Sandra C. Kim

Enterococcus faecalis metalloprotease compromises epithelial barrier and contributes to intestinal inflammation.

BACKGROUND & AIMS Matrix metalloproteases (MMPs) mediate pathogenesis of chronic intestinal inflammation. We characterized the role of the gelatinase (GelE), a metalloprotease from Enterococcus faecalis, in the development of colitis in mice. METHODS Germ-free, interleukin-10-deficient (IL-10(-/-)) mice were monoassociated with the colitogenic E faecalis strain OG1RF and isogenic, GelE-mutant strains. Barrier function was determined by measuring E-cadherin expression, transepithelial electrical resistance (TER), and translocation of permeability markers in colonic epithelial cells and colon segments from IL-10(-/-) and TNF(ΔARE/Wt) mice. GelE specificity was shown with the MMP inhibitor marimastat. RESULTS Histologic analysis (score 0-4) of E faecalis monoassociated IL-10(-/-) mice revealed a significant reduction in colonic tissue inflammation in the absence of bacteria-derived GelE. We identified cleavage sites for GelE in the sequence of recombinant mouse E-cadherin, indicating that it might be degraded by GelE. Experiments with Ussing chambers and purified GelE revealed the loss of barrier function and extracellular E-cadherin in mice susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) before inflammation developed. Colonic epithelial cells had reduced TER and increased translocation of permeability markers after stimulation with GelE from OG1RF or strains of E faecalis isolated from patients with Crohns disease and ulcerative colitis. CONCLUSIONS The metalloprotease GelE, produced by commensal strains of E faecalis, contributes to development of chronic intestinal inflammation in mice that are susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) by impairing epithelial barrier integrity.

Transforming Growth Factor-β1 Inhibits Non-pathogenic Gramnegative Bacteria-induced NF-κB Recruitment to the Interleukin-6 Gene Promoter in Intestinal Epithelial Cells through Modulation of Histone Acetylation

We have shown that non-pathogenic enteric Gramnegative Bacteroides vulgatus induces RelA phosphorylation, NF-κB activation, and proinflammatory gene expression in primary and intestinal epithelial cell (IEC) lines. We now demonstrate the transient induction of nuclear phospho-RelA (day 3) followed by persistent activation of phospho-Smad2 (days 3 and 7) in IEC from mucosal tissue sections of B. vulgatus-monoassociated rats, indicating that both NF-κB and transforming growth factor-β1 (TGF-β1) signaling are induced in vivo following bacterial colonization. Interestingly, TGF-β1 inhibited B. vulgatus- and lipopolysaccharide (LPS)-induced NF-κB transcriptional activity as well as interleukin-6 (IL-6) mRNA accumulation and protein secretion in IEC. The inhibitory effect of TGF-β1 is mediated independently of B. vulgatus/LPS-induced IκBα, Akt, and RelA phosphorylation as well as NF-κB DNA binding activity. Moreover, the specific histone deacetylase inhibitor trichostatin A blocked B. vulgatus/LPS-induced histone acetylation/phosphorylation (Lys-9/Ser-10) and reversed TGF-β1-mediated inhibition of IL-6 gene expression. Chromatin immunoprecipitation analysis revealed that B. vulgatus/LPS-induced RelA recruitment to the IL-6 promoter is inhibited by TGF-β1 treatment. Adenoviral delivery of Smad7 and dominant negative Smad3 (SmadΔ3) reversed the TGF-β1-mediated inhibition of NF-κB transcriptional activity and NF-κB recruitment to the IL-6 promoter. In addition, TGF-β1 and Ad5Smad3/4 prevent B. vulgatus/LPS-induced CBP/p300 and p65 nuclear co-association. We concluded that the TGF-β1/Smad signaling pathway helps maintain normal intestinal homeostasis to commensal luminal enteric bacteria by regulating NF-κB signaling in IEC through altered histone acetylation.

