David M. Rodrigues

Bacterial infection causes stress-induced memory dysfunction in mice

Background The brain–gut axis is a key regulator of normal intestinal physiology; for example, psychological stress is linked to altered gut barrier function, development of food allergies and changes in behaviour. Whether intestinal events, such as enteric bacterial infections and bacterial colonisation, exert a reciprocal effect on stress-associated behaviour is not well established. Objective To determine the effects of either acute enteric infection or absence of gut microbiota on behaviour, including anxiety and non-spatial memory formation. Methods Behaviour was assessed following infection with the non-invasive enteric pathogen, Citrobacter rodentium in both C57BL/6 mice and germ-free Swiss-Webster mice, in the presence or absence of acute water avoidance stress. Whether daily treatment with probiotics normalised behaviour was assessed, and potential mechanisms of action evaluated. Results No behavioural abnormalities were observed, either at the height of infection (10 days) or following bacterial clearance (30 days), in C rodentium-infected C57BL/6 mice. When infected mice were exposed to acute stress, however, memory dysfunction was apparent after infection (10 days and 30 days). Memory dysfunction was prevented by daily treatment of infected mice with probiotics. Memory was impaired in germ-free mice, with or without exposure to stress, in contrast to conventionally reared, control Swiss-Webster mice with an intact intestinal microbiota. Conclusions The intestinal microbiota influences the ability to form memory. Memory dysfunction occurs in infected mice exposed to acute stress, while in the germ-free setting memory is altered at baseline.

Probiotics are effective for the prevention and treatment of Citrobacter rodentium-induced colitis in mice.

BACKGROUND Probiotics prevent disease induced by Citrobacter rodentium, a murine-specific enteric pathogen. Whether probiotics can be used to interrupt the infectious process following initiation of infection was determined. METHODS C57BL/6 adult and neonatal mice were challenged with C. rodentium, and a probiotic mixture containing Lactobacillus helveticus and Lactobacillus rhamnosus was provided 1 week before bacterial challenge, concurrently with infection, or 3 days and 6 days after infection. Mice were sacrificed 10 days after infection, and disease severity was assessed by histological analysis and in vivo intestinal permeability assay. Inflammatory pathways and the composition of the fecal microbiome were assessed in adult mice. RESULTS Preadministration and coadministration of probiotics ameliorated C. rodentium-induced barrier dysfunction, epithelial hyperplasia, and binding of the pathogen to host colonocytes in adults, with similar findings in neonatal mice. Upregulated tumor necrosis factor α and interferon γ transcripts were suppressed in the pretreated probiotic group, whereas interleukin 17 transcription was suppressed with probiotics given up to 3 days after infection. Probiotics promoted transcription of interleukin 10 and FOXP3, and increased follicular T-regulatory cells in pretreatment mice. C. rodentium infection resulted in an altered fecal microbiome, which was normalized with probiotic intervention. CONCLUSIONS This study provides evidence that probiotics can prevent illness and treat disease in an animal model of infectious colitis.

Probiotics normalize the gut-brain-microbiota axis in immunodeficient mice

The gut-brain-microbiota axis is increasingly recognized as an important regulator of intestinal physiology. Exposure to psychological stress causes activation of the hypothalamic-pituitary-adrenal (HPA) axis and causes altered intestinal barrier function, intestinal dysbiosis, and behavioral changes. The primary aim of this study was to determine whether the effects of psychological stress on intestinal physiology and behavior, including anxiety and memory, are mediated by the adaptive immune system. Furthermore, we wanted to determine whether treatment with probiotics would normalize these effects. Here we demonstrate that B and T cell-deficient Rag1(-/-) mice displayed altered baseline behaviors, including memory and anxiety, accompanied by an overactive HPA axis, increased intestinal secretory state, dysbiosis, and decreased hippocampal c-Fos expression. Both local (intestinal physiology and microbiota) and central (behavioral and hippocampal c-Fos) changes were normalized by pretreatment with probiotics, indicating an overall benefit on health conferred by changes in the microbiota, independent of lymphocytes. Taken together, these findings indicate a role for adaptive immune cells in maintaining normal intestinal and brain health in mice and show that probiotics can overcome this immune-mediated deficit in the gut-brain-microbiota axis.

