Shervin Rabizadeh

A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses.

The intestinal flora may promote colon tumor formation. Here we explore immunologic mechanisms of colonic carcinogenesis by a human colonic bacterium, enterotoxigenic Bacteroides fragilis (ETBF). ETBF that secretes B. fragilis toxin (BFT) causes human inflammatory diarrhea but also asymptomatically colonizes a proportion of the human population. Our results indicate that whereas both ETBF and nontoxigenic B. fragilis (NTBF) chronically colonize mice, only ETBF triggers colitis and strongly induces colonic tumors in multiple intestinal neoplasia (Min) mice. ETBF induces robust, selective colonic signal transducer and activator of transcription-3 (Stat3) activation with colitis characterized by a selective T helper type 17 (TH17) response distributed between CD4+ T cell receptor-αβ (TCRαβ)+ and CD4–8–TCRγδ+ T cells. Antibody-mediated blockade of interleukin-17 (IL-17) as well as the receptor for IL-23, a key cytokine amplifying TH17 responses, inhibits ETBF-induced colitis, colonic hyperplasia and tumor formation. These results show a Stat3- and TH17-dependent pathway for inflammation-induced cancer by a common human commensal bacterium, providing new mechanistic insight into human colon carcinogenesis.

Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis.

It is estimated that the etiology of 20–30% of epithelial cancers is directly associated with inflammation, although the direct molecular events linking inflammation and carcinogenesis are poorly defined. In the context of gastrointestinal disease, the bacterium enterotoxigenic Bacteroides fragilis (ETBF) is a significant source of chronic inflammation and has been implicated as a risk factor for colorectal cancer. Spermine oxidase (SMO) is a polyamine catabolic enzyme that is highly inducible by inflammatory stimuli resulting in increased reactive oxygen species (ROS) and DNA damage. We now demonstrate that purified B. fragilis toxin (BFT) up-regulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent generation of ROS and induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in C57BL/6 mice is associated with increased SMO expression and treatment of mice with an inhibitor of polyamine catabolism, N1,N4-bis(2,3-butandienyl)-1,4-butanediamine (MDL 72527), significantly reduces ETBF-induced chronic inflammation and proliferation. Most importantly, in the multiple intestinal neoplasia (Min) mouse model, treatment with MDL 72527 reduces ETBF-induced colon tumorigenesis by 69% (P < 0.001). The results of these studies indicate that SMO is a source of bacteria-induced ROS directly associated with tumorigenesis and could serve as a unique target for chemoprevention.

Induction of Persistent Colitis by a Human Commensal, Enterotoxigenic Bacteroides fragilis, in Wild-Type C57BL/6 Mice

ABSTRACT Enterotoxigenic Bacteroides fragilis (ETBF) causes diarrhea and is implicated in inflammatory bowel diseases and colorectal cancer. The only known ETBF virulence factor is the Bacteroides fragilis toxin (BFT), which induces E-cadherin cleavage, interleukin-8 secretion, and epithelial cell proliferation. A murine model for ETBF has not been characterized. Specific pathogen-free (SPF) C57BL/6J or germfree 129S6/SvEv mice were orally inoculated with wild-type ETBF (WT-ETBF) strains, a nontoxigenic WT strain of B. fragilis (WT-NTBF), WT-NTBF overexpressing bft (rETBF), or WT-NTBF overexpressing a biologically inactive mutated bft (rNTBF). In SPF and germfree mice, ETBF caused colitis but was lethal only in germfree mice. Colonic histopathology demonstrated mucosal thickening with inflammatory cell infiltration, crypt abscesses, and epithelial cell exfoliation, erosion, and ulceration. SPF mice colonized with rETBF mimicked WT-ETBF, whereas rNTBF caused no histopathology. Intestinal epithelial E-cadherin was rapidly cleaved in vivo in WT-ETBF-colonized mice and in vitro in intestinal tissues cultured with purified BFT. ETBF mice colonized for 16 months exhibited persistent colitis. BFT did not directly induce lymphocyte proliferation, dendritic cell stimulation, or Toll-like receptor activation. In conclusion, WT-ETBF induced acute then persistent colitis in SPF mice and rapidly lethal colitis in WT germfree mice. Our data support the hypothesis that chronic colonization with the human commensal ETBF can induce persistent, subclinical colitis in humans.

