Activation of Immune Reserves to Prevent Secondary Bacterial Lung Infections After Trauma and Hemorrhage

Background: Severe trauma and blood loss are major causes for development of organ dysfunction, which contributes to as many as half of all hospital deaths in armed forces Service Members and Veterans. Notably, higher mortality occurs among patients that initially survived injury, but later developed life-threatening immunosuppression and related pneumonias. Effective therapeutic interventions for trauma-related infections are not available for ~750,000 trauma and sepsis survivors in the U.S. Rationale: The major goal of the proposed study is to reduce the severity of trauma-induced lung bacterial infection, in particular, to restore mitochondrial and lysosomal biogenesis, and therefore achieve synergistic benefit for macrophage antimicrobial function after polytrauma/hemorrhage. This concept is directly built on previous and preliminary findings that mitochondrial signaling is coupled with autophagy for macrophage-dependent killing of bacterial pathogens, but is impaired due to severe trauma and sepsis. While the cause(s) of such impairment is not well understood, we found that lung injury is linked to accumulation of ZKSCAN3 (zinc finger/KRAB/SCAN3), the major suppressor of transcription factor EB (TFEB, and homolog TFE3), which promotes mitochondria and lysosome biogenesis. FY23 PRMRP Topic Area addressed by the proposed research project includes the strategic goals to (1) prevent lung injury caused by trauma due to secondary lung bacterial infections; (2) utilize ZKSCAN3 accumulation in macrophages/monocytes as a marker to monitor risk of bacterial infections after trauma and hemorrhage; (3) develop new therapeutic approaches to reduce ZKSCAN3 accumulation and slow the progression and/or reverse the risk of lung injury due to lung bacterial infections secondary to trauma/hemorrhage; (4) Use proteomics and transcriptomics to establish in-depth effects of AMPK activators and ZKSCAN3 deficiency on lung macrophages in polytrauma and bacterial pneumonia. Hypothesis: In polytrauma/hemorrhage, ZKSCAN3-dependent impairment of lysosomal and mitochondrial biogenesis reduces macrophages' anti-microbial capacity, thereby potentiating the severity of bacterial pneumonia after trauma/hemorrhage, a mechanism amplified by aging. We hypothesize that AMPK activation diminishes ZKSCAN3-mediated repression of TFEB/TFE3, thereby restores mitochondrial and lysosomal biogenesis. Specific Aims: (1) Delineate the mechanism by which inflammatory signaling pathways affect ZKSCAN3 accumulation in macrophages after bacterial lung infections and during susceptibility to secondary lung infections after polytrauma/hemorrhage. (2) Determine the mechanism by which ZKSCAN3 deficiency affects mitochondrial and lysosomal biogenesis and clearance of bacterial pathogens, including susceptibility to bacterial lung infections due to polytrauma/hemorrhage. (3) Delineate how AMPK activation affects ZKSCAN3-dependent suppression of mitochondrial and lysosomal biogenesis and severity of bacterial pneumonia, including secondary infections in immunocompromised mice due to polytrauma/hemorrhage and in aging. Study Design: The specific mechanisms of accumulation and action of ZKSCAN3 in lung macrophages will be investigated using wild-type and mice deficient in ZKSCAN3; total deficiency Zkscan3-/- and leukocyte depleted LysM-Zkscan3-/- mice. We will test the feasibility of AMPK activators metformin and MK-8722 (direct allosteric activator) as a preemptive treatment in mice subjected to P. aeruginosa-induced pneumonia via inhibition of ZKSCAN3 accumulation in macrophages. The second model will test if AMPK activators affect macrophage anti-bacterial capacity during the immunosuppression phase induced by polytrauma/hemorrhage. The severity of bacterial infection and lung injury will be tested using lung CFUs, lung resistance and compliance test, histology, inflammation and leukocytes flux. Proteomics and transcriptomics will determine how AMPK activators and ZKSCAN3 deficiency affects lung macrophages in polytrauma and bacterial pneumonia. Impact: Upon successful completion, our results will be used as a springboard to test AMPK activators in a Placebo Controlled Randomized Clinical Trial for patients with trauma-related immunosuppression. We believe therapies that target metabolic maladaptation in immune impairment will save lives of military personnel, Veterans and civilians affected by severe trauma/blood loss. Our studies will not only identify therapeutic targets but also provide a crucial mechanistic explanation for molecular mechanisms that can be tested in other models of injury. Relevance to Military Health: Severe trauma/hemorrhage are especially relevant in combat situations and training-related accidents. Blast explosions, burns, smoke and chemical inhalation are directly linked to injury and secondary pneumonias. Onset of immunosuppression is also linked to complications after major surgery. Notably, lung injury caused by influenza and COVID-19 place military personnel, Veterans and their Families at a high risk of secondary lung infections and respiratory failure. Less