SIGIRR mutation identified in human necrotizing enterocolitis (NEC) disrupts STAT3-dependent microRNA expression in neonatal gut.

Abstract

BACKGROUND AND AIMS\nSingle Immunoglobulin Interleukin-1 Related Receptor (SIGIRR) is a major inhibitor of Toll-like Receptor (TLR) signaling. Our laboratory identified a novel SIGIRR stop mutation (p.Y168X) in an infant who succumbed to severe NEC. Herein, we investigated the mechanisms by which SIGIRR mutations induce TLR hyper-responsiveness in the neonatal gut disrupting postnatal intestinal adaptation.\n\n\nMETHODS\nCRISPR/Cas9 was used to general transgenic mice encoding the SIGIRR p.Y168X mutation. Ileal lysates, mouse intestinal epithelial cell (IEC) lysates and intestinal sections were used to assess inflammation, STAT3 phosphorylation, miRNA and Interleukin-1 Related Associated Kinase 1 (IRAK1) expression. Western blotting, qRT-PCR, and luciferase assays were performed to investigate SIGIRR-STAT3 signaling in human intestinal epithelial cells (HIEC) expressing wildtype or SIGIRR (p.Y168X) plasmids.\n\n\nRESULTS\nSigirrTg mice exhibited increased intestinal inflammation and NF-kB activation concomitant with decreased IEC expression of miR-146a and miR-155. Mechanistic studies in HIEC revealed that while SIGIRR induced STAT3-mediated expression of miR-146a and miR-155, the p.Y168X mutation disrupted SIGIRR mediated STAT3-dependent miRNA expression. ChIP and luciferase assays demonstrated that SIGIRR activation of STAT3-induced miRNA expression is dependent on IRAK1. Both in HIEC and in the mouse intestine, decreased expression of miR-146a observed with the p.Y168X mutation increased expression of IRAK1, a protein whose downregulation is important for postnatal gut adaptation.\n\n\nCONCLUSIONS\nOur results uncover a novel pathway (SIGIRR-STAT3-miRNA-IRAK1 repression) by which SIGIRR regulates postnatal intestine adaptation, which is disrupted by a SIGIRR mutation identified in human NEC. These data provide new insights into how human genetic mutations in SIGIRR identified in NEC result in loss of postnatal intestinal immune tolerance.