Presenter Status
Fellow
Abstract Type
Research
Primary Mentor
Venkatesh Sampath, MD, MRCPCh
Start Date
3-5-2022 11:30 AM
End Date
3-5-2022 1:30 PM
Presentation Type
Poster Presentation
Description
Background: A key event underlying uncontrolled inflammation in necrotizing enterocolitis (NEC) is pathologic activation of Toll-like receptors (TLR). TLR4 recognizes lipopolysaccharide (LPS), from Gramnegative 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.
Objectives/Goal: 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).
Methods/Design: 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).
Conclusions: 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.
MeSH Keywords
Necrotizing enterocolitis; Neonatal; Toll-like receptor; SIGIRR
Additional Files
SIGIRR Variants Identified in NEC Infants Exaggerate Toll-like Receptor Mediated Inflammation.pdf (523 kB)Abstract
Included in
Congenital, Hereditary, and Neonatal Diseases and Abnormalities Commons, Digestive System Diseases Commons, Pediatrics Commons
SIGIRR Variants Identified in NEC Infants Exaggerate Toll-like Receptor Mediated Inflammation
Background: A key event underlying uncontrolled inflammation in necrotizing enterocolitis (NEC) is pathologic activation of Toll-like receptors (TLR). TLR4 recognizes lipopolysaccharide (LPS), from Gramnegative 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.
Objectives/Goal: 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).
Methods/Design: 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).
Conclusions: 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.
