Gonzalez, Michelle

Background & Aims: We examined treatment patterns and clinical outcomes in a contemporary cohort of children with newly diagnosed inflammatory bowel disease (IBD) in a large, commercially insured U.S. population. Methods: Using the OptumLabs® Data Warehouse, we created an inception cohort of physician-diagnosed IBD in children aged 6–17 years between 2007–23. We examined cumulative incidence of hospitalization, surgery, safety events, and medication use in patients with Crohn's disease (CD) and ulcerative colitis (UC), overall and stratified by diagnostic era, race and ethnicity, sex, and age at diagnosis. Results: Among 787 children with newly diagnosed CD (age at diagnosis, 13.4±2.9y; 57% male; 82% White, 5% Black, 7% Hispanic) followed over mean 4.6±3.0y, 5-year risk of IBD-related hospitalization and major surgery was 30% and 10%, respectively. In children diagnosed in 2019–23, 1-year cumulative use of corticosteroids and advanced therapy was 46% and 75%, respectively; advanced therapy use increased significantly over time (aHR, 3.43 [95% CI, 2.71–4.32] for 2019–23 vs. 2007–12), while corticosteroid use declined (0.67 [0.53–0.84]). Risk of IBD-related hospitalization was lower in children diagnosed between 2019–23 (0.65 [0.43–0.97]) compared with those diagnosed between 2007-12; surgical risk did not decline significantly, with confidence intervals compatible with a clinically meaningful reduction. Among 399 children with newly diagnosed UC (age at diagnosis, 14.0±3.0y; 53% male; 78% White, 5% Black, 7% Hispanic) followed over 4.5±3.0y, 5-year risk of IBD-related hospitalization and colectomy was 36% and 8%, respectively. Advanced therapy use increased significantly over time (4.46 [2.85–6.98] for 2019–23 vs. 2007–12), with no significant change in hospitalization or surgical risk across eras. No consistent racial and ethnic, age, or sex differences in advanced therapy use or clinical outcomes were identified in either CD or UC. Conclusions: In a contemporary cohort of commercially insured children with newly diagnosed IBD, early advanced therapy use has increased markedly over time. Surgical risk did not decline significantly in either disease; however, the study was not powered to exclude a clinically meaningful reduction, and these findings are hypothesis-generating rather than conclusive.

Background: The pathogenesis of human Inflammatory Bowel Disease (IBD) is a multifactorial process including interaction between immune, epithelial, and environmental factors. Growing evidence suggests that epithelial cell defects play major role in the pathogenesis of IBD. Epigenetic memory involves lasting, cell-type specific modifications to chromatin at multiple scales, which impacts transcriptional programs required for normal cell functions. A major gap in knowledge are the mechanisms involved in establishing and maintaining memory in cell types and diseases of the intestine. The objective of this study was to determine if intestinal epithelial cells (IECs) retain a memory of altered chromatin architectures and resultant regulation following inflammation. The hypothesis was that the IECs would show lasting altered chromatin structure landscapes (epigenetic memory) in response to inflammation. Methods: We have developed a multicellular model system of IBD using adult stem cell derived organoids from humans with IBD, from regions of the colon with and without active inflammation. We performed ATAC-seq, bulk RNA-seq and single cell RNA-seq on these organoids derived from the uninflamed and inflamed tissues of IBD patients, specifically Ulcerative Colitis (UC). Results: Regions of increased chromatin accessibility are enriched in organoids derived from areas of inflammation in UC patients compared to organoids from patient-matched, uninflamed regions. These open chromatin regions are associated with proinflammatory genes, with modest transcriptional alterations, pointing to an epigenetic inflammatory memory. Motif analysis for accessibly peaks in UC showed an enrichment of the AP-1 family of transcription factors and chromatin remodelers (SMARCA4). This data is consistent with other studies of inflammatory memory from other epithelial cell types, but we show this for the first time in IECs in the context of IBD. Conclusions: This study presents a novel line of investigation and development of model systems to examine epigenetic memory of IECs in human IBD. We expect that our studies will identify new chromatin-based mechanisms of IEC function that are impacted by inflammation. This research was funded by American Gastroenterological Association Research Scholar Award and KL2 Scholar Career Development Award (KL2 TR002379, National Center for Advancing Translational Science). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract IBD affects roughly 1.6 million US adults, with its prevalence increasing since the late 1990s. Characterized by unchecked intestinal inflammation, multiple proinflammatory cytokines including IL6 have been implicated as critical factors in this pathology. It has been established that EZH2 is a gene involved in the production of specific histone methyltransferase enzymes, as well as in the differentiation of Treg cells. The association between the Treg master transcription factor, Foxp3 and EZH2 results in the suppressive function of Tregs, with our previous work revealing impairment of this interaction under the influence of IL6 signaling in an inflamed intestinal setting. We investigated this further in a murine preventative T cell transfer model of colitis that involved injecting donor Treg cells that lacked IL6Ra, into immunocompromised mice. Histological scoring of the mice receiving the IL6Rko Tregs revealed a resolved inflammatory milieu and flow cytometric analysis of cells isolated from the spleens and MLNs showed increased Foxp3+ expression when compared to mice that received WT Tregs. Foxp3IL6Rako mice were subjected to a DSS model of colitis to further characterize the absence of the IL6R on Treg function in the intestinal milieu. Exacerbated signs of colitis were observed through histology and colon shortening of the conditional knockout mice when compared to their WT counterparts. We postulate that Tregs could be acting as a donor of sILR to promote intestinal integrity and healing. Further experiments will be conducted to define a role for the IL6R in the development of Foxp3+ cells, along with outlining the instrumental process of sIL6R donation in the intestinal milieu. Supported by Mayo Clinic Graduate School for Biomedical Sciences

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 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.