Deborah Caldeira Brandt Almeida¹, Ismael Pretto Sauter¹, Lina Borda Samper¹, Patricio Reyes Orrego², Mauro Javier Cortez¹

INTRODUCTION Leishmania amazonensis, part of the Leishmania mexicana complex, thrives within a unique and expansive parasitophorous vacuole (PV) inside macrophages. The formation of this structure must be tightly regulated by the parasite to ensure its survival and proliferation in such a challenging environment. For this L. amazonensis parasites increase the size of the PV, associated with the increase levels of Lyst transcripts, and induce CD200, glycoproteins that bind its receptor CD200R to block the powerful leishmanicidal mechanism of nitric oxide. Calcineurin (CaN), a calcium-dependent phosphatase, plays a crucial role in various cellular processes related to thermotolerance and adaptation to oxidative stress. OBJECTIVE This study investigates the involvement of CaN in Leishmania during the process of macrophage infection, focusing on two distinct infective forms. MATERIALS AND METHODS We treated both promastigote and amastigote forms with calcineurin-specific inhibitors (Cyclosporin A [CsA] and FK506) and assessed parasite viability using standard techniques, including propidium iodide/annexin staining and the MTT assay. Additionally, we infected bone marrow-derived macrophages (BMM) with the different forms of L. amazonensis, both with and without prior treatment with calcineurin inhibitors, to measure intracellular parasite proliferation and the size of the Leishmania-PV over time. At 96 hours post-infection, we collected RNA samples and supernatants from each treatment to quantify macrophage transcripts (Lyst and CD200) as well as cytokine and nitric oxide levels, reflecting the host cellular response to Leishmania. RESULTS The promastigote and amastigote forms of L. amazonensis exhibited different susceptibilities to calcineurin inhibitors. When treated with 80 uM of FK506, over 90% of both infective forms remained viable. Notably, when amastigotes were treated with CsA at 80 uM, nearly 80% stayed viable. In contrast, the promastigotes were more vulnerable to CsA, with viability dropping to 60% at 40 uM and 35% at 80 uM. When promastigotes were pretreated with 25 uM of CsA or FK506—a concentration that is not toxic to the parasites—intracellular parasite proliferation was significantly affected compared to untreated controls. Conversely, treatment of amastigotes with these drugs mainly disrupted growth when using CsA, which also impacted the size of the Leishmania-PV in infected macrophages.CONCLUSION These results indicate that calcineurin has different roles in the infective forms of L. amazonensis. Further experiments will be conducted to deepen our understanding of the specific role calcineurin plays during macrophage infection.

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