Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of two IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20 and Abin-1-deficient IECs were sensitized to TNF-independent, TNFR-1-mediated death in response to lymphotoxin alpha (LT⍺) homotrimers. Blockade of LT⍺ in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LT⍺ all contribute to TNF-independent intestinal injury.Anti-TNF therapy remains one of the most effective approaches for treating Crohn's disease (CD) and ulcerative colitis (UC), but roughly one-third of patients have no response and one-third of patients lose response over time (46)(47)(48)(49). Therefore, understanding the gene products that control TNF-independent IEC injury could have significant translational relevance for anti-TNF non-responders. To better understand the pathways leading to TNF-independent IEC injury we performed in vivo and in vitro analysis of IECs after acute simultaneous deletion of A20 and Abin-1. RESULTS Germ-free A20/Abin-1 T-ΔIEC Tnf -/mice are protected from TNF-independent IEC deathMice with floxed A20 (A20 fl/fl ) and floxed Abin-1 (Abin-1 fl/fl ) on a Vil-cre-ER T2+ background (A20/Abin-1 T-ΔIEC ) undergo acute deletion of A20 and Abin-1 in IECs upon treatment with tamoxifen, culminating in spontaneous apoptotic IEC death, severe enterocolitis, and rapid mouse lethality ( 9). This death occurs on a Tnf +/+ or Tnf -/background, demonstrating the important role of TNF-independent death in this model. Tamoxifen delivery by intraperitoneal (i.p.) oil injection has been reported to cause peritoneal inflammation, foam cell formation, and depletion of resident macrophages (50). To exclude the possibility that sterile peritonitis contributes to TNFindependent death in A20/Abin-1 T-ΔIEC Tnf -/mice, we treated mice with tamoxifen by oral gavage rather than i.p. A higher dose of tamoxifen was required to delete A20 and Abin-1 in IECs from the small intestine and colon by oral gavage (Supplemental Figure 1A), and with this approach A20/Abin-1 T-ΔIEC Tnf -/mice died with similar kinetics as A20/Abin-1 T-ΔIEC mice (Figure 1A). Enteroids derived from A2...
Ulcerative colitis (UC) is an inflammatory intestinal disorder driven by mucosal immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin monoclonal antibody that is effective for treating UC. VDZ is thought to primarily inhibit lymphocyte trafficking to the intestine, but its effect on other cell subsets is less well characterized. To identify the inflammatory cells that contribute to colitis and respond to VDZ, we performed a single-cell transcriptomic and proteomic analysis of peripheral blood and colonic biopsies in healthy controls (HC) and patients with UC on either aminosalicylates or VDZ. We identified mononuclear phagocytes (MNPs) as a primary target of VDZ, with comparatively modest effects on lymphocytes. Spatial proteomics and transcriptomics demonstrated increased density and proximity of MNP and fibroblast subsets in UC biopsies when compared to HC, with inhibition by VDZ. VDZ non-responders were enriched for activated fibroblast and MNP signatures in a validation cohort.
Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of two IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitizes mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion is rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provides only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protects against death after acute deletion of A20 and Abin-1 in IECs. A20 and Abin-1-deficient IECs are sensitized to TNF-independent, TNFR-1-mediated death in response to lymphotoxin alpha (LTα) homotrimers. Blockade of LTα in vivo reduces weight loss and improves survival when combined with partial deletion of MyD88. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.SUMMARYHere we show that germ-free derivation, MyD88 deletion, combined Ripk3 and Casp8 deletion, or anti-LTα, all reduce TNF-independent intestinal injury after A20 and Abin-1 deletion.
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