Prevalence and epidemiology of overweight and obesity in children with inflammatory bowel disease

Background: Obesity is a significant public health threat to children in the United States. The aims were to: 1) Determine the prevalence of obesity in a multicenter cohort of children with inflammatory bowel disease (IBD); 2) Evaluate whether overweight and obese status is associated with patient demographics or disease characteristics. Methods: We used data from the ImproveCareNow Collaborative for pediatric IBD, a multicenter registry of children with IBD, collected between April 2007 and December 2009. Children ages 2–18 years were classified into body mass index (BMI) percentiles. Bivariate analyses and multivariate logistic regression were used to compare demographic and disease characteristics by overweight (BMI >85%) and obese (BMI >95%) status. Results: The population consisted of 1598 children with IBD. The prevalence of overweight/obese status in pediatric IBD is 23.6%, (20.0% for Crohns disease [CD] and 30.1% for ulcerative colitis [UC] and indeterminate colitis [IC]). African American race (odds ratio [OR] 1.64, 95% confidence interval [CI] 1.10–2.48) and Medicaid insurance (OR 1.67, 95% CI 1.19–2.34) were positively associated with overweight/obese status. Prior IBD‐related surgery (OR 1.73, 95% CI 1.07–2.82) was also associated with overweight and obese status in children with CD. Other disease characteristics were not associated with overweight and obesity in children with IBD. Conclusions: Approximately one in five children with CD and one in three with UC are overweight or obese. Rates of obesity in UC are comparable to the general population. Obese IBD patients may have a more severe disease course, as indicated by increased need for surgery. Sociodemographic risk factors for obesity in the IBD population are similar to those in the general population. (Inflamm Bowel Dis 2010;)

IL-10 Gene-Deficient Mice Lack TGF-β/Smad Signaling and Fail to Inhibit Proinflammatory Gene Expression in Intestinal Epithelial Cells after the Colonization with Colitogenic Enterococcus faecalis

Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in IL-10 gene-deficient mice (IL-10−/−). Bacteria-specific effects on the epithelium are difficult to dissect due to the complex nature of the gut microflora. We showed that IL-10−/− mice compared with wild-type mice fail to inhibit proinflammatory gene expression in native intestinal epithelial cells (IEC) after the colonization with colitogenic Gram-positive Enterococcus faecalis. Interestingly, proinflammatory gene expression was transient after 1 wk of E. faecalis monoassociation in IEC from wild-type mice, but persisted after 14 wk of bacterial colonization in IL-10−/− mice. Accordingly, wild-type IEC expressed phosphorylated NF-κB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10−/− mice triggered persistent RelA, but no Smad2 phosphorylation in IEC at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and proinflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice, but persisted in IL-10−/− IEC. Of note, TGF-β1-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-κB-dependent gene expression in IEC lines. In conclusion, E. faecalis-monoassociated IL-10−/−, but not wild-type mice lack protective TGF-β/Smad signaling and fail to inhibit TLR2-mediated proinflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-β in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.

Increased Effectiveness of Early Therapy With Anti-Tumor Necrosis Factor-α vs an Immunomodulator in Children With Crohn's Disease

BACKGROUND & AIMS Standard therapy for children newly diagnosed with Crohns disease (CD) includes early administration of immunomodulators after initial treatment with corticosteroids. We compared the effectiveness of early (≤3 mo after diagnosis) treatment with an anti-tumor necrosis factor (TNF)α with that of an immunomodulator in attaining clinical remission and facilitating growth of pediatric patients. METHODS We analyzed data from the RISK study, an observational research program that enrolled patients younger than age 17 diagnosed with inflammatory (nonpenetrating, nonstricturing) CD from 2008 through 2012 at 28 pediatric gastroenterology centers in North America. Patients were managed by physician dictate. From 552 children (median age, 11.8 y; 61% male; 63% with pediatric CD activity index scores >30; and median C-reactive protein level 5.6-fold the upper limit of normal), we used propensity score methodology to identify 68 triads of patients matched for baseline characteristics who were treated with early anti-TNFα therapy, early immunomodulator, or no early immunotherapy. We evaluated relationships among therapies, corticosteroid and surgery-free remission (pediatric CD activity index scores, ≤10), and growth at 1 year for 204 children. Treatment after 3 months was a covariate. RESULTS Early treatment with anti-TNFα was superior to early treatment with an immunomodulator (85.3% vs 60.3% in remission; relative risk, 1.41; 95% confidence interval [CI], 1.14-1.75; P = .0017), whereas early immunomodulator therapy was no different than no early immunotherapy (60.3% vs 54.4% in remission; relative risk, 1.11; 95% CI, 0.83-1.48; P = .49) in achieving remission at 1 year. Accounting for therapy after 3 months, early treatment with anti-TNFα remained superior to early treatment with an immunomodulator (relative risk, 1.51; 95% CI, 1.20-1.89; P = .0004), whereas early immunomodulator therapy was no different than no early immunotherapy (relative risk, 1.00; 95% CI, 0.75-1.34; P = .99). The mean height z-score increased compared with baseline only in the early anti-TNFα group. CONCLUSIONS In children newly diagnosed with comparably severe CD, early monotherapy with anti-TNFα produced better overall clinical and growth outcomes at 1 year than early monotherapy with an immunomodulator. Further data will be required to best identify children most likely to benefit from early treatment with anti-TNFα therapy.

