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，但革兰氏阴性细菌感染和败血症在危重患者中仍然是普遍存在的问题。脓毒症的高死亡率在一定程度上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肿瘤坏死因子、干扰素-γ、分泌型磷脂酶A2(SPLA2)、一氧化氮和HMGB1。抗肿瘤坏死因子药物只有在预防性给药的情况下才能预防致命的内毒素血症；而能够抑制HMGB1释放或活性的药物可以将小鼠从致命的脓毒症中拯救出来，即使这些抗HMGB1药物是在败血症发生后给予的。我们已经证明，一种内源性的普遍存在的多胺，精胺，聚集在感染和损伤部位，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一种阴性的急性时相蛋白-胎球蛋白的可获得性，在内毒素血症和脓毒症(盲肠结扎和穿孔，CLP)期间，胎球蛋白的循环水平显著降低。值得注意的是，CLP后24小时腹腔注射外源性胎球蛋白显著地将小鼠从致死性败血症中解救出来，促使有必要研究精胺和/或胎球蛋白减少内毒素诱导的HMGB1释放的机制，并提供对致死性内毒素血症和败血症的保护。鉴于新出现的证据表明JNK MAP、sPLA2和诱导型一氧化氮合酶(INOS)在细菌内毒素诱导的HMGB1释放中可能起作用，我们将首先确定精胺和/或胎儿蛋白是否通过JNK MAP、sPLA2或iNOS依赖的机制抑制内毒素诱导的HMGB1释放(特定目标1)。然后，我们将确定外源性胎儿蛋白和/或精胺的应用，或胎儿蛋白表达的遗传干扰，是否会影响内毒素血症和败血症动物的存活(特定目标2)。最后，我们将通过确定给予精胺和/或胎球蛋白是否改变全身致炎细胞因子的积累，或替代地影响缺乏肿瘤坏死因子、干扰素-γ或胎球蛋白的野生型或突变小鼠对入侵病原体和/或凋亡细胞的清除来描述精胺和/或胎球蛋白介导的保护的具体机制(特定目标3)。对这些问题的回答将提高我们对脓毒症的病理生理学的理解，并有助于开发治疗致死性全身炎症性疾病的新的治疗策略。

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.