Role of AMP-activated protein kinase in bacterial endophthalmitis

项目摘要 中性粒细胞等髓系细胞在提供宿主防御微生物感染方面的作用是很好的- 然而，单核/巨噬细胞(M-ϕ)在细菌的病理生理学中的作用 眼内炎不太清楚。我们的初步研究表明，Mϕ耗尽会导致 炎症介质处于解决阶段，表明它们参与了解决 眼内炎。Mϕ执行多项任务，包括感知病原体、组织修复以及作为响应 对于宿主来源的介体，它们分化为不同的功能表型；这一特征被称为“可塑性”。 经典激活的Mϕ(M1)产生炎性细胞因子和一氧化氮，促进宿主组织的生长 损坏。相反，“交替激活”的Mϕ(M2)通过消除 受损的细胞/组织和抗炎分子的产生，以化解炎症。因此， 了解M-ϕ的表型转换机制可以用来开发新的 治疗策略。细菌感染视网膜的转录组和代谢组学分析 腺苷一磷酸活化蛋白激酶(AMPK)是一种代谢基因，它是一种新的基因 调节眼内炎的浸润性髓样细胞表型。我们发现患有全球疾病的小鼠 与野生型相比，AMPKα1(KO)缺失(敲除)可导致严重的眼内炎和病理改变 (WT)小鼠。缺乏AMPK-ϕ-1的M-α维持低代谢状态，即使处于高炎症状态。至 精确检测AMPK在髓系细胞中的作用，我们诱导了髓系细胞特异性KO的眼内炎 AMPKα1(LysM-KO)，观察到LysM-KO表现为炎症加重和视网膜减少 与WT小鼠相比，提示髓系细胞中的AMPK在WT小鼠的发病机制中起着重要作用 细菌性眼内炎。在这些发现的基础上，我们建议测试我们的中心假设AMPK 调节浸润性单核细胞/M-ϕ极化对细菌性眼内炎的保护作用 促进炎症消退，代谢重新编程是其潜在机制 单核细胞/Mϕ表型转换。为了验证我们的假设，在目标1中，我们将研究这些机制 细菌性眼内炎中AMPK活性降低的潜在原因是通过检测LKB1的修饰 亚硝化或化学加合物的形成。Aim 2验证了AMPKα1消融可增强 髓系细胞的激活状态，并在解决期维持其促炎状态(M1) 这种疾病。在目标3中，我们将破译由Mϕ中的AMPK调控的生物能量事件，这些事件极化和 保持其促炎性质。这项研究的预期结果将证明有缺陷的 髓系细胞，主要是单核细胞的AMPK活性/M通过以下途径影响眼内炎的缓解 细胞代谢的调节。此外，它还可能为抗肿瘤药物的发展提供新的治疗靶点。 眼内炎和其他微生物感染的炎症治疗。

Project Summary The role of myeloid cells such as neutrophils in providing host defense to microbial infections is well- established; however, the contribution of monocytes/macrophages (Mϕ) to the pathophysiology of bacterial endophthalmitis is less clear. Our preliminary studies revealed that Mϕ depletion results in increased inflammatory mediators at the resolution phase, suggesting their involvement in the resolution of endophthalmitis. The Mϕ perform multiple tasks, including sensing pathogens, tissue repair, and, in response to host-derived mediators, they differentiate into distinct functional phenotypes; a feature termed "plasticity". The "classically activated" Mϕ (M1) produce inflammatory cytokines and nitric oxide, contributing to host tissue damage. Conversely, the "alternatively activated" Mϕ (M2) mediate tissue repair through the elimination of damaged cells/tissue and the production of anti-inflammatory molecules to resolve inflammation. Therefore, understanding the mechanisms governing the phenotypic switch of Mϕ can be utilized to develop novel therapeutic strategies. Our transcriptome and metabolomics analyses of the bacteria-infected retina directed us to the identification of adenosine monophosphate-activated protein kinase (AMPK), a metabolic gene, which modulates the infiltrating myeloid cell phenotype in endophthalmitis. We discovered that mice with global deletion (knockout) of AMPKα1 (KO) developed severe endophthalmitis and pathology compared to wild type (WT) mice. Mϕ lacking AMPKα1 maintained a low metabolic state, even in the hyper-inflammatory state. To precisely examine the role of AMPK in myeloid cells, we induced endophthalmitis in myeloid cell specific KO of AMPKα1 (LysM-KO) and observed that LysM-KO displayed exacerbated inflammation and reduced retinal function compared to WT mice, suggesting an essential role of AMPK in myeloid cells in the pathogenesis of bacterial endophthalmitis. Building on these findings, we propose to test our central hypothesis that AMPK exerts protective effects in bacterial endophthalmitis by modulating the polarization of infiltrating monocytes/Mϕ to promote inflammation resolution and that metabolic reprograming is an underlying mechanism of the monocytes/Mϕ phenotype switch. To test our hypothesis, in Aim 1, we will investigate the mechanisms underlying reduced AMPK activity in bacterial endophthalmitis by examining the modification of LKB1 via nitrosylation or chemical adduct formation. Aim 2 tests the hypothesis that AMPKα1 ablation enhances the activation state of myeloid cells and maintains their proinflammatory (M1) state during the resolution phase of the disease. In Aim 3, we will decipher the bioenergetic events, regulated by AMPK in Mϕ, that polarize and maintain their pro-inflammatory nature. The anticipated results of this study will demonstrate that defective AMPK activity in myeloid cells, mainly in monocytes/M impacts the resolution of endophthalmitis via regulation of cellular metabolism. Also, it may provide novel therapeutic targets for the development of anti- inflammatory therapies for endophthalmitis and other microbial infections.