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Rationale: Chronic lung allograft dysfunction (CLAD) is the leading cause of death after lung transplant, and azithromycin has variable efficacy in CLAD. The lung microbiome is a risk factor for developing CLAD, but the relationship between lung dysbiosis, pulmonary inflammation, and allograft dysfunction remains poorly understood. Whether lung microbiota predict outcomes or modify treatment response after CLAD is unknown. Objectives: To determine whether lung microbiota predict post-CLAD outcomes and clinical response to azithromycin. Methods: Retrospective cohort study using acellular BAL fluid prospectively collected from recipients of lung transplant within 90 days of CLAD onset. Lung microbiota were characterized using 16S rRNA gene sequencing and droplet digital PCR. In two additional cohorts, causal relationships of dysbiosis and inflammation were evaluated by comparing lung microbiota with CLAD-associated cytokines and measuring ex vivo P. aeruginosa growth in sterilized BAL fluid. Measurements and Main Results: Patients with higher bacterial burden had shorter post-CLAD survival, independent of CLAD phenotype, azithromycin treatment, and relevant covariates. Azithromycin treatment improved survival in patients with high bacterial burden but had negligible impact on patients with low or moderate burden. Lung bacterial burden was positively associated with CLAD-associated cytokines, and ex vivo growth of P. aeruginosa was augmented in BAL fluid from transplant recipients with CLAD. Conclusions: In recipients of lung transplants with chronic rejection, increased lung bacterial burden is an independent risk factor for mortality and predicts clinical response to azithromycin. Lung bacterial dysbiosis is associated with alveolar inflammation and may be promoted by underlying lung allograft dysfunction.
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Azitromicina , Rejeição de Enxerto , Transplante de Pulmão , Microbiota , Humanos , Azitromicina/uso terapêutico , Masculino , Feminino , Pessoa de Meia-Idade , Rejeição de Enxerto/microbiologia , Rejeição de Enxerto/prevenção & controle , Estudos Retrospectivos , Adulto , Microbiota/efeitos dos fármacos , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Pulmão/microbiologia , Doença Crônica , Transplantados/estatística & dados numéricos , Idoso , Disbiose , Estudos de Coortes , Líquido da Lavagem Broncoalveolar/microbiologiaRESUMO
Rationale: Among patients with sepsis, variation in temperature trajectories predicts clinical outcomes. In healthy individuals, normal body temperature is variable and has decreased consistently since the 1860s. The biologic underpinnings of this temperature variation in disease and health are unknown. Objectives: To establish and interrogate the role of the gut microbiome in calibrating body temperature. Methods: We performed a series of translational analyses and experiments to determine whether and how variation in gut microbiota explains variation in body temperature in sepsis and in health. We studied patient temperature trajectories using electronic medical record data. We characterized gut microbiota in hospitalized patients using 16S ribosomal RNA gene sequencing. We modeled sepsis using intraperitoneal LPS in mice and modulated the microbiome using antibiotics, germ-free, and gnotobiotic animals. Measurements and Main Results: Consistent with prior work, we identified four temperature trajectories in patients hospitalized with sepsis that predicted clinical outcomes. In a separate cohort of 116 hospitalized patients, we found that the composition of patients' gut microbiota at admission predicted their temperature trajectories. Compared with conventional mice, germ-free mice had reduced temperature loss during experimental sepsis. Among conventional mice, heterogeneity of temperature response in sepsis was strongly explained by variation in gut microbiota. Healthy germ-free and antibiotic-treated mice both had lower basal body temperatures compared with control animals. The Lachnospiraceae family was consistently associated with temperature trajectories in hospitalized patients, experimental sepsis, and antibiotic-treated mice. Conclusions: The gut microbiome is a key modulator of body temperature variation in both health and critical illness and is thus a major, understudied target for modulating physiologic heterogeneity in sepsis.
