TNF IS REQUIRED FOR SPONTANEOUS INNATE COLITIS IN MICE LACKING ADAPTIVE IMMUNITY

Abstract

Abstract BACKGROUND Colitis involves excessive inflammation driven by the reciprocal interaction of innate and adaptive immune cells. To better understand how innate immunity contributes to colitis, we developed a spontaneous innate model of colitis that is highly penetrant, early onset, and driven by microbes. TNF is a cytokine produced predominantly by T cells but also by innate and non-immune cells and contributes to IBD in many patients. We therefore examined the role of TNF in innate immune mediated colitis. METHODOLOGY We developed a 100% penetrant mouse model of colitis by crossing villin transgenic expression of tnfaip3 (villin-tnfaip3) to rag1 null mice (rag1-/-), designated as TRAG mice (villin-tnfaip3 x rag1-/-). TRAG mice are characterized by inflammation of the colon in the absence of T and B cells. We crossed TRAG mice with tnfa-/- mice (tnfa-/- x TRAG) to elucidate the role of TNF in this model. The histopathological score of disease was evaluated by H&E, and the innate immune populations characterized by multi-color flow cytometry. Apoptosis, necrosis, and cell death were evaluated histologically by localizing cleaved caspase-3, phosho-MLKL and TUNEL positive cells. Secretion of cytokines from intestinal tissue in culture was quantified by ELISA. RESULTS TRAG mice develop early-onset colitis that is driven by microbes but is not transmissible to rag1-/- littermates. TRAG mice showed histological signs of colitis and a high infiltration of leukocytes in the intestinal mucosa without concurrent systemic inflammation. This colitis was not observed in tnfa-/- x TRAG mice. TRAG mice exhibited a pro-inflammatory cytokine signature with elevated secretion of IL1a and IL1b but not IL-18 or IL-33, which was not observed in mucosa from tnfa-/- x TRAG mice. LPLs from TRAG colons indicated TNF production by inflammatory monocytes, monocytes, and neutrophils. Monocytes and macrophages from the TRAG mouse mucosa, but not the tnfa-/- x TRAG mucosa, showed an M1 profile defined by elevated iNOS and decreased arginase. Increased crypt cell death was evident in TRAG colons, and this was significantly diminished in the crypts of tnfa-/- x TRAG colons. Early aggressive treatment of TRAG mice with anti-TNF antibody, but not anti-IL1a antibody, prevented colitis in TRAG mice. CONCLUSIONS TNF drives innate colitis featuring increased infiltration of mucosal leukocytes, increased secretion of IL1a and IL1b, skewing of macrophages to an M1 profile, and increased cell death in colonic crypts. Even in the absence of T cell derived cytokines, including TNF, innate sources of TNF are sufficient to drive colitis and early use of anti-TNF therapy is effective against this innate form of colitis. We are currently investigating the signals and forms of cell death that are driven by TNF in this model.