Project Summary The natural history of human dermal leishmaniasis caused by Leishmania Viannia species provides unequivocal evidence of long-term persistence of parasites in asymptomatically as well as symptomatically infected individuals. Persistent infection can remain asymptomatic, or can be clinically manifested through the development of mucosal or cutaneous leishmaniasis (ML and CL) following either asymptomatic infections, or as recurrent disease long after resolution of primary lesions. Molecular evidence of persistent parasites in the conjunctiva, nasal and oropharyngeal mucosal tissues, skin and blood monocytes has been documented in as many as 40% of infected individuals in endemic areas of L. Viannia transmission, however, the role of the host immune response and parasite subpopulations in promoting and sustaining Leishmania persistence in humans remains unknown. It is also well established that immune tolerance is an underlying factor of latent infection and tissue protection in infections by other protozoa such as Plasmodium and Toxoplasma. Similar host mechanisms of immune tolerance likely contribute to Leishmania persistence in the absence of signs and symptoms, since disruption of the immune homeostasis by immunosuppression or local trauma have been identified as contributing factors in recurrent CL. We hypothesize that Leishmania-host interactions eliciting strong, yet regulated innate immune responses drive the establishment of local environments of immune tolerance (metabolic and immunological), that favor Leishmania persistence in humans. In this project we will characterize the transcriptomes, metabolomes and immunophenotypes of host tissues and cells associated with persistent infection. We will genotype the corresponding Leishmania populations, and will assess the participation of immunological tolerance in human tissues where Leishmania persistence has been detected: nasal mucosa, skin and monocytes. More specifically, we will 1) Identify the molecular signature of Leishmania persistence in a cohort of patients with prior history of CL, 2) Characterize the genotype and phenotype of persister Leishmania sub-populations as they modulate human host cells, and 3) Expand an ex vivo 3D cell culture system to model the metabolic and immunological microenvironment of Leishmania persistence. This project will provide critical knowledge on the metabolic and immunological microenvironments of human tissues permissive for Leishmania persistence, define the signatures and biomarkers of persistence, and advance ex vivo models of persistent Leishmania infection.