Dietary Depletion of Milk Exosomes and Their MicroRNA Cargos Elicits a Depletion of miR-200a-3p and Elevated Intestinal Inflammation and Chemokine (C-X-C Motif) Ligand 9 Expression in Mdr1a−/− Mice

Background: Exosomes transfer regulatory microRNAs (miRs) from donor cells to recipient cells. Exosomes and miRs originate from both endogenous synthesis and dietary sources such as milk. miR-200a-3p is a negative regulator of the proinflammatory chemokine (C-X-C motif) ligand 9 (CXCL9). Male Mdr1a−/− mice spontaneously develop clinical signs of inflammatory bowel disease (IBD). Objectives: We assessed whether dietary depletion of exosomes and miRs alters the severity of IBD in Mdr1a−/− mice owing to aberrant regulation of proinflammatory cytokines. Methods: Starting at 5 wk of age, 16 male Mdr1a−/− mice were fed either milk exosome– and RNA-sufficient (ERS) or milk exosome– and RNA-depleted (ERD) diets. The ERD diet is characterized by a near-complete depletion of miRs and a 60% loss of exosome bioavailability compared with ERS. Mice were killed when their weight loss exceeded 15% of peak body weight. Severity of IBD was assessed by histopathological evaluation of cecum. Serum cytokine and chemokine concentrations and mRNA and miR tissue expression were analyzed by multiplex ELISAs, RNA-sequencing analysis, and qRT-PCR, respectively. Results: Stromal collapse, gland hyperplasia, and additive microscopic disease scores were (mean ± SD) 56.7% ± 23.3%, 23.5% ± 11.8%, and 29.6% ± 8.2% lower, respectively, in ceca of ERS mice than of ERD mice (P \u3c 0.05). The serum concentration of CXCL9 was 35.0% ± 31.0% lower in ERS mice than in ERD mice (P \u3c 0.05). Eighty-seven mRNAs were differentially expressed in the ceca from ERS and ERD mice; 16 of these mRNAs are implicated in immune function. The concentrations of 4 and 1 out of 5 miRs assessed (including miR-200a-3p) were ≤63% lower in livers and ceca, respectively, from ERD mice than from ERS mice. Conclusions: Milk exosome and miR depletion exacerbates cecal inflammation in Mdr1a−/− mice

Experimental Evidence for Adaptation to Species-Specific Gut Microbiota in House Mice

The gut microbial communities of mammals have codiversified with host species, and changes in the gut microbiota can have profound effects on host fitness. Therefore, the gut microbiota may drive adaptation in mammalian species, but this possibility is underexplored. Here, we show that the gut microbiota has codiversified with mice in the genus Mus over the past 6 million years, and we present experimental evidence that the gut microbiota has driven adaptive evolution of the house mouse, Mus musculus domesticus. Phylogenetic analyses of metagenomeassembled bacterial genomic sequences revealed that gut bacterial lineages have been retained within and diversified alongside Mus species over evolutionary time. Transplantation of gut microbiotas from various Mus species into germfree M. m. domesticus showed that foreign gut microbiotas slowed growth rate and upregulated macrophage inflammatory protein in hosts. These results suggest adaptation by M. m. domesticus to its gut microbiota since it diverged from other Mus species

A gut pathobiont synergizes with the microbiota to instigate inflammatory disease marked by immunoreactivity against other symbionts but not itself

Inflammatory bowel diseases (IBD) are likely driven by aberrant immune responses directed against the resident microbiota. Although IBD is commonly associated with a dysbiotic microbiota enriched in putative pathobionts, the etiological agents of IBD remain unknown. Using a pathobiont-induced intestinal inflammation model and a defined bacterial community, we provide new insights into the immune-microbiota interactions during disease. In this model system, the pathobiont Helicobacter bilis instigates disease following sub-pathological dextran sulfate sodium treatment. We show that H. bilis causes mild inflammation in mono-associated mice, but severe disease in the presence of a microbiota, demonstrating synergy between the pathobiont and microbiota in exacerbating pathology. Remarkably, inflammation depends on the presence of H. bilis, but is marked by a predominant Th17 response against specific members of the microbiota and not the pathobiont, even upon the removal of the most immune-dominant taxa. Neither increases in pathobiont burden nor unique changes in immune-targeted microbiota member abundances are observed during disease. Collectively, our findings demonstrate that a pathobiont instigates inflammation without being the primary target of a Th17 response or by altering the microbiota community structure. Moreover, our findings point toward monitoring pathobiont-induced changes in microbiota immune targeting as a new concept in IBD diagnotics

