels are prepared via drop-based microfluidics combined with radical polymerization. GSII and ECL are used as model lectins that bind specifically to the corresponding carbohydrates, namely, GlcNAc and LacNAc. LacNAc microgels bind ECL with a high capacity and high affinity (Kd ≈ 0.5 to 1 µM), suggesting multivalent binding of the lectin to the LacNAc-decorated flexible microgel network. Glycan-functionalized microgels present a useful tool for lectin scavenging in biomedical applications.
Assuntos
Géis/química , Lectinas/química , Polissacarídeos/química , Géis/síntese química , Lectinas/metabolismo , Microfluídica/métodos , Polimerização , Ligação ProteicaRESUMO
UNLABELLED: Nonalcoholic fatty liver disease is seen as the hepatic manifestation of the metabolic syndrome and represents the most common liver disease in Western societies. The G protein-coupled chemokine receptor CX3CR1 plays a central role in several metabolic syndrome-related disease manifestations and is involved in maintaining intestinal homeostasis. Because diet-induced intestinal dysbiosis is a driver for nonalcoholic fatty liver disease, we hypothesized that CX3CR1 may influence the development of steatohepatitis. In two independent models of diet-induced steatohepatitis (high-fat diet and methionine/choline-deficient diet), CX3CR1 protected mice from excessive hepatic steatosis and inflammation, as well as systemic glucose intolerance. Lack of Cx3cr1 expression was associated with significantly altered intestinal microbiota composition, which was linked to an impaired intestinal barrier. Concomitantly, endotoxin levels in portal serum and inflammatory macrophages in liver were increased in Cx3cr1-/- mice, indicating an increased inflammatory response. Depletion of intestinal microbiota by administration of broad-spectrum antibiotics suppressed the number of infiltrating macrophages and promoted macrophage polarization in liver. Consequently, antibiotic-treated mice demonstrated a marked improvement of steatohepatitis. CONCLUSION: Microbiota-mediated activation of the innate immune responses through CX3CR1 is crucial for controlling steatohepatitis progression, which recognizes CX3CR1 as an essential gatekeeper in this scenario.
Assuntos
Homeostase , Mucosa Intestinal/metabolismo , Hepatopatia Gordurosa não Alcoólica/imunologia , Receptores de Quimiocinas/fisiologia , Animais , Antibacterianos/farmacologia , Translocação Bacteriana , Glicemia/análise , Receptor 1 de Quimiocina CX3C , Imunidade Inata , Intestinos/microbiologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
OBJECTIVES: Inflammatory bowel disease (IBD), particularly Crohn's disease (CD), is associated with increased microbial-specific IgG and IgA antibodies, whereas alterations of anti-food antibodies are still disputed. The knowledge about IgG subclass antibodies in IBD is limited. In this study we analysed IgG subclass antibodies specific for nutritional and commensal antigens in IBD patients and controls. METHODS: Serum IgG1, IgG2, IgG3 and IgG4 specific for wheat and milk extracts, purified ovalbumin, Escherichia coli and Bacteroides fragilis lysates and mannan from Saccharomyces cerevisiae were analysed by ELISA in patients with CD (n = 56), ulcerative colitis (UC; n = 29), acute gastroenteritis/colitis (n = 12) as well as non-inflammatory controls (n = 62). RESULTS: Anti-Saccharomyces cerevisiae antibodies (ASCA) of all IgG subclasses and anti-B. fragilis IgG1 levels were increased in CD patients compared to UC patients and controls. The discriminant validity of ASCA IgG2 and IgG4 was comparable with that of ASCA pan-IgG and IgA, whereas it was inferior for ASCA IgG1/IgG3 and anti-B. fragilis IgG1. Complicated CD defined by the presence of perianal, stricturing or penetrating disease phenotypes was associated with increased ASCA IgG1/IgG3/IgG4, anti-B. fragilis IgG1 and anti-E. coli IgG1 levels. Anti-food IgG subclass levels were not different between IBD patients and controls and did not correlate with food intolerance. In contrast to anti-microbial Abs, food-specific IgG responses were predominately of the IgG4 isotype and all food-specific IgG subclass levels correlated negatively with age. CONCLUSION: Our study supports the notion that the adaptive immune recognition of food and commensal antigens are differentially regulated.
