# Crotoxin Upregulating NLRP-3 Inflammasome and IL-18 and Activating CD4+ and CD8+ Lymphocytes in Experimental Encephalitozoon cuniculi Infection

## TL;DR

Crotoxin, a compound from snake venom, boosts immune defenses against a fungal infection in immunosuppressed mice.

## Contribution

Crotoxin's ability to upregulate the NLRP3 inflammasome and activate T lymphocytes in E. cuniculi infection is newly demonstrated.

## Key findings

- Crotoxin reduced fungal burden in infected mice.
- Crotoxin increased IL-18 production and NLRP3 inflammasome activation.
- Crotoxin enhanced activation of CD4+ and CD8+ T lymphocytes.

## Abstract

Encephalitozoon cuniculi is an opportunistic intracellular fungal pathogen capable of causing severe infections in immunosuppressed hosts by evading immune defenses. Crotoxin, a bioactive molecule isolated from rattlesnake venom, has been reported to modulate immune responses. In the present study, we investigated whether crotoxin could enhance the immune response against E. cuniculi infection in immunosuppressed mice. Our results demonstrated that crotoxin treatment reduced the fungal burden and enhanced immune activation by stimulating the NLRP3 inflammasome and increasing IL-18 production. Additionally, crotoxin promoted the activation of macrophages, B cells, and both CD4+ and CD8+ T lymphocytes. Overall, these findings suggest that crotoxin may strengthen host immune defenses against opportunistic fungal infections.

Encephalitozoon cuniculi is an atypical, opportunistic, obligate intracellular fungal pathogen that infects vertebrates. It survives within the host by modulating the host immune response. Crotoxin (CTX), a bioactive compound isolated from the venom of Crotalus durissus terrificus, has been reported to modulate immune responses. This study evaluated the effects of CTX on the immune response of mice infected with E. cuniculi. Mice were immunosuppressed with cyclophosphamide (Cy), infected with E. cuniculi spores, and treated with a single dose of CTX on the day of experimental. The animals were euthanized on day 14 post-infection. Levels of T helper (Th1, Th2, and Th17) cytokines were measured in plasma, and macrophage and lymphocyte populations were analyzed in peritoneal lavage fluid and spleen. In addition, histopathological alterations, hepatic fungal burden, and mRNA expression levels of NLRP3 inflammasome–related genes were assessed. CTX upregulated NLRP3 inflammasome expression and increased IL-18 production, while reducing fungal burden in E. cuniculi-infected mice. Moreover, CTX increased the proportions of macrophages and B cells and enhanced IFN-γ expression in CD4+ and CD8+ T lymphocytes. Collectively, these findings indicate that CTX reduces fungal load in Cy-immunosuppressed mice infected with E. cuniculi by priming the NLRP3 inflammasome complex and upregulating IL-18 production.