RESUMO
Microbiota are thought to influence the development and progression of inflammatory bowel disease (IBD), but determining generalizable effects of microbiota on IBD etiology requires larger-scale functional analyses. We colonized germ-free mice with intestinal microbiotas from 30 healthy and IBD donors and determined the homeostatic intestinal T cell response to each microbiota. Compared to microbiotas from healthy donors, transfer of IBD microbiotas into germ-free mice increased numbers of intestinal Th17 cells and Th2 cells and decreased numbers of RORγt+ Treg cells. Colonization with IBD microbiotas exacerbated disease in a model where colitis is induced upon transfer of naive T cells into Rag1-/- mice. The proportions of Th17 and RORγt+ Treg cells induced by each microbiota were predictive of human disease status and accounted for disease severity in the Rag1-/- colitis model. Thus, an impact on intestinal Th17 and RORγt+ Treg cell compartments emerges as a unifying feature of IBD microbiotas, suggesting a general mechanism for microbial contribution to IBD pathogenesis.
Assuntos
Colite/microbiologia , Microbioma Gastrointestinal/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , RNA Ribossômico 16S/genética , Linfócitos T Reguladores/imunologia , Células Th17/metabolismo , Animais , Diferenciação Celular , Colite/induzido quimicamente , Colite/imunologia , Modelos Animais de Doenças , Progressão da Doença , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismoRESUMO
Functional characterization of the microbiome's influence on host physiology has been dominated by a few characteristic example strains that have been studied in detail. However, the extensive development of methods for high-throughput bacterial isolation and culture over the past decade is enabling functional characterization of the broader microbiota that may impact human health. Characterizing the understudied majority of human microbes and expanding our functional understanding of the diversity of the gut microbiota could enable new insights into diseases with unknown etiology, provide disease-predictive microbiome signatures, and advance microbial therapeutics. We summarize high-throughput culture-dependent platforms for characterizing bacterial strain function and host-interactions. We elaborate on the importance of these technologies in facilitating mechanistic studies of previously unexplored microbes, highlight new opportunities for large-scale in vitro screens of host-relevant microbial functions, and discuss the potential translational applications for microbiome science.
Assuntos
Doença , Saúde , Imunidade , Microbiota , Estado Nutricional , Microbiota/genética , Humanos , Animais , Inflamação/microbiologia , Carcinogênese , MetabolismoRESUMO
Despite identification of numerous associations between microbiomes and diseases, the complexity of the human microbiome has hindered identification of individual species and strains that are causative in host phenotype or disease. Uncovering causative microbes is vital to fully understand disease processes and to harness the potential therapeutic benefits of microbiota manipulation. Developments in sequencing technology, animal models, and bacterial culturing have facilitated the discovery of specific microbes that impact the host and are beginning to advance the characterization of host-microbiome interaction mechanisms. We summarize the historical and contemporary experimental approaches taken to uncover microbes from the microbiota that affect host biology and describe examples of commensals that have specific effects on the immune system, inflammation, and metabolism. There is still much to learn, and we lay out challenges faced by the field and suggest potential remedies for common pitfalls encountered in the hunt for causative commensal microbes.
Assuntos
Microbiota , Animais , Interações entre Hospedeiro e Microrganismos , SimbioseRESUMO
How bacterial strains within a complex human microbiota collectively shape intestinal T cell homeostasis is not well understood. Methods that quickly identify effector strains or species that drive specific mucosal T cell phenotypes are needed to define general principles for how the microbiota modulates host immunity. We colonize germ-free mice with defined communities of cultured strains and profile antigen-specific responses directed toward individual strains ex vivo. We find that lamina propria T cells are specific to bacterial strains at the species level and can discriminate between strains of the same species. Ex vivo restimulations consistently identify the strains within complex communities that induce Th17 responses in vivo, providing the potential to shape baseline immune tone via community composition. Using an adoptive transfer model of colitis, we find that lamina propria T cells respond to different bacterial strains in conditions of inflammation versus homeostasis. Collectively, our approach represents a unique method for efficiently predicting the relative impact of individual bacterial strains within a complex community and for parsing microbiota-dependent phenotypes into component fractions.
