Abstract
Background
Despite numerous therapeutic advancements, inflammatory bowel disease (IBD) remains a major health burden due to the inefficiency of conventional therapies. We recently demonstrated the role of death receptor 3 (DR3), a member of the tumor necrosis factor receptor (TNFR) superfamily and receptor for the cytokine TL1A, in regulating the balance between effector [T helper type 1 (TH)1,TH2,TH17] and regulatory T cells during murine Crohn’s disease (CD)-like ileitis. New evidence suggests a potential role of IL-9-secreting TH9 cells in the pathogenesis of IBD. However, the role of the TL1A/DR3 system in the differentiation and function of TH9 cells in CD is not fully understood. Thus, here, we investigated the role of a functional DR3 receptor in disease progression and activation of downstream signaling pathways relevant to TH9 cell differentiation and pathogenicity.
Methods
Ileal tissues were collected from 20-wk SAMP/YitFc (SAMP,WT) and DR3×SAMP (KO) mice, and analyzed by H&E, RT-qPCR and ELISA. CD4+ cells were purified by MACS sorting from mesenteric lymph nodes (MLNs) and spleens of WT and KO mice. TH9 cell were generated in medium supplemented with IL-4, TGF-β and TL1A. TH9 cells were analyzed by flow cytometry and cell supernatants by ELISA. TH9 cells (5x105) were adoptively transferred by i.p. injection into Rag2-/- mice, which were euthanized after 6-weeks. Colons were analyzed by H&E and MLNs were immunophenotyped by flow cytometry.
Results
Lack of DR3 ameliorated ileitis in SAMP (histologic score:5.2±1.3vs17.2±3.1, P≤0.0001, n=6), reduced of ∼ 50% the expression of Baft3, PU.1, il-9 mRNAs and IL-9 protein (P≤0.003, n=12), and 3-fold increased Id3 mRNA (P<0.0007,n=12) in small intestinal tissues. CD4+-enriched TH9 cells from KO mice secreted 2-fold lower IL-9 and TNF (P≤0.0002,n=8), and 5-fold higher IL-10 protein level (P<0.0001, n=8) than those from WT mice. Using the Rag2-/- T-cell transfer model of chronic colitis, we found that recipients of TH9 cells from KO donors developed less severe colitis (histologic score: 0.9±0.1vs3.9±0.8, P=0.005, n=5) and retained only half of the frequency of circulating TH9 cells in MLNs (P≤0.02,n=5) compared to those receiving WT TH9 cells, at 6-week after adoptive transfer. A similar experiment is currently ongoing to verify the impact of WT and KO TH9 cells on ileo-colitis development in SAMP×Rag2-/- mice.
Conclusions
The TL1A/DR3 system heavily contributes to IL-9-signaling pathway activation and it is required for the secretion of higher amount of IL-9 protein by TH9 cells in SAMP mice. KO TH9 cells were less colitogenic that those isolated from WT mice, indicating a crucial role of functional DR3 receptor in TH9 pathogenicity. Collectively, our results hold great translational significance by showing that modulation of DR3 signaling may be a novel therapeutic target for the treatment of CD.
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