R. Balfour Sartor

Microbial Influences in Inflammatory Bowel Diseases

The predominantly anaerobic microbiota of the distal ileum and colon contain an extraordinarily complex variety of metabolically active bacteria and fungi that intimately interact with the hosts epithelial cells and mucosal immune system. Crohns disease, ulcerative colitis, and pouchitis are the result of continuous microbial antigenic stimulation of pathogenic immune responses as a consequence of host genetic defects in mucosal barrier function, innate bacterial killing, or immunoregulation. Altered microbial composition and function in inflammatory bowel diseases result in increased immune stimulation, epithelial dysfunction, or enhanced mucosal permeability. Although traditional pathogens probably are not responsible for these disorders, increased virulence of commensal bacterial species, particularly Escherichia coli, enhance their mucosal attachment, invasion, and intracellular persistence, thereby stimulating pathogenic immune responses. Host genetic polymorphisms most likely interact with functional bacterial changes to stimulate aggressive immune responses that lead to chronic tissue injury. Identification of these host and microbial alterations in individual patients should lead to selective targeted interventions that correct underlying abnormalities and induce sustained and predictable therapeutic responses.

Mechanisms of Disease: pathogenesis of Crohn's disease and ulcerative colitis

Crohns disease and ulcerative colitis are idiopathic, chronic, relapsing, inflammatory conditions that are immunologically mediated. Although their exact etiologies remain uncertain, results from research in animal models, human genetics, basic science and clinical trials have provided important new insights into the pathogenesis of chronic, immune-mediated, intestinal inflammation. These studies indicate that Crohns disease and ulcerative colitis are heterogeneous diseases characterized by various genetic abnormalities that lead to overly aggressive T-cell responses to a subset of commensal enteric bacteria. The onset and reactivation of disease are triggered by environmental factors that transiently break the mucosal barrier, stimulate immune responses or alter the balance between beneficial and pathogenic enteric bacteria. Different genetic abnormalities can lead to similar disease phenotypes; these genetic changes can be broadly characterized as causing defects in mucosal barrier function, immunoregulation or bacterial clearance. These new insights will help develop better diagnostic approaches that identify clinically important subsets of patients for whom the natural history of disease and response to treatment are predictable.

Specific Microbiota Direct the Differentiation of IL-17-Producing T-Helper Cells in the Mucosa of the Small Intestine

The requirements for in vivo steady state differentiation of IL-17-producing T-helper (Th17) cells, which are potent inflammation effectors, remain obscure. We report that Th17 cell differentiation in the lamina propria (LP) of the small intestine requires specific commensal microbiota and is inhibited by treating mice with selective antibiotics. Mice from different sources had marked differences in their Th17 cell numbers and animals lacking Th17 cells acquired them after introduction of bacteria from Th17 cell-sufficient mice. Differentiation of Th17 cells correlated with the presence of cytophaga-flavobacter-bacteroidetes (CFB) bacteria in the intestine and was independent of toll-like receptor, IL-21 or IL-23 signaling, but required appropriate TGF-beta activation. Absence of Th17 cell-inducing bacteria was accompanied by increase in Foxp3+ regulatory T cells (Treg) in the LP. Our results suggest that composition of intestinal microbiota regulates the Th17:Treg balance in the LP and may thus influence intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases.

VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis.

BACKGROUND AND AIMS:Intestinal bacteria have been implicated in the initiation and perpetuation of IBD; in contrast, “probiotic bacteria” have properties possibly effective in treating and preventing relapse of IBD. We evaluated the safety and efficacy of VSL#3 and the components, and the composition of the biopsy-associated microbiota in patients with active mild to moderate ulcerative colitis (UC).METHODS:Thirty-four ambulatory patients with active UC received open label VSL#3, 3,600 billion bacteria daily in two divided doses for 6 wk. The presence of biopsy-associated bacteria was detected using a nucleic acid-based method and the presence of VSL#3 species confirmed by DNA sequencing of 16S rRNA.RESULTS:Thirty-two patients completed 6 wk of VSL#3 treatment and 2 patients did not have the final endoscopic assessment. Intent to treat analysis demonstrated remission (UCDAI ≤ 2) in 53% (n = 18); response (decrease in UCDAI ≥ 3, but final score ≥3) in 24% (n = 8); no response in 9% (n = 3); worsening in 9% (n = 3); and failure to complete the final sigmoidoscopy assessment in 5% (n = 2). There were no biochemical or clinical adverse events related to VSL#3. Two of the components of VSL#3 were detected by PCR/DGGE in biopsies collected from 3 patients in remission.CONCLUSION:Treatment of patients with mild to moderate UC, not responding to conventional therapy, with VSL#3 resulted in a combined induction of remission/response rate of 77% with no adverse events. At least some of the bacterial species incorporated in the probiotic product reached the target site in amounts that could be detected.

