Project Summary Liver fibrosis/cirrhosis is the 14th leading cause of death worldwide. Despite the prevalence of this disease, there are no drugs to treat liver fibrosis/cirrhosis. Several landmark clinical studies have demonstrated that fibrosis, and even cirrhosis, are reversible in patients. In animal models, during regression of fibrosis, disease causing, “pro-fibrotic macrophages” differentiate into “pro-resolving macrophages”. These macrophages produce matrix metalloproteinases (MMPs) that remove excess extracellular matrix and produce mediators that terminate matrix production by “activated” hepatic stellate cells, the primary matrix producing cell in the liver. The studies proposed in this application aim to exploit the phenotypic plasticity of pro-fibrotic macrophages and identify chemicals/drugs that stimulate their conversion into pro-resolving macrophages. Towards this goal, we have developed a primary screening assay that utilizes high-content imaging to detect macrophage phenotypic transition in a 384 well format. With this assay, we propose to screen a library of 3,000 compounds of known mechanism of action to identify drugs for repurposing as macrophage phenotypic modulators with anti-fibrotic properties. We will screen an additional library of 23,000 compounds with unknown activities to identify novel anti-fibrotics. Positive hits from these screens will be further characterized by using the Nanostring assay, in a medium throughput format, to quantify several macrophage phenotype- specific mRNAs. Top hits from this secondary screen will be further evaluated in biological assays for anti- fibrotic activity. Collectively, these studies have the potential to identify novel anti-fibrotics that not only limit further fibrosis development but stimulate fibrosis reversal by triggering the conversion of pro-fibrotic macrophages into pro-resolving macrophages. Identification of compounds with this novel activity has the potential to tremendously impact treatment of liver fibrosis/cirrhosis.