Specific Pathogen-free Microbiota Research Articles

T cells regulate intestinal motility and shape enteric neuronal responses to intestinal microbiota

ABSTRACT How the gut microbiota and immune system maintain intestinal homeostasis in concert with the enteric nervous system (ENS) remains incompletely understood. To address this gap, we assessed small intestinal transit, enteric neuronal density, enteric neurogenesis, intestinal microbiota, immune cell populations and cytokines in wildtype and T-cell deficient germ-free mice colonized with specific pathogen-free (SPF) microbiota, conventionally raised SPF and segmented filamentous bacteria (SFB)-monocolonized mice. SPF microbiota increased small intestinal transit in a T cell-dependent manner. SPF microbiota increased neuronal density in the myenteric and submucosal plexuses of the ileum and colon, similar to conventionally raised SPF mice, independently of T cells. SFB increased neuronal density in the ileum in a T cell-dependent manner, but independently of T cells in the colon. SPF microbiota stimulated enteric neurogenesis (Sox2 expression in enteric neurons) in the ileum in a T cell-dependent manner, but in the colon this effect was T cell-independent. T cells regulated nestin expression in the ENS. SPF colonization increased Th17 cells, RORγT+ Treg cells, and IL-1β and IL-17A levels in the ileum and colon. By neutralizing IL-1β and IL-17A, we observed that they control microbiota-mediated enteric neurogenesis but were not involved in the regulation of motility. Together, these findings provide new insights into the microbiota-neuroimmune dialog that regulates intestinal physiology.

A51 INTESTINAL MICROBIOTA DETERMINES ARYL HYDROCARBON RECEPTOR ACTIVATION AND SUSCEPTIBILITY TO COLITIS

Abstract Background Intestinal microbiota, diet, and the immune system have been proposed to contribute to the development of inflammatory bowel diseases (IBD). The aryl hydrocarbon receptor (AhR) is a critical regulator of intestinal immunity and mucosal barrier homeostasis that is activated by agonists such as host and microbial tryptophan metabolites. IBD patients have altered microbiota and reduced AhR agonists in intestinal content resulting in the downregulation of AhR. These findings necessitate further study to understand how diet-microbiota interactions contribute to intestinal inflammation. Purpose To study the influence of intestinal microbiota on tryptophan metabolism, AhR activation, and colitis severity. Method 8- to 10-week-old germ-free C57BL/6 mice were colonized with cecal content of mice harbouring specific pathogen-free (SPF) or a limited and well-defined altered Schaedler flora (ASF) microbiota. Germ-free mice were used as controls. Three weeks following colonization, mucosal injury was induced in a subset of mice with dextran sulfate sodium (DSS) in drinking water for five days followed by two days of water recovery. Activation of AhR was measured in stool using an in vitro AhR luciferase reporter assay. Stool AhR agonists were determined using high-performance liquid chromatography coupled to high-resolution mass spectrometry. Colonic expression of AhR pathway genes Cyp1a1, Il22, Ahrr, Ahr, and Il17 was evaluated by RT-qPCR. Susceptibility to colitis was assessed by analysing stool consistency and stool blood, colonic microscopic damage, immune infiltration by immunohistochemistry, and pro-inflammatory gene expression (NanoString). Fecal microbiota was analysed by 16S rRNA gene sequencing (Illumina). Result(s) AhR agonists, AhR activation in vitro, and AhR pathway gene expression were elevated in mice colonized with SPF microbiota in comparison to mice colonized with ASF microbiota and germ-free mice. In ASF-colonized mice, DSS induced more severe inflammation than in SPF-colonized mice, as demonstrated by worsened mucosal injury, greater weight loss, and softer stools. SPF-colonized mice developed less mucosal immune cell infiltration and pro-inflammatory gene signaling. Conclusion(s) Our findings suggest that intestinal microbiota composition determines the metabolic capacity to degrade tryptophan into agonists that homeostatically activate AhR. When mucosal injury is induced, mice with elevated microbiota-derived AhR agonists and AhR activation develop less severe mucosal injury and signs of colitis. This study presents a useful tool for evaluating dietary and microbial therapies in the context of a microbiota with impaired tryptophan metabolism. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR Disclosure of Interest None Declared

Attenuated fibrosis in specific pathogen-free microbiota in experimental cholestasis- and toxin-induced liver injury.