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Microbioma Gastrointestinal , Microbiota , Sepse , Animais , Camundongos , Temperatura Corporal , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , RNA Ribossômico 16S/genéticaRESUMO
RATIONALE: Oral microbiota associate with diseases of the mouth and serve as a source of lung microbiota. However, the role of oral microbiota in lung disease is unknown. OBJECTIVES: To determine associations between oral microbiota and disease severity and death in idiopathic pulmonary fibrosis. METHODS: We analyzed 16S rRNA gene and shotgun metagenomic sequencing data of buccal swabs from 511 patients with idiopathic pulmonary fibrosis in the multicenter CleanUP-IPF trial. Buccal swabs were collected from usual care, and antimicrobial cohorts. Microbiome data was correlated with measures of disease severity using principal component analysis and linear regression models. Associations between the buccal microbiome and mortality were determined using Cox additive models, Kaplan Meier analysis and Cox proportional hazards models. MEASUREMENTS AND MAIN RESULTS: Greater buccal microbial diversity associated with lower forced vital capacity (FVC) at baseline [mean diff -3.60: 95% CI -5.92 to -1.29 percent predicted FVC per 1 unit increment]. The buccal proportion of Streptococcus correlated positively with FVC [mean diff 0.80: 95% CI 0.16-1.43 percent predicted per 10% increase] (n=490). Greater microbial diversity was associated with an increased risk of death [HR 1.73: 95% CI 1.03-2.90] while a greater proportion of Streptococcus was associated with a reduced risk of death [HR 0.85: 95% CI 0.73 to 0.99]. The Streptococcus genus was mainly comprised of Streptococcus mitis species. CONCLUSIONS: Increasing buccal microbial diversity predicts disease severity and death in IPF. The oral commensal Streptococcus mitis spp associates with preserved lung function and improved survival.
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BACKGROUND: Critically ill patients routinely receive antibiotics with activity against anaerobic gut bacteria. However, in other disease states and animal models, gut anaerobes are protective against pneumonia, organ failure and mortality. We therefore designed a translational series of analyses and experiments to determine the effects of anti-anaerobic antibiotics on the risk of adverse clinical outcomes among critically ill patients. METHODS: We conducted a retrospective single-centre cohort study of 3032 critically ill patients, comparing patients who did and did not receive early anti-anaerobic antibiotics. We compared intensive care unit outcomes (ventilator-associated pneumonia (VAP)-free survival, infection-free survival and overall survival) in all patients and changes in gut microbiota in a subcohort of 116 patients. In murine models, we studied the effects of anaerobe depletion in infectious (Klebsiella pneumoniae and Staphylococcus aureus pneumonia) and noninfectious (hyperoxia) injury models. RESULTS: Early administration of anti-anaerobic antibiotics was associated with decreased VAP-free survival (hazard ratio (HR) 1.24, 95% CI 1.06-1.45), infection-free survival (HR 1.22, 95% CI 1.09-1.38) and overall survival (HR 1.14, 95% CI 1.02-1.28). Patients who received anti-anaerobic antibiotics had decreased initial gut bacterial density (p=0.00038), increased microbiome expansion during hospitalisation (p=0.011) and domination by Enterobacteriaceae spp. (p=0.045). Enterobacteriaceae were also enriched among respiratory pathogens in anti-anaerobic-treated patients (p<2.2×10-16). In murine models, treatment with anti-anaerobic antibiotics increased susceptibility to Enterobacteriaceae pneumonia (p<0.05) and increased the lethality of hyperoxia (p=0.0002). CONCLUSIONS: In critically ill patients, early treatment with anti-anaerobic antibiotics is associated with increased mortality. Mechanisms may include enrichment of the gut with respiratory pathogens, but increased mortality is incompletely explained by infections alone. Given consistent clinical and experimental evidence of harm, the widespread use of anti-anaerobic antibiotics should be reconsidered.