Toll-Like Receptor 2 Signaling Protects Mice from Tumor Development in a Mouse Model of Colitis-Induced Cancer

Inflammatory bowel disease (IBD) is a disorder of chronic inflammation with increased susceptibility to colorectal cancer. The etiology of IBD is unclear but thought to result from a dysregulated adaptive and innate immune response to microbial products in a genetically susceptible host. Toll-like receptor (TLR) signaling induced by intestinal commensal bacteria plays a crucial role in maintaining intestinal homeostasis, innate immunity and the enhancement of intestinal epithelial cell (IEC) integrity. However, the role of TLR2 in the development of colorectal cancer has not been studied. We utilized the AOM-DSS model for colitis-associated colorectal cancer (CAC) in wild type (WT) and TLR2−/− mice. Colons harvested from WT and TLR2−/− mice were used for histopathology, immunohistochemistry, immunofluorescence and cytokine analysis. Mice deficient in TLR2 developed significantly more and larger colorectal tumors than their WT controls. We provide evidence that colonic epithelium of TLR2−/− mice have altered immune responses and dysregulated proliferation under steady-state conditions and during colitis, which lead to inflammatory growth signals and predisposition to accelerated neoplastic growth. At the earliest time-points assessed, TLR2−/− colons exhibited a significant increase in aberrant crypt foci (ACF), resulting in tumors that developed earlier and grew larger. In addition, the intestinal microenvironment revealed significantly higher levels of IL-6 and IL-17A concomitant with increased phospho-STAT3 within ACF. These observations indicate that in colitis, TLR2 plays a protective role against the development of CAC.

Early infliximab trough levels are associated with persistent remission in pediatric patients with inflammatory bowel disease.

Background:Low infliximab (IFX) trough levels and high anti-infliximab antibodies (ATI) levels are associated with loss of response to IFX. Optimizing IFX levels to maintain target concentrations before loss of response may improve long-term efficacy. We hypothesized that trough levels at week 14 are predictive of IFX durability. Methods:A prospective observational cohort of pediatric patients with inflammatory bowel disease initiating IFX had IFX and ATI levels drawn at weeks 14 and 54. Primary outcome was week 54 persistent remission (PR), defined as clinical remission without IFX dose intensification. Univariate analyses tested associations of week 14 IFX (IFX14) and ATI (ATI14) levels, clinical and laboratory data with week 54 outcomes. Receiver operating curve analysis and positive and negative predictive values for IFX14 cut-off points were examined. Results:Of 58 patients enrolled, 8 (13%) stopped IFX before week 14 and 4 discontinued IFX between weeks 14 and 54. IFX14 level (P = 0.03), baseline C-reactive protein (CRP) level (P = 0.01), and week 14 CRP (CRP14) level (P = 0.0001) were associated with PR. A model with IFX14 levels predicting PR had an area under the receiver operating curve of 0.68 and a model with both IFX14 level and CRP14 >1.0 mg/dL had an area under the receiver operating curve of 0.74. IFX14 cut points of >3, >4, and >7 µg/mL had positive predictive values of 64%, 76% and 100%, respectively, for predicting PR. Conclusions:Both IFX levels and CRP at week 14 were significantly associated with week 54 efficacy. A model combining both CRP and IFX at week 14 may help predict remission at week 54.

