CaMK: central regulators of the inflammatory response to surgical sepsis

描述（由申请人提供）：对脓毒症复杂的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25年来保持不变的死亡率上。受损的钙（Ca2+）处理已被引用为异常炎症的潜在的细胞死亡和器官功能障碍的中介败血症。令人惊讶的是，这种普遍的（88%的严重脓毒症患者）病理生理状况是如何发展的，也不知道这些改变的Ca2+信号调节的信号通路和细胞反应。最近，我们发现了钙/钙调素依赖性蛋白激酶（CaMK），这是一个响应细胞内Ca2+浓度的丝氨酸/苏氨酸激酶家族，在MF中介导Ca2+依赖性信号传导。我们现在认识到CaMK，特别是CaMKI和IV，在体内起作用，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。本研究将重点研究CaMKI和IV介导LPS诱导的MF中Ca2+信号转导的机制。我们提出，在LPS刺激MF中的TLR4通路后，ryanodine受体（RyR）门控的细胞质Ca2+瞬间激活CaMKI和CaMKIV。CaMKI和CaMKIV反过来调节HMGB1、TNFa和IL-10的释放，这些都是脓毒性炎症的关键介质。CaMKI和CaMKIV在介导这种炎症中扮演着不同的角色，我们将对此进行定义。在Aim 1中，我们将利用高速、深度分辨率的Ca2+瞬态光学图谱和缺乏TLR4和CaMK信号通路关键成分的基因工程小鼠来表征lps诱导的MF中Ca2+信号和CaMK激活的机制。Aim 2将确定CaMKI-和camkiv依赖性调节HMGB1从MF释放的机制，因为HMGB1与败血症死亡率有因果关系。在Aim 3中，我们表明这些机制在手术败血症的体内CLP模型中是有效的。我们将在CaMK RNAi和CaMKIV表达不足的小鼠体内证明，CaMKI和CaMKIV在败血症期间调节炎症反应，我们将确定它们在器官功能障碍和死亡中的作用。这些联合研究可能提供将MF中的Ca2+信号与败血症炎症和器官功能障碍的Ca2+处理失调联系起来的关键见解。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/钙调素依赖性蛋白激酶（CaMK）信号，并确定这些转导系统中的干扰是败血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，证明在设计新型免疫调节疗法方面是有用的，并提供对危重疾病中目前补钙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.