Amy Mackos

Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter rodentium Infection

ABSTRACT Stressor exposure has been shown to enhance host susceptibility and the severity of a plethora of illnesses, including gastrointestinal disease. In mice, susceptibility to Citrobacter rodentium has been shown to be dependent on host genetics as well as the composition of the intestinal microbiota, but the effects of stressor exposure on this gastrointestinal pathogen have not been elucidated fully. Previously, our lab showed that exposure to the prolonged-restraint stressor prior to a challenge with C. rodentium alters the intestinal microbiota community structure, including a reduction of beneficial genera such as Lactobacillus, which may contribute to stressor-enhanced C. rodentium-induced infectious colitis. To test the effects of stressor exposure on C. rodentium infection, we exposed resistant mice to a prolonged-restraint stressor concurrent with pathogen challenge. Exposure to prolonged restraint significantly enhanced C. rodentium-induced infectious colitis in resistant mice, as measured by increases in colonic histopathology, colonic inflammatory mediator gene production, and pathogen translocation from the colon to the spleen. It was further tested if the beneficial bacterium Lactobacillus reuteri could reduce the stressor-enhanced susceptibility to C. rodentium-enhanced infectious colitis. While L. reuteri treatment did not reduce all aspects of stressor-enhanced infectious colitis, it did significantly reduce pathogen translocation from the colon to the spleen. Taken together, these data demonstrate the deleterious effects that prolonged stressor exposure can have at the onset of a gastrointestinal infection by its ability to render a resistant mouse highly susceptible to C. rodentium. Probiotic treatment ameliorated the systemic manifestations of stress on colonic infection.

Social stress-enhanced severity of Citrobacter rodentium-induced colitis is CCL2-dependent and attenuated by probiotic Lactobacillus reuteri.

Psychological stressors are known to affect colonic diseases but the mechanisms by which this occurs, and whether probiotics can prevent stressor effects, are not understood. Because inflammatory monocytes that traffic into the colon can exacerbate colitis, we tested whether CCL2, a chemokine involved in monocyte recruitment, was necessary for stressor-induced exacerbation of infectious colitis. Mice were exposed to a social disruption stressor that entails repeated social defeat. During stressor exposure, mice were orally challenged with Citrobacter rodentium to induce a colonic inflammatory response. Exposure to the stressor during challenge resulted in significantly higher colonic pathogen levels, translocation to the spleen, increases in colonic macrophages, and increases in inflammatory cytokines and chemokines. The stressor-enhanced severity of C. rodentium-induced colitis was not evident in CCL2−/− mice, indicating the effects of the stressor are CCL2-dependent. In addition, we tested whether probiotic intervention could attenuate stressor-enhanced infectious colitis by reducing monocyte/macrophage accumulation. Treating mice with probiotic Lactobacillus reuteri reduced CCL2 mRNA levels in the colon and attenuated stressor-enhanced infectious colitis. These data demonstrate that probiotic L. reuteri can prevent the exacerbating effects of stressor exposure on pathogen-induced colitis, and suggest that one mechanism by which this occurs is through downregulation of the chemokine CCL2.

Stressor exposure has prolonged effects on colonic microbial community structure in Citrobacter rodentium- challenged mice

Stressor exposure significantly affects the colonic mucosa-associated microbiota, and exacerbates Citrobacter rodentium-induced inflammation, effects that can be attenuated with probiotic Lactobacillus reuteri. This study assessed the structure of the colonic mucosa-associated microbiota in mice exposed to a social stressor (called social disruption), as well as non-stressed control mice, during challenge with the colonic pathogen C. rodentium. Mice were exposed to the social stressor or home cage control conditions for six consecutive days and all mice were challenged with C. rodentium immediately following the first exposure to the stressor. In addition, mice received probiotic L. reuteri, or vehicle as a control, via oral gavage following each stressor exposure. The stressor-exposed mice had significant differences in microbial community composition compared to non-stressed control mice. This difference was first evident following the six-cycle exposure to the stressor, on Day 6 post-C. rodentium challenge, and persisted for up to 19 days after stressor termination. Mice exposed to the stressor had different microbial community composition regardless of whether they were treated with L. reuteri or treated with vehicle as a control. These data indicate that stressor exposure affects the colonic microbiota during challenge with C. rodentium, and that these effects are long-lasting and not attenuated by probiotic L. reuteri.

