Neto, Manuel B. Braga

Background While FOXP3 + regulatory T cell (Treg) dysfunction has been linked to human inflammatory bowel disease (IBD), molecular mechanisms for disease pathophysiology are unclear. Tregs play a vital role in maintaining self‐tolerance and homeostasis by suppressing pathological immune responses. We recently documented that the transcription factor FOXP3 via histone methyltransferase EZH2 prevents multi‐organ inflammation in mice. Here, we tested the hypothesis that a physical FOXP3‐EZH2 protein interaction is essential for gene co‐repressive function critical for human Treg physiological functions. Methods Human FOXP3 mutations clinically relevant to IBD and Immune dysregulation, polyendocrinopathy, enteropathy, X‐linked [IPEX] syndrome pathogenesis were generated by site‐directed mutagenesis. T cells were isolated from human blood donors and lamina propria of Crohn's disease (CD) patients. Proximity Ligation Assay (PLA), confocal microscopy, and co‐immunoprecipitation (co‐IP) studies were performed for biochemical analysis. Dual Luciferase Reporter (DLR) Assay, Chromatin‐immunoprecipitation (ChIP) and RT‐PCR were performed to study gene regulation. Results PLA, confocal imaging and co‐IP studies revealed that constitutive FOXP3‐EZH2 interaction was uniquely abrogated by FOXP3 cysteine 232 to glycine mutation (C232G) that was previously associated with early‐onset IBD in contrast to IPEX‐inducing FOXP3 variants and wild‐type. As demonstrated by DLR experiment, C232 mutant consequently displayed impaired repression of model IL2 and IL17 promoters compared to wild‐type FOXP3. Establishing a mechanistic link between disrupted FOXP3‐EZH2 interaction and EZH2‐mediated gene repression, ChIP‐PCR revealed that C232 mutant lacked the capacity to recruit EZH2 to the IFNG locus as evident by diminished tri‐methylation of Histone 3 at lysine 27. Therefore, the impaired gene repressive function of C232 mutant is indicative of compromised Treg‐physiologic function. Exploring a generalizable mechanism, IL‐6 impaired FOXP3‐EZH2 interaction in a manner that correlated with increased tyrosine phosphorylation of FOXP3. Notably, IL‐6‐induced effects were reversed by a clinically available JAK1/2 inhibitor ruxolitinib. Remarkably, in lamina propria‐derived CD4 + T cells, reduced FOXP3‐EZH2 complex was a feature of CD patients in contrast to non‐CD control subjects. Conclusions Destabilized FOXP3‐EZH2 protein interaction and consequent impairment of EZH2‐mediated co‐repressor function may drive the pathological characteristics of intestinal inflammation. Our study indicates potential therapeutic approaches and mechanisms for improving Treg function during IBD pathogenesis via stabilizing FOXP3‐EZH2 interaction. Support or Funding Information This work was supported by NIDDK training grant T32DK007198 and the Pilot & Feasibility Award by the Center for Cell Signaling in Gastroenterology (P30DK084567) (to AOB), the National Institutes of Health NIDDK RO1 grant DK52913 (to RAU), NIAID RO1 grant AI089714 and the Leona Helmsley Charitable Trust (to WAF). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract Background Certolizumab pegol (CZP) has been successfully used for the treatment of Crohn disease (CD); however, real-world data regarding the utility of CZP trough levels (CTLs) are lacking. We aimed to correlate CTL with CD outcomes and to determine frequency of CZP antibodies. Methods Retrospective evaluation of all CD patients on maintenance CZP with CTL obtained between 2016 and 2019. Outcomes included: median CTL, presence of anti-CZP antibodies, biochemical response (BR), clinical response (CR), radiologic response (RR), radiologic healing (RH), and mucosal healing (MH). Results Seventy-seven CD patients were included. Median CTL was 18.9 µg/mL (interquartile range, 7.6–35.4). Twenty-three patients (27.3%) had positive antibody levels, with lower median CTL compared to patients with no antibodies (0.0 vs 29.8; P < 0.0001). Median CTL levels were higher in patients with vs without CR (30.4 vs 10.3 µg/mL; P = 0.0015) and RR (29.6 vs 5.8 µg/mL; P = 0.006). CZP dosing at least every 2 weeks was associated with higher odds of achieving MH (odds ratio, 3.2; 95% confidence interval, 1.03–9.97). CTL resulted in change in clinical management in 62.7% of cases and presence of CMZ antibodies was associated with an odds ratio of 5.83 (95% confidence interval, 1.57–21.73) of change in management. Receiver operating characteristic curve and quartile analysis suggested that CTL >19 µg/mL is associated with increased rates of CR and RR. Conclusions Higher CTL was significantly associated with CR and RR. The rate of CZP antibodies was 27.3%. Our data suggest maintenance CTL of ≥19 µg/mL should be achieved in order to optimize outcomes in clinical practice.

