PROJECT SUMMARY Virus-associated bacterial pneumonia is a leading cause of death in pandemics. Since we use different strategies to fight viruses and bacteria, host innate immune responses are confused and impaired when both types of infection occur simultaneously, resulting in higher mortality in post-viral bacterial pneumonia. Determining the key events in the initial viral infection that disturb the subsequent antibacterial responses will guide therapies in treating post-viral bacterial pneumonia. STAT2, as a critical component of IFN-I signaling, is essential for the antiviral response, but elicits detrimental effects in antibacterial response though yet-unknown mechanisms. We discovered a novel T404 phosphorylation of STAT2, stimulated by virus infections, enabling an efficient antiviral response in infected cells. This proposal sheds new light on the role of STAT2 T404 phosphorylation in the pathogenesis of post-viral bacterial pneumonia from three perspectives: (1) the action of IFN: In response to viral infection, IFN-I is produced to limit viral dissemination, but with enigmatic functions in the subsequent bacterial infection, due to suppressing chemokine production, IL-17-dependent immune responses, and recruitment of macrophages. Considering the positive regulation of T404 phosphorylation in IFN-I signaling, STAT2 will compromise the integrity of lung barriers in post-viral bacterial pneumonia; (2) the regulation of inflammation: We reported that the presence of STAT2 augments the production of IL-6 and other NF-kB-dependent inflammatory cytokines in response to LPS. This subset of cytokines drives inflammation-associated lung injury. In agreement with this finding, our preliminary data show that STAT2 T404 phosphorylation-deficient mice are protected from a challenge with LPS, compared with wild-type siblings. (3) STING-mediated antibacterial functions of macrophages: By detecting bacterial-derived cyclic dinucleotides, STING mediates the antibacterial functions of macrophages, including cytokine production, and bacterial digestion. We found that T404 phosphorylation mediates pervasive functions of STAT2 in STING activation, including reshaping STING- mediated gene expression to a pro-inflammatory profile, and inhibiting subsequent outcomes, including bacterial clearance. In summary, we propose that T404 phosphorylation of STAT2 is a key event, induced by virus infection, that enhances IFN-I-dependent signaling, exaggerates inflammatory-associated acute lung injury, and compromises antibacterial functions of macrophages in secondary bacterial pneumonia. This proposal will carry our understanding from structural and mechanistic analyses forward to phenotypic changes in vitro and in vivo. We will also determine the kinetics of T404 phosphorylation of STAT2 in mice with post- influenza P. aeruginosa pneumonia, and validate the findings in macrophages from patients with ARDS- associated pneumonia. Successful completion of this proposal wi...