CaMK: central regulators of the inflammatory response to surgical sepsis

描述（由申请人提供）：脓毒症的复杂生物学过程知之甚少，这阻碍了脓毒症特异性治疗的发展，反映在死亡率25年来保持不变。受损的钙（Ca2+）处理已被引用为介导的异常炎症的细胞死亡和器官功能障碍的败血症。令人惊讶的是，很少有人知道这种普遍的（88%的严重脓毒症患者）病理生理条件的发展，也没有这些改变的Ca2+信号调节的信号通路和细胞反应。最近，我们确定了钙/钙调素依赖性蛋白激酶（CaMK），一个丝氨酸/苏氨酸激酶家族响应于细胞内Ca 2+浓度[Ca 2 +]，介导MF中的Ca 2+依赖性信号。我们现在认识到，CaMK，特别是CaMKI和IV，在体内是起作用的，并调节与器官功能障碍和死亡有关的脓毒性炎症的关键介质。该建议将集中于CaMKI和IV介导LPS诱导的MF中的Ca 2+信号传导的机制。我们建议，LPS刺激TLR4途径MF，RyR门控胞质Ca 2+瞬时激活CaMKI和CaMKIV。CaMKI和CaMKIV反过来调节脓毒性炎症的关键介质HMGB1、TNF α和IL-10的释放。CaMKI和CaMKIV在介导这种炎症中发挥不同的作用，我们将对此进行定义。在目标1中，我们将利用高速，深度分辨的Ca2+瞬变和缺乏TLR4和CaMK信号通路的关键组分的基因工程小鼠的光学映射，以表征MF中LPS诱导的Ca2+信号传导和CaMK激活的机制。目的2将确定CaMKI和CaMKIV依赖性调节MF释放HMGB1的机制，因为HMGB1与脓毒性死亡率有因果关系。在目标3中，我们表明这些机制在手术脓毒症的体内CLP模型中是可操作的。我们将使用体内CaMK RNAi和CaMKIV表达缺陷的小鼠显示，CaMKI和CaMKIV调节脓毒症期间的炎症反应，并且我们将定义它们在器官功能障碍和死亡中的作用。这些综合研究可能提供关键的见解，将MF中的Ca2+信号传导与脓毒症炎症和器官功能障碍的Ca2+处理失调联系起来。公共卫生相关性：该项目将确定巨噬细胞中TLR4依赖性钙和钙/钙调蛋白依赖性蛋白激酶（CaMK）信号传导，并将这些转导系统中的扰动作为脓毒症炎症和器官功能障碍的生物学机制。对这些机制的理解将增加我们对炎症的理解，证明在设计新形式的免疫调节治疗中是有用的，并提供对目前在危重病中补充钙的做法的潜在危害的见解。

DESCRIPTION (provided by applicant): The complex biological processes of sepsis are poorly understood, which has hindered the development of sepsis-specific therapies, reflected by a mortality rate that has remained unchanged for 25 years. Impaired calcium (Ca2+) handling has been cited as the mediator of aberrant inflammation underlying the cell death and organ dysfunction of sepsis. Surprisingly, little is known of how this prevalent (88% of severely septic patients) pathophysiologic condition develops nor the signaling pathways and cellular responses regulated by these altered Ca2+ signals. Recently, we identified the calcium/calmodulin-dependent protein kinases (CaMK), a family of serine/threonine kinases responsive to intracellular Ca2+ concentration [Ca2+], mediate Ca2+-dependent signaling in the MF. We now recognize that CaMK, specifically CaMKI and IV, are operant in vivo and regulate key mediators of septic inflammation implicated in organ dysfunction and death. This proposal will focus upon the mechanisms by which CaMKI and IV mediate LPS induced Ca2+ signaling in MF. We propose that following LPS stimulation of the TLR4 pathway in MF, a ryanodine receptor (RyR)-gated cytosolic Ca2+ transient activates CaMKI and CaMKIV. CaMKI and CaMKIV, in turn, regulate the release of HMGB1, TNFa, and IL-10, key mediators of septic inflammation. CaMKI and CaMKIV assume distinct roles in mediating this inflammation, which we will define. In Aim 1 we will utilize high-speed, depth resolved optical mapping of Ca2+ transients and genetically engineered mice lacking key components of TLR4 and CaMK signaling pathways to characterize the mechanisms of LPS-induced Ca2+ signaling and CaMK activation in MF. Aim 2 will determine the mechanisms of CaMKI- and CaMKIV-dependent regulation of HMGB1 release from MF, as HMGB1 has been causally associated with septic mortality. In Aim 3 we show that these mechanisms are operant in an in vivo CLP model of surgical sepsis. We will show, using in vivo CaMK RNAi and mice deficient in the expression of CaMKIV, that CaMKI and CaMKIV regulate the inflammatory response during sepsis, and we will define their roles in organ dysfunction and death. The combined studies may provide key insights linking Ca2+ signaling in MF to the dysregulated Ca2+ handling underlying the inflammation and organ dysfunction of sepsis. PUBLIC HEALTH RELEVANCE: This project will determine TLR4-dependent calcium and calcium/calmodulin-dependent protein kinase (CaMK) signaling in macrophages and establish perturbations in these transduction systems as a biological mechanism underlying the inflammation and organ dysfunction of sepsis. An understanding of these mechanisms will increase our understanding of inflammation, prove useful in the design of novel forms of immunomodulatory therapy, and provide insight into the potential detriment of current practices of calcium supplementation in critical illness.