Assuntos
Hiperóxia , Pneumonia Associada à Ventilação Mecânica , Animais , Camundongos , Antibacterianos/efeitos adversos , Estudos de Coortes , Estudos Retrospectivos , Estado Terminal , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , Unidades de Terapia IntensivaRESUMO
Recent studies have implicated lung microbiota in shaping local alveolar immune responses. Toll-like receptors are major sensors of microbiota and determinants of local epithelial homeostasis. The impact of toll-like receptor deficiency on lung microbiota is unknown. To determine whether the absence of toll-like receptors results in altered lung microbiota or dysbiosis, we compared lung microbiota in wild-type and toll-like receptor-deficient experimental mice using 16S ribosomal RNA gene quantification and sequencing. We used a randomized environmental caging strategy to determine the impact of toll-like receptors on lung microbiota. Lung microbiota are detectable in toll-like receptor-deficient experimental mice and exhibit considerable variability. The lung microbiota of toll-like receptor-deficient mice are altered in community composition (PERMANOVA P < 0.001), display reduced diversity (t test P = 0.0075), and bacterial burden (t test P = 0.016) compared with wild-type mice with intact toll-like receptors and associated signaling pathways. The lung microbiota of wild-type mice when randomized to cages with toll-like receptor-deficient mice converged with no significant difference in community composition (PERMANOVA P > 0.05) after 3 wk of cohousing. The lung microbiome of toll-like receptor-deficient mice is distinct from wild-type mice and may be less susceptible to the effects of caging as an environmental variable. Our observations support a role for toll-like receptor signaling in the shaping of lung microbiota.
Assuntos
Bactérias , Disbiose/microbiologia , Pulmão/microbiologia , Microbiota , Receptores Toll-Like/deficiência , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Disbiose/genética , Disbiose/patologia , Pulmão/patologia , Camundongos , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Receptores Toll-Like/metabolismoRESUMO
Rationale: Recent studies have revealed that, in critically ill patients, lung microbiota are altered and correlate with alveolar inflammation. The clinical significance of altered lung bacteria in critical illness is unknown.Objectives: To determine if clinical outcomes of critically ill patients are predicted by features of the lung microbiome at the time of admission.Methods: We performed a prospective, observational cohort study in an ICU at a university hospital. Lung microbiota were quantified and characterized using droplet digital PCR and bacterial 16S ribosomal RNA gene quantification and sequencing. Primary predictors were the bacterial burden, community diversity, and community composition of lung microbiota. The primary outcome was ventilator-free days, determined at 28 days after admission.Measurements and Main Results: Lungs of 91 critically ill patients were sampled using miniature BAL within 24 hours of ICU admission. Patients with increased lung bacterial burden had fewer ventilator-free days (hazard ratio, 0.43; 95% confidence interval, 0.21-0.88), which remained significant when the analysis was controlled for pneumonia and severity of illness. The community composition of lung bacteria predicted ventilator-free days (P = 0.003), driven by the presence of gut-associated bacteria (e.g., species of the Lachnospiraceae and Enterobacteriaceae families). Detection of gut-associated bacteria was also associated with the presence of acute respiratory distress syndrome.Conclusions: Key features of the lung microbiome (bacterial burden and enrichment with gut-associated bacteria) predict outcomes in critically ill patients. The lung microbiome is an understudied source of clinical variation in critical illness and represents a novel therapeutic target for the prevention and treatment of acute respiratory failure.
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Pulmão/microbiologia , Microbiota/genética , Respiração Artificial/estatística & dados numéricos , Síndrome do Desconforto Respiratório/terapia , Adulto , Idoso , Carga Bacteriana , Líquido da Lavagem Broncoalveolar/microbiologia , Clostridiales , Estudos de Coortes , Estado Terminal , Enterobacteriaceae , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Pessoa de Meia-Idade , Pasteurellaceae , Análise de Componente Principal , Prognóstico , Modelos de Riscos Proporcionais , RNA Ribossômico 16S/genética , Síndrome do Desconforto Respiratório/microbiologiaRESUMO
Rationale: Idiopathic pulmonary fibrosis (IPF) causes considerable global morbidity and mortality, and its mechanisms of disease progression are poorly understood. Recent observational studies have reported associations between lung dysbiosis, mortality, and altered host defense gene expression, supporting a role for lung microbiota in IPF. However, the causal significance of altered lung microbiota in disease progression is undetermined. Objectives: To examine the effect of microbiota on local alveolar inflammation and disease progression using both animal models and human subjects with IPF. Methods: For human studies, we characterized lung microbiota in BAL fluid from 68 patients with IPF. For animal modeling, we used a murine model of pulmonary fibrosis in conventional and germ-free mice. Lung bacteria were characterized using 16S rRNA gene sequencing with novel techniques optimized for low-biomass sample load. Microbiota were correlated with alveolar inflammation, measures of pulmonary fibrosis, and disease progression. Measurements and Main Results: Disruption of the lung microbiome predicts disease progression, correlates with local host inflammation, and participates in disease progression. In patients with IPF, lung bacterial burden predicts fibrosis progression, and microbiota diversity and composition correlate with increased alveolar profibrotic cytokines. In murine models of fibrosis, lung dysbiosis precedes peak lung injury and is persistent. In germ-free animals, the absence of a microbiome protects against mortality. Conclusions: Our results demonstrate that lung microbiota contribute to the progression of IPF. We provide biological plausibility for the hypothesis that lung dysbiosis promotes alveolar inflammation and aberrant repair. Manipulation of lung microbiota may represent a novel target for the treatment of IPF.