Matrix metalloproteinase 9 contributes to gut microbe homeostasis in a model of infectious colitis

BackgroundInflammatory bowel diseases are associated with increased expression of zinc-dependent Matrix Metalloproteinase 9 (MMP-9). A stark dysregulation of intestinal mucosal homeostasis has been observed in patients with chronic inflammatory bowel diseases. We therefore sought to determine the contribution of MMP-9 to the pathogenesis of Citrobacter rodentium-induced colitis and its effects on gut microbiome homeostasis.ResultsWild-type and MMP-9−/− mice aged 5–6 weeks were challenged with C. rodentium by orogastric gavage and sacrificed either 10 or 30 days post-infection. Disease severity was assessed by histological analysis of colonic epithelial hyperplasia and by using an in vivo intestinal permeability assay. Changes in the inflammatory responses were measured by using qPCR, and the composition of the fecal microbiome evaluated with both qPCR and terminal restriction fragment length polymorphism. Activation and localization of MMP-9 to the apical surface of the colonic epithelium in response to C. rodentium infection was demonstrated by both zymography and immunocytochemistry. The pro-inflammatory response to infection, including colonic epithelial cell hyperplasia and barrier dysfunction, was similar, irrespective of genotype. Nonmetric multidimensional scaling of terminal restriction fragments revealed a different fecal microbiome composition and C. rodentium colonization pattern between genotypes, with MMP-9−/− having elevated levels of protective segmented filamentous bacteria and interleukin-17, and lower levels of C. rodentium. MMP-9−/− but not wild-type mice were also protected from reductions in fecal microbial diversity in response to the bacterial enteric infection.ConclusionsThese results demonstrate that MMP-9 expression in the colon causes alterations in the fecal microbiome and has an impact on the pathogenesis of bacterial-induced colitis in mice.

Microbial Disruption of Autophagy Alters Expression of the RISC Component AGO2, a Critical Regulator of the miRNA Silencing Pathway

Background:Autophagy is implicated in Crohns disease (CD) pathogenesis. Recent evidence suggests autophagy regulates the microRNA (miRNA)-induced silencing complex (miRISC). Therefore, autophagy may play a novel role in CD by regulating expression of miRISC, thereby altering miRNA silencing. As microbes associated with CD can alter autophagy, we hypothesized that microbial disruption of autophagy affects the critical miRISC component AGO2. Methods:AGO2 expression was assessed in epithelial and immune cells, and intestinal organoids with disrupted autophagy. Microarray technology was used to determine the expression of downstream miRNAs in cells with defective autophagy. Results:Increased AGO2 was detected in autophagy-deficient ATG5−/− and ATG16−/− mouse embryonic fibroblast cells (MEFs) in comparison with wild-type MEFs. Chemical agents and VacA toxin, which disrupt autophagy, increased AGO2 expression in MEFs, epithelial cells lines, and human monocytes, respectively. Increased AGO2 was also detected in ATG7−/− intestinal organoids, in comparison with wild-type organoids. Five miRNAs were differentially expressed in autophagy-deficient MEFs. Pathway enrichment analysis of the differentially expressed miRNAs implicated signaling pathways previously associated with CD. Conclusions:Taken together, our results suggest that autophagy is involved in the regulation of the critical miRISC component AGO2 in epithelial and immune cells and primary intestinal epithelial cells. We propose a mechanism by which autophagy alters miRNA expression, which likely impacts the regulation of CD-associated pathways. Furthermore, as enteric microbial products can manipulate autophagy and AGO2, our findings suggest a novel mechanism by which enteric microbes could influence miRNA to promote disease.