Assuntos
Intestinos , Microbiota , Humanos , Animais , Camundongos , Intestinos/microbiologia , Mucosa , Bactérias , Linfócitos T CD4-Positivos , Fenótipo , Mucosa IntestinalRESUMO
The gut microbiota codevelops with the immune system beginning at birth. Mining the microbiota for bacterial strains responsible for shaping the structure and dynamic operations of the innate and adaptive arms of the immune system represents a formidable combinatorial problem but one that needs to be overcome to advance mechanistic understanding of microbial community and immune system coregulation and to develop new diagnostic and therapeutic approaches that promote health. Here, we discuss a scalable, less biased approach for identifying effector strains in complex microbial communities that impact immune function. The approach begins by identifying uncultured human fecal microbiota samples that transmit immune phenotypes to germ-free mice. Clonally arrayed sequenced collections of bacterial strains are constructed from representative donor microbiota. If the collection transmits phenotypes, effector strains are identified by testing randomly generated subsets with overlapping membership in individually housed germ-free animals. Detailed mechanistic studies of effector strain-host interactions can then be performed.
Assuntos
Trato Gastrointestinal/microbiologia , Sistema Imunitário/imunologia , Consórcios Microbianos/imunologia , Microbiota/imunologia , Imunidade Adaptativa , Animais , Fezes/microbiologia , Vida Livre de Germes/imunologia , Humanos , Imunidade Inata , Camundongos , Linfócitos T Reguladores/imunologiaRESUMO
The building evidence for the contribution of microbiota to human disease has spurred an effort to develop therapies that target the gut microbiota. This is particularly evident in inflammatory bowel diseases (IBDs), where clinical trials of fecal microbiota transplantation have shown some efficacy. To aid the development of novel microbiota-targeted therapies and to better understand the biology underpinning such treatments, we have used gnotobiotic mice to model microbiota manipulations in the context of microbiotas from humans with inflammatory bowel disease. Mice colonized with IBD donor-derived microbiotas exhibit a stereotypical set of phenotypes, characterized by abundant mucosal Th17 cells, a deficit in the tolerogenic RORγt+ regulatory T (Treg) cell subset, and susceptibility to disease in colitis models. Transplanting healthy donor-derived microbiotas into mice colonized with human IBD microbiotas led to induction of RORγt+ Treg cells, which was associated with an increase in the density of the microbiotas following transplant. Microbiota transplant reduced gut Th17 cells in mice colonized with a microbiota from a donor with Crohn's disease. By culturing strains from this microbiota and screening them in vivo, we identified a specific strain that potently induces Th17 cells. Microbiota transplants reduced the relative abundance of this strain in the gut microbiota, which was correlated with a reduction in Th17 cells and protection from colitis.
Assuntos
Transplante de Microbiota Fecal , Doenças Inflamatórias Intestinais/microbiologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Colite/prevenção & controle , Colo/microbiologia , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Citocinas/imunologia , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/microbiologia , Células Th17/microbiologiaRESUMO
Importance: The effect of rationally defined nonpathogenic, nontoxigenic, commensal strains of Clostridia on prevention of Clostridioides difficile infection (CDI) is unknown. Objective: To determine the efficacy of VE303, a defined bacterial consortium of 8 strains of commensal Clostridia, in adults at high risk for CDI recurrence. The primary objective was to determine the recommended VE303 dosing for a phase 3 trial. Design, Setting, and Participants: Phase 2, randomized, double-blind, placebo-controlled, dose-ranging study conducted from February 2019 to September 2021 at 27 sites in the US and Canada. The study included 79 participants aged 18 years or older who were diagnosed with laboratory-confirmed CDI with 1 or more prior CDI episodes in the last 6 months and those with primary CDI at high risk for recurrence (defined as aged ≥75 years or ≥65 years with ≥1 risk factors: creatinine clearance <60 mL/min/1.73 m2, proton pump inhibitor use, remote [>6 months earlier] CDI history). Interventions: Participants were randomly assigned to high-dose VE303 (8.0 × 109 colony-forming units [CFUs]) (n = 30), low-dose VE303 (1.6 × 109 CFUs) (n = 27), or placebo capsules (n = 22) orally once daily for 14 days. Main Outcomes and Measures: The primary efficacy end point was the proportion of participants with CDI recurrence at 8 weeks using a combined clinical and laboratory