Assuntos
Fibrose Pulmonar Idiopática/microbiologia , Inflamação/microbiologia , Pulmão/microbiologia , Microbiota/fisiologia , Idoso , Animais , Líquido da Lavagem Broncoalveolar/microbiologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Citometria de Fluxo , Vida Livre de Germes , Humanos , Fibrose Pulmonar Idiopática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/genética , Pessoa de Meia-Idade , Alvéolos Pulmonares/microbiologia , Alvéolos Pulmonares/patologia , RNA Ribossômico 16S/genéticaRESUMO
RATIONALE: The "gut-lung axis" is commonly invoked to explain the microbiome's influence on lung inflammation. Yet the lungs harbor their own microbiome, which is altered in respiratory disease. The relative influence of gut and lung bacteria on lung inflammation is unknown. OBJECTIVES: To determine whether baseline lung immune tone reflects local (lung-lung) or remote (gut-lung) microbe-host interactions. METHODS: We compared lung, tongue, and cecal bacteria in 40 healthy, genetically identical, 10-week-old mice, using 16S ribosomal RNA gene quantification and sequencing. We measured inflammatory cytokines, using a multiplex assay of homogenized lung tissue. We compared lung bacteria in healthy mice treated with varied durations of systemic antibiotics. MEASUREMENTS AND MAIN RESULTS: Lung bacterial communities are highly variable among mice, cluster strongly by cage, shipment, and vendor, and are altered by antibiotics in a microbiologically predictable manner. Baseline lung concentrations of two key inflammatory cytokines (IL-1α and IL-4) are correlated with the diversity and community composition of lung bacterial communities. Lung concentrations of these inflammatory cytokines correlate more strongly with variation in lung bacterial communities than with that of the gut or mouth. CONCLUSIONS: In the lungs of healthy mice, baseline innate immune tone more strongly reflects local (lung-lung) microbe-host interactions than remote (gut-lung) microbe-host interactions. Our results independently confirm the existence and immunologic significance of the murine lung microbiome, even in health. Variation in lung microbiota is likely an important, underappreciated source of experimental and clinical variability. The lung microbiome is an unexplored therapeutic target for the prevention and treatment of inflammatory lung disease.
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Imunidade Inata/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Microbiota/fisiologia , Animais , Meio Ambiente , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Modelos AnimaisRESUMO
RATIONALE: Sepsis causes brain dysfunction and neuroinflammation. It is unknown whether neuroinflammation in sepsis is initiated by dissemination of bacteria to the brain and sustained by persistent infection, or whether neuroinflammation is a sterile process resulting solely from circulating inflammatory mediators. OBJECTIVES: To determine if gut bacteria translocate to the brain during sepsis, and are associated with neuroinflammation. METHODS: Murine sepsis was induced using cecal ligation and puncture, and sepsis survivor mice were compared with sham and unoperated control animals. Brain tissue of patients who died of sepsis was compared with patients who died of noninfectious causes. Bacterial taxa were characterized by 16S ribosomal RNA gene sequencing in both murine and human brain specimens; compared among sepsis and nonsepsis groups; and correlated with levels of S100A8, a marker of neuroinflammation using permutational multivariate ANOVA. MEASUREMENTS AND MAIN RESULTS: Viable gut-associated bacteria were enriched in the brains of mice 5 days after surviving abdominal sepsis (P < 0.01), and undetectable by 14 days. The community structure of brain-associated bacteria correlated with severity of neuroinflammation (P < 0.001). Furthermore, bacterial taxa detected in brains of humans who die of sepsis were distinct from those who died of noninfectious causes (P < 0.001) and correlated with S100A8/A9 expression (P < 0.05). CONCLUSIONS: Although bacterial translocation is associated with acute neuroinflammation in murine sepsis, bacterial translocation did not result in chronic cerebral infection. Postmortem analysis of patients who die of sepsis suggests a role for bacteria in acute brain dysfunction in sepsis. Further work is needed to determine if modifying gut-associated bacterial communities modulates brain dysfunction after sepsis.
