TARGETING MYD88-JAK2 SIGNALING IN ABERRANTLY DIFFERENTIATED MESENCHYMAL PROGENITORS IN ULCERATIVE COLITIS

Abstract INTRODUCTION Three novel therapies have shown promise in managing moderate-to-severe ulcerative colitis (UC): fecal microbiota transplantation (FMT), mesenchymal stem cell (MSC) therapy, and targeting the JAK/STAT signaling pathway. Individually, these treatment approaches face issues with either safety concerns or limited efficacy due to disease relapses. Yet, the development of better strategies is hampered by limited knowledge about the cellular mechanisms driving chronic inflammation and disease relapse in UC. In UC, the number of aberrantly differentiated tissue resident MSCs in the colonic mucosa is increased. The mechanisms behind this abnormal MSC differentiation and how they affect MSC therapy in UC remain unclear. JAK2 signaling is involved in the differentiation of bone marrow derived (BM)-MSCs and previously we observed that stimulation of BM-MSCs with UC relevant microbial ligand LPS upregulated JAK2. We hypothesized that dysbiotic microbial ligands in UC promote aberrant MSC differentiation through JAK2 activation, reducing the efficacy of MSC therapy. METHODS Mesenchymal progenitor cells were isolated from human UC tissue. Wild-type and MyD88 mesenchymal lineage conditional knockout mice (Grem1CreMyD88KO) were used in two models of experimental colitis (TNBS-induced and oxazolone-induced colitis). Murine FMT and syngeneic MSC transplant were also used in these studies. RESULTS Analysis of publicly available single-cell RNA seq data showed increased numbers of mesenchymal cells co-expressing stem cell (POU5F1) and progenitor markers (Grem1), along with JAK2, in inflamed colon tissue from UC patients. Bulk RNA seq analysis of healthy colonic mesenchymal cells from normal tissue stimulated with the Gram-negative bacteria-derived TLR4 ligand, LPS, revealed upregulation of inflammatory genes IL-6, IL-33, PD-L1, and JAK2 signaling. Deletion of MyD88 in MSCs during the acute phase of TNBS-induced colitis improved symptoms and decreased UC-relevant inflammation concomitantly with downregulation of JAK2. In the oxazolone-induced model of colitis, depletion of dysbiotic microbiota with antibiotics and subsequent FMT resulted in reduced JAK2 expression in the colonic mucosa, and when combined with MSC therapy shown superior efficacy in improvement of clinical sign of colitis, improvement of colonic tissue architecture and reduction of UC-relevant inflammatory responses when compared to FMT or MSCs therapy alone. CONCLUSION Dysbiotic microbial ligands in UC contribute to the generation of abnormally differentiated MSCs with active JAK2 signaling and downstream molecules supporting inflammation in UC and this process involves MSC intrinsic MyD88 signaling. Further, our data suggests depletion of the dysbiotic microbiota in combination of FMT/MSCs therapy could counteract this effect and may offer a more effective approach to treating UC.