Characterization of an animal model to study the role of ferroportin in myeloid leukocytes during Mycobacterium tuberculosis infection

Author(s)

Marina Bonifácio Denadai

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-07-18

Examining board members

Diego Luís Costa; Ana Paula Junqueira Kipnis; Luciane Alarcão Dias Melicio

• Advisor: Diego Luís Costa

Abstract

The absence of an effective vaccine and a therapy that quickly eliminates the pathogenic agent from the host organism, contribute to tuberculosis (TB) being one of the most lethal infectious diseases in the world. New lines of treatment proposed in the literature suggest using host biological processes that favor the replication of bacteria as targets of therapeutic strategies to achieve a faster and more effective cure of the disease. Specifically, iron metabolism in infected cells has been suggested as a promising target for the development of host-directed therapies. The regulation of intracellular iron levels in macrophages, the main cell infected by the bacillus Mycobacterium tuberculosis (Mtb), occurs mainly through the combined action of the enzyme heme oxygenase-1 (HO-1) and the transporter ferroportin (FPN). While the metabolization of heme by HO-1 results in the release of iron into the cytoplasm, FPN acts as a transporter of iron ions to the extracellular space. Thus, the decrease in its expression on the membrane of infected cells may directly contribute to the increase in their intracellular iron concentration, which can impact the control of bacterial replication. Therefore, our aim was to investigate the role of FPN during Mtb infection and to develop an animal model genetically deficient for FPN in macrophages in order to address this question. Our results showed that the infection of bone marrow-derived macrophages (BMDM) with Mtb induced a significant increase in FPN expression in the presence of iron supplementation. We observed that the in vivo Mtb infection induces changes in the relative expression of important markers for iron regulation in the lungs of C57BL/6 mice, such as HO-1, SPIC, ferritin (FTH) and hepcidin (HAMP). More importantly, we identified that in the lungs of animals infected with Mtb there was a significant increase in FPN expression in infected alveolar macrophages (AM) and, mainly, in infected monocyte-derived parenchymal macrophages (PM) compared to non-infected cells from the same populations, at four weeks post-infection (4 wpi). Therefore, we developed an animal model genetically deficient for FPN in AM and PM (FPNfl/fl LyzMCre+), in order to investigate the role of this transporter in the containment of bacterial replication. We observed that FPN-deficient BMDM were more susceptible to Mtb infection in vitro when compared to wild-type (WT) cells, displaying a higher bacterial load and cell death, especially in the presence of iron supplementation. However, this scenario was not reproduced in vivo, since FPNfl/fl LyzMCre+ mice infected with a high dose of Mtb displayed similar pulmonary bacterial loads compared to WT animals; along with lower cell death in pulmonary leukocytes. Concomitant with these results, we observed a significant increase in iNOS production in total and infected PM. Taken together, our results indicate that the absence of FPN results in increased susceptibility to bacterial replication in macrophages in vitro, although, in vivo, in the lungs, the absence of FPN did not interfere with resistance to bacterial replication. However, a more comprehensive investigation using this model is still necessary, in order to identify whether FPN deficiency can impact the dissemination of Mtb to organs in which macrophages play an important role in iron metabolism, such as the spleen and liver. Finally, a better understanding of the role played by this receptor during experimental TB may identify possible new targets for the development of host-directed therapies for the treatment of TB. (AU)

• FAPESP's process: 21/04028-3 - Study on the immunomodulation of ferroportin expression and iron homeostasis in myeloid leukocytes and the related consequences to the pathogenesis of Mycobacterium tuberculosis infection
• Grantee: Marina Bonifácio Denadai
• Support Opportunities: Scholarships in Brazil - Master