Commensal \u3ci\u3eEscherichia coli\u3c/i\u3e Strains Can Promote Intestinal Inflammation via Differential Interleukin-6 Production

Escherichia coli is a facultative anaerobic symbiont found widely among mammalian gastrointestinal tracts. Several human studies have reported increased commensal E. coli abundance in the intestine during inflammation; however, host immunological responses toward commensal E. coli during inflammation are not well-defined. Here, we show that colonization of gnotobiotic mice with different genotypes of commensal E. coli isolated from healthy conventional microbiota mice and representing distinct populations of E. coli elicited strain-specific disease phenotypes and immunopathological changes following treatment with the inflammatory stimulus, dextran sulfate sodium (DSS). Production of the inflammatory cytokines GM-CSF, IL-6, and IFN-y was a hallmark of the severe inflammation induced by E. coli strains of Sequence Type 129 (ST129) and ST375 following DSS administration. In contrast, colonization with E. coli strains ST150 and ST468 caused mild intestinal inflammation and triggered only low levels of pro-inflammatory cytokines, a response indistinguishable from that of E. coli-free control mice treated with DSS. The disease development observed with ST129 and ST375 colonization was not directly associated with their abundance in the GI tract as their levels did not change throughout DSS treatment, and no major differences in bacterial burden in the gut were observed among the strains tested. Data mining and in vivo neutralization identified IL-6 as a key cytokine responsible for the observed differential disease severity. Collectively, our results show that the capacity to exacerbate acute intestinal inflammation is a strain-specific trait that can potentially be overcome by blocking the pro-inflammatory immune responses that mediate intestinal tissue damage

Experimental evaluation of the importance of colonization history in early-life gut microbiota assembly

The factors that govern assembly of the gut microbiota are insufficiently understood. Here, we test the hypothesis that inter-individual microbiota variation can arise solely from differences in the order and timing by which the gut is colonized early in life. Experiments in which mice were inoculated in sequence either with two complex seed communities or a cocktail of four bacterial strains and a seed community revealed that colonization order influenced both the outcome of community assembly and the ecological success of individual colonizers. Historical contingency and priority effects also occurred in Rag1-/- mice, suggesting that the adaptive immune system is not a major contributor to these processes. In conclusion, this study established a measurable effect of colonization history on gut microbiota assembly in a model in which host and environmental factors were strictly controlled, illuminating a potential cause for the high levels of unexplained individuality in host-associated microbial communities. Supplemental figures attached below

Evidence for a Causal Role for \u3ci\u3eEscherichia coli\u3c/i\u3e Strains Identified as Adherent-Invasive (AIEC) in Intestinal Inflammation

Enrichment of adherent-invasive Escherichia coli (AIEC) has been consistently detected in subsets of inflammatory bowel disease (IBD) patients. Although some AIEC strains cause colitis in animal models, these studies did not systematically compare AIEC with non-AIEC strains, and causal links between AIEC and disease are still disputed. Specifically, it remains unclear whether AIEC shows enhanced pathogenicity compared to that of commensal E. coli found in the same ecological microhabitat and if the in vitro phenotypes used to classify strains as AIEC are pathologically relevant. Here, we utilized in vitro phenotyping and a murine model of intestinal inflammation to systematically compare strains identified as AIEC with those identified as non-AIEC and relate AIEC phenotypes to pathogenicity. Strains identified as AIEC caused, on average, more severe intestinal inflammation. Intracellular survival/replication phenotypes routinely used to classify AIEC positively correlated with disease, while adherence to epithelial cells and tumor necrosis factor alpha production by macrophages did not. This knowledge was then applied to design and test a strategy to prevent inflammation by selecting E. coli strains that adhered to epithelial cells but poorly survived/replicated intracellularly. Two E. coli strains that ameliorated AIEC-mediated disease were subsequently identified. In summary, our results show a relationship between intracellular survival/replication in E. coli and pathology in murine colitis, suggesting that strains possessing these phenotypes might not only become enriched in human IBD but also contribute to disease. We provide new evidence that specific AIEC phenotypes are pathologically relevant and proof of principle that such mechanistic information can be therapeutically exploited to alleviate intestinal inflammation

