Dataset Information

SIGIRR Mutation in Human Necrotizing Enterocolitis (NEC) Disrupts STAT3-Dependent microRNA Expression in Neonatal Gut.

ABSTRACT:

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

Sigirr^Tg 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.

SUBMITTER: Yu W

PROVIDER: S-EPMC8688179 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

SIGIRR Mutation in Human Necrotizing Enterocolitis (NEC) Disrupts STAT3-Dependent microRNA Expression in Neonatal Gut.

Yu Wei W Haque Inamul I Venkatraman Aparna A Menden Heather L HL Mabry Sherry M SM Roy Badal C BC Xia Sheng S Prokop Jeremy W JW Umar Shahid S Geurts Aron M AM Sampath Venkatesh V

Cellular and molecular gastroenterology and hepatology 20210923 2

<h4>Background & aims</h4>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.<h4>Methods</h4>Clustered regularly interspaced shor ...[more]

PMID: 34563711

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Project description:ObjectiveBetter measures are needed to identify infants at risk for developing necrotizing enterocolitis (NEC) and facilitate communication about risk across transitions. Although NEC is multi-factorial, quantification of composite risk for NEC in an individual infant is not clearly defined. The objective of this study was to describe the derivation, validation and calibration testing of a novel clinical NEC risk index, GutCheck(NEC). Individual risk factors were weighted to assess composite odds of developing NEC. GutCheck(NEC) is designed to improve communication about NEC risk and coordination of care among clinicians across an infant's clinical course.Study designOn the basis of a synthesis of research evidence about NEC risk and an e-Delphi study including 35 neonatal experts, we identified NEC risk factors believed by the experts to be most relevant for a NEC risk index, then applied a logistic model building process to derive and validate GutCheck(NEC). De-identified data from the Pediatrix BabySteps Clinical Data Warehouse (discharge date 2007 to 2011) were split into three samples for derivation, validation and calibration. By comparing infants with medical NEC, surgical NEC and those who died to infants without NEC, we derived the logistic model using the un-matched derivation set. Discrimination was then tested in a case-control matched validation set and an un-matched calibration set using receiver operating characteristic curves.ResultSampled from a cohort of 58 820 infants, the randomly selected derivation set (n=35 013) revealed nine independent risk factors (gestational age, history of packed red blood cell transfusion, unit NEC rate, late-onset sepsis, multiple infections, hypotension treated with inotropic medications, Black or Hispanic race, outborn status and metabolic acidosis) and two risk reducers (human milk feeding on both days 7 and 14 of life, and probiotics). Unit NEC rate carried the most weight in the summed score. Validation using a 2:1 matched case-control sample (n=360) demonstrated fair to good discrimination. In the calibration set (n=23 447), GutCheck(NEC) scores (range 0 to 58) discriminated those infants who developed surgical NEC (area under the curve (AUC)=0.84, 95% confidence interval (CI) 0.82 to 0.84) and NEC leading to death (AUC=0.83, 95% CI 0.81 to 0.85), more accurately than medical NEC (AUC= 0.72, 95% CI 0.70 to 0.74).ConclusionGutCheck(NEC) represents weighted composite risk for NEC and discriminated infants who developed NEC from those who did not with very good accuracy. We speculate that targeting modifiable NEC risk factors could reduce national NEC prevalence.

Project description:BackgroundNecrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data.ResultsWe found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation.ConclusionsThe identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling.

Dataset Information

SIGIRR Mutation in Human Necrotizing Enterocolitis (NEC) Disrupts STAT3-Dependent microRNA Expression in Neonatal Gut.

Background & aims

Methods

Results

Conclusions

Publications

SIGIRR Mutation in Human Necrotizing Enterocolitis (NEC) Disrupts STAT3-Dependent microRNA Expression in Neonatal Gut.

Similar Datasets

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