Gut microbiota protects against gastrointestinal tumorigenesis caused by epithelial injury.
ABSTRACT: Inflammation is a critical player in the development of both colitis-associated and sporadic colon cancers. Several studies suggest that the microbiota contribute to inflammation and tumorigenesis; however, studies to understand the role of the microbiota in colon tumor development in germ-free (GF) mice are limited. We therefore studied the effects of the microbiota on the development of inflammation and tumors in GF and conventionally raised specific pathogen-free (SPF) mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS). We discovered that GF mice developed significantly more and larger tumors compared with that in SPF mice after AOM and DSS treatment despite the lack of early acute inflammation in response to chemically induced injury by DSS. Although the extent of intestinal epithelial damage and apoptosis was not significantly different in GF and SPF mice, there was a delay in intestinal epithelial repair to DSS-induced injury in GF mice resulting in a late onset of proinflammatory and protumorigenic responses and increased epithelial proliferation and microadenoma formation. Recolonization of GF mice with commensal bacteria or administration of lipopolysaccharide reduced tumorigenesis. Thus, although commensal bacteria are capable of driving chronic inflammation and tumorigenesis, the gut microbiota also have important roles in limiting chemically induced injury and proliferative responses that lead to tumor development.
Project description: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.
Project description:Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.
Project description:Consumption of a Western-type diet has been linked to gut-microbiota-mediated colon inflammation that constitutes a risk factor for colorectal cancer. A high salt diet (HSD) exacerbates IL-17A-induced inflammation in inflammatory bowel disease and other autoimmune diseases. Enterotoxigenic Bacteroides fragilis (ETBF) is a gut commensal bacterium and reported to be a potent initiator of colitis via secretion of the Bacteroides fragilis toxin (BFT). BFT induces ectodomain cleavage of E-cadherin in colonic epithelial cells, consequently leading to cell rounding, epithelial barrier disruption, and the secretion of IL-8, which promotes tumorigenesis in mice via IL-17A-mediated inflammation. A HSD is characteristic of the Western-type diet and can exhibit inflammatory effects. However, a HSD induces effects in ETBF-induced colitis and tumorigenesis remain unknown. In this study, we investigated HSD effects in ETBF-colonized mice with azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced tumorigenesis as well as ETBF colitis mice. Unexpectedly, ETBF-infected mice fed a HSD exhibited decreased weight loss and splenomegaly and reduction of colon inflammation. The HSD significantly decreased the expression of IL-17A and inducible nitric oxide synthase (iNOS) in the colonic tissues of ETBF-infected mice. In addition, serum levels of IL-17A and nitric oxide (NO) were also diminished. However, HT29/C1 colonic epithelial cells treated with sodium chloride showed no changes in BFT-induced cellular rounding and IL-8 expression. Furthermore, HSD did not affect ETBF colonization in mice. In conclusion, HSD decreased ETBF-induced tumorigenesis through suppression of IL-17A and iNOS expression in the colon. HSD also inhibited colonic polyp numbers in the ETBF-infected AOM/DSS mice. Taken together, these findings suggest that a HSD consumption inhibited ETBF-promoted colon carcinogenesis in mice, indicating that a HSD could have beneficial effects under certain conditions.
Project description:Microbiota have been shown to have a great influence on functions of intestinal epithelial cells (ECs). The role of indole as a quorum-sensing (QS) molecule mediating intercellular signals in bacteria has been well appreciated. However, it remains unknown whether indole has beneficial effects on maintaining intestinal barriers in vivo. In this study, we analyzed the effect of indole on ECs using a germ free (GF) mouse model. GF mice showed decreased expression of junctional complex molecules in colonic ECs. The feces of specific pathogen-free (SPF) mice contained a high amount of indole; however the amount was significantly decreased in the feces of GF mice by 27-fold. Oral administration of indole-containing capsules resulted in increased expression of both tight junction (TJ)- and adherens junction (AJ)-associated molecules in colonic ECs in GF mice. In accordance with the increased expression of these junctional complex molecules, GF mice given indole-containing capsules showed higher resistance to dextran sodium sulfate (DSS)-induced colitis. A similar protective effect of indole on DSS-induced epithelial damage was also observed in mice bred in SPF conditions. These findings highlight the beneficial role of indole in establishing an epithelial barrier in vivo.
Project description:Chronic inflammation is involved in the development of colon cancer by inducing mutations and aberrant DNA methylation in colon epithelial cells. Furthermore, there is growing evidence that colonic microbiota modulates the inflammation response in the host and influences colon tumorigenesis. However, the influence of colonic microbiota on aberrant DNA methylation remains unknown. Here, we show the effect of colonic microbes on DNA methylation and tumorigenicity using a mouse model of human ulcerative colitis. Mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS) showed an increase in degree of colitis, as estimated by body weight, occult blood, and stool consistency/diarrhea at 2 weeks after treatment, but treatment with antibiotics markedly reduced the severity of the colitis. Although mucosal hyperplasia and increased inflammation-related genes were observed in the colonic epithelial cells of the AOM/DSS-treated mice, treatment with antibiotics abrogated these changes. In addition, treatment with antibiotics significantly decreased the number of mucosal nodules from 5.9 ± 5.3 to 0.2 ± 0.6 (P < .01) and area of occupancy from 50.1 ± 57.4 to 0.5 ± 1.4 mm<sup>2</sup> (P < .01). Aberrant DNA methylation of three marker CpG islands (Cbln4, Fosb, and Msx1) was induced by AOM/DSS treatment in colonic mucosae, but this increase was suppressed by 50%-92% (P < .05) with antibiotic treatment. Microbiome analysis showed that this change was associated with a decrease of the Clostridium leptum subgroup. These data indicate that antibiotics suppressed tumorigenesis through inhibition of aberrant DNA methylation induced by chronic inflammation.