Interleukin-10 Signaling Blocks Inhibitor of κB Kinase Activity and Nuclear Factor κB DNA Binding

The transcription factor nuclear factor κB (NF-κB) coordinates the activation of numerous genes in response to pathogens and proinflammatory cytokines and is, therefore, pivotal in the development of acute and chronic inflammatory diseases. In its inactive state, NF-κB is constitutively present in the cytoplasm as a p50-p65 heterodimer bound to its inhibitory protein IκB. Proinflammatory cytokines, such as tumor necrosis factor (TNF), activate NF-κB by stimulating the activity of the IκB kinases (IKKs) which phosphorylate IκBα on serine residues 32 and 36, targeting it for rapid degradation by the 26 S proteasome. This enables the release and nuclear translocation of the NF-κB complex and activation of gene transcription. Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines which are known to be transcriptionally controlled by NF-κB. Conflicting data exists on the effects of IL-10 on TNF- and LPS-induced NF-κB activity in human monocytes and the molecular mechanisms involved have not been elucidated. In this study, we show that IL-10 functions to block NF-κB activity at two levels: 1) through the suppression of IKK activity and 2) through the inhibition of NF-κB DNA binding activity. This is the first evidence of an anti-inflammatory protein inhibiting IKK activity and demonstrates that IKK is a logical target for blocking inflammatory diseases.

Promotion of Hepatocellular Carcinoma by the Intestinal Microbiota and TLR4

Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation, and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and TLR4 activation in non-bone-marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC, suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease.

Disease phenotype and genotype are associated with shifts in intestinal-associated microbiota in inflammatory bowel diseases.

Background: Abnormal host–microbe interactions are implicated in the pathogenesis of inflammatory bowel diseases. Previous 16S rRNA sequence analysis of intestinal tissues demonstrated that a subset of Crohns disease (CD) and ulcerative colitis (UC) samples exhibited altered intestinal‐associated microbial compositions characterized by depletion of Bacteroidetes and Firmicutes (particularly Clostridium taxa). We hypothesize that NOD2 and ATG16L1 risk alleles may be associated with these alterations. Methods: To test this hypothesis, we genotyped 178 specimens collected from 35 CD, 35 UC, and 54 control patients for the three major NOD2 risk alleles (Leu 1007fs, R702W, and G908R) and the ATG16L1T300A risk allele, that had undergone previous 16S rRNA sequence analysis. Our statistical models incorporated the following independent variables: 1) disease phenotype (CD, UC, non‐IBD control); 2) NOD2 composite genotype (NOD2R = at least one risk allele, NOD2NR = no risk alleles); 3) ATG16L1T300A genotype (ATG16L1R/R, ATG16L1R/NR, ATG16L1NR/NR); 4) patient age at time of surgery and all first‐order interactions. The dependent variable(s) were the relative frequencies of bacterial taxa classified by applying the RDP 2.1 classifier to previously reported 16S rRNA sequence data. Results: Disease phenotype, NOD2 composite genotype and ATG16L1 genotype were significantly associated with shifts in microbial compositions by nonparametric multivariate analysis of covariance (MANCOVA). Shifts in the relative frequencies of Faecalibacterium and Escherichia taxa were significantly associated with disease phenotype by nonparametric ANCOVA. Conclusions: These results support the concept that disease phenotype and genotype are associated with compositional changes in intestinal‐associated microbiota. (Inflamm Bowel Dis 2011;)

Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice.

