The goal of Project 2 is to determine how Tollip protects airways from viral infection and IL-33 signaling during asthma exacerbations. Asthma exacerbations associated with respiratory infection of viruses (e.g., influenza virus) remain a significant challenge due to lack of effective antiviral therapy. These viruses primarily infect human airway epithelium. The mechanisms underlying viral infection, tissue injury and inflammation have not been well understood. In this project, we will focus on the role of a multifunctional immune regulator Toll- interacting protein (Tollip) in viral infection, particularly influenza A virus (IAV) as it represents a more severe form of viral infection in asthma. One of the key cytokines involved in asthma pathogenesis is IL-33, which is expressed by airway epithelial cells and other structural cells, and can be released during tissue injury following viral infection. Through binding to membrane-bound receptor ST2L, IL-33 exerts a variety of functions involved in asthma pathogenesis. By using a Tollip knockout mouse model of IAV infection, we discovered that Tollip deficiency increased lung viral load, loss of body weight, airway neutrophilic inflammation and importantly the release of cleaved IL-33 into the airway lumen. Mechanistically, Tollip deficiency delayed airway epithelial wound healing, reduced the production of IL-33 decoy receptor soluble ST2 (sST2), and increased mucous goblet cells expressing ST2L in IL-13-stimulated airway epithelial cells. We hereby hypothesize that Tollip is protective against viral exacerbations of asthma by inhibiting the IL-33 signaling. In Aim 1, we will define the role of Tollip in IL-33 release and activation during viral infection in airways with type 2 inflammation by testing if Tollip is protective against airway epithelial injury by promoting epithelial wound healing and reducing ATP release during respiratory viral infection in human airway epithelial cells and mouse models with type 2 inflammation. In Aim 2, we will determine sST2 regulation by Tollip during viral infection in airways with type 2 inflammation by testing if Tollip increases transcriptional activity of the ST2 proximal promoter to induce sST2 expression in part through inhibiting activation of STAT3. In Aim 3, we will determine ST2L regulation by Tollip during viral infection in airways with type 2 inflammation by testing if Tollip inhibits ST2L expression by reducing mucous goblet cells in airway epithelial cells exposed to IL-13/IL-33 and IAV. The proposed studies in Project 2 will reveal novel mechanisms by which Tollip deficiency due to genetic and/or environmental factors promotes airway injury and inflammation in a type 2 inflammation setting. sST2 or agents to reduce released ATP function/activity may serve as a therapy to correct the detrimental effect of Tollip deficiency-mediated IL-33 signaling in airways during viral exacerbations of asthma.