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BACKGROUND & AIMS: Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitis-associated neoplasia. METHODS: Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H2O2) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. RESULTS: Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H2O2. Epithelial H2O2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H2O2 production and tumorigenesis. CONCLUSIONS: Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H2O2. The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The inter-relationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.
Assuntos
Colite Ulcerativa/complicações , Neoplasias Associadas a Colite/patologia , Oxidases Duais/metabolismo , Microbioma Gastrointestinal/imunologia , Receptor 4 Toll-Like/metabolismo , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Carcinogênese/induzido quimicamente , Carcinogênese/imunologia , Carcinogênese/patologia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colite Ulcerativa/microbiologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/microbiologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/microbiologia , Colo/patologia , Conjuntos de Dados como Assunto , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Vida Livre de Germes , Humanos , Peróxido de Hidrogênio/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NADPH Oxidase 1/metabolismo , Receptor 4 Toll-Like/genéticaRESUMO
BACKGROUND & AIMS: Metabolic syndrome (MetS) is characterized by obesity, glucose intolerance, and hepatic steatosis. Alterations in the gut microbiome play important roles in the development of MetS. However, the mechanisms by which this occurs are poorly understood. Dual oxidase 2 (DUOX2) is an antimicrobial reduced nicotinamide adenine dinucleotide phosphate oxidase expressed in the gut epithelium. Here, we posit that epithelial DUOX2 activity provides a mechanistic link between the gut microbiome and the development of MetS. METHODS: Mice carrying an intestinal epithelial-specific deletion of dual oxidase maturation factor 1/2 (DA IEC-KO), and wild-type littermates were fed a standard diet and killed at 24 weeks. Metabolic alterations were determined by glucose tolerance, lipid tests, and body and organ weight measurements. DUOX2 activity was determined by Amplex Red. Intestinal permeability was determined by fluorescein isothiocyanate-dextran, microbial translocation assessments, and portal vein lipopolysaccharide measurements. Metagenomic analysis of the stool microbiome was performed. The role of the microbiome was assessed in antibiotic-treated mice. RESULTS: DA IEC-KO males showed increased body and organ weights accompanied by glucose intolerance and increased plasma lipid and liver enzyme levels, and increased adiposity in the liver and adipose tissue. Expression of F4/80, CD68, uncoupling protein 1, carbohydrate response element binding protein, leptin, and adiponectin was altered in the liver and adipose tissue of DA IEC-KO males. DA IEC-KO males produced less epithelial H2O2, had altered relative abundance of Akkermansiaceae and Lachnospiraceae in stool, and showed increased portal vein lipopolysaccharides and intestinal permeability. Females were protected from barrier defects and MetS, despite producing less H2O2. Antibiotic depletion abrogated all MetS phenotypes observed. CONCLUSIONS: Intestinal epithelial inactivity of DUOX2 promotes MetS in a microbiome-dependent manner.
Assuntos
Microbioma Gastrointestinal , Intolerância à Glucose , Síndrome Metabólica , Animais , Feminino , Masculino , Camundongos , Antibacterianos , Oxidases Duais , Peróxido de Hidrogênio , Lipopolissacarídeos , Obesidade/metabolismoRESUMO
BACKGROUND AND AIMS: Lamina propria phagocytes are key mediators of inflammatory bowel disease (IBD). We aimed to understand the transcriptomic and functional differences in these cells based on location, disease type, inflammation state, and medication use in patients with IBD. METHODS: Phagocytic immune cells in the lamina propria, as defined by the marker CD11b, were isolated from 54 unique patients (n = 111 gut mucosal biopsies). We performed flow cytometry for cell phenotyping (n = 30) and RNA sequencing with differential gene expression analysis (n = 58). We further cultured these cells in vitro and exposed them to janus kinase inhibitors to measure cytokine output (n = 27). Finally, we matched patient genomic data to our RNA sequencing data to perform candidate gene expression quantitative trait locus analysis (n = 34). RESULTS: We found distinct differences in gene expression between CD11b+ cells from the colon vs ileum, as well as in different inflammatory states and, to a lesser degree, IBD types (Crohn's disease or ulcerative colitis). These genes mapped to targetable immune pathways and metabolic and cancer pathways. We further explored the janus kinase-signal transducer and activator of transcription pathway, which was upregulated across many comparisons including in biopsies from anti-tumor necrosis factor refractory patients. We found that isolated CD11b+ cells treated with janus kinase inhibitors had decreased secretion of cytokines tumor necrosis factora and interleukin-8 (P ≤ .05). We also found 3 genetic variants acting as expression quantitative trait loci (P ≤ .1) within our CD11b+ data set. CONCLUSIONS: Lamina propria phagocytes from IBD mucosa provide pathogenetic clues on the nature of treatment refractoriness and inform new targets for therapy.
