Innate Immune Response to S. aureus Biofilm

金黄色葡萄球菌(S.aureus)是生物膜相关假体关节感染的主要原因。 以抗炎细胞环境为代表。白细胞的炎性表型与 他们的代谢状态，其中抗炎MΦS主要依赖氧化磷酸化(OxPhos)和 促炎MΦS利用糖酵解。单核细胞也被极化为抗炎状态。 我们的初步数据显示，这与OxPhos的偏向一致。我们设计了一种创新的 利用细胞靶向代谢重编程生物膜相关单核细胞以促进糖酵解的方法 含有OxPhos抑制剂寡霉素的纳米颗粒。用寡霉素治疗已建立的生物被膜 纳米颗粒在增加中性粒细胞募集的同时促进单核细胞的促炎活性， 导致生物膜清除。在最近的PPG周期中，我们的实验室第一个发现了髓系- 衍生抑制细胞(MDSCs)使生物膜相关的单核细胞偏向抗炎状态，部分地， 通过IL-10的产生。因此，我们筛选了内布拉斯加州转座子突变体库，以鉴定突变体 他们触发IL-10产生的能力受损。确定了与乳酸生物合成有关的重要命中， 我们的初步数据支持金黄色葡萄球菌衍生的乳酸在组织抗炎生物膜中的作用。 环境，从以MDSCs/单核细胞为靶点发展到定义作用的分子机制。第一, 在PJI期间，金黄色葡萄球菌ddh1和ldh1/ldh2突变株的D-乳酸和L-乳酸水平分别降低。 伴随着MDSC渗入和IL-10产生的显著减少，这转化为增强 白细胞募集和生物膜清除。第二，IL-10启动子通过乙酰化和我们的 ChIP-Seq数据表明，组蛋白启动子乙酰化在全基因组范围内显著增加 从WT与ldh1/ldh2感染的小鼠中恢复的白细胞，提供了金黄色葡萄球菌生物被膜的分子证据。 衍生乳酸盐的功能是组蛋白脱乙酰酶抑制物(HDACi)。我们的核心P01假说是S。 金黄色葡萄球菌生物膜的发育创造了独特的代谢环境，促进了免疫抑制 环境。在项目4中，我们将探索金黄色葡萄球菌生物膜、MDSCs、 和单核细胞，由此生物膜衍生的乳酸部分地通过IL-1促进白细胞抗炎特性。 10生产，有助于生物被膜的持久性。这种代谢串扰及其分子机制 对这种相互作用的责任将在以下具体目标中进行探讨。1)建立白细胞 体内代谢可靶向促进促炎活性和生物膜清除；2)研究 金黄色葡萄球菌生物膜衍生乳酸盐促进MDSC和单核细胞抗炎活性的作用 刺激IL-10的产生；以及3)确定金黄色葡萄球菌生物膜衍生的乳酸是否调节IL-10 通过抑制组蛋白脱乙酰酶(HDAC)活性来产生。这些研究将为我们的长期目标提供信息 以新陈代谢途径为靶点，解除抗菌天然免疫防御，促进生物膜根除。

Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infections (PJIs) typified by an anti-inflammatory cellular milieu. The inflammatory phenotype of leukocytes is intimately tied to their metabolic status, where anti-inflammatory MΦs primarily rely on oxidative phosphorylation (OxPhos) and pro-inflammatory MΦs utilize glycolysis. Monocytes are also polarized toward an anti-inflammatory state during S. aureus PJI, which our preliminary data shows coincides with an OxPhos bias. We have devised an innovative approach to metabolically re-reprogram biofilm-associated monocytes to promote glycolysis using cell-targeted nanoparticles containing the OxPhos inhibitor oligomycin. Treatment of established biofilms with oligomycin nanoparticles promoted monocyte pro-inflammatory activity concomitant with increased neutrophil recruitment, leading to biofilm clearance. During the recent PPG cycle, our laboratory was the first to identify that myeloid- derived suppressor cells (MDSCs) skew biofilm-associated monocytes toward an anti-inflammatory state, in part, through IL-10 production. Therefore, we screened the Nebraska Transposon Mutant Library to identify mutants impaired in their ability to trigger IL-10 production. Significant hits involved in lactate biosynthesis were identified, and our preliminary data support a role for S. aureus-derived lactate in organizing the anti-inflammatory biofilm milieu, progressing from MDSCs/monocytes as a target to defining the molecular mechanism of action. First, during PJI, D- and L-lactate levels are reduced in S. aureus ddh1 and ldh1/ldh2 mutants, respectively, concomitant with significant reductions in MDSC infiltrates and IL-10 production, which translates into enhanced leukocyte recruitment, and biofilm clearance. Second, the IL-10 promoter is activated by acetylation and our ChIP-Seq data demonstrate that histone promoter acetylation is dramatically increased genome-wide in leukocytes recovered from WT vs. ldh1/ldh2 infected mice, providing molecular evidence that S. aureus biofilm- derived lactate functions as a histone deacetylase inhibitor (HDACi). Our central P01 hypothesis is that S. aureus biofilm development creates unique metabolic niches that promote an immune suppressive environment. In Project 4, we will explore the existence of a metabolic triad between S. aureus biofilm, MDSCs, and monocytes, whereby biofilm-derived lactate promotes leukocyte anti-inflammatory properties, in part, via IL- 10 production, contributing to biofilm persistence. This metabolic crosstalk and the molecular mechanisms responsible for this interplay will be explored in the following Specific Aims. 1) Establish that leukocyte metabolism can be targeted in vivo to promote pro-inflammatory activity and biofilm clearance; 2) Investigate the role of S. aureus biofilm-derived lactate in promoting MDSC and monocyte anti-inflammatory activity by stimulating IL-10 production; and 3) Determine whether S. aureus biofilm-derived lactate regulates IL-10 production by inhibiting histone deacetylase (HDAC) activity. These studies will inform our long-term goal of targeting metabolic pathways that disarm anti-bacterial innate immune defenses to facilitate biofilm eradication.