Interleukin (IL)-10-deficient (IL-10−/−) mice develop colitis under specific pathogen-free (SPF) conditions and remain disease free if kept sterile (germ free [GF]). We used four different protocols that varied the time-points of oral administration of Lactobacillus plantarum 299v (L. plantarum) relative to colonization with SPF bacteria to determine whether L. plantarum could prevent and treat colitis induced by SPF bacteria in IL-10−/− mice and evaluated the effect of this probiotic organism on mucosal immune activation. Assessment of colitis included blinded histologic scores, measurements of secreted colonic immunoglobulin isotypes, IL-12 (p40 subunit), and interferon (IFN)-&ggr; production by anti-CD3-stimulated mesenteric lymph node cells. Treating SPF IL-10−/− mice with L. plantarum attenuated previously established colonic inflammation as manifested by decreased mucosal IL-12, IFN-&ggr;, and immunoglobulin G2a levels. Colonizing GF animals with L. plantarum and SPF flora simultaneously had no protective effects. Gnotobiotic IL-10−/− mice monoassociated with L. plantarum exhibited mild immune system activation but no colitis. Pretreatment of GF mice by colonization with L. plantarum, then exposure to SPF flora and continued probiotic therapy significantly decreased histologic colitis scores. These results demonstrate that L. plantarum can attenuate immune-mediated colitis and suggest a potential therapeutic role for this agent in clinical inflammatory bowel diseases.

Cytokine messenger RNA profiles in inflammatory bowel disease mucosa detected by polymerase chain reaction amplification

Immunoregulatory properties of cytokines may mediate disordered inflammatory events in ulcerative colitis (UC) and Crohns disease (CD). In the present study, profiles of cytokines produced by activated macrophages were studied in colonic tissue from 43 patients with and without inflammatory bowel disease (IBD). Cytokine messenger RNA (mRNA) extracted from mucosal biopsy specimens was studied using polymerase chain reaction assay techniques. A greater percentage of active UC samples had detectable levels of mRNA for interleukins (IL) 1, 6, and 8 and gro than samples in inactive UC and noninflammatory controls. These cytokines were comparable in active UC and inflammatory controls. Expression of gro mRNA in active UC tissue was significantly higher than in active CD. Tumor necrosis factor was detected in only 7 of 43 samples with no difference between groups. Active and inactive CD did not differ in percentage of cytokine mRNA expression. IL-1 receptor antagonist (IL-1ra) was detected in more inflammatory controls than in CD and was expressed in fewer IBD patients than IL-1. Expression of proinflammatory cytokines in grossly inactive CD and possible defective production of IL-1ra may explain disease reactivation and chronicity.

Mechanisms of acute and chronic intestinal inflammation induced by indomethacin

The objective of this study was to characterize the mechanisms of acute and chronic intestinal mucosal injury and inflammation induced by subcutaneously injected indomethacin (Indo). One injection of Indo (7.5 mg/kg) produced acute injury and inflammation in the distal jejunum and proximal ileum that were maximal at three days and completely resolved within one week. Two daily subcutaneous injections of Indo produced a more extensive and chronic inflammation that lasted in an active form in more than 75% of the rats for at least two weeks. Epithelial injury, as measured by enhanced mucosal permeability, was significantly elevated only at one day in the acute model (one injection) but was persistently elevated in the chronic model (two injections). Bile duct ligation completely attenuated increased mucosal permeability in the acute model, however, depletion of circulating neutrophils had no effect. Neither Indo (0–0.1 mg/ml) nor normal bile was cytotoxic to cultured rat intestinal epithelial cells; however, they synergistically promoted significant cytotoxicity. Bile collected from rats treated with Indo was cytotoxic towards the epithelial cells in a dose-dependent manner. Sulfasalazine and metronidazole (100 mg/kg/day, both) attenuated enhanced mucosal permeability in the chronic model. Massive bacterial translocation into the mesenteric lymph nodes, liver, and spleen following two injections of Indo was significantly attenuated by metronidazole. We conclude that: (1) a single injection of Indo produces acute intestinal mucosal injury and inflammation that resolve completely within three to seven days, whereas two daily injections of Indo produce both acute and chronic injury and inflammation, (2) enterohepatic circulation of Indo is important in promoting the acute phases of injury and inflammation, (3) circulating neutrophils do not play a role in the pathogenesis of this model, and (4) endogenous bacteria play an important role in exacerbating and/or perpetuating the chronic phases of injury and inflammation.