Trauma and hemorrhagic shock are critical surgical conditions that are managed daily by surgeons and physicians in VA medical centers. It rapidly triggers suppression of innate immune system, leading to an increased susceptibility to infections. Previously, physicians and scientists have extensively studied whether and how the function of innate immune cells is disrupted by severe trauma and hemorrhagic shock. In contrast, our knowledge about molecular mechanisms involved in impairment of innate immunity in non-immune cells by severe trauma and hemorrhagic shock is limited. Thus, the objective of our long-term research is to study effects of severe trauma and hemorrhagic shock on non-immune cell function against infections and molecular mechanisms involved. Recent studies have revealed that autophagic machinery is a critical innate immune effector against intracellular microbes for non-phagocytic cells. Particularly, Atg12 has been shown to play an important role in eliminating invaded bacteria by epithelial cells. In this project, we will test our central hypothesis that trauma and hemorrhage induce down-regulation of Atg12 in lung epithelial cells, which in turn impairs Atg12-dependent innate defense system against opportunistic pathogens in lung epithelial cells.
In Specific Aim 1, we will characterize the effect of sublethal hemorrhage stress on Atg12 expression in lungs using a molecular biology approach. A classic murine hemorrhage model will be used. Furthermore, we will study whether hemorrhage-induced alteration of Atg12 is associated with increase in susceptibility to Pseudomonas aeruginosa infection in lungs. In addition, we will investigate whether knockdown of Atg12 results in attenuation of eliminating bacteria by lung epithelial cells in vitro using a lentivirus-based gene silencing approach. Finally, we will delete Atg12 in lung epithelial cells in vivo using a novel gene knockout approach and examine whether lung epithelial-specific down-regulation of Atg12 contributes to impairing pulmonary innate immunity against P. aeruginosa infection in mice.
In Specific Aim 2, we will investigate molecular mechanisms by which surgical insult-derived proinflammatory inflammatory mediators alter Atg12 gene expression in human respiratory epithelial cells. Specifically, we will characterize specific binding motifs in the promoter of human Atg12 gene and their binding proteins, and study their role in alteration of expression of Atg12 during severe surgical circumstances. The standard approach for analysis of the gene promoter function will be applied.
In Specific Aim 3, we investigate whether a nature molecule is able to preserve Atg12 levels in respiratory epithelial cells, which in turn sustains innate immunity in lungs during severe surgical conditions. The studies involve infection of post-hemorrhaged mice with Pseudonumo aeruginosa followed by assessing the clinical course including survival rate, bacteremia, tissue injury, and inflammatory and cytokine response. At the completion of this project, we will provide novel mechanisms by which severe surgical stresses (i.e. trauma and hemorrhage) suppress innate immune function in non-phagocytes. This work will expand our knowledge on understanding and treatment of suppression of innate immunity in patients under severe surgical circumstances.
Infection after hemorrhagic shock remains a common cause of mortality and morbidity in patients admitted to trauma units at VA Medical Centers. It causes extended hospital stay and charges. Individuals with severe trauma and hemorrhage have been found to be susceptible to infections. However, a major gap remains in our understanding how hemorrhage and other severe surgical circumstances result in increase in susceptibility to infections. In this project, thus, we will elucidate a novel mechanism that leads to understanding of increase in susceptibility to infections after hemorrhage and other severe surgical circumstances. We will further explore a new treatment strategy for protection against post hemorrhagic shock infection. This work has potential impact on the development of novel therapeutic interventions against impairment of innate immunity in by trauma or inflammation in VA patients. Thus, the subject matter of this proposal is timely important and has a direct clinical application for management of VA patients with severe trauma/hemorrhage and other surgical diseases.