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BACKGROUND: Inflammatory bowel disease (IBD) involves chronic T cell-mediated inflammatory responses. Vedolizumab (VDZ), a monoclonal antibody against α4ß7 integrin, inhibits lymphocyte extravasation into intestinal mucosae and is effective in ulcerative colitis (UC) and Crohn's disease (CD). AIM: We sought to identify immune cell phenotypic and gene expression signatures that related to response to VDZ. METHODS: Peripheral blood (PBMC) and lamina propria mononuclear cells (LPMCs) were analyzed by flow cytometry and Cytofkit. Sorted CD4â +â memory (Tmem) or regulatory T (Treg) cells from PBMC and LPMC were analyzed by RNA sequencing (RNA-seq). Clinical response (≥2-point drop in partial Mayo scores [UC] or Harvey-Bradshaw index [CD]) was assessed 14 to 22 weeks after VDZ initiation. Machine-learning models were used to infer combinatorial traits that predicted response to VDZ. RESULTS: Seventy-one patients were enrolled: 37 received VDZ and 21 patients remained on VDZâ >2 years. Fourteen of 37 patients (38%; 8 UC, 6 CD) responded to VDZ. Immune cell phenotypes and CD4â +â Tmem and Treg transcriptional behaviors were most divergent between the ileum and colon, irrespective of IBD subtype or inflammation status. Vedolizumab treatment had the greatest impact on Treg metabolic pathways, and response was associated with increased expression of genes involved in oxidative phosphorylation. The strongest clinical predictor of VDZ efficacy was concurrent use of thiopurines. Mucosal tissues offered the greatest number of response-predictive biomarkers, whereas PBMC Treg-expressed genes were the best predictors in combinatorial models of response. CONCLUSIONS: Mucosal and peripheral blood immune cell phenotypes and transcriptional profiles can inform VDZ efficacy and inform new opportunities for combination therapies.
Vedolizumab (VDZ) is effective in the treatment of IBD. Immunophenotyping and RNAseq of T cells were used to inform its mechanism of action. Changes in T regulatory cells in the periphery and mucosa have the greatest relationship to VDZ response.
Assuntos
Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Humanos , Fármacos Gastrointestinais/uso terapêutico , Linfócitos T Reguladores/metabolismo , Leucócitos Mononucleares/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Resultado do TratamentoRESUMO
The human gastrointestinal tract is colonized by trillions of microorganisms that interact with the host to maintain structural and functional homeostasis. Acting as the interface between the site of the highest microbial burden in the human body and the richest immune compartment, a single layer of intestinal epithelial cells specializes in nutrient absorption, stratifies microorganisms to limit colonization of tissues and shapes the responses of the subepithelial immune cells. In this Review, we focus on the expression, regulation and functions of Toll-like receptors (TLRs) in the different intestinal epithelial lineages to analyse how epithelial recognition of bacteria participates in establishing homeostasis in the gut. In particular, we elaborate on the involvement of epithelial TLR signalling in controlling crypt dynamics, enhancing epithelial barrier integrity and promoting immune tolerance towards the gut microbiota. Furthermore, we comment on the regulatory mechanisms that fine-tune TLR-driven immune responses towards pathogens and revisit the role of TLRs in epithelial repair after injury. Finally, we discuss how dysregulation of epithelial TLRs can lead to the generation of dysbiosis, thereby increasing susceptibility to colitis and tumorigenesis.
