Novel regulation of mucosal innate defense by AMPK in Otitis Media

粘蛋白是粘液中的一种主要蛋白质组分，通过提供一种免疫原性，在粘膜先天防御中起着关键作用。 物理屏障和捕获病原体用于粘膜纤毛清除。如果不加控制，则产生过多的粘蛋白 在中耳炎（OM）和粘液性中耳炎中阻碍粘液纤毛清除并导致传导性听力损失 肺部感染引起的阻塞。因此，粘蛋白的生产必须严格控制。然而，分子 粘蛋白的严格调节的潜在机制仍然很大程度上未知。 中耳炎（OM）是最常见的儿童细菌感染和传导性中耳炎的主要原因。 听力损失它仍然是一个重大的健康问题和巨大的社会经济负担。S.肺炎链球菌， 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耐药性。迫切需要 用于开发创新的非抗生素治疗剂以抑制粘液过度产生。我们的长期 目的是阐明OM发病机制的分子机制， 目标的与相对公知的Toll样受体（TLR）依赖性机制相反， 肺炎球菌溶血素（pneumolysin，Pneumolysin）是一种由几乎所有临床分离株产生的关键毒力因子， 在免疫应答中，TLR非依赖性机制，包括关键的调节因子，在很大程度上仍不清楚。 腺苷5 '-单磷酸活化蛋白激酶α1（AMPKα1）是近年来发现的一种主要的调节宿主 能量平衡它在感染性疾病中的作用，特别是在宿主粘膜先天防御反应中的作用， 例如粘液的产生，在很大程度上仍不清楚。我们令人鼓舞的初步数据表明， 通过激活AMPKα1以TLR 2/4非依赖性方式上调粘蛋白MUC 5AC和MUC 5 B， 中耳和气道上皮细胞在体外和小鼠模型的急性和慢性OM。 有趣的是，Sp和peptide可能通过诱导新的非传统（蛋白质降解）激活AMPKα1。 AMPKα1的泛素化可能是通过下调关键的去泛素化酶CYLD。所有这些 因此，令人兴奋的初步数据为我们假设AMPKα1作为一种细胞因子发挥作用提供了坚实的基础。 通过TLR非依赖性信号传导，SP诱导MUC 5AC和MUC 5 B上调的关键调节因子; [2] AMPKα1通过多聚泛素化和磷酸化之间的相互作用而活化，在Sp- 诱导MUC 5AC和MUC 5 B的上调（假设）。为了验证我们的假设，我们将追踪两个具体的 目的是确定（目的1）AMPKα1在AOM和COM的OM发病机制中的作用;以及（目的2）如何 Sp激活AMPKα1。这些研究将大大促进我们对关键监管机构的理解，包括 AMPK在TLR非依赖性宿主粘膜先天防御细菌感染中的作用及其鉴定 用于控制粘液过度产生的新的治疗靶点。我们的AMPK信号研究也可能有助于 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).