MYELOPOIETIC STIMULATION FOLLOWING BURN INJURY

尽管在重症监护方面取得了进展，但由于脓毒症引起的全身炎症反应综合征(SIRS)和多系统器官衰竭，大面积烧伤患者的死亡率仍然很高。严重烧伤患者由于持续的单核/巨噬细胞活化，往往表现出不受控制的促炎细胞因子和前列腺素类物质的过度产生。许多微生物感染和炎性损伤导致单核细胞产量增加，其驱动因素是单核细胞系特异性集落刺激因子(M-CSF)的伴随升高。然而，关于单核细胞产生在热损伤中的调节作用的研究很少。此外，越来越多的单核细胞可用于激活，作为细胞因子过度产生的潜在原因，导致严重烧伤后SIRS的发生，这一点从未被探索过。这一假设是，严重烧伤和脓毒症导致有利于增加单核细胞生成的骨髓环境。这种持续增加的单核/巨噬细胞激活的可能性可能通过失调的细胞因子产生来潜在地推动炎症。我们将在小鼠背部烫伤模型中测试这一前提，在该模型中，脓毒症将在最初烧伤后临床相关的早期和晚期时间点诱导。在第一个目标中，我们将a)通过单核细胞生成发育抗原(ER-MP12和20)的流式细胞术分析和克隆分析来确定骨髓单核细胞的生成得到增强；b)检测血浆和骨髓M-CSF水平；c)M-CSF受体(c-FMS)在整个骨髓细胞和单核细胞发育的不同区域的mRNA和细胞表面表达；d)记录单核细胞发育相关转录因子PU.1和Egr-1在烧伤和脓毒症不同单核细胞生成区域的表达模式。在第二个目标中，利用M1细胞的体外单核细胞分化，我们将评估不同的细胞因子，如IL-1、IL-6和IL-10以及在烧伤中升高的前列腺素E_2是如何影响a)单核细胞分化的：b)c-FMS表达；c)PU.1和Egr-1的激活；d)细胞因子的产生和功能。在第三个目标中，我们将确定在我们的小鼠模型中，取消免疫抑制和骨髓生成调节前列腺素2是否可以使单核细胞生成、c-FMS表达、PU.1和Egr-1表达以及单核细胞释放动力学正常化。这些研究将直接检验我们的假设，并提供关于烧伤和脓毒症中单核细胞生成的状态和机制的关键信息。更重要的是，这些研究将有助于解释严重烧伤患者细胞因子的异常产生，并为合理治疗以提高他们的存活率提供靶点。

Despite advances in critical care, mortality among patients with massive burns still remains high due to sepsis-induced systemic inflammatory response syndrome (SIRS) and multisystem organ failure. Patients with severe burns often manifest uncontrolled overproduction of proinflammatory cytokines and prostanoids due to continued monocyte/macrophage activation. Many microbial infections and inflammatory insults result in increased monocyte production driven by a concomitant elevation in monocytic lineage specific colony-stimulating factor, M-CSF. Yet, little research has been done on the regulation of monocyte production in thermal injury. Furthermore, the availability of increasing number of monocytes for activation as an underlying cause for cytokine overproduction leading to the development of SIRS in severe burn injury has never been explored. The hypothesis of this propsal is that severe burn injury and sepsis result in a bone marrow milieu conducive to increased monocytopoiesis. This continued availa-bility of increasing number of monocytes/macrophages for activation can potentially drive inflammation through dysregulated cytokine production. We will test this premise in a murine dorsal scald burn model in which sepsis will be induced at clinically relevant early and late time points after the initial burn injury. In the first aim, we will a) establish that bone marrow monocyte production is enhanced through flowcytometric analysis of monocytopoietic developmental antigens (ER-MP12 and 20) and clonogenic assays; b) measure plasma and bone marrow M-CSF levels; c) mRNA and cell surface expression of M-CSF receptor (c-fms) in total bone marrow cells and in different compartments of monocytic development; d) document the expression pattern of monocyte development associated transcription factors PU.1 and Egr-1 in different monocytopoietic compartments in burn and sepsis. In the second aim, using an in vitro monocytic differentiation of M1 cells, we will evaluate how different cytokines such as IL-1, IL-6 and IL-10 and the prostanoid PGE2 that are elevated in burn injury influence a) monocytic differentiation: b) c-fms expression; c) PU.l and Egr-1 activation and d) cytokine production and functions. In the third aim, we will determine if abrogation of the immunosuppressive and myelopoiesis regulating prostanoid, PGE2, can normalize monocytopoiesis, c-fms expression, PU.1 and Egr-1 expression and kinetics of monocyte release in our murine model. These studies will directly test our hypothesis and provide crucial information on the status and the mechanisms of monocytopoiesis in burn injury and sepsis. More importantly, these studies will help explain the aberrant cytokine production in severely injured burn patients and provide targets for rational therapies to improve their survival.