TREM2 senses both pathogen- and damage-associated molecular patterns to promote S. aureus craniotomy infection

项目摘要 开颅手术允许神经外科医生进入大脑进行手术，包括肿瘤切除，定位 切除致痫灶，通过切除和术中置换动脉瘤夹闭术， 颅骨碎片（骨瓣）。开颅手术后感染的发生率约为1 - 3%，尽管有些人 报告显示，这一比率超过10%。近一半的开颅手术感染是由金黄色葡萄球菌引起的 （S.金黄色葡萄球菌）在骨瓣上形成生物膜。由于生物膜本身由于抗生素而难以根除， 耐受性和毒力因子允许免疫逃避，开颅手术感染携带显著的发病率， 需要进行第二次手术治疗。当开颅手术并发感染时， 整个大脑、皮下帽盖和骨瓣组织中的细胞和浸润白细胞暴露于 损伤和病原体相关的分子模式（DAMP和PAMP）均源自外科手术 程序和细菌。髓样细胞上表达的触发受体-2（TREM2）是一种主要的 病理诱导的信号受体参与许多神经系统疾病期间的免疫重塑。一个 TREM2的重要特征是据报道其结合一系列阴离子配体的能力， 脂蛋白、糖脂和磷脂）和PAMP（脂磷壁酸、肽聚糖和 脂多糖）。该提议将检验TREM2在促进细胞增殖中起关键作用的假设。 S.金黄色葡萄球菌开颅手术感染通过联合DAMP和 PAMP信号。为了支持这一假设，我的初步研究表明TREM2表达增加， 并且在暴露于S.金黄色。这一拟议 工作将寻求揭示开颅感染期间TREM2信号传导背后的机制。在具体目标1中， 将利用S.金黄色葡萄球菌开颅感染与TREM2 KO动物，以确定相对 DAMP与PAMP信号传导的贡献，手术后TREM2激活的时间方面， 膜相关与可溶性TREM2的作用，以及TREM2信号转导参与代谢的程度， 小胶质细胞和单个白细胞群体的重编程。在Specific Aim 2中，一种新的细菌scRNA- seq方法将用于鉴定S.金黄色葡萄球菌TREM2靶向毒力因子， 逃避我将是第一个使用这种测序技术在单细胞水平上研究生物膜并阐明 细菌病原体，特别是生物膜，如何劫持宿主免疫力以持续存在。这项工作的结果将 在双重DAMP和PAMP的背景下，为以前未研究的TREM2信号传导主题提供见解 暴露在开颅手术感染和阐明未来的治疗干预的目标。

PROJECT SUMMARY A craniotomy allows neurosurgeons to access the brain for procedures that include tumor resection, localization and resection of epileptogenic foci, and aneurysm clipping via the removal and intraoperative replacement of a skull fragment (bone flap). Infection following craniotomy occurs at rates of approximately 1-3%, although some reports indicate rates exceeding 10%. Nearly half of craniotomy infections are caused by Staphylococcus aureus (S. aureus) forming a biofilm on the bone flap. As biofilms are inherently difficult to eradicate due to antibiotic tolerance and virulence factors allowing immune evasion, craniotomy infections carry significant morbidity since a second surgery is required for treatment. When craniotomies are complicated by infection, resident immune cells and infiltrating leukocytes throughout the brain, subcutaneous galea, and bone flap tissues are exposed to both damage- and pathogen-associated molecular patterns (DAMPs and PAMPs) originating from the surgical procedure and bacteria, respectively. Triggering receptor expressed on myeloid cells-2 (TREM2) is a major pathology-induced signaling receptor involved in immune remodeling during many neurological conditions. An important feature of TREM2 is its reported ability to bind an array of anionic ligands including DAMPs (DNA, lipoproteins, glycolipids, and phospholipids) and PAMPs (lipoteichoic acid, peptidoglycan, and lipopolysaccharide). This proposal will test the hypothesis that TREM2 plays a critical role in promoting S. aureus craniotomy infection by eliciting an anti-inflammatory response through combined DAMP and PAMP signaling. In support of this hypothesis, my preliminary studies showed increased TREM2 expression and shifts toward an anti-inflammatory metabolic state in microglia after exposure to S. aureus. This proposed work will seek to uncover mechanisms behind TREM2 signaling during craniotomy infection. In Specific Aim 1, I will utilize a mouse model of S. aureus craniotomy infection with TREM2 KO animals to determine the relative contribution of DAMP vs. PAMP signaling, temporal aspects of TREM2 activation post-surgery, the comparative roles of membrane-associated vs. soluble TREM2, and the extent of TREM2 signaling involved in metabolic reprogramming of microglia and individual leukocyte populations. In Specific Aim 2, a novel bacterial scRNA- seq approach will used to identify S. aureus TREM2-targeted virulence factors that are leveraged for immune evasion. I will be the first to use this sequencing technique to study biofilm on a single-cell level and elucidate how bacterial pathogens, specifically biofilm, can hijack host immunity to persist. Results from this work will provide insights into previously unstudied topics on TREM2 signaling in the context of dual DAMP and PAMP exposure during craniotomy infection and elucidate targets for future therapeutic intervention.