Role of AMP-activated protein kinase in bacterial endophthalmitis

项目摘要 骨髓细胞如中性粒细胞在提供宿主防御微生物感染中的作用是很好的- 建立;然而，单核细胞/巨噬细胞（M β）对细菌感染的病理生理学的贡献 眼内炎不太清楚。我们的初步研究表明，M β耗竭导致 炎症介质在解决阶段，表明他们参与解决 眼内炎M执行多项任务，包括感应病原体、组织修复，并作为响应 对于宿主衍生的介质，它们分化成不同的功能表型;称为“可塑性”的特征。 “经典活化”的M β（M1）产生炎性细胞因子和一氧化氮，有助于宿主组织 损害相反，“替代性激活”的M2通过消除 受损的细胞/组织和抗炎分子的产生来解决炎症。因此，我们认为， 了解控制M β表型转换的机制可用于开发新的 治疗策略我们对细菌感染视网膜的转录组和代谢组学分析， 我们鉴定了腺苷一磷酸活化蛋白激酶（AMPK），一种代谢基因， 调节眼内炎中的浸润性骨髓细胞表型。我们发现， AMPKα1缺失（敲除）（KO）与野生型相比发生了严重的眼内炎和病理学改变 (WT)小鼠缺乏AMPKα1的M β维持低代谢状态，即使在高度炎症状态下。到 为了精确地检测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），并观察到LysM-KO显示炎症加重和视网膜病变减少。 与WT小鼠相比，AMPK在骨髓细胞中的功能，表明AMPK在骨髓细胞中的发病机制中起着重要作用。 细菌性眼内炎基于这些发现，我们建议测试我们的中心假设，AMPK 通过调节浸润性单核细胞/巨噬细胞的极化在细菌性眼内炎中发挥保护作用 促进炎症消退，代谢重编程是炎症消退的潜在机制。 单核细胞/M β表型转换。为了验证我们的假设，在目标1中，我们将研究 通过检测LKB 1的修饰， 亚硝基化或化学加合物形成。目的2：验证AMPKα1消融可增强 骨髓细胞的活化状态，并在缓解期维持其促炎（M1）状态。 这种疾病在目标3中，我们将破译由M中AMPK调节的生物能量事件， 保持其促炎性。本研究的预期结果将证明， 髓系细胞（主要是单核细胞/单核细胞）中AMPK活性通过以下途径影响眼内炎的消退： 调节细胞代谢。此外，它可能为开发抗肿瘤药物提供新的治疗靶标。 用于眼内炎和其他微生物感染的炎性疗法。

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.