PROJECT SUMMARY Interstitial lung diseases (ILDs) are a class of pulmonary diseases pathologically defined by interstitial fibrosis and inflammation. Owing to our limited understanding of the upstream initiators of pathogenesis, ILDs have poor prognoses and limited therapeutics. Mutations in the Alveolar Epithelial Type 2 Cell (AEC2) restricted Surfactant Protein C (SP-C) gene (SFTPC) in a subset of ILD patients supports a growing hypothesis that AEC2 dysfunction is a driver of disease. When we model these disease-related SFTPC mutations in vitro they segregate into two major classes based upon how the mutated SP-C isoform stresses cellular pathways that manage abnormal proteins: mutations that inhibit macroautophagy and mutations that cause endoplasmic reticulum (ER) stress. However, how these AEC2 stress phenotypes, which have also been identified in ILD patients without SFTPC mutations, relate to ILD development remains poorly understood. To understand this relationship, we have generated two unique Sftpc mutation knock-in mouse models, one expressing an SP-C isoform that induces AEC2 macroautophagy dysfunction (SP-CI73T) and the other inducing ER stress (SP-CC121G). Each of these mutations when expressed in the adult mouse lung results in spontaneous alveolitis and lung injury followed by aberrant repair with resultant fibrotic ILD. These models thus provide proof of concept that AEC2 stress is capable of driving spontaneous lung pathology and are robust preclinical platforms. These models also support a second emerging theory of ILD pathogenesis: that in ILD AEC2s, which must act as critical facultative progenitor cells after lung injury by both proliferating and differentiating to repair damaged epithelium, develop dysfunction in their progenitor cell capacity. We discovered that while similar lung pathology develops in each of our Sftpc models, there are divergent AEC2 proliferation phenotypes following lung injury: SP-CI73T AEC2s become hyperpoliferative and SP-CC121G AEC2s become apoptotic and hypoproliferative. Thus, our models support the hypothesis that abnormal AEC2 progenitor cell function plays a central role in ILD development, and also create a platform to develop a mechanistic understanding of how discrete AEC2 stress signatures result in distinct defects in progenitor cell capacity. This proposal has three interrelated aims that seek to understand the molecular and cellular mechanisms that relate AEC2 cell stress, dysfunctional progenitor cell capacity, and ILD. Aim 1 uses bioinformatics and in vivo linage-tracing to characterize the AEC2 stress signaling and progenitor function pathways involved in each model. Aim 2 provides a mechanistic link between cell stress signaling and progenitor cell dysfunction through ex vivo organoid culture and in vivo modeling. In Aim 3 we will generate the first ILD patient-derived iPSC culture model of an ER stress associated SFTPC mutation as a humanized platform to study the pathways identifi...