SIGIRR in the Neonatal Intestine

ABSTRACT Developmentally regulated programs that inhibit pathological activation of intestinal Toll like receptors (TLR) by gut microbiota are deficient in preterm infants. A life-threatening consequence of intestinal dysmaturity in preterm infants is a loss in barrier function to gut microbiota resulting in necrotizing enterocolitis (NEC), characterized by inflammation, bacterial invasion, and necrosis. A lack of insight into the immunogenetic mechanisms regulating intestinal barrier function and TLR sensitivity during postnatal gut adaptation has limited our knowledge of NEC. Our laboratory has a long-standing interest in studying how genetic mutations program deviant host-pathogen interactions underlying NEC. We have reported that Single Immunoglobulin Interleukin-1 Related Receptor (SIGIRR) inhibits TLR-mediated intestinal inflammation by inhibiting NFκB and inducing STAT3 signaling in neonatal mice. SIGIRR variants identified in human NEC exaggerate inflammation in intestinal epithelial cells (IEC) exposed to bacterial ligands, while SigirrMut mice encoding a SIGIRR mutation (p.Y168X) identified in NEC exhibit TLR hypersensitivity and impaired intestinal adaptation. Ongoing studies reveal that SIGIRR deficiency leads to a marked loss in the IEC adherens junction (AJ) and tight junction (TJ) proteins, E-cadherin and zona occludens 1, which programs increased gut permeability to pathogenic Gram-negative bacteria (GNB). Seeking mechanistic insight into the loss of E cadherin in SigirrMut mice, we investigated TWIST1, a bHLH family transcription factor that transcriptionally represses E-cadherin enabling cellular metastasis in cancer. IEC TWIST1 expression and nuclear translocation were induced in SigirrMut mice and in intestinal autopsy samples obtained from preterm infants with NEC. These pilot data inform our central hypothesis that SIGIRR promotes AJ assembly and IEC barrier function in the neonatal intestine by inhibiting TWIST1-mediated repression of E-cadherin. In sequential aims, we will the test this hypothesis by studying whether IEC-SIGIRR: 1) regulates AJ assembly and function in the neonatal gut, 2) inhibits TWIST1-mediated repression of IEC AJ and TJ function, and 3) prevents experimental NEC in mice by inhibiting TLR4 and TWIST1 signaling. SIGIRR regulation of IEC AJ and TJ component members, TWIST1 inhibition, and gut permeability to GNB will be evaluated in transgenic mice with global or IEC-specific loss/gain of SIGIRR function. Surgical pathology NEC samples and preterm infant enteroids transduced with SIGIRR variants identified in NEC will provide human corroboration to SIGIRR’s role in preserving AJ function through TWIST1 inhibition. We will also examine the opposing effects of SIGIRR and TWIST1 in regulating NEC vulnerability in Sigirr transgenic mice and preterm intestinal enteroids. We study SIGIRR as a paradigm to reveal how host genetics regulates intestinal barrier function during postnatal adaptation. This proposal impacts the field by discovering SIGIRR-TWIST1’s role in regulating IEC-AJ assembly and function using innovative strategies. The significance lies in revealing novel mechanisms by which genetic variants program the defective mucosal barrier in NEC with the potential for clinical translation.