Assuntos
Anticorpos Antibacterianos/sangue , Anticorpos Antifúngicos/sangue , Hipersensibilidade Alimentar/sangue , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Doenças Inflamatórias Intestinais/sangue , Adulto , Idoso , Animais , Bacteroides fragilis , Biomarcadores/sangue , Estudos de Casos e Controles , Escherichia coli , Feminino , Alemanha , Humanos , Doenças Inflamatórias Intestinais/imunologia , Masculino , Microbiota , Pessoa de Meia-Idade , Leite/efeitos adversos , Curva ROC , Saccharomyces cerevisiae , Triticum/efeitos adversos , Adulto JovemRESUMO
The Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium-T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri, via its type III secretion system, impairs the migration pattern of CD4(+) T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4(+) T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4(+) T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses.
Assuntos
Imunidade Adaptativa , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Movimento Celular/imunologia , Disenteria Bacilar/imunologia , Interações Hospedeiro-Patógeno/imunologia , Shigella flexneri/fisiologia , Animais , Disenteria Bacilar/genética , Feminino , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: Development and function of tissue resident mast cells (MCs) is tightly controlled by various cytokines, most of which belong to the typical T helper (Th) 2-type cytokines such as IL-3 and IL-4. The effects of the Th1-type cytokine IFN-γ on human MCs is less clear. RESULTS: Here, we analyzed the effects of IFN-γ on tissue-derived, mature human MCs. We found that INF-γ decreases proliferation, without affecting apoptosis in human intestinal MCs cultured in the presence of optimal concentrations of stem cell factor (SCF) or SCF and IL-4. However, in the absence of growth factors or at suboptimal concentrations of SCF, INF-γ promotes survival through inhibition of MC apoptosis. Interestingly, we found that INF-γ has no effect on FcϵRI expression and FcϵRI-mediated release of histamine and leukotriene (LT)C4, while it has profound effects on FcγR expression and activation. We show that intestinal MCs express FcγRI, FcγRIIa, and FcγRIIc, whereas FcγRIIb expression was found in only 40% of the isolates and FcγRIII was never detectable. INF-γ strongly increases FcγRI and decreases FcγRIIa expression. INF-γ-naïve MCs produce LTC4 but fail to degranulate upon crosslinking of surface-bound monomeric IgG. In contrast, INF-γ-treated MCs rapidly release granule-stored histamine in addition to de novo-synthesized LTC4. CONCLUSION: In summary, we identify INF-γ as an important regulator of tissue-resident human MCs. IFN-γ displays a dual function by blocking extensive MC proliferation, while decreasing apoptosis in starving MCs and enhancing FcγRI expression and activation. These results emphasize the involvement of mucosal MCs in Th1-mediated disorders.
Assuntos
Interferon gama/farmacologia , Intestinos/imunologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Receptores de IgG/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histamina/biossíntese , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Mastócitos/citologia , Mastócitos/imunologia , Ligação Proteica , Receptores de IgE/metabolismo , Receptores de IgG/genéticaRESUMO
UNLABELLED: In human and murine models of nonalcoholic steatohepatitis (NASH), increased hepatocyte apoptosis is a critical mechanism contributing to inflammation and fibrogenesis. Caspase 8 (Casp8) is essential for death-receptor-mediated apoptosis activity and therefore its modulation might be critical for the pathogenesis of NASH. The aim was to dissect the role of hepatocyte Casp8 in a murine model of steatohepatitis. We generated hepatocyte-specific Casp8 knockout (Casp8(Δhep) ) mice. Animals were fed with a methionine-choline-deficient (MCD) diet. Liver injury was assessed by histopathological analysis, apoptotic death, serum alanine aminotransferase (ALT), fluorescent-activated cell sorter (FACS), analysis of liver infiltration and inflammation, reactive oxygen species (ROS), and liver fibrosis. MCD feeding triggered steatosis, hepatic lipid storage, and accumulation of free fatty acid (FFA) in wildtype (WT) livers, which were significantly reduced in Casp8(Δhep) animals. Additionally, lack of Casp8 expression in hepatocytes reduced the MCD-dependent increase in apoptosis and decreased expression of proinflammatory cytokines as well as hepatic infiltration. As a consequence, ROS production was lower, leading to a reduction in the progression of liver fibrosis in Casp8(Δhep) livers. CONCLUSION: Selective ablation of Casp8 in hepatocytes ameliorates development of NASH by modulating liver injury. Casp8-directed therapy might be a plausible treatment for patients with steatohepatitis. (HEPATOLOGY 2013;57:2189-2201).
