PROJECT SUMMARY/ABSTRACT Dermatomyositis (DM) is an acquired autoimmune muscle disorder with poorly understood causes. DM has no specific FDA-approved treatments and off-label use of immunosuppressive medications used in other autoimmune conditions has proven ineffective or infeasible. Up to 20% of patients die of their disease within 5 years, similar to stage III breast cancer. The goals of this grant proposal are to gain an understanding of which parts of the immune system are involved in causing DM and find which features predict the course of disease in each patient. Our long-term goals are to better understand how DM begins, identify which parts of the immune system are involved, predict the course of disease in each patient, and find new treatments to improve patient outcomes. The objective of this grant is to identify if macrophages – a specific immune cell type - participate in muscle inflammation in DM. Our central hypothesis is that macrophages recruit other immune cells into the muscles of patients with DM, causing disease. Our rationale is that by identifying the specific type of macrophages involved in inflammation, we will be able to better understand the main immune pathways involved in DM. This will identify new targets for future therapies that will improve patient outcomes. Our specific aims will test the following hypotheses: (Aim 1) Identify macrophage populations in normal skeletal muscle and dermatomyositis; (Aim 2) Elucidate macrophage signatures of DM severity. Upon conclusion of this project we will, for the first time, have the ability to isolate immune cells from frozen patient biopsies. This advance is necessary as current single cell analyses depend on fresh biopsy samples, which are not available due to the rarity of DM. Using this, we will understand the main macrophage types involved in DM and find out which specific muscle biopsy feature(s) influence clinical disease course. This contribution is significant because it will, for the first time, not only open up many frozen tissues for research purposes, but will also provide a broad overview of all macrophages participating in inflammation, identifying specific new targets for therapy. The proposed research is innovative as we will use cutting edge immunology and genetic technologies, previously limited to the use of fresh tissues, to achieve these goals. In addition, we will confirm a panel of commercial antibodies to use in immunofluorescent microscopy to identify specific macrophages associated with worse DM disease. This method will be clinically applicable and may have a large impact on current approaches to treatment.