Assuntos
Microbioma Gastrointestinal/imunologia , Trato Gastrointestinal/imunologia , Mucosa Intestinal/imunologia , Receptores Toll-Like/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Trato Gastrointestinal/microbiologia , Homeostase , Humanos , Mucosa Intestinal/microbiologiaRESUMO
BACKGROUND: Patients with inflammatory bowel disease (IBD) have intestinal inflammation and are treated with immune-modulating medications. In the face of the coronavirus disease-19 pandemic, we do not know whether patients with IBD will be more susceptible to infection or disease. We hypothesized that the viral entry molecules angiotensin I converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are expressed in the intestine. We further hypothesized that their expression could be affected by inflammation or medication usage. METHODS: We examined the expression of Ace2 and Tmprss2 by quantitative polymerase chain reacion in animal models of IBD. Publicly available data from organoids and mucosal biopsies from patients with IBD were examined for expression of ACE2 and TMPRSS2. We conducted RNA sequencing for CD11b-enriched cells and peripheral and lamina propria T-cells from well-annotated patient samples. RESULTS: ACE2 and TMPRSS2 were abundantly expressed in the ileum and colon and had high expression in intestinal epithelial cells. In animal models, inflammation led to downregulation of epithelial Ace2. Expression of ACE2 and TMPRSS2 was not increased in samples from patients with compared with those of control patients. In CD11b-enriched cells but not T-cells, the level of expression of ACE2 and TMPRSS2 in the mucosa was comparable to other functional mucosal genes and was not affected by inflammation. Anti-tumor necrosis factor drugs, vedolizumab, ustekinumab, and steroids were linked to significantly lower expression of ACE2 in CD11b-enriched cells. CONCLUSIONS: The viral entry molecules ACE2 and TMPRSS2 are expressed in the ileum and colon. Patients with IBD do not have higher expression during inflammation; medical therapy is associated with lower levels of ACE2. These data provide reassurance for patients with IBD.
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Regulação da Expressão Gênica , Imunossupressores/farmacologia , Síndrome do Intestino Irritável/fisiopatologia , Peptidil Dipeptidase A/genética , Serina Endopeptidases/genética , Adolescente , Adulto , Idoso , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/metabolismo , Biópsia , COVID-19 , Colo/efeitos dos fármacos , Colo/metabolismo , Biologia Computacional , Infecções por Coronavirus/fisiopatologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Imunossupressores/uso terapêutico , Inflamação/fisiopatologia , Mucosa Intestinal/metabolismo , Síndrome do Intestino Irritável/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/fisiopatologia , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Transcriptoma , Adulto JovemRESUMO
BACKGROUND & AIMS: The interaction between intestinal microbiota and the immune system plays a vital role in inflammatory bowel disease (IBD). Although numerous deep-sequencing studies have suggested dysbiosis in IBD, identifying specific bacteria from the stool or mucosa that are responsible for disease susceptibility or severity has remained a challenge. Lamina propria phagocytes ideally are localized to interact with bacteria that are in close proximity to, or have invaded, the tissue. Thus, we examined the microbial populations associated with the lamina propria phagocytes in 20 Crohn's disease and 12 ulcerative colitis patients. Specifically, we aimed to address whether the phagocyte-associated microbiota differed from the mucosa-associated microbiota and whether this varied based on IBD type or the state of inflammation. METHODS: 16S ribosomal RNA gene sequencing and innate immune gene expression profiling was done on CD11b+ lamina propria phagocytes isolated from the biopsies obtained from IBD patients. RESULTS: Phagocyte-associated microbiota was enriched in bacterial species belonging to phylum Proteobacteria, whereas species belonging to phylum Bacteroidetes were enriched in the mucosal microbiota of IBD patients. Disease type was the most influential factor in driving differences in the microbiota of both the mucosa and the lamina propria phagocytes, irrespective of inflammation state o`r anatomic location. Crohn's disease and ulcerative colitis specimens showed similar patterns of increased inflammatory gene expression in phagocytes isolated from inflamed areas compared with those isolated from uninflamed regions. CONCLUSIONS: This pilot study shows the feasibility of using lamina propria phagocytes to characterize the microbiota in IBD patients. The approach used in this study can narrow the spectrum of potentially dysbiotic bacterial populations and clinically relevant gene expression signatures in IBD patients.
