Regulation of HMGB1 Release in Endotoxemia

描述（由申请人提供）：尽管最近抗生素治疗和重症监护取得了进展，但革兰氏阴性细菌感染和败血症是危重患者普遍存在的问题。脓毒症的高死亡率部分是由细菌内毒素介导的，细菌内毒素刺激巨噬细胞依次释放TNF、ifn - γ、分泌性磷脂酶A2 （sPLA2）、一氧化氮和HMGB1。抗肿瘤坏死因子只有在预防性给予时才对致死性内毒素血症具有保护作用；而能够抑制HMGB1释放或活性的药物可以使小鼠免于致命的败血症，即使这些抗HMGB1药物是在败血症发作后给予的。我们已经证明，一种内源性普遍存在的多胺，精胺，在感染和损伤部位积累，可以减弱内毒素诱导的HMGB1释放。精胺介导的细胞因子抑制依赖于阴性急性期蛋白，胎儿蛋白的可用性，其循环水平在内毒素血症和败血症（由盲肠结扎和穿刺引起，CLP）期间显着降低。值得注意的是，CLP后24小时腹腔注射外源性胎儿素可显著拯救小鼠致死性败血症，因此有必要研究精胺和/或胎儿素减轻内毒素诱导的HMGB1释放的机制，并赋予对致死性内毒素血症和败血症的保护作用。鉴于新出现的证据暗示JNK MAP激酶、sPLA2和诱导型一氧化氮合酶（iNOS）在细菌内毒素诱导的HMGB1释放中的潜在作用，我们将首先确定精胺和/或胎儿素是否通过JNK MAP激酶、sPLA2或iNOS依赖机制抑制内毒素诱导的HMGB1释放（Specific Aim 1）。然后，我们将确定外源性胎儿素和/或精胺的施用，或胎儿素表达的遗传破坏，是否会影响内毒素血症和败血症的动物存活（特异性目标2）。最后，我们将描述精胺和/或胎儿蛋白介导的保护的具体机制，通过确定精胺和/或胎儿蛋白的管理是否会改变促炎细胞因子的系统性积累，或者在缺乏TNF、ifn - γ或胎儿蛋白的野生型或突变型小鼠中影响入侵病原体和/或凋亡细胞的清除（specific Aim 3）。这些问题的答案将提高我们对败血症病理生理学的理解，并为治疗致命性全身炎症性疾病的新治疗策略的发展提供启示。

DESCRIPTION (provided by applicant): Despite recent advances in antibiotic therapy and intensive care, Gram-negative bacterial infection and sepsis are widespread problems in critically ill patients. The high mortality of sepsis is in part mediated by bacterial endotoxin, which stimulates macrophages to sequentially release TNF, IFN-gamma, secretory phospholipase A2 (sPLA2), nitric oxide, and HMGB1. Anti-TNF agents are protective against lethal endotoxemia only if given prophylatically; whereas agents capable of inhibiting HMGB1 release or activity can rescue mice from lethal sepsis even when these anti-HMGB1 agents are given after the onset of sepsis. We have demonstrated that, an endogenous ubiquitous polyamine, spermine, accumulates at sites of infection and injury, can attenuate endotoxin-induced HMGB1 release. The spermine-mediated cytokine suppression is dependent on the availability of a negative acute phase protein, fetuin, whose circulating levels are significantly decreased during endotoxemia and sepsis (induced by cecal ligation and puncture, CLP). Notably, intraperitoneal administration of exogenous fetuin 24 hours after CLP significantly rescues mice from lethal sepsis, prompting the necessity to investigate the mechanisms by which spermine and/or fetuin attenuate endotoxin-induced HMGB1 release, and confer protection against lethal endotoxemia and sepsis. In light of emerging evidence implicating a potential role for JNK MAP kinase, sPLA2, and inducible nitric oxide synthase (iNOS) in bacterial endotoxin-induced HMGB1 release, we will first determine whether spermine and/or fetuin inhibit endotoxin-induced HMGB1 release through a JNK MAP kinase, sPLA2, or iNOS-dependent mechanism (Specific Aim 1). We will then determine whether administration of exogenous fetuin and/or spermine, or genetic disruption of fetuin expression, influences animal survival in endotoxemia and sepsis (Specific Aim 2). Lastly, we will delineate the specific mechanisms of spermine- and/or fetuin- mediated protection by determining whether administration of spermine and/or fetuin alters systemic accumulation of pro-inflammatory cytokines, or alternatively influences clearance of invading pathogens and/or apoptotic cells in wild-type or mutant mice deficient in TNF, IFN-gamma, or fetuin (Specific Aim 3). Answers to these questions will improve our understanding of the pathophysiology of sepsis, and shed light on the development of novel therapeutic strategies for treatment of lethal systemic inflammatory diseases.