TLR4 and cardiac inflammatory response to I/R

描述（由申请人提供）： 与心脏手术相关的强制性心肌缺血/再灌注（I/R）诱导炎症反应，导致促炎细胞因子和趋化因子的过度表达。这些因素加重缺血后心功能不全。因此，调节这种炎症反应对于心脏手术中的心肌保护至关重要。然而，参与这种反应的信号机制仍不清楚。尽管Toll样受体4（TLR 4）调节细胞对内毒素的炎症反应，但目前尚不清楚这种先天性免疫应答受体是否在I/R的心脏炎症反应中起作用。我们的初步研究发现，在缺乏功能性TLR 4的心脏中，I/R后炎症介质的产生被消除，并且这种炎症介质产生的减少与缺血后心脏功能恢复的显著改善相关。可能是I/R期间产生和/或释放的内源性试剂激活心肌TLR 4信号通路。在我们的初步研究中，我们发现缺血后心脏的冠状流出物中约50至100 kD的蛋白质是促炎性的。在这些蛋白中，我们鉴定了热休克同源蛋白70（HSC）。此外，我们发现用多克隆抗体中和细胞外HSC抑制TLR 4依赖性心肌炎症反应，并且当细胞外应用HSC时，HSC与TLR 4相互作用并以TLR 4依赖性方式诱导分离的巨噬细胞中细胞因子的产生。我们假设TLR 4信号传导参与缺血后心脏炎症反应，并且细胞外HSC作为内源性配体激活TLR 4信号传导级联反应。研究I/R过程中心肌TLR 4信号通路和确定I/R激活TLR 4信号通路的分子机制是本研究的主要目标。本项目的具体目的是：1）确定TLR 4信号在缺血后心肌炎症反应中的作用，并确定HSC对心肌TLR 4活化的贡献，2）确定心肌释放HSC的机制，3）确定HSC对TLR 4信号通路的影响。拟议的研究应该提供新的见解，涉及心肌先天性免疫反应的I/R的信号机制，并可能提出新的治疗目标和方法，以改善心脏手术后的结果与强制性I/R。

DESCRIPTION (provided by applicant): Obligatory myocardial ischemia/reperfusion (I/R) associated with cardiac surgery induces an inflammatory response, leading to over-expression of proinflammatory cytokines and chemokines. These factors exacerbate post-ischemic cardiac dysfunction. Thus, regulation of this inflammatory response is critical for myocardial protection in cardiac surgery. However, the signaling mechanisms involved in this response remain unclear. While Toll-like receptor 4 (TLR4) regulates cellular inflammatory response to endotoxin, it is presently unknown whether this innate immunoresponsive receptor plays a role in cardiac inflammatory response to I/R. Our preliminary studies found that the production of inflammatory mediators following I/R is abrogated in heart lacking functional TLR4 and that this reduction of inflammatory mediator production correlates to a marked improvement in post-ischemic cardiac functional recovery. It is likely that endogenous agents generated and/or released during I/R activate myocardial TLR4 signaling pathways. In our preliminary studies, we found that proteins of approximately 50 to 100 kD in coronary effluent from post-ischemic heart are proinflammatory. Among these proteins, we identified heat shock cognate protein 70 (HSC). Furthermore, we found that neutralization of extracellular HSC with a polyclonal antibody suppresses TLR4-dependent myocardial inflammatory response and that HSC, when applied extracellularly, interacts with TLR4 and induces cytokine production in isolated macrophages in a TLR4-dependent manner. We hypothesize that TLR4 signaling is involved in post-ischemic cardiac inflammatory response and that extracellular HSC functions as an endogenous ligand to activate TLR4 signaling cascade. Investigation of myocardial TLR4 signaling during I/R and determination of the molecular mechanisms through which I/R activates TLR4 signaling cascade are the major goals of the proposed studies. The Specific Aims of this project are: 1) to define the role of TLR4 signaling in post-ischemic myocardial inflammatory response and to determine the contribution of HSC to activation of myocardial TLR4, 2) to determine the mechanisms through which the myocardium releases HSC, and 3) to determine the effect of HSC on TLR4 signaling pathways. The proposed studies should provide novel insights into the signaling mechanisms involved in myocardial innate immunoresponse to I/R and could suggest new therapeutic targets and approaches for improving outcome following cardiac surgery with obligatory I/R.