Assuntos
Caspase 8/metabolismo , Fígado Gorduroso/enzimologia , Hepatócitos/enzimologia , Metabolismo dos Lipídeos , Animais , Apoptose , Fígado Gorduroso/patologia , Fibrose , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Estresse OxidativoRESUMO
Although a number of studies have examined the development of T-helper cell type 2 (Th2) immunity in different settings, the mechanisms underlying the initiation of this arm of adaptive immunity are not well understood. We exploited the fact that immunization with antigen plus either nucleotide-binding oligomerization domain-containing proteins 1 (Nod1) or 2 (Nod2) agonists drives Th2 induction to understand how these pattern-recognition receptors mediate the development of systemic Th2 immune responses. Here, we show in bone-marrow chimeric mice that Nod1 and Nod2 expression within the stromal compartment is necessary for priming of effector CD4(+) Th2 responses and specific IgG1 antibodies. In contrast, sensing of these ligands by dendritic cells was not sufficient to induce Th2 immunity, although these cells contribute to the response. Moreover, we determined that CD11c(+) cells were the critical antigen-presenting cells, whereas basophils and B cells did not affect the capacity of Nod ligands to induce CD4(+) Th2 effector function. Finally, we found that full Th2 induction upon Nod1 and Nod2 activation was dependent on both thymic stromal lymphopoietin production by the stromal cells and the up-regulation of the costimulatory molecule, OX40 ligand, on dendritic cells. This study provides in vivo evidence of how systemic Th2 immunity is induced in the context of Nod stimulation. Such understanding will influence the rational design of therapeutics that could reprogram the immune system during an active Th1-mediated disease, such as Crohn's disease.
Assuntos
Citocinas/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Células Th2/imunologia , Animais , Linfócitos B/imunologia , Basófilos/imunologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Doença de Crohn/terapia , Citocinas/genética , Células Dendríticas/imunologia , Imunidade Celular/fisiologia , Imunização , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Ligante OX40 , Estrutura Terciária de Proteína , Células Th1/imunologia , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/imunologia , Linfopoietina do Estroma do TimoRESUMO
BACKGROUND & AIMS: Acetaminophen (AAP) overdose is the most frequent cause of drug-induced liver failure. c-Jun N-terminal kinase (JNK) is thought to play a central role in AAP-induced hepatocellular necrosis. The apoptosis repressor with caspase recruitment domain (ARC) is a death repressor that inhibits death receptor and mitochondrial apoptotic signaling. Here, we investigated ARC's therapeutic effect and molecular mechanisms on AAP-induced hepatocellular necrosis. METHODS: We tested the in vivo and in vitro effects of ARC fused with the transduction domain of HIV-1 (TAT-ARC) on murine AAP hepatotoxicity. RESULTS: Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by AAP overdose in C57BL/6 mice. AAP triggered caspase-independent necrosis, as evidenced by liver histology, elevated serum transaminases, and secreted HMGB1 that was inhibited by ARC. ARC-mediated hepatoprotection was not caused by an alteration of AAP metabolism, but resulted in reduced oxidative stress. AAP overdose led to induction of RIP-dependent signaling with subsequent JNK activation. Ectopic ARC inhibited JNK activation by specific interactions between ARC and JNK1 and JNK2. Importantly, survival of mice was even preserved when ARC therapy was initiated in a delayed manner after AAP administration. CONCLUSIONS: This work identifies for the first time ARC-JNK-binding with subsequent inhibition of JNK signaling as a specific mechanism of ARC to interfere with AAP-dependent necrosis. Our data suggests that AAP-mediated induction of RIP signaling serves as a critical switch for hepatocellular necrosis. The efficacy of TAT-ARC protein transduction in murine AAP hepatotoxicity suggests its therapeutic potential for reversing AAP intoxication also in humans.