Assuntos
Colite Ulcerativa/microbiologia , Doença de Crohn/microbiologia , Disbiose/diagnóstico , Microbioma Gastrointestinal/imunologia , Imunidade Inata/genética , Fagócitos/microbiologia , Biópsia , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Doença de Crohn/imunologia , Doença de Crohn/patologia , DNA Bacteriano/isolamento & purificação , Disbiose/imunologia , Disbiose/microbiologia , Disbiose/patologia , Estudos de Viabilidade , Feminino , Microbioma Gastrointestinal/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Separação Imunomagnética , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Tipagem Molecular/métodos , Fagócitos/metabolismo , Projetos Piloto , RNA Ribossômico 16S/genéticaRESUMO
The dextran sulfate sodium (DSS) model of colitis is a common animal model of inflammatory bowel disease that causes pain and distress. In this study, we aimed to determine whether fluid supplementation can be used as a welfare-based intervention to minimize animal suffering. C57Bl/6 females undergoing acute colitis by administration of 3% DSS in drinking water were supplemented with 1 mL intraperitoneal injections of NaCl and compared to non-supplemented control mice. Mouse behavior and locomotive activity were assessed on days 5-6 after DSS initiation by means of tail suspension, novel object recognition and open field activity tests. Mice were euthanized after either the acute (day 7) or the recovery phase (day 12) of colitis and inflammation, epithelial proliferation, and differentiation were assessed by means of histology, immunohistochemistry, quantitative PCR, and western blot. We found that fluid-supplemented mice had reduced signs of colitis with no alterations in behavior or locomotive activity. Furthermore, we observed an accelerated epithelial repair response after fluid hydration during the acute phase of colitis, characterized by increased crypt proliferation, activation of ERK1/2, and modulation of TGF-ß1 expression. Consistent with these findings, fluid-supplemented mice had increased numbers of goblet cells, upregulated expression of differentiation markers for absorptive enterocytes, and reduced inflammation during the recovery phase. Our results show that fluid hydration does not reduce stress in DSS-treated mice but alters colitis evolution by reducing clinical signs and accelerating epithelial repair. These results argue against the routine use of fluid supplementation in DSS-treated mice.
Assuntos
Colite/terapia , Mucosa Intestinal/patologia , Solução Salina/farmacologia , Animais , Colite/induzido quimicamente , Colite/fisiopatologia , Sulfato de Dextrana , Modelos Animais de Doenças , Feminino , Hidratação/métodos , Injeções Intraperitoneais , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Solução Salina/administração & dosagem , Cicatrização/fisiologiaRESUMO
The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS, however, has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here, we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H2O2 and the potential role of Duox2, a NADPH oxidase that is an important source of H2O2. We found that colonoids respond to interferon-γ and flagellin by enhancing production of H2O2 in a Duox2-mediated fashion. To extend these findings, we analyzed ex vivo production of H2O2 by IECs after acute and chronic inflammation, as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H2O2, chronic inflammation caused IECs to release higher levels of H2O2. Furthermore, colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H2O2 production compared with healthy controls. Collectively, these data suggest that IECs are capable of H2O2 production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H2O2 by IECs as a read-out of innate defense by the mucosa.