The original premise of our K01 proposal was that airway host-defense protein Short Palate Lung Nasal epithelium Clone-1 (PLUNC, SPLUNC1) regulates inflammation and therefore could contribute to acute lung injury (ALI) and tissue damage in CF and respiratory infections. We defined a novel immunomodulatory effect of SPLUNC1 in enhancing LPS-induced IFNγ/IFNλ-associated inflammation that led us to refocus the last years of the K01 award on an Influenza A virus (IAV) model of pneumonia, where IFN responses are critical. This led to the submission of our R01 proposal "SPLUNC1-controlled mechanisms of ALI during influenza A infection". IAV mortality is largely due to respiratory failure, precipitated by lung inflammation and ALI. Therefore, a better understanding of the mechanisms that drive lung inflammation and ALI caused by IAV is a critical unmet need. IAV activates endosomal Toll-Like Receptors (TLR3/7/8/9) to induce Interferons (IFN) in airway and immune cells, driving inflammation and causing ALI. The mechanisms underlying this process are not fully understood and there are no specific therapies to prevent ALI or accelerate its resolution. We recently discovered that airway host defense protein PLUNC may increase lung inflammation and ALI by enhancing IFNλ expression. IFNλ increases immune cell recruitment through IFN-stimulated genes, that increase lung inflammation and ALI. PLUNC may modulate IFN by serving as a scaffold for nucleic acids and immune signaling proteins, similar to other host defense peptides with TLR-binding motifs. PLUNC has dual immunomodulatory properties and antiviral effects that decrease early epithelial inflammation but may enhance lung inflammation and ALI later in the clinical course. In recent work we showed that Plunc-/- mice have impaired IFNλ induction that protects them from LPS- induced lung inflammation and ALI. Our preliminary data now show that Plunc-/- mice are similarly protected during IAV infection and that PLUNC may modulate this effect through interactions with TLR3. We will test the hypothesis that PLUNC regulates lung inflammation through interactions with TLR3, and that blocking PLUNC-controlled inflammation decreases ALI during IAV infection. We will define PLUNC-controlled ALI mechanisms that can be modulated to decrease the morbidity and mortality of IAV. We will accomplish this through these aims: Aim 1. Define the mechanisms by which PLUNC increases IFN responses and lung inflammation during IAV infection; Aim 2. Define the therapeutic potential of inhibiting PLUNC to protect against IAV-ALI and accelerate its recovery; and Aim 3. Define PLUNC-regulated immune response and single-cell transcriptome profiles associated with ALI and its resolution in humans. Understanding the role of PLUNC-TLR3 interactions in ALI will provide therapeutic targets to limit IAV morbidity. The mechanisms proposed here offer an opportunity for developments that would limit the impact of IAV on millions of individua...