ImproveCareNow: The development of a pediatric inflammatory bowel disease improvement network

&NA; There is significant variation in diagnostic testing and treatment for inflammatory bowel disease. Quality improvement science methods can help address unwarranted variations in care and outcomes. Methods: The ImproveCareNow Network was established under the sponsorship of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the American Board of Pediatrics as a prototype for a model of improving subspecialty care that included three components: 1) creating enduring multicenter collaborative networks of pediatric subspecialists, 2) sharing of performance data collected in patient registries, and 3) training in quality improvement. The network began with a focus on improving initial diagnostic testing and evaluation, the classification of the severity and extent of disease, the detection and treatment of inadequate nutrition and growth, and the appropriate dosing of immunomodulator medications. Changes are based on an evidence‐based model of chronic illness care involving the use of patient registries for population management, previsit planning, decision support, promoting self‐management, and auditing of care processes. Results: Currently, patients are being enrolled at 23 sites. Through 2009, data have been analyzed on over 2500 patients from over 7500 visits. Initial results suggest improvements in both care processes (e.g., appropriate medication dosing and completion of a classification bundle that includes the patients diagnosis, disease activity, distribution and phenotype, growth status, and nutrition status) and outcomes (e.g., the percentage of patients in remission). Conclusions: These improvements suggest that practice sites are learning how to apply quality improvement methods to improve the care of patients. (Inflamm Bowel Dis 2011;)

Improved Outcomes in a Quality Improvement Collaborative for Pediatric Inflammatory Bowel Disease

OBJECTIVES: Unintended variation in the care of patients with Crohn disease (CD) and ulcerative colitis (UC) may prevent achievement of optimal outcomes. We sought to improve chronic care delivery and outcomes for children with inflammatory bowel disease by using network-based quality improvement methods. METHODS: By using a modified Breakthrough Series collaborative structure, 6 ImproveCareNow Network care centers tested changes in chronic illness care and collected data monthly. We used an interrupted time series design to evaluate the impact of these changes. RESULTS: Data were available for 843 children with CD and 345 with UC. Changes in care delivery were associated with an increase in the proportion of visits with complete disease classification, measurement of thiopurine methyltransferase (TPMT) before initiation of thiopurines, and patients receiving an initial thiopurine dose appropriate to their TPMT status. These were significant in both populations for all process variables (P < .01) except for measurement of TPMT in CD patients (P = .12). There were significant increases in the proportion of CD (55%–68%) and UC (61%–72%) patients with inactive disease. There was also a significant increase in the proportion of CD patients not taking prednisone (86%–90%). Participating centers varied in the success of achieving these changes. CONCLUSIONS: Improvements in the outcomes of patients with CD and UC were associated with improvements in the process of chronic illness care. Variation in the success of implementing changes suggests the importance of overcoming organizational factors related to quality improvement success.

Dual-association of gnotobiotic Il-10−/− mice with 2 nonpathogenic commensal bacteria induces aggressive pancolitis

