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）是糖尿病足最具挑战性的并发症之一。 糖尿病由于发病率和相关死亡率高，且先于大多数非创伤性下肢 成年人中的截肢。在溃疡发作之前，完整皮肤的糖尿病足微生物组是 其特征在于表皮葡萄球菌（SE）水平低和金黄色葡萄球菌水平高 （SA）。溃疡组织中持续存在的细菌水平，导致炎症延长和失调， DFU患者小腿截肢的主要原因。更深入地了解 先天免疫反应与伤口愈合结果之间的关系，我们建议研究如何 肠道微生物SE可阻止SA在细胞内的积累，加速糖尿病创面的形成 愈合过程本项目的长期目标是通过了解DFI的机制和 开发针对DFU患者皮肤细胞内病原体的新治疗策略。我们 已经表明，角质形成细胞和γ-氨基丁酸中天然抗菌蛋白P-2的下调， δ（GD）T细胞导致DFU中细胞内MRSA的积累，导致持续未解决的 炎症此外，小鼠模型中P-2的缺失与较低的抗微生物活性 以及细胞内MRSA的积累和受损的上皮形成。重要的是，我们已经证明， 暴露于SE后，细胞内MRSA在皮肤中的表达增强。基于可靠的初步数据，我们假设 SE定植通过以下方式改变糖尿病皮肤和伤口环境以防止细胞内感染： 致病性SA。我们的假设是细胞内细菌改变P-2的表达和功能， 细菌清除和炎症反应，直接影响DFU的愈合。本项目的目标 确定SE预防糖尿病小鼠中致病性SA引起的细胞内感染的机制 人类的伤口。我们假设SE定植改变了伤口环境，以防止或 解决持续性细菌伤口感染。为了验证我们的假设，我们将评估SE介导的诱导， 急性糖尿病和非糖尿病伤口愈合过程中专业和非专业吞噬细胞中的P-2 使用多个动物和人类模型以及从DFU患者获得的样品进行体内处理。我们将 我们还鉴定了这些细胞中的基因表达特征和途径，它们在“低表达”中受到差异调节。 细胞内SA不愈合”与“高细胞内SA愈合”DFU（目标1）。我们将描述SE保护 在糖尿病伤口感染中对抗细胞内MRSA的机制（目的2）。我们的发现将提供 重要的新知识的作用和机制，通过这种作用和机制，口腔SE可以防止持续性 糖尿病伤口感染靶向细胞内细菌生态位加速高危糖尿病患者的愈合 皮肤和伤口感染有可能在促进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.