ObjectiveThe purpose of this study was to investigate the impact of obesity on disease activity and disease outcome in patients with inflammatory bowel disease.Patients and methodsThe impact of obesity on inflammatory bowel disease disease activity and outcome was retrospectively assessed in 3075 patients enrolled in the prospective nation‐wide Swiss inflammatory bowel disease cohort between July 2006 and September 2018. Baseline characteristics, disease activity and disease course in 325 obese inflammatory bowel disease patients (body mass index ≥30 kg/m2) were compared to 1725 normal weight inflammatory bowel disease individuals (body mass index 18.5‐24.9).ResultsAmong 3075 patients in the prospective Swiss inflammatory bowel disease cohort, 325 patients (10.6%) were obese, namely, 194 Crohn’s disease patients, 131 ulcerative colitis, and inflammatory bowel disease‐unclassified patients. Disease activity scores were elevated in obese Crohn’s disease (Crohn’s Disease Activity Index 33 vs 20, p = 0.001), but not ulcerative colitis patients. Obese Crohn’s disease, but not ulcerative colitis patients were less likely to be in remission based on a Crohn’s Disease Activity Index less than 100 and a calprotectin less than 100 ug/g. In a multivariate regression model, obesity was negatively associated with disease remission in Crohn’s disease (odds ratio 0.610, 95% confidence interval 0.402–0.926, p = 0.020), but not ulcerative colitis. Increased soft stool frequency was observed in both obese Crohn’s disease and ulcerative colitis patients. Adjusted Cox regression models revealed increased risk of complicated disease course in obese Crohn’s disease patients (hazard ratio 1.197, 95% confidence interval 1.046–1.370, p = 0.009). No association between obesity and disease progression, index treatment failure was seen neither in Crohn’s disease nor ulcerative colitis.ConclusionObesity is associated with decreased rates of disease remission and increased risk of complicated disease course in Crohn’s disease over a six‐year follow‐up period. No effects were seen on disease progression and index treatment failure neither in Crohn’s disease nor ulcerative colitis.

AbstractBackgroundAn association between inflammatory bowel disease (IBD) and obesity has been observed. Little is known about the effect of weight loss on IBD course. Our aim was to determine the impact of bariatric surgery on long-term clinical course of obese patients with IBD, either Crohn's disease (CD) or ulcerative colitis (UC).MethodsPatients with IBD who underwent bariatric surgery subsequent to IBD diagnosis were identified from 2 tertiary IBD centers. Complications after bariatric surgery were recorded. Patients were matched 1:1 for age, sex, IBD subtype, phenotype, and location to patients with IBD who did not undergo bariatric surgery. Controls started follow-up at a time point in their disease similar to the disease duration in the matched case at the time of bariatric surgery. Inflammatory bowel disease medication usage and disease-related complications (need for corticosteroids, hospitalizations, and surgeries) among cases and controls were compared.ResultsForty-seven patients met inclusion criteria. Appropriate matches were found for 25 cases. Median follow-up among cases (after bariatric surgery) and controls was 7.69 and 7.89 years, respectively. Median decrease in body mass index after bariatric surgery was 12.2. Rescue corticosteroid usage and IBD-related surgeries were numerically less common in cases than controls (24% vs 52%; odds ratio [OR], 0.36; 95% confidence interval [CI], 0.08–1.23; 12% vs 28%; OR, 0.2; 95% CI, 0.004–1.79). Two cases and 1 control were able to discontinue biologics during follow-up.ConclusionsInflammatory bowel disease patients with weight loss after bariatric surgery had fewer IBD-related complications compared with matched controls. This observation requires validation in a prospective study design.

Abstract Background The aetiology of Crohn’s disease [CD] involves immune dysregulation in a genetically susceptible individual. Genome-wide association studies [GWAS] have identified 200 loci associated with CD, ulcerative colitis, or both, most of which fall within non-coding DNA regions. Long non-coding RNAs [lncRNAs] regulate gene expression by diverse mechanisms and have been associated with disease activity in inflammatory bowel disease. However, disease-associated lncRNAs have not been characterised in pathogenic immune cell populations. Methods Terminal ileal samples were obtained from 22 CD patients and 13 controls. RNA from lamina propria CD4+ T cells was sequenced and long intergenic non-coding RNAs [lincRNAs] were detected. Overall expression patterns, differential expression [DE], and pathway and gene enrichment analyses were performed. Knockdown of novel lincRNAs XLOC_000261 and XLOC_000014 was performed. Expression of Th1 or Th17-associated transcription factors, T-bet and RORγt, respectively, was assessed by flow cytometry. Results A total of 6402 lincRNAs were expressed, 960 of which were novel. Unsupervised clustering and principal component analysis showed that the lincRNA expression discriminated patients from controls. A total of 1792 lincRNAs were DE, and 295 [79 novel; 216 known] mapped to 267 of 5727 DE protein-coding genes. The novel lincRNAs were enriched in inflammatory and Notch signalling pathways [p <0.05]. Furthermore, DE lincRNAs in CD patients were more frequently found in DNA regions with known inflammatory bowel disease [IBD]-associated loci. The novel lincRNA XLOC_000261 negatively regulated RORγt expression in Th17 cells. Conclusions We describe a novel set of DE lincRNAs in CD-associated CD4+ cells and demonstrate that novel lincRNA XLOC_000261 appears to negatively regulate RORγt protein expression in Th17 cells.