Prediction of complicated disease course for children newly diagnosed with Crohn's disease: a multicentre inception cohort study

BACKGROUND Stricturing and penetrating complications account for substantial morbidity and health-care costs in paediatric and adult onset Crohns disease. Validated models to predict risk for complications are not available, and the effect of treatment on risk is unknown. METHODS We did a prospective inception cohort study of paediatric patients with newly diagnosed Crohns disease at 28 sites in the USA and Canada. Genotypes, antimicrobial serologies, ileal gene expression, and ileal, rectal, and faecal microbiota were assessed. A competing-risk model for disease complications was derived and validated in independent groups. Propensity-score matching tested the effect of anti-tumour necrosis factor α (TNFα) therapy exposure within 90 days of diagnosis on complication risk. FINDINGS Between Nov 1, 2008, and June 30, 2012, we enrolled 913 patients, 78 (9%) of whom experienced Crohns disease complications. The validated competing-risk model included age, race, disease location, and antimicrobial serologies and provided a sensitivity of 66% (95% CI 51-82) and specificity of 63% (55-71), with a negative predictive value of 95% (94-97). Patients who received early anti-TNFα therapy were less likely to have penetrating complications (hazard ratio [HR] 0·30, 95% CI 0·10-0·89; p=0·0296) but not stricturing complication (1·13, 0·51-2·51; 0·76) than were those who did not receive early anti-TNFα therapy. Ruminococcus was implicated in stricturing complications and Veillonella in penetrating complications. Ileal genes controlling extracellular matrix production were upregulated at diagnosis, and this gene signature was associated with stricturing in the risk model (HR 1·70, 95% CI 1·12-2·57; p=0·0120). When this gene signature was included, the models specificity improved to 71%. INTERPRETATION Our findings support the usefulness of risk stratification of paediatric patients with Crohns disease at diagnosis, and selection of anti-TNFα therapy. FUNDING Crohns and Colitis Foundation of America, Cincinnati Childrens Hospital Research Foundation Digestive Health Center.

Platelet-activating factor induces TLR4 expression in intestinal epithelial cells: implication for the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in neonatal intensive care units, however its pathogenesis is not completely understood. We have previously shown that platelet activating factor (PAF), bacteria and TLR4 are all important factors in the development of NEC. Given that Toll-like receptors (TLRs) are expressed at low levels in enterocytes of the mature gastrointestinal tract, but were shown to be aberrantly over-expressed in enterocytes in experimental NEC, we examined the regulation of TLR4 expression and signaling by PAF in intestinal epithelial cells using human and mouse in vitro cell lines, and the ex vivo rat intestinal loop model. In intestinal epithelial cell (IEC) lines, PAF stimulation yielded upregulation of both TLR4 mRNA and protein expression and led to increased IL-8 secretion following stimulation with LPS (in an otherwise LPS minimally responsive cell line). PAF stimulation resulted in increased human TLR4 promoter activation in a dose dependent manner. Western blotting and immunohistochemical analysis showed PAF induced STAT3 phosphorylation and nuclear translocation in IEC, and PAF-induced TLR4 expression was inhibited by STAT3 and NFκB Inhibitors. Our findings provide evidence for a mechanism by which PAF augments inflammation in the intestinal epithelium through abnormal TLR4 upregulation, thereby contributing to the intestinal injury of NEC.

Clostridium difficile carriage and serum antitoxin responses in children with inflammatory bowel disease.

Background:Adults with inflammatory bowel disease (IBD) have a high prevalence of Clostridium difficile carriage, but little data exist regarding pediatric patients with IBD. Serum antibody responses to C. difficile toxins in correlation with organism carriage are not described in IBD. This study determines the prevalence of C. difficile carriage and compares serum antibody responses to C. difficile toxins in pediatric outpatients with IBD and controls. Methods:Fecal and serum samples were prospectively collected from pediatric outpatients with IBD (n = 85) and age-matched controls (n = 78). Initial and follow-up stool samples were tested using cytotoxigenic C. difficile culture and PCR to detect the toxin B gene. Pulsed-field gel electrophoresis determined the strain type. Enzyme-linked immunosorbent assay determined serum immunoglobulin responses to C. difficile toxins. Results:Asymptomatic C. difficile carriage was significantly greater in IBD (17%) versus controls (3%) (P = 0.012). IBD type, disease severity, IBD therapy, recent antibiotics, and hospitalizations were not associated with carriage. Proton pump inhibitor use was significantly higher in patients with C. difficile carriage (54% versus 25%, P < 0.05). North American pulsed-field (NAP) strain carriage varied over time in patients colonized with C. difficile. A significantly greater proportion of patients with IBD had a positive serum antibody response to toxin A (69%) compared with controls (53%) (P < 0.05). Conclusions:Asymptomatic toxigenic C. difficile carriage was increased in pediatric outpatients with IBD compared with controls. Proton pump inhibitor use was associated with increased carriage. Antibody responses to C. difficile toxins were increased in IBD, potentially promoting asymptomatic colonization. Future studies should identify the risk factors for symptomatic C. difficile in pediatric IBD.