In advanced chronic liver disease (CLD), the translocation of intestinal bacteria and the resultant increase of proinflammatory cytokines in the splanchnic and systemic circulation may contribute to the progression of fibrosis. We therefore speculated that fibrosis and portal hypertension (PHT) would be attenuated in a mouse model of limited intestinal colonization with altered Schaedler flora (ASF) compared to a more complex colonization with specific pathogen-free (SPF) flora. We induced liver fibrosis in ASF and SPF mice by common bile duct ligation (BDL) or by carbon tetrachloride (CCl4) treatment. We then measured portal pressure (PP), portosystemic shunts (PSSs), and harvested tissues for further analyses. There were no differences in PP between sham-treated ASF or SPF mice. After BDL or CCl4 treatment, PP, PSSs, and hepatic collagen deposition increased in both groups. However, the increase in PP and the degree of fibrosis was significantly higher in ASF than SPF mice. Expression of fibrotic markers α-smooth muscle actin, desmin, and platelet-derived growth factor receptor β were significantly higher in ASF than SPF mice. This was associated with higher activation of hepatic immune cells (macrophages, neutrophils) and decreased expression of the intestinal epithelial tight junction proteins (claudin-1, occludin-1). In 2 models of advanced CLD, SPF mice presented significantly attenuated liver injury, fibrosis, and PHT compared to ASF mice. In contrast to our hypothesis, these findings suggest that a complex intestinal microbiota may play a "hepato-protective" role.-Moghadamrad, S., Hassan, M., McCoy, K. D., Kirundi, J., Kellmann, P., De Gottardi, A. Attenuated fibrosis in specific pathogen-free microbiota in experimental cholestasis- and toxin-induced liver injury.

Microbiota regulates bone marrow mesenchymal stem cell lineage differentiation and immunomodulation

Health is dependent on the homeostasis of both inner and external microenvironments. The microbiota as the external microenvironment plays a critical role in regulation of several organ systems in mammals. However, it is unclear whether the microbiota regulates homeostasis of the skeletal system and bone marrow mesenchymal stem cells (BMMSCs). Here, using a well-established germ-free (GF) mouse model, we show that the microbiota significantly alters the stemness of BMMSCs in comparison to specific-pathogen-free (SPF)-derived BMMSCs. Colonization of GF mice with SPF microbiota (conventionalized (ConvD)) normalizes the proliferation and differentiation abilities of BMMSCs. On the other hand, normal microbiota is required to maintain immunomodulatory properties of BMMSCs through induction of activated T-cell apoptosis and cytokine secretion. GF-derived BMMSCs lose the capacity to ameliorate disease phenotypes in dextran sulfate sodium-induced experimental colitis mice. Mechanistically, single-cell RNA-sequencing analysis shows that ConvD BMMSCs have a similar gene expression pattern to SPF-derived BMMSCs, which have a distinct gene distribution from GF-derived BMMSCs.

Regulation of gut luminal serotonin by commensal microbiota in mice.

Gut lumen serotonin (5-hydroxytryptamine: 5-HT) contributes to several gastrointestinal functions such as peristaltic reflexes. 5-HT is released from enterochromaffin (EC) cells in response to a number of stimuli, including signals from the gut microbiota. However, the specific mechanism by which the gut microbiota regulates 5-HT levels in the gut lumen has not yet been clarified. Our previous work with gnotobiotic mice showed that free catecholamines can be produced by the deconjugation of conjugated catecholamines; hence, we speculated that deconjugation by bacterial enzymes may be one of the mechanisms whereby gut microbes can produce free 5-HT in the gut lumen. In this study, we tested this hypothesis using germ-free (GF) mice and gnotobiotic mice recolonized with specific pathogen-free (SPF) fecal flora (EX-GF). The 5-HT levels in the lumens of the cecum and colon were significantly lower in the GF mice than in the EX-GF mice. Moreover, these levels were rapidly increased, within only 3 days after exposure to SPF microbiota. The majority of 5-HT was in an unconjugated, free form in the EX-GF mice, whereas approximately 50% of the 5-HT was found in the conjugated form in the GF mice. These results further support the current view that the gut microbiota plays a crucial role in promoting the production of biologically active, free 5-HT. The deconjugation of glucuronide-conjugated 5-HT by bacterial enzymes is likely one of the mechanisms contributing to free 5-HT production in the gut lumen.

