Association of antifungals with inhibitory mechanisms of immunosuppression in Paracoccidioidomycosis: depletion of Myeloid-derived Suppressor Cells (MDSCs) and inhibition of PD-1 and CTLA-4 checkpoint receptors.

Abstract

In Paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America countries, previous studies revealed that host immunity is regulated by several suppressive mechanisms mediated by plasmacytoid dendritic cells, regulatory T cells, myeloid-derived suppressor cells (MDSCs), as well as, by inhibitory molecules such as CTLA-4 and PD-1. Immunotherapy with monoclonal antibodies has proven to be an effective therapeutic approach in the treatment of some types of tumors, immunological disorders, and more recently, they have also shown potential in combating infectious diseases by expanding the effectiveness of current antimicrobial drugs. Studies have shown that immune checkpoint inhibitors, such as those targeting PD-1 or CTLA-4 receptors, can modify the course of fungal infections, promoting a more effective immune response and reducing the severity of the infection. In a previous study, we were able to demonstrate that blocking CTLA-4 and PD-1 reduced the fungal load in affected organs, limited the extent of lung lesions and increased the survival of treated animals by strengthening protective immune responses. In relation to MDSCs, the administration of 5-Fluorouracil (5-FU), a chemotherapy agent, promoted partial but selective depletion of MDSCs in Paracoccidioides brasiliensis infected mice, which resulted in a controlled disease, with reduced fungal load in target organs and longer animal survival time. Improvement in the disease was associated with an increase in Th1 and Th17 type immunological responses. Now, we intend to establish an immunotherapeutic procedure in PCM involving the inhibition of MDSCs using 5-FU, or blocking CTLA-4 and PD-1 receptors, in association with antifungals, with the aim of reversing the immunosuppression characteristic of this chronic infection and promoting host cure. Accordingly, C57BL/6 mice will be inoculated with 1x106 P. brasiliensis yeasts, and first different concentrations of amphotericin B, fluconozole or itraconazole will be tested in order to establish the best antifungal in our murine model. Next, the antifungal will be used in association or not with anti-CTLA-4, or with anti-PD1, or with 5-FU. After 8 weeks of infection, the last two weeks being in treatment, the animals will be evaluated for the severity of the disease by CFU, histopathology and morbidity. Additionally, ELISA assays will be used, for the characterization of cytokines, and flow cytometry, for the phenotyping of leukocytes that migrate to the lungs, in order to verify the immunological response resulting from the use of this new immunotherapeutic procedure. In an additional protocol, the animals will also be evaluated four weeks after the end of the treatments. Thus, we intend to develop a protocol that aims to interrupt the progression of the disease through the control of fungal growth associated with the restoration of the immune response which, together, could result in significant improvement or even cure of this chronic infection. (AU)