Novel regulation of mucosal innate defense by AMPK in Otitis Media

粘蛋白是粘液中的主要蛋白质成分，通过提供一种 物理屏障和诱捕病原体清除粘液纤毛。如果不加控制，就会产生过量的粘蛋白 在中耳炎(OM)和粘液中抑制粘液纤毛清除并导致传导性听力损失 肺部感染中的梗阻。因此，粘蛋白的生产必须受到严格的监管。然而，分子 粘蛋白的严格调控机制在很大程度上仍不清楚。 中耳炎(OM)是儿童最常见的细菌感染，也是传导性耳炎的主要原因 听力损失。它仍然是一个重大的健康问题和重大的社会经济负担。肺炎链球菌，Sp. 代表OM的主要革兰氏阳性细菌病原体。目前可用的Sp疫苗具有有限的 对OM的影响。此外，不适当的抗生素使用增加了抗生素的耐药性。有一个迫切的需要 用于开发创新的非抗生素治疗药物，以抑制粘液过度生产。我们的长期合作 目的是阐明OM发病的分子机制，并寻找新的治疗方法 目标。与相对广为人知的Toll样受体(TLR)依赖的机制相比，Sp和Sp 肺炎溶血素(PLY--几乎所有临床分离的Sp产生的关键毒力因子)诱导宿主粘膜 在免疫反应方面，包括关键监管机构在内的TLR独立机制在很大程度上仍不清楚。 腺苷5‘-单磷酸活化蛋白激酶α1(AMPKα1)已成为宿主的主要调节因子 能量平衡。它在传染病中的作用，特别是在宿主粘膜的天然防御反应中， 例如粘液的产生，在很大程度上仍然不清楚。我们令人鼓舞的初步数据表明，Sp和Ply可能 非依赖TLR2/4激活AMPKα1上调粘蛋白MUC5AC和MUC5B 在体外培养的中耳和呼吸道上皮细胞以及急性和慢性OM的小鼠模型中。 有趣的是，Sp和Ply可能通过诱导新的非传统的(蛋白质降解--)激活AMPKα1 独立)AMPKα1的泛素化可能是通过下调一个关键的去泛素酶CyLD来实现的。加在一起，这些 因此，令人兴奋的初步数据为我们假设[1]AMPKα1作为 Sp通过TLR非依赖信号诱导MUC5AC和MUC5B上调的关键调节因子；[2] 多泛素化和磷酸化相互作用激活AMPKα1在Sp. 诱导MUC5AC和MUC5B上调(假设)。为了验证我们的假设，我们将研究两个具体的 目的：(目的1)确定AMPKα1在急性呼吸窘迫综合征和COM的OM发病机制中的作用；以及(目的2)如何 SP激活AMPKα1。这些研究将极大地促进我们对关键调控因子的理解，包括 AMPK在TLR非依赖性宿主黏膜先天防御细菌感染中的作用 控制粘液过度生产的新治疗靶点。我们对AMPK信号的研究也可能会有所帮助 了解其他AMPK相关疾病的分子机制(意义和影响)。

Mucin, a major protein component in mucus, plays a critical role in mucosal innate defense by providing a physical barrier and trapping pathogens for mucociliary clearance. If uncontrolled, excessive mucin production overwhelms mucociliary clearance and causes conductive hearing loss in otitis media (OM) and mucus obstruction in lung infections. Therefore, mucin production must be tightly regulated. However, the molecular mechanisms underlying the tight regulation of mucin remain largely unknown. Otitis media (OM) is the most common childhood bacterial infection and the leading cause of conductive hearing loss. It remains a major health problem and a substantial socioeconomic burden. S. pneumoniae, Sp, represents a major gram-positive bacterial pathogen for OM. Currently available Sp vaccines have a limited impact on OM. Moreover, inappropriate antibiotic use increased antibiotic-resistance. There is an urgent need for developing innovative non-antibiotic therapeutic agent for suppressing mucus overproduction. Our long-term goal is to elucidate the molecular mechanisms underlying OM pathogenesis and identify novel therapeutic targets. In contrast to the relatively well-known toll-like receptor (TLR)-dependent mechanisms by which Sp and pneumolysin (PLY – a key virulence factor produced by virtually all clinical Sp isolates) induce host mucosal immune response, the TLR-independent mechanisms including the key regulators remain largely unclear. Adenosine 5’-monophosphate-activated protein kinase α1 (AMPKα1) has emerged as a master regulator of host energy homeostasis. Its role in infectious diseases, in particular in the host mucosal innate defense response, e.g. mucus production, remains largely unclear. Our encouraging preliminary data suggest that Sp and PLY may up-regulate mucin MUC5AC and MUC5B via activation of AMPKα1 in a TLR2/4-independent manner in the middle ear and airway epithelial cells in vitro and in the mouse models of both acute and chronic OM. Interestingly, Sp and PLY may activate AMPKα1 by inducing novel non-traditional (protein degradation- independent) ubiquitination of AMPKα1 likely via downregulating a key deubiquitinase CYLD. Together, these exciting preliminary data have thus provided a solid foundation for us to hypothesize that [1] AMPKα1 acts as a key regulator for Sp-induced up-regulation of MUC5AC and MUC5B via TLR-independent signaling; [2] Activation of AMPKα1 by interplay between polyubiquitination and phosphorylation plays a critical role in Sp- induced up-regulation of MUC5AC and MUC5B (hypothesis). To test our hypothesis, we will pursue two specific aims to determine (Aim 1) the role of AMPKα1 in OM pathogenesis in both AOM and COM; and (Aim 2) how Sp activates AMPKα1. These studies will significantly advance our understanding of the key regulators including AMPK in TLR-independent host mucosal innate defense in bacterial infections and lead to the identification of novel therapeutic targets for controlling mucus overproduction. Our AMPK signaling studies may also help understand molecular mechanisms of other AMPK-related diseases (Significance and Impact).