Role of miRNAs in the regulation of the polyamine/NO production pathways of THP-1 derived macrophages infected by Leishmania amazonensis

Author(s)

Juliane Cristina Ribeiro Fernandes

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Faculdade de Medicina (FM/SBD)
• Defense date: 2023-05-02

Examining board members

Lucile Maria Floeter Winter; Hiro Goto; Marcelo Alves da Silva Mori

• Advisor: Lucile Maria Floeter Winter; Sandra Marcia Muxel

Abstract

Leishmania amazonensis is a protozoan that causes mainly cutaneous leishmaniasis in humans. The amino acid arginine exerts a central role in the parasite survival within the macrophages and in the establishment of que infection since it supplies ornithine via arginase 1 as a precursor metabolite of the polyamines pathway, that are essential molecules for the parasite proliferation. On the other hand, arginine can lead to nitric oxide (NO) production via nitric oxide synthase 2 (NOS2), the main effector leishmanicidal molecule. Different from what occurs in murine macrophages, there are studies pointing to the absence of NOS2 activity in human macrophages. Some studies show the importance of microRNAs in the regulation of immune response in the infection by different Leishmania and hosts species. In this study, we investigated the polyamines/NO pathway and miRNA-mediated regulations that change L. amazonensis infectivity in human macrophages derived from the monocytic cell line THP-1. Initially, we quantified the expression profile of miRNAs involved in the inflammatory response and we identified positive modulation of miRNAs from the miR-372/373/520 family, that share the seed sequence AAGUGCU, responsible for the recognition of the target mRNA. This sequence is identical to que murine miR-294s seed, previously demonstrated as an important factor promoting infection by reducing the mRNA Nos2. Studies in collaboration demonstrated positive regulation of those miRNAs also in THP-1 infected with L. braziliensis and L. infantum and in the serum of visceral leishmaniasis patients. Our results show that the infection by arginase knockout-L. amazonensis is reduced after 24h and correlates with the absence of regulation of the studied miRNAs. To understand the importance of those miRNAs in the infection by L. amazonensis, we inhibited the miR-372, miR-373 and the miR-520d either alone or in combination and observed that the inhibition of the miR-372, but not the miR-373 and miR-520d alone, was able to reduce the proportion of infected macrophages and the number of amastigotes per macrophage. The simultaneous inhibition of the 3 miRNAs reduced even further the infection, while the use of mimics increased the proportion of infected macrophages, indicating a synergistic mechanism of the miR-372/373/520d promoting infectivity. These miRNAs share putative target-mRNAs encoding proteins involved in the uptake of amino acids, polyamine metabolism and urea cycle, including the main arginine transporter in macrophages, CAT2, whose mRNA is increased with the simultaneous inhibition of the miR-372/373/520d. On the other hand, the target site of mice NOS2 is not conserved in humans. In general, our results showed low modulation of the NO/polyamines pathway at transcriptional level and, to access the macrophages phenotype, we quantified the metabolites levels using mass spectrometry-based metabolomics. Our data show that the infection increases production of urea cycle components and putrescine, while the inhibition of miR-372/373/520d reduces the production of putrescine, spermine and trypanothione. Our results evidence that the coregulation of the miR-372/373/520d family in the Leishmania infection acts in the regulation of genes involved in the L-arginine metabolism, culminating in the production of polyamines and favoring the establishment and maintaining the infection. The molecular mechanisms described could be the basis to the development of host-directed therapies (AU)

• FAPESP's process: 17/21906-9 - Role of miRNAs in the regulation of polyamines/NO production pathways in THP-1 derived macrophages infected with Leishmania amazonensis
• Grantee: Juliane Cristina Ribeiro Fernandes
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)