Microbiota of De-Novo Pediatric IBD: Increased Faecalibacterium Prausnitzii and Reduced Bacterial Diversity in Crohn's But Not in Ulcerative Colitis

OBJECTIVES: The gastrointestinal microbiota is considered important in inflammatory bowel disease (IBD) pathogenesis. Discoveries from established disease cohorts report reduced bacterial diversity, changes in bacterial composition, and a protective role for Faecalibacterium prausnitzii in Crohn's disease (CD). The majority of studies to date are however potentially confounded by the effect of treatment and a reliance on established rather than de-novo disease.

METHODS: Microbial changes at diagnosis were examined by biopsying the colonic mucosa of 37 children: 25 with newly presenting, untreated IBD with active colitis (13 CD and 12 ulcerative colitis (UC)), and 12 pediatric controls with a macroscopically and microscopically normal colon. We utilized a dual-methodology approach with pyrosequencing (threshold >10,000 reads) and confirmatory real-time PCR (RT-PCR).

RESULTS: Threshold pyrosequencing output was obtained on 34 subjects (11 CD, 11 UC, 12 controls). No significant changes were noted at phylum level among the Bacteroidetes, Firmicutes, or Proteobacteria. A significant reduction in bacterial alpha-diversity was noted in CD vs. controls by three methods (Shannon, Simpson, and phylogenetic diversity) but not in UC vs. controls. An increase in Faecalibacterium was observed in CD compared with controls by pyrosequencing (mean 16.7% vs. 9.1% of reads, P = 0.02) and replicated by specific F. prausnitzii RT-PCR (36.0% vs. 19.0% of total bacteria, P = 0.02). No disease-specific clustering was evident on principal components analysis.

CONCLUSIONS: Our results offer a comprehensive examination of the IBD mucosal microbiota at diagnosis, unaffected by therapeutic confounders or changes over time. Our results challenge the current model of a protective role for F. prausnitzii in CD, suggesting a more dynamic role for this organism than previously described.

Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis

Mutations that result in loss of function of Nod2, an intracellular receptor for bacterial peptidoglycan, are associated with Crohn's disease. Here we found that the E3 ubiquitin ligase Pellino3 was an important mediator in the Nod2 signaling pathway. Pellino3-deficient mice had less induction of cytokines after engagement of Nod2 and had exacerbated disease in various experimental models of colitis. Furthermore, expression of Pellino3 was lower in the colons of patients with Crohn's disease. Pellino3 directly bound to the kinase RIP2 and catalyzed its ubiquitination. Loss of Pellino3 led to attenuation of Nod2-induced ubiquitination of RIP2 and less activation of the transcription factor NF-?B and mitogen-activated protein kinases (MAPKs). Our findings identify RIP2 as a substrate for Pellino3 and Pellino3 as an important mediator in the Nod2 pathway and regulator of intestinal inflammation. © 2013 Nature America, Inc. All rights reserved.

FOXP3+ regulatory T-cell counts correlate with histological response in Crohn's colitis treated with infliximab

Crohn's disease is a chronic inflammatory bowel disease of unknown aetiology. Mucosal inflammatory dysregulation is likely important, with increased production of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNFα). The chimeric monoclonal antibody, infliximab, inhibits TNFα and promotes intestinal mucosal healing. Despite this, many patients still require surgical intervention. Patients who have undergone colonic resection post-infliximab therapy, show markedly variable morphological response to treatment. FOXP3+ CD4+ regulatory T-cells have been shown to have a protective role in autoimmune/inflammatory diseases and their sequestration to the bowel is found in those treated with infliximab. We examined the immunohistochemical profile of lymphoid aggregates in tissue sections from post-infliximab Crohn's colitis resection specimens, classified as morphological responders or non-responders, defined in relation to the absence/presence of mucosal ulceration and active inflammation, and a control group. Results indicated no significant diffences in CD68-positive cell counts but increased FOXP3-positive (P = 0.02) and CD4-positive (P = 0.05) cell counts in responders versus non-responders. Untreated control scores were similar to non-responders. Although based on small study numbers, our results suggest an association between upregulation of FOXP3+/CD4+ regulatory T-cells and morphological response to infliximab therapy. This represents a possible quantitative methodology for monitoring therapeutic response to infliximab therapy, based on immunohistochemical evaluation of endoscopic biopsy specimens.