Resistant starch can improve insulin sensitivity independently of the gut microbiota

Background: Obesity-related diseases, including type 2 diabetes and cardiovascular disease, have reached epidemic proportions in industrialized nations, and dietary interventions for their prevention are therefore important. Resistant starches (RS) improve insulin sensitivity in clinical trials, but the mechanisms underlying this health benefit remain poorly understood. Because RS fermentation by the gut microbiota results in the formation of physiologically active metabolites, we chose to specifically determine the role of the gut microbiota in mediating the metabolic benefits of RS. To achieve this goal, we determined the effects of RS when added to a Western diet on host metabolism in mice with and without a microbiota. Results: RS feeding of conventionalized mice improved insulin sensitivity and redressed some of the Western diet-induced changes in microbiome composition. However, parallel experiments in germ-free littermates revealed that RS-mediated improvements in insulin levels also occurred in the absence of a microbiota. RS reduced gene expression of adipose tissue macrophage markers and altered cecal concentrations of several bile acids in both germ-free and conventionalized mice; these effects were strongly correlated with the metabolic benefits, providing a potential microbiota-independent mechanism to explain the physiological effects of RS. Conclusions: This study demonstrated that some metabolic benefits exerted by dietary RS, especially improvements in insulin levels, occur independently of the microbiota and could involve alterations in the bile acid cycle and adipose tissue immune modulation. This work also sets a precedent for future mechanistic studies aimed at establishing the causative role of the gut microbiota in mediating the benefits of bioactive compounds and functional foods

Deciphering Interactions Between the Gut Microbiota and Host Immune System During Intestinal Inflammation

Interactions between the gut microbiota and the host immune system are very complex, ranging from commensalism and mutualism all the way to parasitism, depending on the organism and the status of the gut (i.e., healthy or inflamed). Although the majority of the bacterial members of the intestinal microbiota actively react with the immune system in a mutually beneficial relationship, the disruption of this equilibrium during inflammation allows for the emergence and enrichment of potentially pathogenic microbes (i.e. pathobionts) that are thought to contribute to the development of intestinal inflammation. In this work, we have shown that commensal gut-resident E. coli elicit strain-specific host immunological responses during acute gastrointestinal inflammation independent of their colonization levels in the gut. Amelioration of the intestinal inflammation induced by select commensal E. coli strains was achieved via neutralization of the pro-inflammatory cytokine interleukin-6. We also have investigated the causative role of adherent and invasive E. coli (AIEC), a newly-designated E. coli pathotype frequently isolated from intestinal biopsies of ileal Crohn’s disease patients, in the development of colitis. Utilizing a gnotobiotic mouse model devoid of Escherichia species, we have shown that AIEC directly contribute to the exacerbation of intestinal inflammation following chemical perturbation of the intestinal barrier. Moreover, we show that some of the in vitro phenotypes often used to describe AIEC strains predicts their ability to cause disease in vivo. Also in this work, we evaluated the ability of the prebiotic fiber galactooligosaccharide (GOS) to prevent the pro-inflammatory immune responses and colitis mediated by the intestinal pathogen Citrobacter rodentium. GOS feeding protected colonic tissues from the intestinal damage caused by C. rodentium independently of the well-described anti-adherence effects of GOS. Altogether, this work provides critical insight into the relationships between both resident and pathogenic Enterobacteriaceae and their host. Moreover, it also describes potential therapeutic interventions for treating the intestinal inflammation exacerbated or induced by these bacteria. Such knowledge, if applicable to humans, serves to strategically inform clinical diagnosis and therapeutics for patients suffering from the effects of gastrointestinal inflammation.

