Leishmaniasis is a parasitic disease transmitted by sand flies and is endemic in 88 countries worldwide including Iraq and Afghanistan. Macrophages are a major site of leishmania parasite replication. Macrophages can adopt a range of polarized phenotypes. M1 (classically)-activated macrophages have increased microbicidal activity. However, phagocytosis of leishmania parasites induces a non-M1 activation pattern in macrophages that supports parasite replication. MicroRNAs (miRNAs) are small RNAs that repress large numbers of genes and are important in many cellular responses including immunomodulation. The research plan described herein will test the following hypothesis: As important modulators of gene expression, miRNAs are essential modifiers of macrophage gene expression that regulate, in part, the progression or cure of leishmaniasis. The specific aims will evaluate the role of miRNAs in macrophages. Experimental overexpression or antagonism of selected miRNAs will be achieved by transfecting monocyte-derived macrophages (MDMs) mimics and antagonists, respectively. In specific aim 1, a non-biased approach will be used to identify targets of biologically-important miRNAs in macrophages. Putative miRNA targets will be compiled by merging results from microarray analysis of repressed genes in miRNA mimic-transfected MDMs with computationally-predicted miRNA targets. RT-qPCR, western blots, and luciferase reporter assays will be used to validate miRNA targets. In specific aim 2, the ability of specific microRNAs to modify gene expression programs during macrophage polarization will be characterized. RT-qPCR data suggests a set of miRNAs alters M1-biomarker expression in MDMs treated with M1-polarizing conditions. This set of miRNAs will be thoroughly evaluated i) to determine the influence each miRNA has on expression of additional M1-biomarkers and ii) to interrogate M1-associated signal transduction pathways using RT-qPCR, western blots, and immunofluorescence microscopy. In specific aim 3, changes in leishmania-macrophage interactions following alteration of macrophage miRNA activity will be evaluated. Parasite phagocytosis, macrophage gene expression responses to infection, and leishmanicidal activity will be evaluated by microscopic analyses and RT-qPCR. miRNA targets will be identified to mechanistically explain miRNA-mediated changes in macrophage responses. The long-term goal of this project is to develop novel RNA-based therapeutics that promotes leishmanicidal activity in macrophages.