Mycoplasma Model of Infection-mediated Sterile Inflammation

描述（由申请人提供）：与通常微妙的支原体病不同，短吻鳄支原体感染可引起易感宿主的致命多系统炎症性疾病。我们的总体目标是了解其分泌的糖苷酶的作用，这是支原体中前所未有的，在其生态和显著的毒力。在其他感染中，细菌来源的外源性配体显示病原体相关分子模式（PAMPs），通过TLR信号刺激宿主促炎细胞因子的产生。自我限制的反应可以清除病原体，但不受控制的TLR激活会导致过度的炎症，这对宿主是毁灭性的。无菌炎症不是对外源性PAMPs的反应，而是对从受损宿主组织中释放的内源性TLR激动剂的反应。因此，具有讽刺意味的是，感染期间分泌的细菌糖苷酶降解的ECM成分可能介导无菌炎症。感染介导的无菌性炎症仍未被研究，因为细菌固有的PAMPs如肽聚糖或脂多糖的存在不可避免地混淆。它们使得区分无菌炎症对PAMPs的反应变得困难，以便评估它们在细菌感染中的相对重要性。然而，像短吻肌这样的支原体模型可以避免这种混淆，因为它天生缺乏相关的PAMPs，但能够通过其糖苷酶活性从宿主ECM中释放内源性TLR激动剂，如降解的透明质酸寡糖（HA）。我们将用野生型和敲除透明质酸酶的短吻吻猴感染原代肺成纤维细胞，目的是：1)鉴定感染后激活的TLR信号，重点研究HA激活和关键TLR适配蛋白MyD88的信号转导；2)确定短吻鳄感染过程中TLR信号的下游效应物，重点研究NF？B活化和TNF-？作为TLR激活与无菌性炎症机制相关的关键事件的标志物。预期的结果是证明细菌糖苷酶可以主动引发TLR信号参与无菌炎症。

DESCRIPTION (provided by applicant): In contrast to usually subtle mycoplasmosis, Mycoplasma alligatoris infection causes lethal multisystemic inflammatory disease of susceptible hosts. Our overall objective is to understand the roles of its secreted glycosidases, which are unprecedented among mycoplasmas, in its ecology and remarkable virulence. During other infections, exogenous ligands of bacterial origin that display pathogen-associated molecular patterns (PAMPs) stimulate host pro-inflammatory cytokine production via TLR signaling. Self-limiting responses can clear pathogens, but uncontrolled TLR activation results in exaggerated inflammation that is devastating to the host. Sterile inflammation is a response not to exogenous PAMPs but to endogenous TLR agonists liberated from damaged host tissues. Thus, ironically, ECM components degraded by bacterial glycosidases secreted during infection may mediate sterile inflammation. Infection-mediated sterile inflammation remains unexplored because of the inextricably confounding presence of intrinsic bacterial PAMPs like peptidoglycan or lipopolysaccharide. They make it difficult to partition responses to PAMPs from sterile inflammation, in order to assess their relative significance in bacterial infections. However, a mycoplasma model like M. alligatoris can avoid that confounding because it naturally lacks the relevant PAMPs, but is capable of liberating endogenous TLR agonists like degraded hyaluronan oligosaccharides (HA) from host ECM enzymatically through its glycosidase activity. We will infect primary pulmonary fibroblasts with wild type and hyaluronidase-knockout M. alligatoris to: 1) Identify TLR signaling actively elicited by infection, with focus on activation by HA and signal transduction via the key TLR adapter protein MyD88; and 2) Determine downstream effectors of TLR signaling during M. alligatoris infection, with focus on NF?B activation and TNF-? secretion as markers of the key events that mechanistically link TLR activation to sterile inflammation. The expected outcome is proof that a bacterial glycosidase can actively elicit TLR signaling involved in sterile inflammation. The work is significant because it will distinguish TLR signaling in infection-mediated sterile inflammation from responses to bacterial PAMPs. Once this becomes possible, the complete cascade of events during infection-mediated sterile inflammation in vivo can be determined much more effectively. Beyond improving our understanding of M. alligatoris' virulence, this distinction can be expected to provide a stepping stone toward evidence-based rationales for antagonists of TLRs and/or glycosidases as therapeutic interventions for management of human and animal infections caused by many species of bacteria.