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Translocação Bacteriana/fisiologia , Encéfalo/microbiologia , Encefalite/etiologia , Microbioma Gastrointestinal/fisiologia , Sepse/complicações , Animais , Cadáver , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Índice de Gravidade de DoençaRESUMO
RATIONALE: Hematopoietic cell transplant (HCT) is a common treatment for hematological neoplasms and autoimmune disorders. Among HCT recipients, pulmonary complications are common, morbid, and/or lethal, and they have recently been associated with gut dysbiosis. The role of lung microbiota in post-HCT pulmonary complications is unknown. OBJECTIVES: To investigate the role of lung microbiota in post-HCT pulmonary complications using animal modeling and human BAL fluid. METHODS: For animal modeling, we used an established murine model of HCT with and without postengraftment herpes virus infection. For human studies, we characterized lung microbiota in BAL fluid from 43 HCT recipients. Lung bacteria were characterized using 16S ribosomal RNA gene sequencing and were compared with lung histology (murine) and with alveolar inflammation and pulmonary function testing (human). MEASUREMENTS AND MAIN RESULTS: Both HCT and viral infection independently altered the composition of murine lung microbiota, but they had no effect on lung microbial diversity. By contrast, combined HCT and viral infection profoundly altered lung microbiota, decreasing community diversity with an associated pneumonitis. Among human HCT recipients, increased relative abundance of the Proteobacteria phylum was associated with impaired pulmonary function, and lung microbiota were significantly associated with alveolar concentrations of inflammatory cytokines. CONCLUSIONS: In animal models and human subjects, lung dysbiosis is a prominent feature of HCT. Lung dysbiosis is correlated with histologic, immunologic, and physiologic features of post-HCT pulmonary complications. Our findings suggest the lung microbiome may be an unappreciated target for the prevention and treatment of post-HCT pulmonary complications.
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Disbiose/epidemiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Inflamação/epidemiologia , Pneumopatias/epidemiologia , Complicações Pós-Operatórias/epidemiologia , Animais , Comorbidade , Modelos Animais de Doenças , Feminino , Microbioma Gastrointestinal , Humanos , Inflamação/microbiologia , Pulmão/microbiologia , Pneumopatias/microbiologia , Masculino , Camundongos , Pessoa de Meia-Idade , Complicações Pós-Operatórias/microbiologiaAssuntos
Microbiota , Humanos , Imunidade Inata/imunologia , Pulmão/imunologia , Microbiota/imunologiaRESUMO
The inflammatory response to the colonic pathogen Clostridium difficile is characterized by the induction of inflammatory cytokines including Interleukin-23 (IL-23) and interferon-γ (IFN-γ) and the recruitment of myeloid cells including Ly6CHigh monocytes. IL-23 knockout mice showed reduced expression of the monocyte chemokines Ccl4 and Ccl7, but not Ccl2, as well as reduced Ly6CHigh Ly6GMid monocyte recruitment to the colon in response to C. difficile colitis. Clostridium difficile-infected CCR2-/- (CCR2 KO) mice showed a significant defect in Ly6CHigh Ly6GMid monocyte recruitment to the colon in response to C. difficile. Although there was no decrease in expression of the inflammatory cytokines Il1b, Il6 or Tnf or reduction in the severity of colonic histopathology associated with ablation of monocyte recruitment, Slpi and Inos expression was significantly reduced in the colons of these animals. Additionally, neutralization of IFN-γ through the administration of anti-IFN-γ monoclonal antibody resulted in a significant reduction in the expression of the IFN-γ-inducible chemokines Cxcl9 and Cxcl10, but not a reduction in the neutrophil chemokines Cxcl1, Cxcl2 and Ccl3 or the monocyte chemokine Ccl2. Consistently, monocyte and neutrophil recruitment were unchanged following anti-IFN-γ treatment. Additionally, Inos and Slpi expression were unchanged following anti-IFN-γ treatment, suggesting that Inos and Slpi regulation is independent of IFN-γ during C. difficile colitis. Taken together, these data strongly suggest that IL-23 and CCR2 signalling are required for monocyte recruitment during C. difficile colitis. Additionally, these studies also suggest that monocytes, but not IFN-γ, are necessary for full expression of Inos and Slpi in the colon.
