ABSTRACT A primary insult that precipitates sepsis can induce a profound state of immunosuppression, which renders an individual highly susceptible to secondary infections by bacteria such as Pseudomonas aeruginosa (PA). PA- induced pneumonia can cause acute respiratory distress syndrome (ARDS). We have established a 2-hit model in mice involving an initial peripheral exposure to bacterial lipopolysaccharide (LPS) followed by pulmonary infection with PA that induces immune suppression in the lung. The lungs of these mice show high levels of expression of the anti-inflammatory cytokine IL-10 but barely detectable expression of the pro- inflammatory cytokine IL-6. Compared to mice subjected to a single hit with PA only, those with 2-hits show increased morbidity, lung injury and lung bacterial dissemination. Pseudomonas secretes different virulence factors and the host in turn expresses enzymes called paraoxonases (PONs) to defend against these factors. PON2 was shown to induce a switch from pro-inflammatory M1 to suppressive M2 phenotype in macrophages (Mφs). We previously demonstrated increased numbers of regulatory myeloid cells resembling myeloid-derived suppressor cells (MDSCs) in the lung in response to LPS or bacterial infection that produce high levels of IL- 10. Studies of human sepsis have implicated peripheral blood MDSCs in chronic immune suppression and high plasma IL-10 levels were associated with poor prognosis in severely ill ARDS patients. Among cell types that defend against bacterial infections, a subset of innate-like T cells, MAIT cells, control infections of the lung by different bacteria including PA. We failed to detect MAIT cells in the peripheral blood of critically ill patients raising the possibility that susceptibility to secondary infections during sepsis is caused by a decline in host protective cells. Single cell RNA-seq (scRNA-seq) analysis of peripheral blood mononuclear cells (PBMCs) of patients show dynamic changes in gene expression, which may aid in prognosis. Collectively, these observations lead us to hypothesize that immune suppression in the lung during bacterial pneumonia involves dominance of anti-inflammatory mechanisms such as PPARγ and mTOR that induce increased generation of regulatory myeloid cells and increased expression of the anti-inflammatory mediators IL-10 and PON2 but decreased expression of host-protective mediators (IL-6, TNF-α) and cell types (MAIT cells). To prove this hypothesis, we will: Aim 1. Determine immune cell dynamics and gene signatures in PBMCs from critically ill patients and the role of mTOR in immune suppression. Aim 2. Determine the role of PPARγ and PON2 in persistent anti-inflammatory cytokine gene expression in the lungs of mice subjected to the 2-hit model.