Microbial signals, MyD88, and lymphotoxin drive TNF-independent intestinal epithelial tissue damage

Abstract

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...

Distinct roles for LTalpha3 and LTalpha1beta2 produced by B cells contribute to their multi-faceted impact on ileitis

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