Prevention of intracellular infection in diabetic wounds by commensal Staphylococcus epidermidis

摘要 糖尿病足溃疡(DFU)和糖尿病足感染(DFI)是最具挑战性的并发症之一 糖尿病的发病率和相关死亡率较高，先于大多数非创伤性肢体 成年人口中的截肢。糖尿病足的完整皮肤微生物菌群，在溃疡发病前，是 以低水平的表皮葡萄球菌和高水平的金黄色葡萄球菌为特征 (Sa)。溃疡组织中细菌的持续水平，导致长期和松散的炎症是其中之一 DFU患者小腿截肢的主要原因。为了更深入地了解 先天免疫反应和伤口愈合结果之间的关系，我们建议研究如何 共生微生物SE防止SA在细胞内积累并加速糖尿病伤口 治愈的过程。该项目的长期目标是通过了解DFI的机制和 开发针对DFUS患者皮肤细胞内病原体的新治疗策略。我们 已经证明角质形成细胞和伽马细胞中天然抗菌蛋白P-2的下调 Delta(GD)T细胞导致DFU细胞内MRSA积聚，导致持续未解决 发炎。此外，在小鼠模型中，P-2的丢失与两者的低抗菌活性有关 细胞内MRSA积聚，上皮化受损。重要的是，我们已经证明了杀戮 暴露于SE后，皮肤细胞内MRSA的含量增加。基于稳健的初步数据，我们假设 SE定植通过改变糖尿病皮肤和伤口环境来预防细胞内感染 致病性金黄色葡萄球菌。我们的假设是，细胞内的细菌通过改变P-2的表达和功能来影响 细菌清除和炎症反应，直接影响DFU的愈合。这个项目的目标是 目的是确定SE预防糖尿病小鼠致病SA细胞内感染的机制 和人体伤口。我们假设SE定植改变伤口环境，以防止或 解决持续的细菌性伤口感染。为了检验我们的假设，我们将评估SE介导的 急性糖尿病和非糖尿病创面愈合过程中专业和非专业吞噬细胞中P-2的表达 体内过程，使用多种动物和人类模型以及从DFU患者获得的样本。我们会 同时识别这些细胞中差异表达的基因表达特征和途径。 细胞内SA不愈合与高细胞内SA愈合DFU(目标1)。我们将以SE保护性为特征 糖尿病创面感染中抗细胞内MRSA的机制(目标2)。我们的发现将提供 关于共生SE可防止持久的作用和机制的重要新知识 糖尿病伤口感染。靶向细胞内细菌生态位加速糖尿病高危患者的愈合 皮肤和伤口感染的治疗有可能在促进愈合和伤口愈合方面产生重大临床影响 减少截肢。

Abstract Diabetic foot ulcers (DFUs) and diabetic foot infections (DFI) are one of the most challenging complications of diabetes due to high morbidity and associated mortality and precede the majority of non-traumatic lower limb amputations in the adult population. Diabetic foot microbiome of intact skin, prior to the onset of the ulcer, is characterized by the low level of Staphylococcus epidermidis (SE) and high levels of Staphylococcus aureus (SA). Persistent level of bacteria in ulcer tissue, resulting in prolonged and deregulated inflammation is one of the leading causes of lower leg amputations in patients suffering from DFUs. To gain greater insight into relationship between innate immune responses and wound healing outcomes, we propose to study how commensal microorganism SE prevents intracellular accumulation of SA and accelerates the diabetic wound healing process. The long term goal of this project is to prevent DFI by understanding the mechanism and developing new therapeutic strategies targeting cutaneous intracellular pathogens in patients with DFUs. We have already shown that downregulation of an innate-antimicrobial protein P-2 in keratinocytes and gamma delta (GD) T cells results in accumulation of intracellular MRSA in DFUs, contributing to persistent unresolved inflammation. Furthermore, loss of P-2 in murine models is associated with both, lower antimicrobial activity and accumulation of intracellular MRSA, and impaired epithelialization. Importantly, we have shown that killing of intracellular MRSA is enhanced in skin after exposure to SE. Based on robust preliminary data we postulate that SE colonization modifies diabetic skin and wound environment to prevent intracellular infections by pathogenic SA. Our hypothesis is that intracellular bacteria modify expression and function of P-2 to affect bacterial clearance and inflammatory response, directly impacting healing in DFU. The objective of this project is to determine mechanism by which SE prevents intracellular infections by pathogenic SA in a diabetic mouse and human wounds. We hypothesized that SE colonization modifies wound environment in order to prevent or resolve persistent bacterial wound infections. To test our hypothesis, we will evaluate SE-mediated induction of P-2 in professional and non-professional phagocytes during acute diabetic and non-diabetic wound healing process in vivo, using multiple animal and human models and samples obtained from DFU patients. We will also identify gene expression signatures and pathways in these cells that are differentially regulated in “low intracellular SA non-healing” vs “high intracellular SA healing” DFU (Aim 1). We will characterize SE protective mechanisms against intracellular MRSA in diabetic wound infections (Aim 2). Our findings will provide important new knowledge regarding the role and mechanisms by which commensal SE may prevent persistent diabetic wound infections. Targeting intracellular bacteria niche to accelerate healing in diabetic patients at risk of cutaneous and wound infections has potential to achieve a major clinical impact in promoting healing and reducing amputations.