definition. The primary efficacy end point was analyzed in 3 prespecified analyses, using successively broader definitions for an on-study CDI recurrence: (1) diarrhea consistent with CDI plus a toxin-positive stool sample; (2) diarrhea consistent with CDI plus a toxin-positive, polymerase chain reaction-positive, or toxigenic culture-positive stool sample; and (3) diarrhea consistent with CDI plus laboratory confirmation or (in the absence of a stool sample) treatment with a CDI-targeted antibiotic. Results: Baseline characteristics were similar across the high-dose VE303 (n = 29; 1 additional participant excluded from efficacy analysis), low-dose VE303 (n = 27), and placebo (n = 22) groups. The participants' median age was 63.5 years (range, 24-96); 70.5% were female; and 1.3% were Asian, 1.3% Black, 2.6% Hispanic, and 96.2% White. CDI recurrence rates through week 8 (using the efficacy analysis 3 definition) were 13.8% (4/29) for high-dose VE303, 37.0% (10/27) for low-dose VE303, and 45.5% (10/22) for placebo (P = .006, high-dose VE303 vs placebo). Conclusions and Relevance: Among adults with laboratory-confirmed CDI with 1 or more prior CDI episodes in the last 6 months and those with primary CDI at high risk for recurrence, high-dose VE303 prevented recurrent CDI compared with placebo. A larger, phase 3 study is needed to confirm these findings. Trial Registration: ClinicalTrials.gov Identifier: NCT03788434.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Probióticos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Infecções por Clostridium/complicações , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Infecções por Clostridium/terapia , Diarreia/etiologia , Diarreia/microbiologia , Diarreia/prevenção & controle , Diarreia/terapia , Fezes/química , Fezes/microbiologia , Microbioma Gastrointestinal , Probióticos/administração & dosagem , Probióticos/uso terapêutico , Recidiva , Reinfecção/prevenção & controle , Simbiose , Resultado do Tratamento , Método Duplo-Cego , Toxinas Bacterianas/análise , Adulto Jovem , Idoso , Idoso de 80 Anos ou maisRESUMO
BACKGROUND & AIMS: Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance. METHODS: Human intestinal biopsy tissues were obtained from patients with COVID-19 (n = 19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (N = 634) and Europe (N = 287) using multivariate logistic regressions. RESULTS: COVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 15 of 17 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms. CONCLUSIONS: These data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2-associated inflammation needs to be further examined.
Assuntos
COVID-19/virologia , Gastroenteropatias/virologia , Imunidade nas Mucosas , Mucosa Intestinal/virologia , SARS-CoV-2/patogenicidade , Idoso , Idoso de 80 Anos ou mais , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/mortalidade , Estudos de Casos e Controles , Células Cultivadas , Citocinas/sangue , Feminino , Gastroenteropatias/diagnóstico , Gastroenteropatias/imunologia , Gastroenteropatias/mortalidade , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/sangue , Mucosa Intestinal/imunologia , Itália , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque , Prognóstico , Medição de Risco , Fatores de Risco , SARS-CoV-2/imunologia , Carga ViralRESUMO
Medullary thymic epithelial cells (mTECs) induce T cell tolerance in the thymus through the elimination of self-reactive thymocytes. Commensal bacteria are also critical for shaping T cell responses in the gut and distal organs. We previously showed that mice depleted of mTECs (Traf6ΔTEC) generated autoreactive T cells and developed autoimmune hepatitis (AIH). In this report, we found that Toll-like receptor (TLR)-mediated microbial sensing on liver hematopoietic cells and the gut microbiota contributed to AIH development in Traf6ΔTEC mice. While adoptive transfer of thymic Traf6ΔTEC T cells in immune-deficient mice was sufficient for AIH development, colonization of germ-free mice with Traf6ΔTEC microbiota failed to induce AIH, suggesting that the gut microbiota contributes to but is not sufficient for AIH development. Microbiota-mediated exacerbation of AIH associated with increased numbers of hepatic Foxp3+ T cells and their increase was proportional to the degree of inflammation. The contribution of the gut microbiota to AIH development associated with an altered microbial signature whose composition was influenced by the qualitative nature of the thymic T cell compartment. These results suggest that aberrant selection of T cells in the thymus can induce changes in the gut microbiota that lead to exacerbation of organ-specific autoimmunity and AIH. Our results add to our understanding of the mechanisms of AIH development and create a platform towards developing novel therapeutic approaches for treating this disease.
