494 - SIGIRR regulates intestinal epithelial cell tight junctions and thereby maintains intestinal homeostasis in neonates

Poster Number: 494
Publication Number: 494.424

Aparna Venkatraman, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Wei Yu, Children's Mercy Kansas City, Kansas city, MO, United States; Sherry Mabry, Children's Mercy, Kansas City, MO, United States; Venkatesh Sampath, Childrens Mercy Kansas City, Kansas City, KS, United States

Background: Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by Toll-like receptor 4 (TLR4)-dependent intestinal inflammation and enterocyte death. Though disruption of tight junctions and loss of barrier function has been implicated in pathophysiology of neonatal NEC, the signaling mechanisms orchestrating these events remain elusive. We had earlier identified a novel stop mutation (p. Y168X) in Single Immunoglobin Interleukin-1 Related receptor (SIGIRRmu/mu), an inhibitor of toll-like receptor (TLR) signaling, in preterm infants with severe NEC. We tested the hypothesis that SIGIRR regulates neonatal intestinal homeostasis by regulating enterocyte tight junctions in SIGIRRmu/mu mice.

Objective: 1: To determine whether SIGIRRmu/mu mice demonstrate phenotypic changes at the molecular and cellular level in the terminal ileum. 2 To determine whether SIGIRRmu/mu mice have altered tight junction proteins in the terminal ileum.

Design/Methods: Intestine was excised from SIGIRRmu/mu mice and from wild type (Sigirr+/+) mice on post-natal day 14. Gross morphology and histological changes were compared between wild type and mutant mice. Confocal imaging was carried out on intestinal sections for immunofluorescence and co-localization studies. Single cells isolated using collagenase digestion from the terminal ileum were used for quantitation of absolute numbers and analysis of gene and protein expression. Rescue experiments were performed by crossing Sigirrmu/mu mice with TLR4 deficient mice (TLR4-/-) to obtain double mutants (Sigirrmu/mu:Tlr4-/-).

Results: Phenotypic analysis of Sigirrmu/mu mice revealed a decrease in intestinal length with reduction in villus height and increase in crypt depth. Single cell isolation of intestinal mucosal cells revealed a dramatic reduction in total cell number (Figure 1). Defects in enterocyte proliferation and increased cell death was seen restricted to the crypt compartment in Sigirrmu/mu mice (Fig2). These mice also demonstrate defects in formation of membrane proteins such as Epcam and E cadherin and tight junction proteins like Claudin in the crypt. Mechanistically, we noted that Sigirrmu/mu mice had increased intestinal epithelial expression and nuclear accumulation of transcription factor Twist 1 (a regulator of tight junction proteins) (Fig 3). Genetic depletion of TLR4-/- in Sigirrmu/mu mice rescued all the phenotypic changes partially or completely (Fig 1-3).Conclusion(s): Sigirr regulates IEC homeostasis and barrier function by orchestrating formation of tight junction proteins in a Twist1 dependent manner.
. Sigirr regulates intestinal size and crypt-villus axis(A) Measurement of body and intestinal weight and intestinal length. (B) Representative histopathological image of terminal ileum from Sigirr+/+ and Sigirrmu/mu mice. (C) Morphometric analysis of crypt-villus axis in terminal ileum. (D) Representative flow cytometric analysis of mucosal isolation as single cells and its quantification. All experiments are biological replicates of n=5 and p < .05
Sigirr regulates crypt cell proliferation and survival in terminal ileum(A) Gene expression analysis of cell death and cell cycle genes. (B) Representative image of Tunel assay demonstrating increased cell death in Sigirrmu/mu mice. (C) Immunofluorescent image depicting rescue of crypt cell proliferation in Tlr4-/- Sigirrmu/mu mice. All experiments are biological replicates of n=5, p < .05