Regulation of HMGB1 Release in Endotoxemia

描述(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，脓毒症仍然是重症监护病房最常见的死亡原因，仅在美国每年就有225,000名患者死亡。其发病机制尚不清楚，但部分原因是由早期细胞因子(如肿瘤坏死因子和干扰素？)传播的失调的炎症反应。并由后期行动的调解人(例如，HMGB1)维持。如果预防性给予针对早期细胞因子的药物(如肿瘤坏死因子)，则可以起到保护作用；而能够抑制HMGB1释放的药物即使在脓毒症发生后给予，也可以将动物从致命的脓毒症中拯救出来。我们的开创性发现HMGB1是致死性脓毒症的晚期介体，这促使我们进一步研究其释放的调节机制。一方面，早期细胞因子(如肿瘤坏死因子、白介素1、白介素6或干扰素？)直接刺激天然免疫细胞(如巨噬细胞和单核细胞)释放HMGB1，从而促进内毒素诱导的HMGB1释放。另一方面，它们改变了肝脏衍生的急性时相蛋白(APP)的表达，这些APP可能类似地参与HMGB1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SAA1，在体外有效地诱导HMGB1的释放，并在体内加剧内毒素介导的动物死亡。相反，SAA特异性中和抗体和多肽拮抗剂可减弱SAA诱导的HMGB1释放，并在致死性内毒素血症和败血症动物模型中提供保护作用。这些令人兴奋的发现提出了几个重要的问题，涉及SAA介导的HMGB1释放的调控机制，以及SAA在致死性全身炎症(LSI)发病机制中的可能作用。目标1中概述的实验将验证SAA通过TLR4/RAGE受体作为内毒素诱导的HMGB1释放的正调节因子的假设。具体地说，我们将确定抑制SAA表达(通过基因敲除)或活性(通过使用中和抗体、高密度脂蛋白或多肽拮抗剂)是否会影响 在野生型和SAA受体(如CD36、TLR2、TLR4或RAGE)缺乏的巨噬细胞中，脂多糖、TSST-1、干扰素或SAA可诱导HMGB1的释放。在目标2中，我们将通过检测SAA水平的改变(通过基因敲除或蛋白质补充)或活动(通过使用中和抗体、高密度脂蛋白或多肽拮抗剂)是否影响内毒素血症或败血症动物的存活率来检验SAA在致死性全身炎症的发病机制中的作用这一假设。AIM 3中概述的实验将测试SAA或抑制剂通过改变系统细胞因子(例如，HMGB1、sPLA2或其他)积聚、白细胞渗透和细菌清除，或过度自噬或淀粉样变性造成的肝损伤来影响LSI的假设。这些激动人心的研究的完成将进一步提高我们对HMGB1释放调控机制的理解，并为未来治疗致死性炎症性疾病的治疗策略的发展提供指导。

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-?) 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-?) 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-?-, 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.