Abstract 4140535: High throughput screen reveals that atypical antipsychotics promote continual efferocytosis by antagonizing dopamine signaling and promoting vitamin A-dependent Arginase1 upregulation

Introduction: Programmed cell removal, or efferocytosis, has been proposed as a method to dispose of unwanted cells that accumulate to promote diseases such as cancer, infection, and atherosclerosis. The defective efferocytosis process in advanced atherosclerotic plaque causes secondary necrosis and contributes to plaque vulnerability. Blockade of ‘don’t eat me’ molecules such as CD47 induces favorable vascular effects, but can induce the clearance of some healthy tissue such as RBCs. Accordingly, we conducted a high-throughput screen of about 3000 FDA-approved drugs and compounds, to identify new pro-efferocytic therapies that may have translational potential. Methods&Results: Using flow cytometry-based phagocytosis assays with RAW 264.7 murine macrophages, we identified 8 candidate compounds that reproducibly increased efferocytosis. Subsequent validation assays using Incucyte live-cell imaging assay in RAW cells, primary cultured murine macrophages, and human macrophages allowed us to identify the atypical antipsychotic drug, thiothixene, as a promising candidate for clinical translation, which induced cellular clearance without changing macrophage apoptosis, migration or proliferation. Leveraging thiothixene’s known inhibitory effects on a wide range of neurotransmitters and catecholamines, we found that dopamine has a potent inhibitory effect on efferocytosis, which thiothixene could partially offset. Thiothixene induced macrophage polarization and Arginase1 upregulation, promoting continual efferocytosis. Mechanistically, RNA-sequencing revealed that thiothixene upregulated Stra6L, a receptor for retinol-binding protein that mediates cellular uptake of retinol. Retinol and all-trans retinoic acid upregulated Arginase1 expression and promoted efferocytosis, which could be reversed via knockdown of Stra6l. Conclusion: This unbiased screen identified unanticipated anti-efferocytic properties of dopamine, while highlighting the translational potential of a generic, FDA-approved anti-psychotic drug. This therapy may represent a novel method for promoting continual efferocytosis and the disposal of unwanted cells that otherwise can promote a range of clinical disorders including atherosclerosis.