Assuntos
Acetaminofen/efeitos adversos , Proteínas Reguladoras de Apoptose/uso terapêutico , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Musculares/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Proteínas Reguladoras de Apoptose/farmacologia , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Glutationa/metabolismo , HIV-1 , Neoplasias Hepáticas/patologia , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/farmacologia , Necrose/induzido quimicamente , Necrose/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
UNLABELLED: Acute liver failure (ALF) is associated with massive hepatocyte cell death and high mortality rates. Therapeutic approaches targeting hepatocyte injury in ALF are hampered by the activation of distinct stimulus-dependent pathways, mechanism of cell death, and a limited therapeutic window. The apoptosis repressor with caspase recruitment domain (ARC) is a recently discovered death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. Here, we investigated the in vivo effects of ARC fused with the transduction domain of human immunodeficiency virus 1 (HIV-1) (TAT-ARC) on Fas- and tumor necrosis factor (TNF)-mediated murine models of fulminant liver failure. Treatment with TAT-ARC protein completely abrogated otherwise lethal liver failure induced by Fas-agonistic antibody (Jo2), concanavalin A (ConA), or D-galactosamine/lipopolysaccharide (GalN/LPS) administration. Importantly, survival of mice was even preserved when TAT-ARC therapy was initiated in a delayed manner after stimulation with Jo2, ConA, or GalN/LPS. ARC blocked hepatocyte apoptosis by directly interacting with members of the death-inducing signaling complex. TNF-mediated liver damage was inhibited by two independent mechanisms: inhibition of jun kinase (JNK)-mediated TNF-α expression and prevention of hepatocyte apoptosis by inhibition of both death receptor and mitochondrial death signaling. We identified JNK as a novel target of ARC. ARC's caspase recruitment domain (CARD) directly interacts with JNK1 and JNK2, which correlates with decreased JNK activation and JNK-dependent TNF-α production. CONCLUSION: This work suggests that ARC confers hepatoprotection upstream and at the hepatocyte level. The efficacy of TAT-ARC protein transduction in multiple murine models of ALF demonstrates its therapeutic potential for reversing liver failure.
Assuntos
Proteínas do Citoesqueleto/genética , Terapia Genética/métodos , Falência Hepática Aguda/genética , Falência Hepática Aguda/terapia , Proteínas do Tecido Nervoso/genética , Proteínas Recombinantes de Fusão/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Animais , Apoptose/fisiologia , Caspases/química , Caspases/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Hepatócitos/citologia , Hepatócitos/fisiologia , Falência Hepática Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Estrutura Terciária de Proteína , Transdução Genética/métodos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The T cell response to Shigella, the causative agent of bacillary dysentery, remains poorly understood. Using a murine model of infection, we report that Shigella flexneri primes predominately IL-17A- and IL-22-producing Th17 cells. Shigella-specific Th1 cells are only significantly induced on secondary infection, whereas specific Th2 and CD8(+) T cells are undetectable. Apart from Th17 cells that are primed in a MHC class II- and IL-6-dependent, but IL12/23p40-independent manner, we identified gammadelta T cells as an additional but minor source of IL-17A. Priming of IL-17A(+) gammadelta T cells is dependent on IL12/23p40, but independent of MHC-class II and IL-6. Th17 cells have emerged as important players in inflammatory, autoimmune, and infectious diseases. Among the yet unresolved questions is their role in long-term immunity to pathogens. In this study, we show that the elicited S. flexneri-specific Th17 pool gives rise to an enhanced recall response up to 12 mo after priming, suggesting the presence of a long-term memory state. The clearance of primary infection is impaired in the absence of T cells, but independently of IL-17A. However, after reinfection, IL-17A produced by S. flexneri-specific Th17 cells becomes important to ultimately restrict bacterial growth. These findings bring new insights into the adaptive immune response to Shigella infection and highlight the importance of pathogen-specific Th17 cell immunity for secondary immune protection.