Locoregional Effects of Microbiota in a Preclinical Model of Colon Carcinogenesis.

Inflammation and microbiota are critical components of intestinal tumorigenesis. To dissect how the microbiota contributes to tumor distribution, we generated germ-free (GF) ApcMin/+ and ApcMin/+ ;Il10-/- mice and exposed them to specific-pathogen-free (SPF) or colorectal cancer-associated bacteria. We found that colon tumorigenesis significantly correlated with inflammation in SPF-housed ApcMin/+ ;Il10-/- , but not in ApcMin/+ mice. In contrast, small intestinal neoplasia development significantly correlated with age in both ApcMin/+ ;Il10-/- and ApcMin/+ mice. GF ApcMin/+ ;Il10-/- mice conventionalized by an SPF microbiota had significantly more colon tumors compared with GF mice. Gnotobiotic studies revealed that while Fusobacterium nucleatum clinical isolates with FadA and Fap2 adhesins failed to induce inflammation and tumorigenesis, pks+Escherichia coli promoted tumorigenesis in the ApcMin/+ ;Il10-/- model in a colibactin-dependent manner, suggesting colibactin is a driver of carcinogenesis. Our results suggest a distinct etiology of cancers in different locations of the gut, where colon cancer is primarily driven by inflammation and the microbiome, while age is a driving force for small intestine cancer. Cancer Res; 77(10); 2620-32. ©2017 AACR.

P065 Effects of gut bacteria on the intestinal immune cell composition and barrier function

Background: Inflammatory bowel diseases (IBD) go along with a dysbiosis of intestinal microbiota. Little is known about the effect of these changes on the local immune cell subsets and intestinal integrity. The aim was to determine the consequences of changes in the intestinal microbiota compositions for epithelial integrity and immune cell composition in the gut. Methods: The immune cell phenotype in the gut of germ free (GF), specific pathogen free (SPF) mice and GF mice colonised with SPF microbiota at the age of 5 weeks was assessed in health and inflammation. The phenotype of lamina propria mononuclear cells (LPMC) was determined by flow cytometry and immunohistochemistry. LPMC were stimulated with lipopolysaccharides LPS) in the presence of Brefeldin A to assess intracellular tumor necrosis factor (TNF) α-expression by flow cytometry. Acute colitis was induced by dextran sodium sulphate (DSS). Colonic barrier function was assessed by electrophysiology using the Ussing chamber. Supernatants of cultures of ex vivo isolated colon tissue were analysed regarding cytokine production by Cytometric Bead Array. To confirm successful colonisation ceacal content was sequenced for 16 S ribosomal RNA. Mucins were analysed using periodic acid-Schiff reagent/Alcian blue staining on histological sections. Results: In the terminal ileum of GF mice the number of T cells was profoundly decreased. The amount of Ly6C+ monocytes as well as F4/80+ CD11b+ macrophages were decreased in the distal part of the ileum. The immune cell composition in the healthy colon was similar in GF and SPF mice. However, colonic macrophages from GF mice showed an increased TNFα-expression after LPS-stimulation compared to macrophages from SPF mice. Furthermore, in GF mice the total resistance of the colon was decreased accompanied by an increased 3H-Mannitol flux suggesting a barrier dysfunction. Less mucin was expressed by the intestine of GF mice. GF mice died following the exposure to DSS, whereas SPF mice survived and showed signs of colitis. Colonisation rescued GF mice from death. However, the inflammation score and the expression of monocyte chemoattractant protein-1 and interleukin-6 in the colon were higher in SPF than in colonised mice whereas the immune cell composition as well as intestinal microbiota was similar in these mice. Conclusions: The microbiota is essential for the development of the colonic integrity and local immune cell composition in the ileum. The dysbiosis might play a role in the pathogenesis of IBD and could provide a potential target for therapeutic intervention.

Depletion of Cultivatable Gut Microbiota by Broad-Spectrum Antibiotic Pretreatment Worsens Outcome After Murine Stroke.