Assuntos
Microbioma Gastrointestinal , Hepatite Autoimune , Animais , Tolerância Central , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores , TimoAssuntos
Abandono do Hábito de Fumar , Humanos , Fumar/efeitos adversos , Fumar Tabaco , Aumento de PesoRESUMO
Blood polymorphonuclear neutrophils provide immune protection against pathogens, but may also promote tissue injury in inflammatory diseases. Although neutrophils are generally considered to be a relatively homogeneous population, evidence for heterogeneity is emerging. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and replenishment by newly released neutrophils from the bone marrow. Aged neutrophils upregulate CXCR4, a receptor allowing their clearance in the bone marrow, with feedback inhibition of neutrophil production via the IL-17/G-CSF axis, and rhythmic modulation of the haematopoietic stem-cell niche. The aged subset also expresses low levels of L-selectin. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties. Here, using in vivo ageing analyses in mice, we show that neutrophil pro-inflammatory activity correlates positively with their ageing whilst in circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMß2 integrin activation and neutrophil extracellular trap formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptor and myeloid differentiation factor 88-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle-cell disease or endotoxin-induced septic shock. These results identify a role for the microbiota in regulating a disease-promoting neutrophil subset.
Assuntos
Senescência Celular/imunologia , Microbiota/imunologia , Neutrófilos/citologia , Neutrófilos/imunologia , Anemia Falciforme/sangue , Anemia Falciforme/microbiologia , Anemia Falciforme/patologia , Animais , Modelos Animais de Doenças , Eritrócitos Anormais/patologia , Inflamação/imunologia , Inflamação/patologia , Antígeno de Macrófago 1/metabolismo , Masculino , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , Choque Séptico/imunologia , Choque Séptico/microbiologia , Choque Séptico/patologia , Transdução de Sinais , Receptores Toll-Like/imunologiaRESUMO
BACKGROUND AND AIMS: Prenatal and early life bacterial colonisation is thought to play a major role in shaping the immune system. Furthermore, accumulating evidence links early life exposures to the risk of developing IBD later in life. We aimed to assess the effect of maternal IBD on the composition of the microbiome during pregnancy and on the offspring's microbiome. METHODS: We prospectively examined the diversity and taxonomy of the microbiome of pregnant women with and without IBD and their babies at multiple time points. We evaluated the role of maternal IBD diagnosis, the mode of delivery, antibiotic use and feeding behaviour on the microbiome composition during early life. To assess the effects of IBD-associated maternal and infant microbiota on the enteric immune system, we inoculated germ-free mice (GFM) with the respective stool and profiled adaptive and innate immune cell populations in the murine intestines. RESULTS: Pregnant women with IBD and their offspring presented with lower bacterial diversity and altered bacterial composition compared with control women and their babies. Maternal IBD was the main predictor of the microbiota diversity in the infant gut at 7, 14, 30, 60 and 90 days of life. Babies born to mothers with IBD demonstrated enrichment in Gammaproteobacteria and depletion in Bifidobacteria. Finally, GFM inoculated with third trimester IBD mother and 90-day infant stools showed significantly reduced microbial diversity and fewer class-switched memory B cells and regulatory T cells in the colon. CONCLUSION: Aberrant gut microbiota composition persists during pregnancy with IBD and alters the bacterial diversity and abundance in the infant stool. The dysbiotic microbiota triggered abnormal imprinting of the intestinal immune system in GFM.
