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SIGIRR Mutation in Human Necrotizing Enterocolitis (NEC) Disrupts STAT3-Dependent microRNA Expression in Neonatal Gut
Wei Yu1, Inamul Haque1, Aparna Venkatraman1
1Division of Neonatology, Department of Pediatrics, Children's Mercy Hospital, Kansas City, Missouri.
Background & Aims:
Single immunoglobulin interleukin-1-related receptor (SIGIRR) is a major inhibitor of Toll-like receptor signaling. Our laboratory identified a novel SIGIRR stop mutation (p.Y168X) in an infant who died of severe necrotizing enterocolitis (NEC). Herein, we investigated the mechanisms by which SIGIRR mutations induce Toll-like receptor hyper-responsiveness in the neonatal gut, disrupting postnatal intestinal adaptation.
Methods:
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was used to generate transgenic mice encoding the SIGIRR p.Y168X mutation. Ileal lysates, mouse intestinal epithelial cell (IEC) lysates, and intestinal sections were used to assess inflammation, signal transducer and activator of transcription 3 (STAT3) phosphorylation, microRNA (miRNA), and interleukin-1-related-associated kinase 1 (IRAK1) expression. Western blot, quantitative reverse-transcription polymerase chain reaction(qRT-PCR), and luciferase assays were performed to investigate SIGIRR-STAT3 signaling in human intestinal epithelial cells (HIEC) expressing wild-type or SIGIRR (p.Y168X) plasmids.
Results:
SigirrTg mice showed increased intestinal inflammation and nuclear factor-κB activation concomitant with decreased IEC expression of miR-146a and miR-155. Mechanistic studies in HIECs showed that although SIGIRR induced STAT3-mediated expression of miR-146a and miR-155, the p.Y168X mutation disrupted SIGIRR-mediated STAT3-dependent miRNA expression. Chromatin immunoprecipitation and luciferase assays showed that SIGIRR activation of STAT3-induced miRNA expression is dependent on IRAK1. Both in HIECs and in the mouse intestine, decreased expression of miR-146a observed with the p.Y168X mutation increased expression of IRAK1, a protein whose down-regulation is important for postnatal gut adaptation.
Conclusions:
Our 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.
Insights
A novel Single immunoglobulin interleukin-1-related receptor (SIGIRR) mutation disrupts immune tolerance in the neonatal gut, leading to necrotizing enterocolitis (NEC). This SIGIRR-STAT3-miRNA-IRAK1 pathway is crucial for postnatal intestinal adaptation.
Area of Science:
- Immunology
- Gastroenterology
- Genetics
Background:
- Single immunoglobulin interleukin-1-related receptor (SIGIRR) inhibits Toll-like receptor (TLR) signaling.
- A novel SIGIRR stop mutation (p.Y168X) was identified in an infant with severe necrotizing enterocolitis (NEC).
Purpose of the Study:
- Investigate mechanisms of SIGIRR mutations in neonatal gut TLR hyper-responsiveness.
- Determine how SIGIRR mutations disrupt postnatal intestinal adaptation.
Main Methods:
- Generated transgenic mice with the SIGIRR p.Y168X mutation using CRISPR/Cas9.
- Assessed inflammation, STAT3 phosphorylation, miRNA, and IRAK1 expression in mouse and human intestinal cells.
- Utilized Western blot, qRT-PCR, and luciferase assays to study SIGIRR-STAT3 signaling.
Main Results:
- SigirrTg mice exhibited increased intestinal inflammation and NF-κB activation with decreased miR-146a/miR-155 expression.
- The p.Y168X mutation impaired SIGIRR-mediated STAT3-dependent miRNA expression.
- Decreased miR-146a led to increased IRAK1 expression, hindering gut adaptation.
Conclusions:
- Uncovered a novel SIGIRR-STAT3-miRNA-IRAK1 repression pathway regulating postnatal intestinal adaptation.
- SIGIRR mutations identified in NEC disrupt this pathway, causing loss of intestinal immune tolerance.
- Provides insights into genetic mutations in SIGIRR contributing to NEC pathogenesis.
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