TRAUMA & SEPSIS INDUCED CHANGES IN IMMUNE CELL MEMBRANE RECEPTOR TRAFFICKING

描述（由申请方提供）：单核细胞对于根除入侵微生物至关重要。这些先天免疫细胞对这些入侵者做出反应的机制是通过激活一系列模式识别受体或Toll样受体（TLR）。这些受体在专门的质膜微区上的激活是复杂的，并且难以阐明。基于我们以前的工作，我们假设这些复合物的形成需要质膜鞘脂分解成神经酰胺，导致形成脂筏大结构域和TLR复合物的形成。因此，特定的感染因子被呈递给这些模式识别受体，导致细胞活化。虽然这些反应可能挽救生命，但已知严重创伤会导致先天免疫的重新编程和改变。这些改变的表型，而不是导致宿主保护，是负责增加的易感性，入侵的生物体，导致脓毒症和器官衰竭的发展。这种状态已经在体外通过使单核细胞经受由创伤诱导的因子（包括血小板活化因子、氧化应激和补体5a）来重建。虽然负责这种重编程的机制仍然未知，但以前的工作已经证明，这一过程与质膜内脂质和蛋白质含量的改变有关。这些改变被假设发生在脂筏上。损伤后，我们假设创伤引起的因素导致神经酰胺的产生，但程度低于激活过程中看到的。神经酰胺一旦产生就在筏内融合，导致形成类似于活化时发生的宏域。此外，神经酰胺导致钙的动员，导致CaMK II的活化。这些细胞信使的激活与包含一些但不是全部TLR组分的局灶性粘附样复合物的形成相关。我们推测，这些复合物的组装和脂筏神经酰胺含量的变化是负责随后的重编程，诱导增强激活响应随后的感染。因此，该提案旨在通过探索神经酰胺、钙和CaMK II在体外和严重创伤患者中的作用，更充分地确定创伤后重编程和激活的分子机制。

DESCRIPTION (provided by applicant): Mononuclear cells are critical to the eradication of invading organisms. The mechanism in which these innate immune cells respond to these invaders is through the activation of a series of pattern recognition receptors or Toll-like receptors (TLRs). Activation of these receptors, on specialized plasma membrane microdomains is complex and poorly elucidated. Based on previous work by us, we hypothesize that formation of these complexes requires breakdown of plasma membrane sphingolipids into ceramide leading to the formation of lipid raft macrodomains and the formation of TLR complexes. As a result, specific infectious factors are presented to these pattern recognition receptors leading to cellular activation. Although these responses may be life saving, severe trauma is know to result in reprogramming and alterations in innate immunity. These altered phenotypes, rather than leading to host protection, are responsible for increased susceptibility to invading organisms leading to the development of sepsis and organ failure. This state has been recreated in vitro by subjecting mononuclear cells to factors induced by trauma, including platelet activating factor, oxidant stress and complement 5a. Although the mechanism(s) responsible for this reprogramming remain unknown, previous work has demonstrated that this process is associated with alterations in the lipid and protein content within the plasma membrane. These alterations are hypothesized to occur on lipid rafts. Following injury, we hypothesize that factors induced by trauma result in the production of ceramide, but to a lesser degree than that seen during activation. Ceramide once produced fuses within rafts leading to the formation of macrodomains similar to that which occurs with activation. Additionally, ceramide leads to the mobilization of calcium leading to the activation of CaMK II. Activation of these cellular messengers is associated with the formation of focal adhesion-like complexes that contain some but not all of the TLR components. We hypothesize that assembly of these complexes and changes in lipid raft ceramide content are responsible for subsequent reprogramming that induces enhanced activation in response to subsequent infection. Thus, this proposal sets out to determine more fully the molecular mechanisms responsible for reprogramming and activation following trauma by exploring the effects of ceramide, calcium and CaMK II in vitro, and in severely injured trauma patients.