Project description:The composition of the gut microbiota is influenced by sex hormones and colorectal cancer (CRC). Previously, we reported that 17?-estradiol (E2) inhibits azoxymethane/dextran sulfate sodium (AOM/DSS)-induced tumorigenesis in male mice. Here, we investigated whether the composition of the gut microbiota is different between male and female, and is regulated by estrogen as a secondary outcome of previous studies. We established four groups of mice based on the sex and estrogen status [ovariectomized (OVX) female and E2-treated male]. Additionally, three groups of males were established by treating them with AOM/DSS, and E2, after subjecting them to AOM/DSS treatment. The mice were sacrificed at 21 weeks old. The composition of the gut microbiota was analyzed using 16S rRNA metagenomics sequencing. We observed a significant increase in the microbial diversity (Chao1 index) in females, males supplemented with E2, and males treated with AOM/DSS/E2 compared with normal males. In normal physiological condition, sex difference and E2 treatment did not affect the ratio of Firmicutes/Bacteroidetes (F/B). However, in AOM/DSS-treated male mice, E2 supplementation showed significantly lower level of the F/B ratio. The ratio of commensal bacteria to opportunistic pathogens was higher in females and E2-treated males compared to normal males and females subjected to OVX. Unexpectedly, this ratio was higher in the AOM/DSS group than that determined in other males and the AOM/DSS/E2 group. Our findings suggest that estrogen alters the gut microbiota in ICR (CrljOri:CD1) mice, particularly AOM/DSS-treated males, by decreasing the F/B ratio and changing Shannon and Simpson index by supply of estrogen. This highlights another possibility that estrogen could cause changes in the gut microbiota, thereby reducing the risk of developing CRC.
Project description:Gut microbiota and their metabolites are instrumental in regulating intestinal homeostasis. However, early-life microbiota associated influences on intestinal development remain incompletely understood. Here we demonstrate that co-housing of germ-free (GF) mice with specific-pathogen free (SPF) mice at weaning (exGF) results in altered intestinal gene expression. Our results reveal that one highly differentially expressed gene, erythroid differentiation regulator-1 (Erdr1), is induced during development in SPF but not GF or exGF mice and localizes to Lgr5<sup>+</sup> stem cells and transit amplifying (TA) cells. Erdr1 functions to induce Wnt signaling in epithelial cells, increase Lgr5<sup>+</sup> stem cell expansion, and promote intestinal organoid growth. Additionally, Erdr1 accelerates scratch-wound closure in vitro, increases Lgr5<sup>+</sup> intestinal stem cell regeneration following radiation-induced injury in vivo, and enhances recovery from dextran sodium sulfate (DSS)-induced colonic damage. Collectively, our findings indicate that early-life microbiota controls Erdr1-mediated intestinal epithelial proliferation and regeneration in response to mucosal damage.
Project description:It has been suggested that manipulation of gut microbiota using antibiotics can inhibit colitis-associated colorectal cancer (CAC) in a mouse model. We investigated whether timing of gut microbial manipulation using antibiotics affects colon tumorigenesis in the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC model. CAC was induced in C57BL/6 mice by injection of 12.5 mg/kg AOM followed by three rounds of 1.7% DSS exposure. There were six groups based on timing of antibiotic administration. Colonic inflammation, proliferation, and tumorigenesis were evaluated after animal sacrifice. High-throughput sequencing of the mice feces was performed to characterize changes in gut microbiota. Full-time antibiotic treatment significantly decreased the number and size of tumors, histological scores, and expression of pro-inflammatory cytokines compared to the AOM/DSS group without antibiotic treatment. The early and late antibiotic groups, antibiotic administration from the first and second rounds of DSS to the end of the study, showed significantly lower histological scores and tumor burden. In contrast, the pretreatment antibiotic group, antibiotic administration from 3 weeks prior to AOM to the first round of DSS, did not exhibit decreased tumorigenesis. Principal coordinate analysis showed similar gut microbial community structures among the full-time, early, and late antibiotic groups, whereas other groups showed distinct gut microbial profiles. There was a positive correlation between number of tumors and number of operational taxonomic units. Colonic tumorigenesis was attenuated by antibiotic administration, except for that only prior to DSS administration, suggesting that gut microbial changes should be maintained throughout the entire period of inflammation to suppress tumorigenesis.
Project description:Commensal microbiota colonize the surface of our bodies. The inside of the gastrointestinal tract is one such surface that provides a habitat for them. The gastrointestinal tract is a long organ system comprising of various parts, and each part possesses various functions. It has been reported that the composition of intestinal luminal metabolites between the small and large intestine are different; however, comprehensive metabolomic and commensal microbiota profiles specific to each part of the gastrointestinal lumen remain obscure. In this study, by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome and 16S rRNA gene-based microbiome analyses of specific pathogen-free (SPF) and germ-free (GF) murine gastrointestinal luminal profiles, we observed the different roles of commensal microbiota in each part of the gastrointestinal tract involved in carbohydrate metabolism and nutrient production. We found that the concentrations of most amino acids in the SPF small intestine were higher than those in the GF small intestine. Furthermore, sugar alcohols such as mannitol and sorbitol accumulated only in the GF large intestine, but not in the SPF large intestine. On the other hand, pentoses, such as arabinose and xylose, gradually accumulated from the cecum to the colon only in SPF mice, but were undetected in GF mice. Correlation network analysis between the gastrointestinal microbes and metabolites showed that niacin metabolism might be correlated to Methylobacteriaceae. Collectively, commensal microbiota partially affects the gastrointestinal luminal metabolite composition based on their metabolic dynamics, in cooperation with host digestion and absorption.