MAPK/ERK Activation in Macrophages Promotes Leishmania Internalization and Pathogenesis

The obligate intracellular protozoan parasite Leishmania binds several host cell receptors to trigger its uptake by phagocytic cells, ultimately resulting in visceral or cutaneous leishmaniasis. After Leishmania engages receptors on macrophages and other phagocytes, a series of signaling pathways in the host cell are activated during its internalization, which are critical for establishment and persistence of Leishmania infection. Thus, preventing Leishmania internalization by phagocytes could be a novel therapeutic strategy for leishmaniasis. However, the host cellular machinery that mediates promastigote and amastigote uptake is not well understood. Here, using small molecule inhibitors of Mitogen-activated protein kinases/Extracellular signal regulated kinases (MAPK/ERK), we demonstrate that ERK1/2 mediates Leishmania amazonensis uptake and (to a lesser extent) phagocytosis of beads by macrophages. We find that inhibition of MEK1/2 or ERK1/2 leads to inefficient amastigote uptake by macrophages. Moreover, using inhibitors and primary macrophages lacking spleen tyrosine kinase (SYK) or Abl family kinases, we show that SYK and Abl family kinases mediate Raf, MEK, and ERK1/2 activity and are necessary for efficient uptake. Finally, we demonstrate that trametinib, a MEK1/2 inhibitor used clinically to treat certain cancers, significantly reduces disease severity and parasite burden in Leishmania -infected mice, even if it is started significantly after lesions develop. Our results show that maximal Leishmania infection requires MAPK/ERK and highlight the potential for MAPK/ERK-mediated signaling pathways to be novel therapeutic targets for leishmaniasis. Lay summary Leishmania is a single-celled parasite that causes skin ulcers or a disseminated disease in humans. Our goal is to identify new drugs to treat Leishmania infection. Leishmania must live inside human immune cells to cause disease. If Leishmania is not able to enter human immune cells, it dies. Our studies demonstrate how Leishmania infection permits a set of proteins called MAP kinases to pass signals from one protein to the next within mammalian immune cells. The resulting signals allow Leishmania to enter into these immune cells and survive within its host. Importantly, trametinib, a drug that prevents these signals from MAP kinases, decreases the development of skin ulcers when it is given to mice that are infected with Leishmania . Our findings suggest that leishmaniasis could be treated with drugs that act on kinases found in humans rather than the parasites themselves. One sentence summary An Abl2-SYK-Raf-MEK-ERK pathway facilitates Leishmania uptake by phagocytic cells and promotes disease severity in Leishmania -infected mice. ### Competing Interest Statement The authors have declared no competing interest. * Abbreviations: BCA : Bicinchoninic acid BMDM : bone marrow-derived macrophages CC : cytotoxic concentration CR3 : complement receptor 3 FcγR : Fc gamma Receptor h : hour IC : inhibitory concentration IL : Interleukin INFγ : Interferon gamma Mϕ : macrophage m : minutes MAPK/ERK : Mitogen-activated protein kinases/Extracellular signal regulated kinases PKC : Protein Kinase C PVDF : polyvinylidene difluoride RAF : Rapidly Accelerated Fibrosarcoma RIPA : radioimmunoprecipitation assay p- : phosphorylated PMA : Phorbol 12-myristate 13-acetate SD : standard deviation SFK : Src family kinases SYK : spleen tyrosine kinase TNF : Tumor Necrosis Factor