Therapeutic effect of curdlan in experimental visceral leishmaniasis is mediated by activation of NLRP3 inflammasome.

Curdlan, a naturally occurring immunomodulator, significantly reduced the parasite load in experimental visceral through nitric oxide (NO) generation and interleukin 1β (IL-1β) production. Splenocyte supernatant from curdlan-treated mice infected with Leishmania donovani, when treated with anti-IL-1β antibody, showed a reduction in NO generation. Treatment with anti-IL-1β antibody to infected curdlan-treated mice reversed the protective effects against the infection. However, the underlying signaling events mediated by curdlan during infection remain to be elucidated. Curdlan strongly induced pro (inactive) form (signal 1) and then active form of IL-1β (signal 2) in parasite-infected macrophages, as revealed by immunoblot analysis. Given that caspase-1 is essential for the maturation of IL-1β from pro-IL-1β, we examined both forms of IL-1β in the presence of the caspase-1-specific inhibitor AcYVAD-Fmk in infected curdlan-treated cells. Notably, AcYVAD-Fmk treatment significantly inhibited the formation of active IL-1β following curdlan treatment, while the expression of pro-IL-1β remained unchanged. Curdlan significantly induced NLRP3 (nod-like receptor pyrin domain containing 3) inflammasome and adaptor protein, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), two critical regulators of IL-1β pathway in infected curdlan-treated cells. The gene silencing of NLRP3 in curdlan-treated infected cells failed to alter the pro-IL-1β expression. However, NLRP3 gene silencing inhibited IL-1β activation and parasite clearance, suggesting that a distinct second signal is necessary for maturation of IL-1β in the context of curdlan-mediated protection. Curdlan significantly increased the production of reactive oxygen species (ROS) and activated nuclear factor κB (NF-κB), two additional prerequisites for signal 1 and signal 2, respectively, in infected cells. Treatment of NF-κB inhibitor, BAY 11-7085 or ROS scavenger NAC (N-acetylcysteine) failed to release mature IL-1β and increased the parasite survival confirmed our observation. Our results suggest that curdlan-mediated curative effect may associate with the expression and activation of NLRP3 inflammasome resulting in the release of IL-1β secretion.