Background: Monoassociating gnotobiotic IL‐10‐deficient (−/−) mice with either nonpathogenic Enterococcus faecalis or a nonpathogenic Escherichia coli strain induces T‐cell‐mediated colitis with different kinetics and anatomical location (E. faecalis: late onset, distal colonic; E. coli: early onset, cecal). Hypothesis: E. faecalis and E. coli act in an additive manner to induce more aggressive colitis than disease induced by each bacterial species independently. Methods: Germ‐free (GF) inbred 129S6/SvEv IL‐10−/− and wildtype (WT) mice inoculated with nonpathogenic E. faecalis and/or E. coli were killed 3–7 weeks later. Colonic segments were scored histologically for inflammation (0 to 4) or incubated in media overnight to measure spontaneous IL‐12/IL‐23p40 secretion. Bacterial species were quantified by serial dilution and plated on culture media. Mesenteric lymph node (MLN) CD4+ cells were stimulated with antigen‐presenting cells pulsed with bacterial lysate (E. faecalis, E. coli, Bacteroides vulgatus) or KLH (unrelated antigen control). IFN‐&ggr; and IL‐17 levels were measured in the supernatants. Results: Dual‐associated IL‐10−/− (but not WT) mice developed mild‐to‐moderate pancolitis by 3 weeks that progressed to severe distal colonic‐predominant pancolitis with reactive atypia and duodenal inflammation by 7 weeks. NF‐&kgr;B was activated in the duodenum and colon in dual‐associated IL‐10−/− × NF‐&kgr;BEGFP mice. The aggressiveness of intestinal inflammation and the degree of antigen‐specific CD4+ cell activation were greater in dual‐ versus monoassociated IL‐10−/− mice. Conclusion: Two commensal bacteria that individually induce phenotypically distinct colitis in gnotobiotic IL‐10−/− mice act additively to induce aggressive pancolitis and duodenal inflammation. (Inflamm Bowel Dis 2007)

Short pediatric Crohn's disease activity index for quality improvement and observational research†

Background: Practical and objective instruments to assess pediatric Crohns disease (CD) activity are required for observational research and quality improvement. The objectives were: 1) to determine the feasibility of completing the Pediatric Crohns Disease Activity Index (PCDAI) and the Abbreviated PCDAI (APCDAI); and 2) to create a Short PCDAI by retaining and reweighting the most practical and informative components. Methods: Physicians in the ImproveCareNow Collaborative for pediatric inflammatory bowel disease (IBD) were asked to record components of the PCDAI and assign a Physician Global Assessment (PGA) of disease severity at each patient encounter. We assessed the feasibility of the PCDAI, the APCDAI, and the individual index components by determining the proportion of visits in which data were recorded. We created a short index by retaining and reweighting components of the PCDAI completed in ≥80% of visits. The feasibility of the Short PCDAI and its ability to discriminate between PGA categories were evaluated using descriptive statistics. Results: This study population included 1355 subjects with CD (6373 visits). The PCDAI and APCDAI were complete in 16.7% and 44.1% of visits, respectively. A Short PCDAI, including general well‐being, abdominal pain, stools, weight, abdominal exam, and extraintestinal manifestations were completed in 66.5% of visits. The correlation between the Short PCDAI and PGA was similar to that of the PCDAI (r = 0.60, P < 0.001 versus 0.61, P < 0.001). Conclusions: The Short PCDAI is a practical and valid tool to measure pediatric CD activity. Its use should facilitate quality improvement and observational research. (Inflamm Bowel Dis 2011;)

IL-10 gene-deficient mice lack TGF-beta/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation.

Abstract:  Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in interleukin‐10 geneeficient mice (IL‐10‐/‐). Bacteria‐specific effects on the epithelium are difficult to distinguish because of the complex nature of the gut microflora. We showed that IL‐10‐/‐ mice compared to wild‐type mice fail to inhibit pro‐inflammatory gene expression in native intestinal epithelial cells after the colonization with colitogenic Gram‐positive Enterococcus faecalis. Of interest, pro‐inflammatory gene expression was transient after 1 week of E. faecalis monoassociation in IECs from wild‐type mice but persisted after 14 weeks of bacterial colonization in IL‐10‐/‐ mice. Accordingly, wild‐type IECs expressed phosphorylated NF‐kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis‐monoassociated IL‐10‐/‐ mice triggered persistent RelA but no Smad2 phosphorylation in IECs at days 3, 7, 14, and 28. Consistent with the induction of TLR2‐mediated RelA phosphorylation and pro‐inflammatory gene expression in E. faecalis‐stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis‐monoassociated wild‐type mice but persisted in IL‐10‐/‐ IECs. Of note, TGF‐beta‐activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF‐kappa Bependent gene expression in E. faecalis‐stimulated IEC lines. In conclusion, E. faecalis‐monoassociated IL‐10‐/‐ but not wild‐type mice lack protective TGF‐beta/Smad signaling and fail to inhibit TLR2‐mediated pro‐inflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL‐10 and TGF‐beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.