B. Sovran

IL-22-STAT3 Pathway Plays a Key Role in the Maintenance of Ileal Homeostasis in Mice Lacking Secreted Mucus Barrier

Background:Muc2-deficient mice show no signs of ileal pathology but the mechanisms remained unknown. Methods:Wild-type (WT), Muc2+/−, and Muc2−/− mice were killed at 2, 4, and 8 weeks of age. Total RNA from ileum was used for full genome transcriptome analysis and qPCR. Microbiota composition was determined using a mouse intestinal chip (MITChip). Morphological and immunohistological studies were performed on segments of ileum. Results:The ileum was colonized by more diverse microbiota in young (week 4) WT than in Muc2−/− mice, and composition was influenced by genotype. Weaning was associated with major changes in the transcriptome of all mice, and the highest number of differentially expressed genes compared with adults, reflecting temporal changes in microbiota. Although the spatial compartmentalization of bacteria was compromised in Muc2−/− mice, gene set enrichment analysis revealed a downregulation of Toll-like receptor, immune, and chemokine signaling pathways compared to WT mice. The predicted effects of enhanced IL-22 signaling were identified in the Muc2−/− transcriptome as the upregulation of epithelial cell proliferation altered expression of mitosis and cell-cycle control pathways. This is consistent with increased villus length and number of Ki67+ epithelial cells in Muc2−/− mice. Additionally, expression of the network of IL-22 regulated defense genes, including Fut2, Reg3&bgr;, Reg3&ggr;, Relmb, and the Defensin Defb46 were increased in Muc2−/− mice. Conclusions:These findings highlight a role for the IL-22-STAT3 pathway in maintaining ileal homeostasis when the mucus barrier is compromised and its potential as a target for novel therapeutic strategies in inflammatory bowel disease.

Identification of Commensal Species Positively Correlated with Early Stress Responses to a Compromised Mucus Barrier.

Background:Our aims were (1) to correlate changes in the microbiota to intestinal gene expression before and during the development of colitis in Muc2−/− mice and (2) to investigate whether the heterozygote Muc2+/− mouse would reveal host markers of gut barrier stress. Methods:Colon histology, transcriptomics, and microbiota profiling of faecal samples was performed on wild type, Muc2+/−, and Muc2−/− mice at 2, 4, and 8 weeks of age. Results:Muc2−/− mice develop colitis in proximal colon after weaning, resulting in inflammatory and adaptive immune responses, and expression of genes associated with human inflammatory bowel disease. Muc2+/− mice do not develop colitis, but produce a thinner mucus layer. The transcriptome of Muc2+/− mice revealed differential expression of genes participating in mucosal stress responses and exacerbation of a transient inflammatory state around the time of weaning. Young wild type and Muc2+/− mice have a more constrained group of bacteria as compared with the Muc2−/− mice, but at 8 weeks the microbiota composition is more similar in all mice. At all ages, microbiota composition discriminated the groups of mice according to their genotype. Specific bacterial clusters correlated with altered gene expression responses to stress and bacteria, before colitis development, including colitogenic members of the genus Bacteroides. Conclusions:The abundance of Bacteroides pathobionts increased before histological signs of pathology suggesting they may play a role in triggering the development of colitis. The Muc2+/− mouse produces a thinner mucus layer and can be used to study mucus barrier stress in the absence of colitis.