A 10-year, single tertiary care center experience on the durability of infliximab in pediatric inflammatory bowel disease.

Background:Despite increasing use of infliximab (IFX) in children with Crohns disease (CD) and ulcerative colitis (UC), long-term durability and safety of IFX beyond 1 year is limited in pediatric inflammatory bowel disease. Methods:We performed a 10-year single-center retrospective cohort study of 188 patients initiating IFX at <21 years of age with 1-year minimum follow-up. Data were retrieved from medical records. IFX outcomes were defined as sustained remission in the absence of dose modification (sustained durable remission), recaptured response, and treatment failure. Adverse events, anti-infliximab antibodies (ATI), and role of concomitant low-dose oral methotrexate (<10 mg/wk) on IFX durability were analyzed. Univariate associations and survival analysis were performed. Results:As of the last follow-up, 39% of patients with CD and 29% of patients with UC achieved sustained durable remission and another 60% recaptured and maintained response. For CD, 88% remained on IFX at 1 year, 80% at 2 years, and 82% at 5 years. In UC, 70% avoided colectomy at 1 year. Of IFX failures, 25% with CD and 11% with UC developed ATI. The most common adverse event causing cessation of therapy was infusion reactions. Treatment limiting recurrent infections occurred in <1%, and 1 patient developed lymphoproliferative disease. Low-dose methotrexate did not influence any IFX outcomes. Conclusions:IFX is safe and effective for long-term maintenance therapy in pediatric patients with inflammatory bowel disease. IFX dose intensification can optimize durability and overcome loss of response. Loss of response is likely affected by development of ATI. Higher doses of oral methotrexate may be needed to optimize IFX.

Multi-Center Experience of Vedolizumab Effectiveness in Pediatric Inflammatory Bowel Disease.

Background:Though vedolizumab has received regulatory approval for the treatment of Crohns disease (CD) and ulcerative colitis (UC) in adults, there is increasing off-label use in children. Aims:To describe the experience with vedolizumab in pediatric inflammatory bowel disease (IBD) patients at 3 tertiary IBD centers and examine predictors of remission. Methods:A retrospective review identified pediatric IBD patients (age < 18 yrs) receiving vedolizumab. Data on demographics, disease behavior, location, activity, and previous treatments/surgeries were collected. Disease activity was assessed using the weighted pediatric CD activity index or pediatric UC activity index. Primary outcome was week 14 remission, defined as pediatric UC activity index <10 or weighted pediatric CD activity index <12.5. Descriptive statistics and univariate analyses were performed to examine associations of clinical characteristics with efficacy. Results:Fifty-two patients, 58% CD and 42% UC, initiated vedolizumab between June 2014 and August 2015. Median age at vedolizumab initiation was 14.9 (range 7–17) years. Ninety percent had failed ≥1 anti-tumor necrosis factor (TNF) agent. Week 14 remission rates for UC and CD were 76% and 42%, respectively (P < 0.05). Eighty percent of anti–TNF-naive patients experienced week 14 remission. At week 22, anti–TNF-naive patients had higher remission rates than TNF-exposed patients (100% versus 45%, P = 0.04). There were no infusion reactions or serious adverse events/infections. Conclusions:Our results suggest that vedolizumab is efficacious and safe in pediatric IBD patients, with UC patients experiencing earlier and higher rates of remission than CD patients. Anti–TNF-naive patients experienced higher remission rates than those with anti-TNF exposure. Controlled clinical trial data are needed to confirm these observations.