Assuntos
Clostridioides difficile/imunologia , Colo/imunologia , Enterocolite Pseudomembranosa/imunologia , Interferon gama/metabolismo , Interleucina-23/metabolismo , Mucosa Intestinal/imunologia , Monócitos/fisiologia , Receptores CCR2/metabolismo , Doença Aguda , Animais , Anticorpos Bloqueadores/administração & dosagem , Antígenos Ly/metabolismo , Movimento Celular/genética , Células Cultivadas , Quimiocinas/metabolismo , Feminino , Mediadores da Inflamação/metabolismo , Interferon gama/imunologia , Interleucina-23/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/genética , Receptores CCR2/genéticaRESUMO
Our objective was to determine the role of the inflammatory cytokine interleukin-23 (IL-23) in promoting neutrophil recruitment, inflammatory cytokine expression and intestinal histopathology in response to Clostridium difficile infection. Wild-type (WT) and p19(-/-) (IL-23KO) mice were pre-treated with cefoperazone in their drinking water for 5 days, and after a 2-day recovery period were challenged with spores from C. difficile strain VPI 10463. Interleukin-23 deficiency was associated with significant defects in both the recruitment of CD11b(High) Ly6G(H) (igh) neutrophils to the colon and the expression of neutrophil chemoattractants and stabilization factors including Cxcl1, Cxcl2, Ccl3 and Csf3 within the colonic mucosa as compared with WT animals. Furthermore, the expression of inflammatory cytokines including Il33, Tnf and Il6 was significantly reduced in IL-23-deficient animals. There was also a trend towards less severe colonic histopathology in the absence of IL-23. The induction of Il17a and Il22 was also significantly abrogated in IL-23KO mice. Inflammatory cytokine expression and neutrophilic inflammation were not reduced in IL-17a-deficient mice or in mice treated with anti-IL-22 depleting monoclonal antibody. However, induction of RegIIIg was significantly reduced in animals treated with anti-IL-22 antibody. Taken together, these data indicate that IL-23, but not IL-17a or IL-22, promotes neutrophil recruitment and inflammatory cytokine and chemokine expression in the colon in response to C. difficile infection.