Assuntos
Microbioma Gastrointestinal/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Complicações na Gravidez/microbiologia , Efeitos Tardios da Exposição Pré-Natal/microbiologia , Imunidade Adaptativa , Adulto , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Disbiose/imunologia , Disbiose/microbiologia , Transplante de Microbiota Fecal/métodos , Fezes/microbiologia , Feminino , Seguimentos , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Vida Livre de Germes , Humanos , Recém-Nascido , Doenças Inflamatórias Intestinais/imunologia , Masculino , Troca Materno-Fetal , Gravidez , Complicações na Gravidez/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Estudos ProspectivosRESUMO
BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) can induce remission in patients with ulcerative colitis (UC). In a randomized controlled trial of FMT in patients with active UC, we aimed to identify bacterial taxonomic and functional factors associated with response to therapy. METHODS: We performed a double-blind trial of 81 patients with active UC randomly assigned to groups that received an initial colonoscopic infusion and then intensive multidonor FMT or placebo enemas, 5 d/wk for 8 weeks. Patients in the FMT group received blended homogenized stool from 3-7 unrelated donors. Patients in the placebo group were eligible to receive open-label FMT after the double-blind study period. We collected 314 fecal samples from the patients at screening, every 4 weeks during treatment, and 8 weeks after the blinded or open-label FMT therapy. We also collected 160 large-bowel biopsy samples from the patients at study entry, at completion of 8 weeks of blinded therapy, and at the end of open-label FMT, if applicable. We analyzed 105 fecal samples from the 14 individual donors (n = 55), who in turn contributed to 21 multidonor batches (n = 50). Bacteria in colonic and fecal samples were analyzed by both 16S ribosomal RNA gene and transcript amplicon sequencing; 285 fecal samples were analyzed by shotgun metagenomics, and 60 fecal samples were analyzed for metabolome features. RESULTS: FMT increased microbial diversity and altered composition, based on analyses of colon and fecal samples collected before vs after FMT. Diversity was greater in fecal and colon samples collected before and after FMT treatment from patients who achieved remission compared with patients who did not. Patients in remission after FMT had enrichment of Eubacterium hallii and Roseburia inulivorans compared with patients who did not achieve remission after FMT and had increased levels of short-chain fatty acid biosynthesis and secondary bile acids. Patients who did not achieve remission had enrichment of Fusobacterium gonidiaformans, Sutterella wadsworthensis, and Escherichia species and increased levels of heme and lipopolysaccharide biosynthesis. Bacteroides in donor stool were associated with remission in patients receiving FMT, and Streptococcus species in donor stool was associated with no response to FMT. CONCLUSIONS: In an analysis of fecal and colonic mucosa samples from patients receiving FMT for active UC and stool samples from donors, we associated specific bacteria and metabolic pathways with induction of remission. These findings may be of value in the design of microbe-based therapies for UC. ClinicalTrials.gov, Number NCT01896635.
Assuntos
Bactérias/metabolismo , Colite Ulcerativa/terapia , Microbioma Gastrointestinal , Bactérias/classificação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Biomarcadores/metabolismo , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/microbiologia , Método Duplo-Cego , Transplante de Microbiota Fecal/efeitos adversos , Fezes/microbiologia , Humanos , Metabolômica , New South Wales , Indução de Remissão , Ribotipagem , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: It is not clear how the complex interactions between diet and the intestinal microbiota affect development of mucosal inflammation or inflammatory bowel disease. We investigated interactions between dietary ingredients, nutrients, and the microbiota in specific pathogen-free (SPF) and germ-free (GF) mice given more than 40 unique diets; we quantified individual and synergistic effects of dietary macronutrients and the microbiota on intestinal health and development of colitis. METHODS: C56BL/6J SPF and GF mice were placed on custom diets containing different concentrations and sources of protein, fat, digestible carbohydrates, and indigestible carbohydrates (fiber). After 1 week, SPF and GF mice were given dextran sulfate sodium (DSS) to induce colitis. Disease severity was determined based on the percent weight change from baseline, and modeled as a function of the concentration of each macronutrient in the diet. In unchallenged mice, we measured intestinal permeability by feeding mice labeled dextran and measuring levels in blood. Feces were collected and microbiota were analyzed by 16S rDNA sequencing. We collected colons from mice and performed transcriptome analyses. RESULTS: Fecal microbiota varied with diet; the concentration of protein and fiber had the strongest effect on colitis development. Among 9 fiber sources tested, psyllium, pectin, and cellulose fiber reduced the severity of colitis in SPF mice, whereas methylcellulose increased severity. Increasing dietary protein increased the density of the fecal microbiota and the severity of colitis in SPF mice, but not in GF mice or mice given antibiotics. Psyllium fiber reduced the severity of colitis through microbiota-dependent and microbiota-independent mechanisms. Combinatorial perturbations to dietary casein protein and psyllium fiber in parallel accounted for most variation in gut microbial density and intestinal permeability in unchallenged mice, as well as the severity of DSS-induced colitis; changes in 1 ingredient could be offset by changes in another. CONCLUSIONS: In an analysis of the effects of different dietary components and the gut microbiota on mice with and without DSS-induced colitis, we found complex mixtures of nutrients affect intestinal permeability, gut microbial density, and development of intestinal inflammation.