17. Probiotic Lactobacillus abrogates stressor-induced increases in colonic inflammation and shifts to commensal microbe abundance during pathogen challenge

Exposure to psychological stressors can aggravate gastrointestinal inflammatory disorders and enteric infections. Previous work by our lab has shown that mice exposed to a social disruption (SDR) stressor during oral challenge with the colonic pathogen Citrobacter rodentium exhibited increases in colitic pathology, as well as TNF-α and iNOS mRNA levels. Daily treatment with the probiotic bacterium Lactobacillus reuteri ameliorated the heightened inflammation, but the mechanisms by which this occurred are not known. This proposal tested the hypothesis that L. reuteri exerts its beneficial effects by restoring community structure to the commensal gut microbiota. In the first study, germfree mice were colonized with L. reuteri prior to oral challenge with C. rodentium . This monoassociation did not prevent pathogen-induced colitis. Because these mice do not contain commensal microbiota, this indicated that the microbiota are necessary for the protective effects of probiotics to manifest. Thus, a second study was conducted in which the impact of probiotic L. reuteri administration on the commensal gut microbiota was assessed. SDR-exposed mice challenged with C. rodentium had a reduction in commensal genus Lactobacillus . This was abolished in pathogen-challenged mice treated with probiotic Lactobacillus during stressor exposure. Thus, our data indicate that the stressor-induced increases in colitic inflammation during pathogen challenge are associated with shifts to microbiota population abundances and probiotic bacteria ameliorate the inflammatory increases by preventing changes to the gut microbiota.

Prolonged restraint stressor exposure in outbred CD-1 mice impacts microbiota, colonic inflammation, and short chain fatty acids

Stressor-exposure has been shown to exacerbate inflammation and change the composition of the gastrointestinal microbiota; however stressor-induced effects on microbiota-derived metabolites and their receptors are unknown. Thus, bacterial-produced short chain fatty acids (SCFAs), as well as microbial community composition, were assessed in the colons of mice exposed to stress during infection with Citrobacter rodentium. Mice were exposed to overnight restraint on 7 consecutive nights, or left undisturbed as a control. After the first exposure of restraint, mice were orally challenged with C. rodentium or with vehicle. Microbial community composition was assessed using 16S rRNA gene sequencing and SCFA levels measured using gas chromatography-mass spectrometry (GC-MS). Pathogen levels and colonic inflammation were also assessed 6 days post-infection. Results demonstrated that the microbial community structure and SCFA production were significantly affected by both stressor exposure and C. rodentium-infection. Exposure to prolonged restraint in the absence of infection significantly reduced SCFAs (acetic acid, butyric acid, and propionic acid). Multiple bacterial taxa were affected by stressor exposure, with the relative abundance of Lactobacillus being significantly reduced and directly correlated with propionic acid. Lactobacillus abundances were inversely correlated with colonic inflammation, supporting the contention that Lactobacillus helps to regulate mucosal inflammatory responses. Our data indicates that restraint stressor can have significant effects on pathogen-induced colonic inflammation and suggest that stressor-induced changes in the microbiota, microbial-produced SCFAs and their receptors may be involved.

Mo1803 Stressor-Induced Dysbiosis Is Associated With an Aggravated Inflammatory Response to a Colonic Pathogen

Background The etiology and pathogenesis of collagenous colitis (CC) are incompletely known. Similar to other inflammatory bowel diseases (IBD), an aberrant immune response to various unidentified luminal factors, particularly the intestinal microbiota, seems to play an important role. Aim We aimed to compare the microbiotic profile between patients with CC, ulcerative colitis (UC), Crohns disease (CD) and healthy controls (HC). Methods Fecal samples were collected from patients at the out-patient clinic; UC (n=32), CD (n=32) and CC (n=29). Healthy controls (n=29) were matched by sex and age (+/-5 years) to the CC group. DNA was extracted through the Arrow Stool DNA cartridge using the Arrow Instrument (NorDiag). The hypervariable V3-V4 regions of the 16S rRNA genes were amplified by PCR and analyzed using high-throughput 454 pyrosequencing (Roche). The sequence data was filtered from bad quality sequences, denoised using AmpliconNoise and taxonomically classified using the SILVA database. Alpha diversity was assessed by Shannons diversity index and beta diversity by a Bonferroni corrected ANOSIM analysis, using Bray Curtis metrics as distance index. Differences in operative taxonomic units (OTUs) between groups of patients were analyzed by Wilcoxons test, False discovery rate was applied to adjust for multiple comparisons. Results The dataset contained 349 963 sequences with an average of 2 869 sequences/sample. Three samples (2 CC and 1 UC) were excluded due to low number of sequences. There was no difference in Shannons diversity index between patients with CC and HC (p=0.54). Analysis of the beta diversity using ANOSIM showed that the CC group segregated from the HC with increasing taxonomic resolution, reaching significance at the highest taxonomic resolution when operative taxonomic unit data (OTU) were compared (p=0.03). Wilcoxons test was further used to identify which OTUs that contributed to this difference. We found that several OTUs belonging to the Ruminococcaceae family were underrepresented in the CC group compared with the controls. Similarly, the beta diversity of patients with CD differed significantly from the HC and segregated already at a phylum level (p=0.007). In addition, the CD cohort was characterized by a lower Shannons diversity index (p<0.001). The beta diversity of patients with UC did not significantly differ from the HC (p=0.08 p=1.0), but the UC cohort was characterized by a lower alpha diversity (p=0.02). Conclusion The fecal microbiotic profile of CC differs from HC and is characterized by a lower abundance of OTUs belonging to the Ruminococcaceae family. Intriguingly, underrepresentation of the Ruminococcaceae family has previously been associated with CD indicating that the microbiotic profile of CC share features with the profile of CD. 1.Morgan XC, Et. al. Genome Biol. 2012; 13(9): R79