Deciphering Interactions Between the Gut Microbiota and Host Immune System During Intestinal Inflammation

Interactions between the gut microbiota and the host immune system are very complex, ranging from commensalism and mutualism all the way to parasitism, depending on the organism and the status of the gut (i.e., healthy or inflamed). Although the majority of the bacterial members of the intestinal microbiota actively react with the immune system in a mutually beneficial relationship, the disruption of this equilibrium during inflammation allows for the emergence and enrichment of potentially pathogenic microbes (i.e. pathobionts) that are thought to contribute to the development of intestinal inflammation. In this work, we have shown that commensal gut-resident E. coli elicit strain-specific host immunological responses during acute gastrointestinal inflammation independent of their colonization levels in the gut. Amelioration of the intestinal inflammation induced by select commensal E. coli strains was achieved via neutralization of the pro-inflammatory cytokine interleukin-6. We also have investigated the causative role of adherent and invasive E. coli (AIEC), a newly-designated E. coli pathotype frequently isolated from intestinal biopsies of ileal Crohn’s disease patients, in the development of colitis. Utilizing a gnotobiotic mouse model devoid of Escherichia species, we have shown that AIEC directly contribute to the exacerbation of intestinal inflammation following chemical perturbation of the intestinal barrier. Moreover, we show that some of the in vitro phenotypes often used to describe AIEC strains predicts their ability to cause disease in vivo. Also in this work, we evaluated the ability of the prebiotic fiber galactooligosaccharide (GOS) to prevent the pro-inflammatory immune responses and colitis mediated by the intestinal pathogen Citrobacter rodentium. GOS feeding protected colonic tissues from the intestinal damage caused by C. rodentium independently of the well-described anti-adherence effects of GOS. Altogether, this work provides critical insight into the relationships between both resident and pathogenic Enterobacteriaceae and their host. Moreover, it also describes potential therapeutic interventions for treating the intestinal inflammation exacerbated or induced by these bacteria. Such knowledge, if applicable to humans, serves to strategically inform clinical diagnosis and therapeutics for patients suffering from the effects of gastrointestinal inflammation.

Deciphering Interactions Between the Gut Microbiota and Host Immune System During Intestinal Inflammation

Interactions between the gut microbiota and the host immune system are very complex, ranging from commensalism and mutualism all the way to parasitism, depending on the organism and the status of the gut (i.e., healthy or inflamed). Although the majority of the bacterial members of the intestinal microbiota actively react with the immune system in a mutually beneficial relationship, the disruption of this equilibrium during inflammation allows for the emergence and enrichment of potentially pathogenic microbes (i.e. pathobionts) that are thought to contribute to the development of intestinal inflammation. In this work, we have shown that commensal gut-resident E. coli elicit strain-specific host immunological responses during acute gastrointestinal inflammation independent of their colonization levels in the gut. Amelioration of the intestinal inflammation induced by select commensal E. coli strains was achieved via neutralization of the pro-inflammatory cytokine interleukin-6. We also have investigated the causative role of adherent and invasive E. coli (AIEC), a newly-designated E. coli pathotype frequently isolated from intestinal biopsies of ileal Crohn’s disease patients, in the development of colitis. Utilizing a gnotobiotic mouse model devoid of Escherichia species, we have shown that AIEC directly contribute to the exacerbation of intestinal inflammation following chemical perturbation of the intestinal barrier. Moreover, we show that some of the in vitro phenotypes often used to describe AIEC strains predicts their ability to cause disease in vivo. Also in this work, we evaluated the ability of the prebiotic fiber galactooligosaccharide (GOS) to prevent the pro-inflammatory immune responses and colitis mediated by the intestinal pathogen Citrobacter rodentium. GOS feeding protected colonic tissues from the intestinal damage caused by C. rodentium independently of the well-described anti-adherence effects of GOS. Altogether, this work provides critical insight into the relationships between both resident and pathogenic Enterobacteriaceae and their host. Moreover, it also describes potential therapeutic interventions for treating the intestinal inflammation exacerbated or induced by these bacteria. Such knowledge, if applicable to humans, serves to strategically inform clinical diagnosis and therapeutics for patients suffering from the effects of gastrointestinal inflammation.