Single Immunoglobulin Interleukin-1 Related Receptor and necrotizing enterocolitis in premature infants

坏死性小肠结肠炎(NEC)是早产儿最常见的急性胃肠道急症， 死亡率为20%-35%。尽管免疫受体Toll样受体(TLR)家族的异常激活 肠道微生物区系是NEC发病机制中的一个中心事件，TLR途径激活的机制 对于患有NEC的婴儿，选择性地仍不清楚。我们的实验室使用了一种免疫遗传学方法 发现单个免疫球蛋白白介素1相关受体(SIGIRR)，TLR信号的负调节因子， 可能是NEC的易感基因。我们实验室的初步研究表明，发现了SIGIRR变体 在人类中，NEC导致失去对肠道上皮细胞TLR和白细胞介素1受体信号的抑制。 有趣的是，带有我们在人类NEC中发现的突变的未断奶小鼠表现出自发的 促炎性TLR信号的激活与肠道生物失调。根据这些初步研究，我们 假设SIGIRR是早产儿NEC易感性的致病基因，并且有害 SIGIRR突变使TLR4-微生物区系异常相互作用成为NEC发病机制的基础。这一假设 将通过以下方式进行测试：1)确定SIGIRR变异是否会增加早产儿患NEC的风险，并检查 致病变异体扰乱分子功能的机制，以及2)研究机制 通过SIGIRR突变在NEC中编程TLR高反应和生物失调 在人类NEC中发现的SIGIRR突变的敲入。在目标1中，将对SIGIRR遗传基因座进行测序 在患有和不患有NEC的早产儿中确定稀有或新的SIGIRR变异是否与NEC有关 易感性或严重性。结合生物信息学的SIGIRR序列-结构-功能分析 用遗传变异的体外功能分析来分析SIGIRR蛋白结构将揭示分子 具有NEC变体的改变的SIGIRR函数的基础。对于目标2，我们创建了SIGIRRTIR-/-小鼠，敲入了 在人NEC中发现p.Y168X突变以阐明微生物区系异常的机制-TLR4 NEC中的相互作用。我们将描述指导原生TLR的分子参与者和微生物区系特征 高响应性和NEC脆弱性。我们还将测试益生菌和TLR4拮抗剂对 减轻SIGIRRTIR-/-小鼠的NEC。这里的基本原理是研究遗传中介的范式 NEC中SIGIRR功能程序中异常微生物区系-TLR相互作用的缺陷。 我们采取了床边平移的方法来描述SIGIRR的机械作用 NEC发病机制中的突变。这项研究的结果将极大地促进对SIGIRR领域的了解 NEC易感性的生物学、遗传学基础和SIGIRR对新生儿肠道微生物群的调节。这些 科学成果将对目前缺乏个性化筛查工具的现状产生积极影响 用于预测NEC，以及预防早产儿NEC的新治疗策略。

Necrotizing enterocolitis (NEC) is the most common acute gastrointestinal emergency in premature infants with a mortality of 20-35%. Although aberrant activation of the Toll-Like Receptor (TLR) family of immune receptors by the gut microbiota is a central event in NEC pathogenesis, the mechanisms underlying TLR pathway activation selectively in infants who develop NEC remain unknown. Our laboratory used an immunogenetics approach to discover Single Immunoglobulin Interleukin-1-Related Receptor (SIGIRR), a negative regulator of TLR signaling, as a potential locus for NEC susceptibility. Pilot studies from our lab demonstrate that SIGIRR variants identified in human NEC result in a loss of inhibition of TLR and interleukin-1 receptor signaling in intestinal epithelial cells. Interestingly, unweaned mice with a knock-in of the mutation we identified in human NEC exhibit spontaneous activation of pro-inflammatory TLR signaling and intestinal dysbiosis. Based on these preliminary studies, we hypothesize that SIGIRR is a pathogenic locus for NEC susceptibility in premature infants, and deleterious SIGIRR mutations program deviant TLR4-microbiota interactions underlying NEC pathogenesis. This hypothesis will be tested by: 1) determining whether SIGIRR variants increase NEC risk in premature infants, and examining the mechanisms by which pathogenic variants disrupt molecular function, and 2) investigating the mechanisms by which SIGIRR mutations program TLR hyperresponsiveness and dysbiosis in NEC using novel mice with a knock-in of the SIGIRR mutation identified in human NEC. In Aim 1, the SIGIRR genetic locus will be sequenced in premature infants with and without NEC to determine whether rare or novel SIGIRR variants contribute to NEC susceptibility or severity. A sequence-to-structure-to-function analysis of SIGIRR combining bioinformatics analysis of SIGIRR protein structure with in vitro functional assays of genetic variants will reveal the molecular basis of altered SIGIRR function with NEC variants. For Aim 2, we created SIGIRRTIR-/- mice with a knock-in of the p.Y168X mutation identified in human NEC to elucidate the mechanisms underlying deviant microbiota-TLR4 interactions in NEC. We will characterize the molecular players and microbiota signatures that direct native TLR hyperresponsiveness and NEC vulnerability. We will also test the efficacy of probiotics and TLR4-antagonists to mitigate NEC in SIGIRRTIR-/- mice. The rationale here is to investigate the paradigm that genetically-mediated defects in SIGIRR function program deviant microbiota-TLR interactions in NEC. We have taken a translational bedside-bench approach to delineate the mechanistic role of SIGIRR mutations in NEC pathogenesis. Results of this study will significantly advance knowledge in the areas of SIGIRR biology, genetic basis of NEC susceptibility, and regulation of neonatal gut microbiome by SIGIRR. These scientific gains will positively impact the existing status quo relating to the paucity of personalized screening tools for NEC prediction, and new treatment strategies for NEC prevention in premature infants.