Assuntos
Disenteria Bacilar/imunologia , Interleucina-17/biossíntese , Shigella flexneri/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/microbiologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/microbiologia , Animais , Células Cultivadas , Disenteria Bacilar/microbiologia , Disenteria Bacilar/prevenção & controle , Epitopos Imunodominantes/imunologia , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/microbiologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/microbiologiaRESUMO
BACKGROUND: Fecal microbiota transfer (FMT) has become a standard of care in the prevention of multiple recurrent Clostridioides difficile (rCDI) infection. AIM: While primary cure rates range from 70-80% following a single treatment using monodirectional approaches, cure rates of combination treatment remain largely unknown. METHODS: In a retrospective case-control study, outcomes following simultaneous bidirectional FMT (bFMT) with combined endoscopic application into the upper and lower gastrointestinal tract, compared to standard routes of application (endoscopy via upper or lower gastrointestinal tract and oral capsules; abbreviated UGIT, LGIT and CAP) on day 30 and 90 after FMT were assessed. Statistical matching partners were identified using number of recurrences (<3; ≥3), age and gender. RESULTS: Primary cure rates at D30 and D90 for bFMT were 100% (p=.001). The matched control groups showed cure rates of 81.3% for LGIT (p=.010), 62.5% for UGIT (p=.000) and 78.1% for CAP (p=.005) on D30 and 81.3% for LGIT (p=.010), 59.4% for UGIT (p=.000) and 71.9% for CAP (p=.001) on D90. CONCLUSION: In our analysis, bFMT on the same day significantly increased primary cure rate at D30 and D90. These data require prospective confirmation but suggest that route of application may play a significant role in optimizing patient outcomes. ClinicalTrials.gov no: NCT02681068.
Assuntos
Infecções por Clostridium/terapia , Transplante de Microbiota Fecal/métodos , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Sistema de Registros , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Mast cells (MCs) are recognized to play an important role in bacterial host defense in the murine system. In this study, we studied the interaction of human MCs, isolated from the intestine and purified to homogeneity, with different Escherichia coli and Shigella flexneri strains. We show that alpha-hemolysin (Hly)-producing E. coli strains induce the release of histamine, leukotrienes, and proinflammatory cytokines in intestinal MCs. In contrast, MCs were virtually unresponsive to S. flexneri and several Hly-negative E. coli strains, including the isogenic Hly-deficient mutants of Hly(+) strains. Hly(+) E. coli but not Hly(-) E. coli caused an increase in intracellular Ca(2+) levels. Blocking of extracellular Ca(2+) and of the calmodulin/calcineurin pathway by cyclosporin A inhibited the response to Hly(+) E. coli. Furthermore, inhibition of MAPKs p38 and ERK reduces activation of MCs by Hly(+) E. coli. In addition, using an ex vivo system, we directly record the histamine release by MCs located in the lamina propria after infection with Hly(+) E. coli. Our data indicate that human intestinal mast cells interact with selected Gram-negative bacteria, establish E. coli Hly as a factor regulating MC effector functions, and argue further for a role of human MCs in innate immunity.
