Immunometabolic profiling of in vitro and ex vivo Leishmania-infected macrophages (LIMs) reveals unique polarization and bioenergetic signatures

Macrophages are the major host cells of the protozoan parasite Leishmania in mammalian infection. These key innate immune cells display remarkable phenotypic plasticity ranging from pro-inflammatory M1 to anti-inflammatory M2 macrophages that can control infection and tissue homeostasis, respectively. It has been recognized that Leishmania exploits macrophage phenotypic plasticity to establish chronic infection. However, the current notion that these parasites simply trigger an M2-like phenotype seems over-simplified considering the immunopathology observed during leishmaniasis, is which often characterized by a mixed Th1/Th2 immune response. Here we combined a series of systems-level analyses to shed new light on the phenotype of Leishmania -infected macrophages (LIMs). Immunometabolic profiling by RNAseq, quantitative RT-qPCR, cytokine immunoassays, and bioenergetic flux analysis of Leishmania amazonensis -infected bone marrow-derived macrophages (BMDMs) revealed a highly complex, mixed polarization phenotype and a unique bioenergetic signature. In vitro LIMs were characterized by (i) co-expression of both M1 and M2 markers at RNA and protein levels, (ii) expression changes of a unique set of inflammatory genes (e.g. RELA, IGFBP3, VEGFA), and (iii) modulation of metabolic gene expression, including up regulation of glycolytic genes. This LIMs-specific phenotype persisted for at least 30 days and was confirmed in vivo in lesion-isolated LIMs. Likewise, in vitro LIMs showed a mixed bioenergetic profile, increasing both glycolytic activity and oxidative phosphorylation (OXPHOS) compared to uninfected, nonpolarized macrophages (M0). Surprisingly, the elimination of intracellular parasites using the fast-acting, anti-leishmanial drug L-leucine-O-methyl ester (LME) enhanced both bioenergetic pathways to levels comparable to M1 (glycolysis) and M2 (OXPHOS) macrophages as soon as one hour after treatment, suggesting that L. amazonensis limits in real-time energy metabolism and ATP production in LIMs. In conclusion, L. amazonensis infection establishes a mixed, immunometabolomic polarization profile in infected BMDMs and lesional macrophages that on one hand can promote an M1-regulated Th1 response required for the recruitment of new host cells to infected tissues, while at the same time promoting an M2 phenotype that dampens pro-inflammatory immune-signaling and shields intracellular parasites from leishmanicidal Nitric Oxide (NO). ### Competing Interest Statement The authors have declared no competing interest.