Antibiotics disturbing microbiota are often used in treatment of poststroke infections. A bidirectional brain-gut microbiota axis was recently suggested as a modulator of nervous system diseases. We hypothesized that gut microbiota may be an important player in the course of stroke. We investigated the outcome of focal cerebral ischemia in C57BL/6J mice after an 8-week decontamination with quintuple broad-spectrum antibiotic cocktail. These microbiota-depleted animals were subjected to 60 minutes middle cerebral artery occlusion or sham operation. Infarct volume was measured using magnetic resonance imaging, and mice were monitored clinically throughout the whole experiment. At the end point, tissues were preserved for further analysis, comprising histology and immunologic investigations using flow cytometry. We found significantly decreased survival in the middle cerebral artery occlusion microbiota-depleted mice when the antibiotic cocktail was stopped 3 days before surgery (compared with middle cerebral artery occlusion specific pathogen-free and sham-operated microbiota-depleted mice). Moreover, all microbiota-depleted animals in which antibiotic treatment was terminated developed severe acute colitis. This phenotype was rescued by continuous antibiotic treatment or colonization with specific pathogen-free microbiota before surgery. Further, infarct volumes on day one did not differ between any of the experimental groups. Conventional microbiota ensures intestinal protection in the mouse model of experimental stroke and prevents development of acute and severe colitis in microbiota-depleted mice not given antibiotic protection after cerebral ischemia. Our experiments raise the clinically important question as to whether microbial colonization or specific microbiota are crucial for stroke outcome.

Carbon Monoxide and Heme Oxygenase-1 Prevent Intestinal Inflammation in Mice by Promoting Bacterial Clearance

Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish. Germ-free, wild-type, and interleukin (Il)10(-/-) mice and germ-free zebrafish embryos were colonized with specific pathogen-free (SPF) microbiota. Germ-free or SPF-raised wild-type and Il10(-/-) mice were given intraperitoneal injections of cobalt(III) protoporphyrin IX chloride (CoPP), which up-regulates HO-1, the CO-releasing molecule Alfama-186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with Salmonella typhimurium. In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of nuclear factor erythroid 2-related factor 2-, IL-10-, and Toll-like receptor-dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10(-/-) mice. Administration of CoPP to Il10(-/-) mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10(-/-) mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes. In mice with S typhimurium-induced enterocolitis, CoPP reduced the numbers of live S typhimurium recovered from the lamina propria, mesenteric lymph nodes, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E coli, E faecalis, and S typhimurium, whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes. Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.

Host gene expression in the colon of gnotobiotic interleukin-2-deficient mice colonized with commensal colitogenic or noncolitogenic bacterial strains: Common patterns and bacteria strain specific signatures

Specific pathogen-free (SPF), but not germfree (GF), interleukin (IL)-2-deficient (IL-2-/-) mice develop inflammatory bowel disease (IBD) at 10 to 15 weeks of age. Gnotobiotic IL-2-/- mice monocolonized with E. coli mpk develop IBD at 25 to 33 weeks of age but not B. vulgatus mpk, E. coli Nissle 1917, or mice cocolonized with both E. coli mpk and B. vulgatus. To determine genes regulated by these commensal bacteria, host gene expression in the colon of 8-week-old IL-2-/- mice was compared by using microarrays and semiquantitative reverse-transcription polymerase chain reaction. Colonization with E. coli mpk/B. vulgatus or SPF microbiota altered the gene expression profile more profoundly than monocolonization with either B. vulgatus, E. coli mpk or E. coli Nissle indicating that the complexity of the gene expression pattern is influenced by the diversity of the microbiota. A small but distinct group of genes could be defined which might be associated with colitis development. Thus, 8 week old E. coli mpk IL-2-/- mice rone to colitis compared to E. coli Nissle, B. vulgatus and E. coli mpk/B. vulgatus IL-2-/- mice displayed a lower expression of the anti-inflammatory RegIII family genes such as RegIII[gamma] and pancreatitis associated protein (PAP) and peroxisome proliferator-activated receptor-[gamma] regulated genes such as adipsin and adiponectin. The increased expression of these genes in B. vulgatus colonized mice might be associated with prevention of E. coli mpk triggered colitis in E. coli mpkM/B. vulgatus IL-2-/- mice.