512 - SIGIRR Variants Identified in NEC Infants Exaggerate Toll-like Receptor Mediated Inflammation

Poster Number: 512
Publication Number: 512.426

Jennie Godwin, Children's Mercy Hospital Kansas City, Prairie Village, KS, United States; Heather Menden, Childrens Mercy, Kansas City, MO, United States; Craig Smail, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Sheng Xia, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Venkatesh Sampath, Childrens Mercy Kansas City, Kansas City, KS, United States

Background: A key event underlying uncontrolled inflammation in necrotizing enterocolitis (NEC) is pathologic activation of Toll-like receptors (TLR). TLR4 recognizes lipopolysaccharide (LPS), from Gram-negative bacteria, and TLR2 peptidoglycans (PAM3Csyk4, PAM), from Gram-positive bacteria. TLR4 and TLR2 signaling events induce an inflammatory cascade through NF-kB, a cytokine inducing transcription factor. Single-immunoglobulin interleukin-1-related receptor (SIGIRR) is a major negative regulator of TLR-mediated NF-kB activation. Previous work from our lab suggests that loss of function in SIGIRR may predispose to NEC. Our lab has identified four variants in NEC patients predicted to alter function of SIGIRR. We hypothesized that identified SIGIRR variants will lead to excessive TLR4 and TLR2-mediated NF-kB activation and cytokine expression.

Objective: To investigate the effect of SIGIRR variants on TLR4 and TLR2-mediated NF-kB activation and cytokine expression in a human monocyte-macrophage cell line (THP-1 cells).

Design/Methods: THP-1 cells were transfected with SIGIRR variants (p.S80Y, p.P115R, p.P168X, and p.P366S) or wild type plasmids (SIGIRR-WT). To examine TLR4 and TLR2-dependent NF-kB activation, cells were treated with LPS or PAM respectively. THP-1 cells were engineered to express Secreted Alkaline Phosphatase (SEAP) when NF-kB is activated. RNA extracted from THP-1 cells after LPS or PAM treatment was used for quantifying expression of inflammatory cytokines (ICAM-1, iNOS, IL-8, IL-6, IL-1b, TNFα, IFNg) by qRT-PCR. Changes in NF-kB activation quantified from SEAP and RNA cytokine levels were expressed as a fold-change relative to control (θ). Statistical comparisons were calculated using PRISM software.

Results: LPS-induced TLR4-mediated NF-kB activation was strongly inhibited in SIG-WT and exaggerated in mutant SIGIRR variants (Fig 1A). PAM-induced TLR2-mediated NF-kB activation was also suppressed in SIG-WT and increased in mutant SIGIRR variants (Fig 1B). LPS-induced ICAM-1, iNOS, and IL-8 RNA expression were significantly inhibited in SIG-WT and exaggerated by the mutant variants (Fig 2). PAM-induced IL-8, IL-6, IL-1b, TNFα, and IFNg RNA expression were also strongly inhibited in SIG-WT and increased in mutant variants (Fig 3).Conclusion(s): SIGIRR variants identified in infants with NEC alter SIGIRR function leading to loss of TLR suppression and excessive inflammation. This study supports the hypothesis that inherited defects in the regulation of innate immune signaling can contribute to increased intestinal inflammation and NEC susceptibility.
Figure 1Effect of SIGIRR variants on LPS (Figure 1A) and PAM (Figure 1B)-induced inflammation. THP-1 cells transfected overnight with plasmids encoding empty plasmid, the reference allele (SIG-WT), and four SIGIRR variants (p.S80Y, p.P115R, p.P168X, and p.P366S) were treated with LPS (100 ng/mL) or PAM (100 ng/mL). Culture supernatants or cell lysate protein were used for experiments. NF-kB activation was quantified in culture supernatants 18 hours after LPS or PAM. Fold-increase in NF-kB activation relative to control is shown. p < 0.01 for all comparisons.
Figure 2ICAM, iNOS, and IL-8 RNA expression were quantified in cell lysates by quantitative reverse transcription PCR 18 hours after LPS treatment (100 ng/mL). Fold-increase in expression relative to control is shown. p < 0.02 for all comparisons ($ control vs LPS; * LPS vs SIGIRR mutant + LPS). Nf4.