Assuntos
Escherichia coli/imunologia , Proteínas Hemolisinas/imunologia , Liberação de Histamina , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mastócitos/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Mediadores da Inflamação/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Leucotrienos/imunologia , Leucotrienos/metabolismo , Ativação Linfocitária , Mastócitos/metabolismo , Mastócitos/microbiologiaRESUMO
While a number of microbial-associated molecular patterns have been known for decades to act as adjuvants, the mechanisms and the signaling pathways underlying their action have remained elusive. Here, we examined the unfolding of the adaptive immune response induced by Nod2 in vivo upon activation by its specific ligand, muramyl dipeptide, a component of peptidoglycan. Our findings demonstrate that this bacterial sensor triggers a potent Ag-specific immune response with a Th2-type polarization profile, characterized by the induction of IL-4 and IL-5 by T cells and IgG1 Ab responses. Nod2 was also found to be critical for the induction of both Th1- and Th2-type responses following costimulation with TLR agonists. Importantly, the synergistic responses to Nod2 and TLR agonists seen in vivo were recapitulated by dendritic cells in vitro, suggesting that these cells likely play a central role in the integration of Nod2- and TLR-dependent signals for driving the adaptive immune response. Taken together, our results identify Nod2 as a critical mediator of microbial-induced potentiation and polarization of Ag-dependent immunity. Moreover, these findings affect our understanding of Crohn's diseases pathogenesis, where lack of Nod2-dependent Th2 signaling in a subset of these patients might explain heightened Th1-mediated inflammation at the level of the intestinal mucosa.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/imunologia , Antígenos de Bactérias/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Peptidoglicano/imunologia , Transdução de Sinais/imunologia , Células Th2/imunologia , Receptores Toll-Like/imunologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Antígenos de Bactérias/farmacologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Doença de Crohn/patologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Camundongos , Proteína Adaptadora de Sinalização NOD2/genética , Peptidoglicano/farmacologia , Transdução de Sinais/genética , Células Th1/imunologia , Células Th1/patologia , Células Th2/patologia , Receptores Toll-Like/genéticaRESUMO
BACKGROUND AND AIMS: Faecal biomarkers are used as indicators of disease activity in inflammatory bowel diseases [IBD], which include Crohn's disease [CD] and ulcerative colitis [UC]. Micro-RNAs [miRNAs] are small non-coding RNAs detectable in extracellular fluids and can be used as clinical biomarkers. The aim of this study was to determine if faecal miRNA composition is altered in IBD. METHODS: More than 800 different human faecal miRNAs were measured in stool samples from control individuals and patients with active CD by using NanoString technology. Selected miRNAs were quantified by qRT-PCR in faeces, serum and intestinal tissue of controls [n = 23] and patients with inactive or active CD [n = 22, n = 22] or UC [n = 11, n = 24] as well as patients with Clostridium difficile infection [CDI, n = 8]. RESULTS: In total, 150 miRNAs were significantly detected in faeces from controls and patients, and multivariate analyses showed that CD patients with high disease activities had a distinct miRNA profile and that miR-223 and miR-1246 were distinct from other faecal miRNAs. In a larger cohort, active UC patients displayed significantly higher levels of miR-223 and miR-1246 than controls while patients with CDI had higher levels of faecal miR-1246 but not miR-223. No differences were noted in serum samples. CONCLUSIONS: To our knowledge, this is the first comprehensive screen of faecal miRNAs performed in IBD. Further investigation will aim to confirm these findings in a larger cohort and to understand the biological function and cellular sources of faecal miRNAs.
Assuntos
Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Fezes/química , MicroRNAs/metabolismo , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
BACKGROUND AND AIMS: Fecal biomarkers are important non-invasive markers monitoring disease activity in inflammatory bowel disease (IBD). We compared the significance of fecal eosinophil cationic protein (fECP) and fecal calprotectin (fCal). METHODS: fECP and fCal were measured in patients with Crohn's disease (CD, n = 97), ulcerative colitis (UC, n = 53), Clostridioides difficile infection (CDI, n = 9), primary food allergy (PFA, n = 11), pollen-associated food allergy (n = 25) and non-inflammatory controls (n = 78). Results were correlated with clinical and endoscopic IBD activity scores. RESULTS: fECP was significantly elevated in CD, UC, CDI and PFA compared to controls. fCal was significantly increased in CD, UC and CDI. fECP had lower diagnostic accuracy than fCal (area under the curve (AUC) = 0.88) in differentiating between endoscopically active and inactive patients with IBD (AUC = 0.77, ROC analysis). In contrast to fCal, fECP correlated negatively with age and levels were also elevated in clinically and endoscopically inactive patients with IBD <45 years (endoscopically inactive IBD vs controls; AUC for fECP = 0.86; AUC for fCal = 0.62). However, in those patients with low inflammatory activity (fCal <250 mg/kg), high fECP indicated the need for treatment modification or surgery (fECP <200 µg/kg = 22%; 200-600 µg/kg = 44%; >600 µg/kg = 82%) at month 48 of follow-up. CONCLUSIONS: fECP is a diagnostic and prognostic marker in young patients with IBD in remission.
