mTOR inhibition in Trypanosoma cruzi infected macrophages actives NLRP3 and induces mitochondrial ROS production that regulate parasite survival

ROJAS MÁRQUEZ, JD; ANA, Y; STEMPIN, CC; CERBAN FM

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

We have previously shown that Trypanosoma cruzi infection in macrophages (Mo) activates mTOR pathway and its inhibition decreased parasite replication. However, in rapamycin (Rap) pretreated and T. cruzi infected Mo, we observed reduced nitric oxide production and iNOS expression compared to control cells. Therefore, the aim of this work was to determine alternative activated mechanisms involved in controlling parasite during mTOR inhibition. In this sense, we found that cytoplasmic ROS (cROS) as well as IDO activity were not involved as inflammatory mechanisms. Consequently, to study possible mediators involved in parasite killing, we obtained bone marrow derived Mo (BMDM) from different KO mice pretreated them with Rap and then infected. Parasite load was evaluated 72h post infection (pi) by immunofluorescencia (IF). The results showed a significant increase in parasite load in BMDM from IL-6-KO, TNFa-R-KO and NLRP3-KO compared to WT BMDM. However, parasite number stands out in BMDM from NLRP3 KO (p<0.05). Taking into account that NLRP3 is a key component of the inflammasome, our next aim was to determine whether this pathway is involved. We observed that Rap-pretreated and infected BMDM showed a significant increase in NLRP3 and IL-1β expression at 6h pi (p<0.05). Besides, TNF-a, IL-6, and IL12 expression were also increase at 12, 18 and 24h pi (p<0.05). It has been shown that inflammasome uses the mitochondria as platform of assembly. We found a significantly increase in mitochondrial ROS (mtROS) production at 3 and 6h pi (p<0.05). To evaluate the relevance of mtROS, BMDM were treated with DPI (NADPH oxidase inhibitor), then infected and parasite load was studied by IF. We observed that Rap+DPI pretreated BMDM had significantly higher parasite load compared to Rap pretreated BMDM (p<0.05). Our findings strongly support that mTOR inhibition during T. cruzi infection in Mo induced inflammasome activation that leads to mtROS production controlling parasite survival.