Regulation of C. difficile colitis by host genetic and immune factors

项目概要： 艰难梭菌是美国主要的医院感染。宿主因素是疾病的关键预测因子 结果C.艰难梭菌感染（CDI）。我们已经发现瘦素受体（LEPR）通路的关键作用， 促炎细胞因子巨噬细胞抑制因子（MIF）在CDI发病机制中的作用。我们的数据显示， LEPR基因中常见的单核苷酸多态性（SNP）（Q223 R;存在于高达50%的人类中） 与人类CDI后MIF的过度产生有关。突变体纯合子小鼠中的CDI SNP（RR）导致更高的MIF产生，更多的结肠嗜中性粒细胞和组织损伤，而对 病原体我们报道了使用抗体中和MIF导致减少组织损伤和改善 小鼠存活率，对C.艰难病原体最后，阻断MIF的关键受体CXC趋化因子 受体2（CXCR 2）也减少组织中CDI诱导的中性粒细胞积聚。这项建议是基于这些 已发表的结果和新的初步数据表明，LEPR Q到R的变化增加了Bcl-2样蛋白， 11（Bim）的表达，其通过调节自噬/线粒体自噬/凋亡途径来控制MIF释放。 本研究的总体假设是：LEPR Q到R的取代增强了CDI诱导的、Bim依赖的 MIF的释放增加CXCR 2依赖性组织中性粒细胞的积累和疾病的严重程度。测试 根据这一假设，我们提出了三个具体目标。目标1将定义MIF的关键病原体相关驱动因素 和CDI中产生MIF的主要细胞来源。我们将使用WT和毒素缺陷型C。艰难菌株 确定毒素和非毒素因子在驱动MIF表达中的作用。MIF的细胞来源将是 通过细胞内染色和免疫组织化学在WT小鼠中的CDI后定义， 通过在肠上皮细胞和造血细胞中使用选择性MIF缺失进行测试。目标2将检查 CDI诱导MIF释放的机制。我们将确定LEPR Q到R的取代在调节中的作用。 Bim表达与随后的自噬、线粒体自噬和凋亡途径。研究将使用Bim报告器 和Bim缺陷型小鼠，以及由诱导的多能造血干细胞产生的Bim+/+和Bim-/-人肠类器官。 QQ和RR背景中的干细胞。目的3研究MIF-CXCR 2轴在CDI诱导的细胞凋亡中的作用。 QQ和RR小鼠的组织嗜中性粒细胞。我们将比较MIF对中性粒细胞运输的影响，并对它们的 寿命我们将在体外（迁移试验）和体内使用来自WT和CXCR 2-/-小鼠的中性粒细胞 （内源性中性粒细胞耗竭后，感染前用标记的中性粒细胞重建） 实验我们希望这些研究能够更好地理解宿主遗传学在调节 CDI发病过程中的MIF。我们的研究有可能确定新的基于宿主的靶标， 在设计新型CDI疗法方面。

Project Summary: Clostridium difficile is the leading nosocomial infection in the U.S. Host factors are a key predictor of disease outcomes after C. difficile infection (CDI). We have discovered a key role for leptin receptor (LEPR) pathway and the pro-inflammatory cytokine Macrophage Inhibitory Factor (MIF) in CDI pathogenesis. Our data reveal that a common single nucleotide polymorphism (SNP) in the gene for LEPR (Q223R; present in up to 50% of humans) is associated with exaggerated MIF production after CDI in humans. CDI in mice homozygous for the mutant SNP (RR) resulted in higher MIF production, more colonic neutrophilia and tissue damage, without effects on pathogen. We reported that neutralizing MIF using antibody resulted in reduced tissue injury and improved survival in mice, without effects on C. difficile pathogen. Finally, blocking a key receptor for MIF, CXC chemokine receptor 2 (CXCR2) also reduced CDI-induced neutrophil accrual in tissue. This proposal is based on these published results and new preliminary data which indicate that LEPR Q to R change augments Bcl-2 like protein 11 (Bim) expression which controls MIF release by modulating pathways of autophagy/mitophagy/apoptosis. The overall hypothesis of our studies is that: LEPR Q to R substitution exaggerates CDI-induced, Bim-dependent MIF release that augments CXCR2-dependent tissue neutrophil accrual and worsens disease severity. To test this hypothesis, we propose three specific aims. Aim 1 will define the key pathogen-associated drivers of MIF and the main cellular sources of MIF production in CDI. We will use WT and toxin-deficient C. difficile strains to determine the role of toxins and non-toxin factors in driving MIF expression. Cellular sources of MIF will be defined after CDI in WT mice by intracellular staining and immunohistochemistry and their role in CDI will be tested by using selective MIF deletion in intestinal epithelial cells and in hematopoietic cells. Aim 2 will examine the mechanism of CDI-induced MIF release. We will determine the role of LEPR Q to R substitution in regulating Bim expression and subsequent autophagy, mitophagy and apoptosis pathways. Studies will use Bim-reporter and Bim-deficient mice, and Bim+/+ and Bim-/- human intestinal organoids generated from induced pluripotent stem cells in both QQ and RR background. Aim 3 will investigate the role of MIF-CXCR2 axis in CDI-induced tissue neutrophilia in QQ and RR mice. We will compare the effect of MIF on neutrophil trafficking and on their lifespan. We will utilize neutrophils derived from WT and CXCR2-/- mice in in vitro (migration assays) and in vivo (after depletion of endogenous neutrophils and reconstitution with labeled neutrophils prior to infection) experiments. We expect these studies to provide a greater understanding of the role of host genetics in regulating MIF during CDI pathogenesis. Our studies have the potential to identify new host-based targets that can be used in the design of novel CDI therapies.