Regulation of HMGB1 Release in Endotoxemia

描述（由申请人提供）：尽管最近在抗生素治疗和重症监护方面取得了进展，但脓毒症仍然是重症监护病房中最常见的死亡原因，仅在美国每年就有> 225，000名受害者。其发病机制仍然知之甚少，但部分归因于由早期细胞因子（例如，TNF和IFN-γ）并通过迟效介质（例如，HMGB1）。靶向早期细胞因子（例如，TNF）可以是保护性的，如果给药，而能够抑制HMGB 1释放的药物可以拯救动物免于致命的脓毒症，即使在脓毒症发作后给予。我们开创性的发现HMGB 1作为一个致命的败血症的晚期介质，促使其释放的调节机制的进一步调查。一方面，早期细胞因子（例如，TNF、IL-1、IL-6或IFN-γ）直接刺激先天性免疫细胞（例如，巨噬细胞和单核细胞）释放HMGB 1，从而有助于内毒素诱导的HMGB 1释放。另一方面，它们改变肝源性急性期蛋白（APPs）的表达，这可能类似地参与HMGB 1释放的调节。我们已经产生了令人兴奋的初步数据表明，人血清淀粉样蛋白A（SAA），而不是SAA 1，有效地诱导HMGB 1释放在体外，并加剧内毒素介导的动物体内致死。相反，SAA特异性中和抗体和肽拮抗剂减弱SAA诱导的HMGB 1释放，并在致死性内毒素血症和脓毒症的动物模型中提供保护。这些令人兴奋的发现提出了几个重要的问题，关于SAA介导的HMGB 1释放调节的新机制，以及SAA在致死性全身炎症（LSI）发病机制中的可能作用。目的1中概述的实验将检验SAA作为内毒素诱导的HMGB 1通过TLR 4/TLR 4受体释放的正调节剂的假设。具体地，我们将确定SAA表达（通过基因敲除）或活性（通过使用中和抗体、HDL或肽拮抗剂）的抑制是否会影响野生型中LPS、TSST-1、IFN-γ或SAA诱导的HMGB 1释放，以及SAA或SAA受体（例如，CD 36、TLR 2、TLR 4或CD 36）缺陷型巨噬细胞。在目标2中，我们将通过检查SAA水平（通过基因敲除或蛋白质补充）或活性（通过使用中和抗体，HDL或肽拮抗剂）的改变是否影响内毒素血症或脓毒症中动物的存活率来检验SAA有助于致死性全身性炎症发病机制的假设。目的3中概述的实验将检验SAA或抑制剂通过改变全身细胞因子（例如，HMGB 1、sPLA 2或其他）积累、白细胞浸润和细菌清除，或来自过度自噬或淀粉样变性的肝损伤。这些令人兴奋的研究的完成将进一步提高我们对HMGB 1释放调节机制的理解，并为未来开发治疗致命炎症性疾病的治疗策略提供指导。

DESCRIPTION (provided by applicant): Despite recent advance in antibiotic therapy and intensive care, sepsis remains the most common cause of death in the intensive care unit, claiming > 225,000 victims annually in the U.S. alone. Its pathogenesis remains poorly understood, but is partly attributable to dysregulated inflammatory responses that are propagated by early cytokines (e.g., TNF and IFN-γ) and sustained by late-acting mediators (e.g., HMGB1). Agents targeting early cytokines (e.g., TNF) could be protective if given prophylactically; whereas agents capable of inhibiting HMGB1 release could rescue animals from lethal sepsis even if given after the onset of sepsis. Our seminal discovery of HMGB1 as a late mediator of lethal sepsis has prompted further investigation of the mechanisms underlying the regulation of its release. On one hand, early cytokines (e.g., TNF, IL-1, IL-6, or IFN-γ) directly stimulate innate immune cells (e.g., macrophages and monocytes) to release HMGB1, thereby contributing to endotoxin-induced HMGB1 release. On the other hand, they alter the expression of liver-derived acute phase proteins (APPs), which may similarly participate in the regulation of HMGB1 release. We have generated exciting preliminary data indicating that human serum amyloid A (SAA), but not SAA1, effectively induced HMGB1 release in vitro, and exacerbated endotoxin-mediated animal lethality in vivo. In contrast, SAA-specific neutralizing antibodies and peptides antagonists attenuated SAA-induced HMGB1 release, and conferred protection in animal models of lethal endotoxemia and sepsis. These exciting findings raised several important questions regarding the novel mechanisms underlying the regulation of SAA-mediated HMGB1 release, as well as the possible roles of SAA in the pathogenesis of lethal systemic inflammation (LSI). The experiments outlined in Aim 1 will test the hypothesis that SAA functions as a positive regulator of endotoxin-induced HMGB1 release through TLR4/RAGE receptors. Specifically, we will determine whether inhibition of SAA expression (by gene knockout) or activities (by using neutralizing antibodies, HDL, or peptide antagonists) will affect LPS-, TSST-1-, IFN-γ-, or SAA-induced HMGB1 release in wild-type, and SAA- or SAA receptor (e.g., CD36, TLR2, TLR4, or RAGE)-deficient macrophages. In Aim 2, we will test the hypothesis that SAA contributes to the pathogenesis of lethal systemic inflammation by examining whether alteration of SAA levels (by gene knockout or protein supplementation) or activities (by using neutralizing antibodies, HDL, or peptide antagonists) influences animal survival rates in endotoxemia or sepsis. The experiments outlined in Aim 3 will test the hypothesis that that SAA or inhibitors affect LSI through altering systemic cytokine (e.g., HMGB1, sPLA2, or others) accumulation, leukocyte infiltration and bacterial clearance, or hepatic injury from excessive autophagy or amyloidosis. The completion of these exciting studies will further improve our understanding of the mechanisms underlying the regulation of HMGB1 release, and provide guidance for future development of therapeutic strategies to treat lethal inflammatory diseases.