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

宿主遗传和免疫因素对艰难梭菌结肠炎的调节

• 批准号: 10362805
• 负责人: Rajat Madan
• 金额: $ 41.11万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-17 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10362805
• 关键词: Address; Affect; Antibodies; Apoptosis; Arginine; Automobile Driving; Autophagocytosis; BCL2 gene; Bacteria; Biological; Biological Response Modifiers; Blood; Cells; Centers for Disease Control and Prevention (U.S.); Chemotactic Factors; Chemotaxis; Clinical; Clostridium difficile; Colitis; Colon; Confocal Microscopy; DNA Sequence Alteration; Data; Development; Disease; Disease Outcome; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Exhibits; Exposure to; Flow Cytometry; Genes; Genetic; Glutamine; Grant; Hematopoietic; Human; Image; Immune; Immune response; Immunoglobulin G; Immunohistochemistry; Immunologic Factors; In Vitro; Induction of Apoptosis; Infection; Inflammation Mediators; Inflammatory; Inflammatory Response; Integration Host Factors; Interleukin-8B Receptor; Intestines; Label; Leptin; Longevity; LoxP-flanked allele; Migration Assay; Migration Inhibitory Factor; Modeling; Mus; Mutation; Neutrophilia; Nosocomial Infections; Organoids; Outcome; Pathogenesis; Pathology; Pathway interactions; Peptide Signal Sequences; Pharmacology; Plasma; Production; Proteins; Public Health; Publishing; Regulation; Reporter; Reporting; Risk; Role; Severities; Severity of illness; Shapes; Single Nucleotide Polymorphism; Source; Stains; Testing; Tissues; Toxin; Western Blotting; adverse outcome; base; cytokine; design; experimental study; improved; in vivo; induced pluripotent stem cell; insight; intestinal epithelium; leptin receptor; macrophage; mortality; mutant; neutrophil; new therapeutic target; novel; pathogen; receptor; reconstitution; release factor; tissue injury; trafficking

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.

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素 艰难梭菌感染（CDI）后的结果。我们发现瘦素受体 (LEPR) 通路的关键作用 CDI 发病机制中的促炎细胞因子巨噬细胞抑制因子 (MIF)。我们的数据显示， LEPR 基因中常见的单核苷酸多态性 (SNP)（Q223R；高达 50% 的人类中存在） 与人类 CDI 后 MIF 的过度产生有关。突变体纯合小鼠中的 CDI SNP (RR) 导致更高的 MIF 产量、更多的结肠中性粒细胞增多和组织损伤，但对 病原。我们报道了使用抗体中和 MIF 可减少组织损伤并改善 小鼠存活，对艰难梭菌病原体没有影响。最后，阻断 MIF 的关键受体 CXC 趋化因子 受体 2 (CXCR2) 还减少了组织中 CDI 诱导的中性粒细胞累积。这个提议是基于这些 已发表的结果和新的初步数据表明 LEPR Q 至 R 的变化增强了 Bcl-2 样蛋白 11 (Bim) 表达通过调节自噬/线粒体自噬/凋亡途径来控制 MIF 释放。 我们研究的总体假设是： LEPR Q 到 R 的替代夸大了 CDI 诱导的、Bim 依赖性的 MIF 的释放会增加 CXCR2 依赖性组织中性粒细胞的增长，并使疾病严重程度恶化。测试 针对这一假设，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 以及 CDI 中 MIF 产生的主要细胞来源。我们将使用 WT 和毒素缺陷的艰难梭菌菌株 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 小鼠中进行 CDI 后定义，它们在 CDI 中的作用将是 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 替代在调节中的作用 Bim 表达以及随后的自噬、线粒体自噬和凋亡途径。研究将使用 Bim-reporter 和 Bim 缺陷小鼠，以及诱导多能生成的 Bim+/+ 和 Bim-/- 人肠道类器官 QQ 和 RR 背景下的干细胞。目标 3 将研究 MIF-CXCR2 轴在 CDI 诱导的中的作用 QQ 和 RR 小鼠组织中性粒细胞增多。我们将比较 MIF 对中性粒细胞运输的影响及其对中性粒细胞运输的影响。 寿命。我们将在体外（迁移测定）和体内利用来自 WT 和 CXCR2-/- 小鼠的中性粒细胞 （在感染前耗尽内源性中性粒细胞并用标记的中性粒细胞重建后） 实验。我们期望这些研究能够更好地理解宿主遗传学在调节中的作用 CDI 发病过程中的 MIF。我们的研究有可能确定可使用的新的基于宿主的目标 参与新型 CDI 疗法的设计。