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Clostridioides difficile/imunologia , Colo/imunologia , Enterocolite Pseudomembranosa/imunologia , Imunidade Inata , Mediadores da Inflamação/imunologia , Interleucina-17/imunologia , Subunidade p19 da Interleucina-23/imunologia , Interleucinas/imunologia , Mucosa Intestinal/imunologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Clostridioides difficile/patogenicidade , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/patologia , Feminino , Imunidade Inata/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interleucina-17/deficiência , Interleucina-17/genética , Subunidade p19 da Interleucina-23/deficiência , Subunidade p19 da Interleucina-23/genética , Interleucinas/antagonistas & inibidores , Interleucinas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Proteínas Associadas a Pancreatite , Proteínas/imunologia , Proteínas/metabolismo , Transdução de Sinais , Interleucina 22RESUMO
The host response to Clostridium difficile infection in antibiotic-treated mice is characterized by robust recruitment of Gr-1(+) cells, increased expression of inflammatory cytokines including tumour necrosis factor-α (TNF-α), and the development of severe epithelial damage. To investigate the role of Gr-1(+) cells and TNF-α during C. difficile colitis, we treated infected mice with monoclonal antibodies against Gr-1 or TNF-α. Mice were challenged with vegetative cells of C. difficile strain VPI 10463 following treatment with the third-generation cephalosporin ceftriaxone. Ceftriaxone treatment alone was associated with significant changes in cytokine expression within the colonic mucosa but not overt inflammatory histopathological changes. In comparison, C. difficile infection following ceftriaxone treatment was associated with increased expression of inflammatory cytokines and chemokines including Cxcl1, Cxcl2, Il1b, Il17f and Tnfa, as well as robust recruitment of Ly6C(Mid) Gr-1(High) neutrophils and Ly6C(High) Gr-1(Mid) monocytes and the development of severe colonic histopathology. Anti-Gr-1 antibody treatment resulted in effective depletion of both Ly6C(Mid) Gr-1(High) neutrophils and Ly6C(High) Gr-1(Mid) monocytes: however, we observed no protection from the development of severe pathology or reduction in expression of the pro-inflammatory cytokines Il1b, Il6, Il33 and Tnfa following anti-Gr-1 treatment. By contrast, anti-TNF-α treatment did not affect Gr-1(+) cell recruitment, but was associated with increased expression of Il6 and Il1b. Additionally, Ffar2, Ffar3, Tslp, Tff and Ang4 expression was significantly reduced in anti-TNF-α-treated animals, in association with marked intestinal histopathology. These studies raise the possibility that TNF-α may play a role in restraining inflammation and protecting the epithelium during C. difficile infection.
Assuntos
Clostridioides difficile/patogenicidade , Colo/metabolismo , Enterocolite Pseudomembranosa/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Ceftriaxona , Clostridioides difficile/imunologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/genética , Enterocolite Pseudomembranosa/imunologia , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/patologia , Enterocolite Pseudomembranosa/prevenção & controle , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Microbiota , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/microbiologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/imunologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Our previous work has shown the significant up-regulation of Il22 and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the mucosal inflammatory response to Clostridium difficile infection in mice. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes interleukin-22 (IL-22) and CD160-mediated components. The current study sought to determine the potential role(s) of IL-22 and/or CD160 in the mucosal response to C. difficile infection. Clostridium difficile-infected mice treated with anti-IL-22, anti-CD160 or a combination of the two showed significantly reduced STAT3 phosphorylation in comparison to C. difficile-infected mice that had not received either antibody. In addition, C. difficile-infected mice treated with anti-IL-22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile-infected mice. The affected genes included pro-inflammatory chemokines and cytokines, and anti-microbial peptides. Furthermore, histopathological and flow cytometric assessments both showed a significantly reduced influx of neutrophils in C. difficile-infected mice treated with anti-IL-22/CD160. These data demonstrate that IL-22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL-22 and CD160 in mediating both the pro-inflammatory and pro-survival aspects of the host mucosal response in this infection.
Assuntos
Antígenos CD/imunologia , Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/imunologia , Imunidade nas Mucosas , Interleucinas/imunologia , Mucosa Intestinal/imunologia , Receptores Imunológicos/imunologia , Animais , Antibacterianos , Anticorpos/farmacologia , Antígenos CD/genética , Antígenos CD/metabolismo , Clostridioides difficile/imunologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/genética , Enterocolite Pseudomembranosa/metabolismo , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/prevenção & controle , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica , Imunidade nas Mucosas/efeitos dos fármacos , Interleucinas/antagonistas & inibidores , Interleucinas/genética , Interleucinas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Fosforilação , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Tempo , Interleucina 22RESUMO
Enteral nutrient deprivation via total parenteral nutrition (TPN) administration leads to local mucosal inflammatory responses, but the underlying mechanisms are unknown. Wild-type (WT) and MyD88(-/-) mice underwent jugular vein cannulation. One group received TPN without chow, and controls received standard chow. After 7 d, we harvested intestinal mucosally associated bacteria and isolated small-bowel lamina propria (LP) cells. Bacterial populations were analyzed using 454 pyrosequencing. LP cells were analyzed using quantitative PCR and multicolor flow cytometry. WT, control mucosally associated microbiota were Firmicutes-dominant, whereas WT TPN mice were Proteobacteria-domiant. Similar changes were observed in MyD88(-/-) mice with TPN administration. UniFrac analysis showed divergent small bowel and colonic bacterial communities in controls, merging toward similar microbiota (but distinct from controls) with TPN. The percentage of LP T regulatory cells significantly decreased with TPN in WT mice. F4/80(+)CD11b(+)CD11c(dull/-) macrophage-derived proinflammatory cytokines significantly increased with TPN. These proinflammatory immunologic changes were significantly abrogated in MyD88(-/-) TPN mice. Thus, TPN administration is associated with significant expansion of Proteobacteria within the intestinal microbiota and increased proinflammatory LP cytokines. Additionally, MyD88 signaling blockade abrogated decline in epithelial cell proliferation and epithelial barrier function loss.