Adjustment to inflammatory bowel disease: The relative influence of illness perceptions and coping

Background: The Common Sense Model (CSM) of illness representations was used in the current study to examine the relative contribution of illness perceptions and coping strategies in explaining adjustment to inflammatory bowel disease (IBD). Methods: Participants were 80 adults consecutively attending an outpatients' clinic with a diagnosis of either Crohn's disease or ulcerative colitis. Respondents completed and returned a questionnaire booklet that assessed illness perceptions, coping, and adjustment. Adjustment was measured from the perspectives of psychological distress, quality of life, and functional independence. Results: Illness perceptions (particularly perception of consequences of IBD) were uniformly the most consistent variables explaining adjustment to IBD. Coping did not significantly add to predicting adjustment once illness perceptions were controlled for and therefore did not mediate the relationship between illness perceptions and adjustment, as proposed in the CSM. Conclusions: The results suggest the importance of addressing illness perceptions in developing appropriate psychological interventions for IBD.

Posttranslational Inhibition of Proinflammatory Chemokine Secretion in Intestinal Epithelial Cells Implications for Specific IBD Indications

Background and Aim: Inflammatory bowel diseases (IBD) are immune-mediated chronic diseases that are characterized by an overreaction of the intestinal immune system to the intestinal microbiota. VSL#3, a mixture of 8 different lactic acid bacteria, is a clinically relevant probiotic compound in the context of IBD, but the bacterial structures and molecular mechanisms underlying the observed protective effects are largely unknown. The intestinal epithelium plays a very important role in the maintenance of the intestinal homeostasis, as the intestinal epithelial cells (IEC) are capable of sensing, processing, and reacting upon signals from the luminal microbiota and the intestinal immune system. This immune regulatory function of the IEC is lost in IBD owing to dysregulated activation of the IEC. Thus, the aim of this study was to reveal protective mechanisms of VSL#3 on IEC function.

Results: In vitro, VSL#3 was found to selectively inhibit activation-induced secretion of the T-cell chemokine interferon-inducible protein (IP)-10 in IEC. Cell wall-associated proteins of VSL#3-derived Lactobacillus casei (L. casei) were identified to be the active anti-inflammatory component of VSL#3. Mechanistically, L. casei did not impair initial IP-10 protein production, but induced posttranslational degradation of IP-10 in IEC. Feeding studies in tumor necrosis factor (TNF)(Delta ARE/+) mice, a mouse model for experimental ileitis, revealed that neither VSL#3 nor L. casei is capable of reducing ileal inflammation. Even preweaning feeding of VSL#3 did not prevent the development of severe ileitis in TNF Delta ARE/+ mice. In contrast, VSL#3 feeding studies in IL-10-/- mice, a model for experimental colitis, revealed that VSL#3 has local, intestinal compartment-specific protective effects on the development of inflammation. Reduced histopathologic inflammation in the cecum of IL-10-/- mice after VSL#3 treatment was found to correlate with reduced levels of IP-10 protein in primary cecal epithelial cells.

Conclusion and Outlook: These results suggest that the inhibitory effect of VSL#3-derived L. casei on IP-10 secretion in IEC is an important probiotic mechanism that contributes to the anti-inflammatory effects of VSL#3 in specific subsets of patients with IBD. An important future aim is the identification of the active probiotic protein, which could serve as a basis for the development of new efficient therapies in the context of IBD.

Inflammatory bowel disease, gut bacteria and probiotic therapy

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD) and both diseases lead to high morbidity and health care costs. Complex interactions between the immune system, enteric commensal bacteria and host genotype are thought to underlie the development of IBD although the precise aetiology of this group of diseases is still unknown. The understanding of the composition and complexity of the normal gut microbiota has been greatly aided by the use of molecular methods and is likely to be further increased with the advent of metagenomics and metatranscriptomics approaches, which will allow an increasingly more holistic assessment of the microbiome with respect to both diversity and function of the commensal gut microbiota. Studies thus far have shown that the intestinal microbiota drives the development of the gut immune system and can induce immune homeostasis as well as contribute to the development of IBD. Probiotics which deliver some of the beneficial immunomodulatory effects of the commensal gut microbiota and induce immune homeostasis have been proposed as a suitable treatment for mild to moderate IBD. This review provides an overview over the current understanding of the commensal gut microbiota, its interactions with the mucosal immune system and its capacity to induce both gut homeostasis as well as dysregulation of the immune system. Bacterial-host events, including interactions with pattern recognition receptors (PRRs) expressed on epithelial cells and dendritic cells (DCs) and the resultant impact on immune responses at mucosal surfaces will be discussed. (C) 2009 Elsevier GmbH. All rights reserved.