Role of a common leptin receptor polymorphism in regulating neutrophil heterogeneity after C. difficile infection

艰难梭菌是美国主要的医院感染，是VA急性和长期感染的主要问题. 定期护理设施。目前治疗艰难梭菌感染(CDI)的方法是以艰难梭菌为靶标使用抗生素。 但这会导致共生菌的死亡，从而降低对艰难梭菌的定植抵抗力 反过来又可以促进CDI的复发。因此，CDI治疗迫切需要微生物区系的保留方法。 宿主中性粒细胞增多是CDI后疾病转归的关键调节因素。我们之前已经报道过 瘦素-瘦素受体(LEPR)轴在调节CDI诱导的中性粒细胞增多中的关键作用：我们的研究表明 LEPR(Q223R)中存在一个SNP，它存在于多达50%的人类中，调节两只小鼠的中性粒细胞数量 以及CDI患者。突变的LEPR等位基因(RR基因型)的纯合性与 在急性CDI期间，中性粒细胞计数伴随着显著的组织损伤和更高的死亡率，但更早 组织中性粒细胞减少和临床疾病。由于异质性中性粒细胞群对 由于组织损伤和组织在调节传染病结果中都是关键的，我们假设不同的 中性粒细胞类型是对艰难梭菌的反应而形成的，并有助于组织损伤和修复。 我们现在有令人信服的初步数据，显示骨髓和 艰难梭菌感染小鼠的结肠组织。我们已经根据细胞内颗粒定义了这些种群 2整合素(CD11b)的表达。在RR小鼠中，组织数量的增加 CDI急性期中性粒细胞(总和激活群体，SSChiCD11bhi细胞)相关 强烈伴有严重的组织损伤和临床疾病。随后，组织中性粒细胞下降(两者合计 和活跃人群)与较轻的肠道病理和较早的临床恢复有关 这些小鼠中的疾病。此外，共生肠道微生物区系在不同的 小鼠骨髓中性粒细胞亚群及其动员。 我们的中心假设是LEPR Q到R的变化与肠道微生物群的变化有关 以STAT3依赖的方式影响中性粒细胞对艰难梭菌的组织反应。我们现在 建议全面定义CDI和CDI过程中中性粒细胞群的演变 了解LEPR SNP和与SNP相关的肠道微生物区系调控形成的机制 CDI后不同的中性粒细胞。 我们将回答以下主要问题： 1)急性期和缓解期形成的中性粒细胞的功能和表型特征是什么？ 具有QQ和RR基因的患者和小鼠的CDI时相？ 2)这些中性粒细胞如何诱导结肠组织损伤，调节CDI的发病机制？ 3)特定的肠道细菌在调节这种中性粒细胞群的发展中起什么作用？ 4)CDI后LEPR下游调控中性粒细胞异质性的信号通路有哪些？ 我们研究的目标是确定在这个过程中促进中性粒细胞亚群的致病和分解。 并了解其发展的机制。组织损伤和组织损伤的识别 CDI后修复性中性粒细胞类型有可能为设计确定新的微生物区系保留靶点 未来的CDI疗法。此外，了解一种常见的人类基因变异在调节 中性粒细胞异质性和CDI结果可用于LEPR SNP为 作为一种新的遗传生物标志物用于CDI患者的风险分层。

Clostridium difficile is the leading nosocomial infection in the U.S and a major concern for VA acute and long- term care facilities. The current therapies for C. difficile infection (CDI) target C. difficile bacterium with antibiotics. But this can leads to killing of commensal bacteria and thus reduce colonization resistance to C. difficile which can in turn promote recurrent CDI. Thus, microbiota sparing approaches for CDI therapy are urgently needed. Magnitude of host neutrophilia is a key regulator of disease outcomes after CDI. We have previously reported a key role for leptin-leptin receptor (LEPR) axis in regulating CDI-induced neutrophilia: our studies revealed that a SNP in LEPR (Q223R), which is present in up to 50% of humans, regulates neutrophil numbers in both mice and patients with CDI. Homozygosity for the mutant LEPR allele (RR genotype) was associated with increased neutrophil counts along with significant tissue damage and higher mortality during acute CDI, but earlier resolution of tissue neutrophilia and clinical disease. Since heterogenous neutrophil populations that contribute to both tissue damage and tissue are critical in regulating infectious disease outcomes, we postulate that different neutrophil types develop in response to C. difficile and contribute to tissue damage and repair. We now have compelling preliminary data that reveals distinct neutrophil populations in bone marrow and colonic tissue of C. difficile infected mice. We have defined these populations based on intra-cellular granules (side scatter on FACS) and 2 integrin (CD11b) expression. In RR mice, an increase in the number of tissue neutrophils (total as well as the activated population, SSChiCD11bhi cells) during acute phase of CDI correlated strongly with severe tissue damage and clinical disease. Subsequently, decline in tissue neutrophils (both total and activated population) was associated with less severe intestinal pathology and earlier recovery from clinical disease in these mice. Further, commensal gut microbiota play an important role in the generation of different neutrophil populations and their mobilization from bone marrow compartment of mice. Our central hypothesis is that LEPR Q to R change is associated with alterations in the gut microbiome that influence the effect of neutrophils on tissue responses to C. difficile in a STAT3-dependent manner. We now propose to comprehensively define the evolution of neutrophil populations during the course of CDI and understand the mechanisms by which LEPR SNP and gut microbiota associated with the SNP regulate formation of distinct neutrophils after CDI. We will answer the following main questions: 1) What are the functional and phenotypic characteristics of neutrophils formed during acute and resolution phase of CDI in both patients and mice with the QQ and RR genotype? 2) How do these neutrophils induce colonic tissue damage and regulate CDI pathogenesis? 3) What is the role of specific gut bacteria in regulating the development of such neutrophil populations? 4) What are the signaling pathways downstream of LEPR that regulate neutrophil heterogeneity after CDI? The goal of our studies is to identify pathogenic and resolution promoting neutrophil subsets during the course of CDI and understand the mechanisms of their development. Identification of the tissue damaging and reparative neutrophil types after CDI has the potential to identify new microbiota-sparing targets for the design of future CDI therapies. In addition, understanding the role of a common human genetic variant in regulating neutrophil heterogeneity and CDI outcomes can be used in precision medicine approaches where LEPR SNP is utilized as a novel genetic biomarker for risk stratification of CDI patients.