The effect of age on the intestinal mucus thickness, microbiota composition and immunity in relation to sex in mice

A mucus layer covers and protects the intestinal epithelial cells from direct contact with microbes. This mucus layer not only prevents inflammation but also plays an essential role in microbiota colonization, indicating the complex interplay between mucus composition-microbiota and intestinal health. However, it is unknown whether the mucus layer is influenced by age or sex and whether this contributes to reported differences in intestinal diseases in males and females or with ageing. Therefore, in this study we investigated the effect of age on mucus thickness, intestinal microbiota composition and immune composition in relation to sex. The ageing induced shrinkage of the colonic mucus layer was associated with bacterial penetration and direct contact of bacteria with the epithelium in both sexes. Additionally, several genes involved in the biosynthesis of mucus were downregulated in old mice, especially in males, and this was accompanied by a decrease in abundances of various Lactobacillus species and unclassified Clostridiales type IV and XIV and increase in abundance of the potential pathobiont Bacteroides vulgatus. The changes in mucus and microbiota in old mice were associated with enhanced activation of the immune system as illustrated by a higher percentage of effector T cells in old mice. Our data contribute to a better understanding of the interplay between mucus-microbiota-and immune responses and ultimately may lead to more tailored design of strategies to modulate mucus production in targeted groups.

Supplementation with Lactobacillus plantarum WCFS1 Prevents Decline of Mucus Barrier in Colon of Accelerated Aging Ercc1(-/Δ7) Mice

Although it is clear that probiotics improve intestinal barrier function, little is known about the effects of probiotics on the aging intestine. We investigated effects of 10-week bacterial supplementation of Lactobacillus plantarum WCFS1, Lactobacillus casei BL23, or Bifidobacterium breve DSM20213 on gut barrier and immunity in 16-week-old accelerated aging Ercc1−/Δ7 mice, which have a median lifespan of ~20 weeks, and their wild-type littermates. The colonic barrier in Ercc1−/Δ7 mice was characterized by a thin (< 10 μm) mucus layer. L. plantarum prevented this decline in mucus integrity in Ercc1−/Δ7 mice, whereas B. breve exacerbated it. Bacterial supplementations affected the expression of immune-related genes, including Toll-like receptor 4. Regulatory T cell frequencies were increased in the mesenteric lymph nodes of L. plantarum- and L. casei-treated Ercc1−/Δ7 mice. L. plantarum- and L. casei-treated Ercc1−/Δ7 mice showed increased specific antibody production in a T cell-dependent immune response in vivo. By contrast, the effects of bacterial supplementation on wild-type control mice were negligible. Thus, supplementation with L. plantarum – but not with L. casei and B. breve – prevented the decline in the mucus barrier in Ercc1−/Δ7 mice. Our data indicate that age is an important factor influencing beneficial or detrimental effects of candidate probiotics. These findings also highlight the need for caution in translating beneficial effects of probiotics observed in young animals or humans to the elderly.

Tryptophan restriction arrests B cell development and enhances microbial diversity in WT and prematurely aging Ercc1−/Δ7 mice

With aging, tryptophan metabolism is affected. Tryptophan has a crucial role in the induction of immune tolerance and the maintenance of gut microbiota. We, therefore, studied the effect of dietary tryptophan restriction in young wild‐type (WT) mice (118‐wk life span) and in DNA‐repair deficient, premature‐aged (Ercc1−/Δ7) mice (20‐wk life span). First, we found that the effect of aging on the distribution of B and T cells in bone marrow (BM) and in the periphery of 16‐wk‐old Ercc1−/Δ7 mice was comparable to that in 18‐mo‐old WT mice. Dietary tryptophan restriction caused an arrest of B cell development in the BM, accompanied by diminished B cell frequencies in the periphery. In general, old Ercc1−/Δ7 mice showed similar responses to tryptophan restriction compared with young WT mice, indicative of age‐independent effects. Dietary tryptophan restriction increased microbial diversity and made the gut microbiota composition of old Ercc1−/Δ7 mice more similar to that of young WT mice. The decreased abundances of Alistipes and Akkermansia spp. after dietary tryptophan restriction correlated significantly with decreased B cell precursor numbers. In conclusion, we report that dietary tryptophan restriction arrests B cell development and concomitantly changes gut microbiota composition. Our study suggests a beneficial interplay between dietary tryptophan, B cell development, and gut microbial composition on several aspects of age‐induced changes.