Assuntos
Inflamação/patologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Fator 88 de Diferenciação Mieloide/imunologia , Nutrição Parenteral Total/efeitos adversos , Animais , Citometria de Fluxo , Inflamação/etiologia , Inflamação/microbiologia , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Mucosa/microbiologia , Mucosa/patologia , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase em Tempo RealRESUMO
ABSTRACT: Sepsis is a common, heterogeneous, and frequently lethal condition of organ dysfunction and immune dysregulation due to infection. The causes of its heterogeneity, including the contribution of the pathogen, remain unknown. Using cecal slurry, a widely used murine model of intraperitoneal polymicrobial sepsis, as well as 16S ribosomal RNA sequencing and measurement of immune markers, we performed a series of translational analyses to determine whether microbial variation in cecal slurry composition (representing intra-abdominal pathogens) mediated variation in septic response. We found wide variation in cecal slurry community composition that changed markedly over the 24-h course of infection. This variation in cecal slurry bacteria led to large variation in physiologic and inflammatory responses. Severity of inflammatory response was positively correlated with intraperitoneal enrichment with Enterobacteriaceae. Likewise, in a human cohort of patients with intra-abdominal abscesses, Enterobacteriaceae was also associated with increased inflammatory markers. Taken together, these data demonstrate that intra-abdominal Enterobacteriaceae drives inflammation in sepsis both in animal models and human subjects. More broadly, our results demonstrate that pathogen identity is a major driver of the host response in polymicrobial sepsis and should not be overlooked as a major source of phenotypic heterogeneity.
Assuntos
Enterobacteriaceae , Sepse , Animais , Sepse/microbiologia , Sepse/imunologia , Camundongos , Humanos , Masculino , Feminino , Inflamação/microbiologia , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Camundongos Endogâmicos C57BL , Ceco/microbiologia , Pessoa de Meia-IdadeRESUMO
The current study sought to delineate the gene expression profile of the host response in the caecum and colon during acute infection with Clostridium difficile in a mouse model of infection, and to investigate the nature of the unfolded protein response in this process. The infected mice displayed a significant up-regulation in the expression of chemokines (Cxcl1, Cxcl2 and Ccl2), numerous pro-inflammatory cytokines (Ifng, Il1b, Il6, and Il17f), as well as Il22 and a number of anti-microbial peptides (Defa1, Defa28, Defb1, Slpi and Reg3g) at the site(s) of infection. This was accompanied by a significant influx of neutrophils, dendritic cells, cells of the monocyte/macrophage lineage and all major subsets of lymphocytes to these site(s). However, CD4 T cells of the untreated and C. difficile-infected mice expressed similar levels of CD69 and CD25. Neither tissue had up-regulated levels of Tbx21, Gata3 or Rorc. The caeca and colons of the infected mice showed a significant increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, but neither the splicing of Xbp1 nor the up-regulation of endoplasmic reticulum chaperones, casting doubt on the full-fledged induction of the unfolded protein response by C. difficile. They also displayed significantly higher phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), an indication of pro-survival signalling. These data underscore the local, innate, pro-inflammatory nature of the response to C. difficile and highlight eIF2α phosphorylation and the interleukin-22-pSTAT3-RegIIIγ axis as two of the pathways that could be used to contain and counteract the damage inflicted on the intestinal epithelium.