Jessica Friton

Autologous Mesenchymal Stem Cells, Applied in a Bioabsorbable Matrix, for Treatment of Perianal Fistulas in Patients With Crohn's Disease

In patients with Crohns disease, perianal fistulas recur frequently, causing substantial morbidity. We performed a 12-patient, 6-month, phase 1 trial to determine whether autologous mesenchymal stem cells, applied in a bioabsorbable matrix, can heal the fistula. Fistula repair was not associated with any serious adverse events related to mesenchymal stem cells or plug placement. At 6 months, 10 of 12 patients (83%) had complete clinical healing and radiographic markers of response. We found placement of mesenchymal stem cell-coated matrix fistula plugs in 12 patients with chronic perianal fistulas to be safe and lead to clinical healing and radiographic response in 10 patients. ClinicalTrials.gov Identifier: NCT01915927.

The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD)

Regulatory T (Treg) cells expressing the transcription factor FOXP3 play a pivotal role in maintaining immunologic self-tolerance. We and others have shown previously that EZH2 is recruited to the FOXP3 promoter and its targets in Treg cells. To further address the role for EZH2 in Treg cellular function, we have now generated mice that lack EZH2 specifically in Treg cells (EZH2Δ/ΔFOXP3+). We find that EZH2 deficiency in FOXP3+ T cells results in lethal multiorgan autoimmunity. We further demonstrate that EZH2Δ/ΔFOXP3+ T cells lack a regulatory phenotype in vitro and secrete proinflammatory cytokines. Of special interest, EZH2Δ/ΔFOXP3+ mice develop spontaneous inflammatory bowel disease. Guided by these results, we assessed the FOXP3 and EZH2 gene networks by RNA sequencing in isolated intestinal CD4+ T cells from patients with Crohns disease. Gene network analysis demonstrates that these CD4+ T cells display a Th1/Th17-like phenotype with an enrichment of gene targets shared by FOXP3 and EZH2. Combined, these results suggest that the inflammatory milieu found in Crohns disease could lead to or result from deregulation of FOXP3/EZH2-enforced T cell gene networks contributing to the underlying intestinal inflammation.

O-015 YI Alterations in the FOXP3-EZH2 Pathway Associates with Increased Susceptibility to Colitis in Both Mice and Human.

Background:Crohns disease is a common intestinal inflammatory disorder of uncertain etiology and incomplete treatment options. It is characterized by lesions infiltrated by inflammatory CD4+ lymphocytes; yet the mechanism of CD4+ mediated pathophysiology is unclear. Through whole genome approaches on clinical cohorts of CD patients, combined with functional in-vivo and in-vitro murine data, we sought to identify and evaluate aberrant transcriptional gene networks in disease-associated CD4+ cells. Methods:We comparatively studied the expression profile in CD4+ lymphocytes isolated from the Ileum of 21 CD-affected individuals and 12 age/gender matched control individuals. Utilizing RNA-seq, we conducted upstream target analysis to identify top disease associated regulatory networks. Potential coordinated function between FOXP3 and EZH2 was identified, and common gene targets were interrogated for differential expression and pathway analysis.We crossed FOXP3-driven CRE recombinase expressing mice (B6129S-Tg(Foxp3-EGFP/cre)1aJbs/J) with EZH2fl/fl animals to better define the effect of EZH2 ablation on Treg function. Splenocytes were analyzed by cell surface markers for T cell distribution and phenotype. EZH2&Dgr;/&Dgr; FOXP3+ cell function was determined by in-vitro Treg suppressor function assay. Cytokine analyses were performed on supernatants of stimulated EZH2&Dgr;/&Dgr;FOXP3+ splenocytes and serum from 14 to 17 days old FOXP3-CRE;EZH2&Dgr;/&Dgr; mice. FOXP3-CRE;EZH2&Dgr;/&Dgr; or animals treated with EZH2 inhibitors received 3% DSS in an acute model of colitis. Furthermore, RB45high transfer utilizing WT or EZH2&Dgr;/&Dgr;Tregs was used in a chronic model of colitis. Results:Differential expression analysis between CD and CTRL samples revealed 5328 statistically significant Differentially Expressed Genes (DEGs). Principle component analysis, and hierarchical clustering, separated patients from control subjects. Among the DEGs, 83 were well-defined transcription factors (TFs) identified by Master Regulatory Analysis. The transcription factor FOXP3 and its associated regulatory network emerged as a highly differentially expressed pathway in diseased samples by Master Regulatory Analysis, Ingenuity Pathway Analysis, and Gene Set Enrichment Analysis. Additionally, common gene targets for both FOXP3 and EZH2 were concurrently up regulated in disease. Genetic ablation of EZH2 in mouse FOXP3+ cells resulted in Treg cells converted to a TH1/TH17- like effector phenotype, a pattern shared by disease associated CD4+ T cells. These cells exhibited pro-inflammatory cell surface markers, cytokine expression, and reduced in-vitro suppressive capacity. EZH2&Dgr;/&Dgr; Tregs failed to mitigate both DSS and T cell mediated colitis, and mice treated with the EZH2 inhibitor DZNep were also more susceptible to DSS colitis. Conclusions:In conclusion, we compare EZH2/Foxp3 co-regulation of gene networks between Crohns lesions and controls, and in multiple mouse models, to clarify the role of epigenetic regulation in this disease process. Multiple colitis disease models show us that interfering with EZH2 by genetic or pharmaceutical methods results in increased susceptibility to colitis. We postulate the inflammatory milieu found in IBD modulates EZH2 function, thereby making it less able to co-repress non-Treg differentiation programs even in the presence of upregulated FOXP3. Further investigation into EZH2 mutations, signaling cascades and relevant post-translation modifications to the FOXP3-EZH2 gene network may assist to elucidate and predict disease progression and/or severity.