Assuntos
Bactérias/crescimento & desenvolvimento , Colite/microbiologia , Colo/microbiologia , Dieta , Microbioma Gastrointestinal , Ração Animal , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Caseínas/administração & dosagem , Colite/metabolismo , Colite/fisiopatologia , Colite/prevenção & controle , Colo/metabolismo , Colo/fisiopatologia , Sulfato de Dextrana , Dieta/efeitos adversos , Fibras na Dieta/administração & dosagem , Proteínas Alimentares/administração & dosagem , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Proteínas de Homeodomínio/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estado Nutricional , Valor Nutritivo , Permeabilidade , Psyllium/administração & dosagem , Índice de Gravidade de Doença , Fatores de TempoRESUMO
BACKGROUND & AIMS: Several studies have shown that signaling via the interleukin 23 (IL23) receptor is required for development of colitis. We studied the roles of IL23, dietary factors, alterations to the microbiota, and T cells in the development and progression of colitis in mice. METHODS: All mice were maintained on laboratory diet 5053, unless otherwise noted. We generated mice that express IL23 in CX3CR1-positive myeloid cells (R23FR mice) upon cyclic administration of tamoxifen dissolved in diet 2019. Diets 2019 and 5053 have minor differences in the overall composition of protein, fat, fiber, minerals, and vitamins. CX3CR1CreER mice (FR mice) were used as controls. Some mice were given antibiotics, and others were raised in a germ-free environment. Intestinal tissues were collected and analyzed by histology and flow cytometry. Feces were collected and analyzed by 16S rDNA sequencing. Feces from C57/Bl6, R23FR, or FR mice were fed to FR and R23FR germ-free mice in microbiota transplant experiments. We also performed studies with R23FR/Rag-/-, R23FR/Mu-/-, and R23FR/Tcrd-/- mice. R23FR mice were given injections of antibodies against CD4 or CD8 to deplete T cells. Mesenteric lymph nodes and large intestine CD4+ cells from R23FR or FR mice in remission from colitis were transferred into Rag-/- mice. CD4+ cells were isolated from donor R23FR mice and recipient Rag-/- mice, and T-cell receptor sequences were determined. RESULTS: Expression of IL23 led to development of a relapsing-remitting colitis that was dependent on the microbiota and CD4+ T cells. The relapses were caused by switching from the conventional diet used in our facility (diet 5053) to the diet 2019 and were not dependent on tamoxifen after the first cycle. The switch in the diet modified the microbiota but did not alter levels of IL23 in intestinal tissues compared with mice that remained on the conventional diet. Mesenteric lymph nodes and large intestine CD4+ cells from R23FR mice in remission, but not from FR mice, induced colitis after transfer into Rag-/- mice, but only when these mice were placed on the diet 2019. The CD4+ T-cell receptor repertoire of Rag-/- mice with colitis (fed the 2019 diet) was less diverse than that from donor mice and Rag-/- mice without colitis (fed the 5053 diet) because of expansion of dominant T-cell clones. CONCLUSIONS: We developed mice that express IL23 in CX3CR1-positive myeloid cells (R23FR mice) and found that they are more susceptible to diet-induced colitis than mice that do not express IL23. The R23FR mice have a population of CD4+ T cells that becomes activated in response to dietary changes and alterations to the intestinal microbiota. The results indicate that alterations in the diet, intestinal microbiota, and IL23 signaling can contribute to pathogenesis of inflammatory bowel disease.
Assuntos
Ração Animal , Linfócitos T CD4-Positivos/metabolismo , Colite/dietoterapia , Colo/metabolismo , Microbioma Gastrointestinal , Interleucina-23/metabolismo , Células Mieloides/metabolismo , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Receptor 1 de Quimiocina CX3C/metabolismo , Colite/imunologia , Colite/metabolismo , Colite/microbiologia , Colo/imunologia , Colo/microbiologia , Modelos Animais de Doenças , Progressão da Doença , Fezes/microbiologia , Interação Gene-Ambiente , Interações Hospedeiro-Patógeno , Interleucina-23/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Valor Nutritivo , Transdução de Sinais , Fatores de TempoRESUMO
It has been 35 y since Carl Woese reported in PNAS how sequencing ribosomal RNA genes could be used to distinguish the three domains of life on Earth. During the past decade, 16S rDNA sequencing has enabled the now frequent enumeration of bacterial communities that populate the bodies of humans representing different ages, cultural traditions, and health states. A challenge going forward is to quantify the contributions of community members to wellness, disease risk, and disease pathogenesis. Here, we explore a theoretical framework for studies of the inheritance of bacterial strains and discuss the advantages and disadvantages of various study designs for assessing the contribution of strains to complex diseases.
