Objectives:

Our aims were to analyze the immunomodulatory effects of a scorpion-venom derived antimicrobial peptide (AMP) ToAP2 on bone marrow-derived murine dendritic cells (BMDCs) and macrophages(BMM) as well as during the interaction of these cells with Cryptococcus neoformans.


Methods:

Dendritic cells (BMDCs) and Macrophages (BMM - M1-like) derived from the bone marrow of C57Bl/6 mice using GM-CSF (Lutz et al., 1999). We evaluated the potential cytotoxicity of the scorpion-derived antimicrobial peptide ToAP2 to BMDCs and BMM cells using the LDH Assay. After that, BMDCs and BMM were incubated with the peptide, and the cultures supernatant were collected for cytokine production evaluation. The production of cytokines produced after the treatment of both cell types with two different concentrations of ToAP2 was evaluated by a multiplex assay that measured 14 different cytokines (TNF-a,INFg, IL1a, IL1b, Il4, Il6, IL10, 12(p70), IL13)/chemokines (IP-10, MCP-1, MIP-1a, MIP-1b, MIP-2). Additionally, we assessed the potential effects of ToAP2 during the interaction of both cell types with C. neoformans H99. More specifically, we evaluated the phagocytosis of C. neoformans by BMDCs and BMM in the presence of ToAP2, and their cultures supernatant were collected for further analysis.

Results:

ToAP2 presented BMDCs and BMM cytotoxicity in concentrations above 2.5 µM in both cells. The cytokine/chemokine analysis of BMDCs and BMM stimulated with this peptide revealed a pro-inflammatory effect with increased production of the cytokines TNF-a, IL1a, Il6, and chemokines IP-10, MCP-1, MIP-1a, MIP-1b, MIP-2. The peptide also produced a decreased expression of IP-10, MCP-1, MIP-2 by BMM cells, especially at the lower concentration.

Pre-incubation of BMDCs and BMM with TOAP 2 also seems to increase the number of internalized C. neoformans in comparison with the control group for both cell types and to increase the percentage of phagocytosis in BMDCs cells.

Conclusion:

AMPs can be used in anti-fungal therapy acting not only as fungicidal/fungistatic molecules but also by helping the immune system to deal with fungal infections. Further characterization of ToAP immunomodulatory activity will help to better evaluate the full potential of AMPs in antifungal therapy.

6 / Immunomodulatory effects of a scorpion-venom derived antimicrobial peptide during the interaction of Cryptococcus neoformans with murine macrophages and dendritic cells