RESUMO
Oral tolerance to soluble antigens is critically important for the maintenance of immunological homeostasis in the gut. The mechanisms of tolerance induction to antigens of the gut microbiota are still less well understood. Here, we investigate whether the subcellular localization of antigens within non-pathogenic E. coli has a role for its ability to induce antigen-specific tolerance. E. coli that express an ovalbumin (OVA) peptide in the cytoplasm, at the outer membrane or as secreted protein were generated. Intestinal colonization of mice with non-pathogenic E. coli expressing OVA at the membrane induced the expansion of antigen-specific Foxp3+ Tregs and mediated systemic immune tolerance. In contrast, cytoplasmic OVA was ignored by antigen-specific CD4+ T cells and failed to induce tolerance. In vitro experiments revealed that surface-displayed OVA of viable E. coli was about two times of magnitude more efficient to activate antigen-specific CD4+ T cells than soluble antigens, surface-displayed antigens of heat-killed E. coli or cytoplasmic antigen of viable or heat-killed E. coli. This effect was independent of the antigen uptake efficiency in dendritic cells. In summary, our results show that subcellular antigen localization in viable E. coli strongly influences antigen-specific CD4+ cell expansion and tolerance induction upon intestinal colonization.
Assuntos
Escherichia coli/fisiologia , Microbioma Gastrointestinal/fisiologia , Linfócitos T Reguladores/imunologia , Administração Oral , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Tolerância Imunológica , Espaço Intracelular , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microrganismos Geneticamente Modificados , Ovalbumina/genética , Ovalbumina/imunologia , Peptídeos/genética , Peptídeos/imunologia , SimbioseRESUMO
The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly regulated, dynamic event in IBD pathogenesis. Using a lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The ulcerative colitis (UC) samples appeared only in the hypo-responsive Cluster 1, and in Cluster 2. Conversely, Crohn's disease (CD) patients appeared in all Clusters with their percentage being higher in the hyper-responsive Cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and the concentrations of cytokines released into medium from stimulated macrophages, but not with serum or biopsy cytokine levels. Confocal imaging of lentivirally-expressed p65 activation revealed that a higher proportion of macrophages from CD patients responded to endotoxin lipid A compared to controls. In contrast, cells from UC patients exhibited a shorter duration of NF-κB p65 subunit nuclear localization compared to healthy controls, and CD donors. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between CD patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-κB activation in blood-derived macrophages have the potential to segregate IBD patients into groups with different phenotypes and may therefore help determine response to therapy.
Assuntos
Núcleo Celular/imunologia , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Fator de Transcrição RelA/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Adulto , Animais , Núcleo Celular/genética , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Feminino , Humanos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Transdução de Sinais/genética , Fator de Transcrição RelA/genéticaRESUMO
Clostridium difficile infection (CDI) represents one of the most common healthcare-associated infections. Due to increasing numbers of recurrences and therapy failures, CDI has become a major disease burden. Studies have shown that fecal microbiota transplantation (FMT) can both be a safe and highly efficacious therapy for patients with therapy-refractory CDI. However, patients undergoing solid organ transplantation are at high risk for CDI due to long-term immunosuppression, previous antibiotic therapy, and proton pump inhibitor use. Additionally, these patients may be especially prone to adverse events related to FMT. Here, we report a successful FMT in a patient with severe therapy-refractory CDI after liver transplantation.
RESUMO
Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2ΔIEC; DSC2ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2ΔIEC, but not DSC2ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.