Assuntos
Microbiota , DNA Ribossômico/genética , Humanos , Microbiota/genética , Modelos Teóricos , RNA Ribossômico 16S/genéticaRESUMO
High-throughput sequencing has revolutionized microbial ecology, but read quality remains a considerable barrier to accurate taxonomy assignment and α-diversity assessment for microbial communities. We demonstrate that high-quality read length and abundance are the primary factors differentiating correct from erroneous reads produced by Illumina GAIIx, HiSeq and MiSeq instruments. We present guidelines for user-defined quality-filtering strategies, enabling efficient extraction of high-quality data and facilitating interpretation of Illumina sequencing results.
Assuntos
Biodiversidade , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Controle de Qualidade , Análise de Sequência de DNA/métodos , HumanosRESUMO
Despite the wealth of metagenomic sequencing data, the functions of most bacterial genes from the mammalian microbiota have remained poorly understood. In their recent study (Yaung et al 2015), Wang, Gerber, and colleagues present a platform which allows functional mining of bacterial genomes for genes that contribute to fitness in vivo and holds great potential for forward engineering microbes with enhanced colonization abilities in the microbiota.
Assuntos
Bacteroides/genética , Trato Gastrointestinal/microbiologia , Metagenômica/métodos , Análise de Sequência de DNA/métodos , AnimaisRESUMO
We lack a deep understanding of genetic and metabolic attributes specializing in microbial consortia for initial and subsequent waves of colonization of our body habitats. Here we show that phylogenetically interspersed bacteria in Clostridium cluster XIVa, an abundant group of bacteria in the adult human gut also known as the Clostridium coccoides or Eubacterium rectale group, contains species that have evolved distribution patterns consistent with either early successional or stable gut communities. The species that specialize to the infant gut are more likely to associate with systemic infections and can reach high abundances in individuals with Inflammatory Bowel Disease (IBD), indicating that a subset of the microbiota that have adapted to pioneer/opportunistic lifestyles may do well in both early development and with disease. We identified genes likely selected during adaptation to pioneer/opportunistic lifestyles as those for which early succession association and not phylogenetic relationships explain genomic abundance. These genes reveal potential mechanisms by which opportunistic gut bacteria tolerate osmotic and oxidative stress and potentially important aspects of their metabolism. These genes may not only be biomarkers of properties associated with adaptation to early succession and disturbance, but also leads for developing therapies aimed at promoting reestablishment of stable gut communities following physiologic or pathologic disturbances.
Assuntos
Bactérias/genética , Bactérias/metabolismo , Intestinos/microbiologia , Metagenoma/fisiologia , Simbiose , Bactérias/classificação , Clostridium/classificação , Clostridium/genética , Clostridium/metabolismo , Genoma Bacteriano , Humanos , Redes e Vias Metabólicas , Dados de Sequência Molecular , FilogeniaRESUMO
The proportion of the human gut bacterial community that is recalcitrant to culture remains poorly defined. In this report, we combine high-throughput anaerobic culturing techniques with gnotobiotic animal husbandry and metagenomics to show that the human fecal microbiota consists largely of taxa and predicted functions that are represented in its readily cultured members. When transplanted into gnotobiotic mice, complete and cultured communities exhibit similar colonization dynamics, biogeographical distribution, and responses to dietary perturbations. Moreover, gnotobiotic mice can be used to shape these personalized culture collections to enrich for taxa suited to specific diets. We also demonstrate that thousands of isolates from a single donor can be clonally archived and taxonomically mapped in multiwell format to create personalized microbiota collections. Retrieving components of a microbiota that have coexisted in single donors who have physiologic or disease phenotypes of interest and reuniting them in various combinations in gnotobiotic mice should facilitate preclinical studies designed to determine the degree to which tractable bacterial taxa are able to transmit donor traits or influence host biology.