Mammalian Target of Rapamycin Inhibition in Trypanosoma cruzi-Infected Macrophages Leads to an Intracellular Profile That Is Detrimental for Infection

Abstract

The causative agent of Chagas’ disease, Trypanosoma cruzi, affects approximately 10 million people living mainly in Latin America, with macrophages being one of the first cellular actors confronting the invasion during T. cruzi infection and their function depending on their proper activation and polarization into distinct M1 and M2 subtypes. Macrophage polarization is thought to be regulated not only by cytokines and growth factors but also by environmental signals. The metabolic checkpoint kinase mammalian target of rapamycin (mTOR)-mediated sensing of environmental and metabolic cues influences macrophage polarization in a complex and as of yet incompletely understood manner. Here, we studied the role of the mTOR pathway in macrophages during T. cruzi infection. We demonstrated that the parasite activated mTOR, which was beneficial for its replication since inhibition of mTOR in macrophages by different inhibitors decreased parasite replication. Moreover, in rapamycin pretreated and infected macrophages, we observed a decreased arginase activity and expression, reduced IL-10 and increased interleukin-12 production, compared to control infected macrophages treated with DMSO. Surprisingly, we also found a reduced iNOS activity and expression in these macrophages. Therefore, we investigated possible alternative mechanisms involved in controlling parasite replication in rapamycin pretreated and infected macrophages. Although, cytoplasmic ROS and the enzyme indoleamine 2, 3-dioxygenase (IDO) were not involved, we observed a significant increase in IL-6, TNF-α, and IL-1β production. Taking into account that IL-1β is produced by activation of the cytoplasmic receptor NLRP3, which is one of the main components of the inflammasome, we evaluated NLRP3 expression during mTOR inhibition and T. cruzi infection. We observed that rapamycin-pretreated and infected macrophages showed a significant increase in NLRP3 expression and produced higher levels of mitochondrial ROS (mtROS) compared with control cells. Moreover, inhibition of mtROS production partially reversed the effect of rapamycin on parasite replication, with there being a significant increase in parasite load in rapamycin pretreated and infected macrophages from NLRP3 KO mice compared to wild-type control cells. Our findings strongly suggest that mTOR inhibition during T. cruzi infection induces NLRP3 inflammasome activation and mtROS production, resulting in an inflammatory-like macrophage profile that controls T. cruzi replication.

Rapamycin Treatment Reduces Acute Myocarditis Induced by <b><i>Trypanosoma cruzi</i></b> Infection

“…Host autophagy machinery is crucial for cellular pathways in immune responses, including lymphocyte activation and intracellular parasite infections, as previously demonstrated [16]. In the case of in vitro T. cruzi infection, some reports described the association of pathogen control with host autophagy and mTORC1 pathway [18,26,28,29], even with some conflicting results. In this work, we evaluated the upregulation of autophagy in T. cruzi infection in vivo and autophagy upmodulation/ mTORC1 inhibition by rapamycin as a mechanism of controlling inflammation and cardiac damage triggered by the parasite.…”

“…The upregulation of autophagy observed in infected cardiac tissue and immune cells from secondary target tissues, like the spleen, prompted us to evaluate if autophagy modulation, particularly the mTOR pathway, could impair parasite damage in mammalian host cells. As rapamycin and other autophagy inducers as a starvation medium have shown to control infection in vitro [18], we evaluated whether rapamycin also had a protective role in vivo. Rapamycin inhibits especially mTORC1, by interacting with FKBP12 [34] and is already used in heart transplant, promoting cardioprotection [35,36].…”

“…In acutely T. cruzi infected-mice, the ratio of LC3-II/I is increased and p62, an adaptor protein of autophagosomes, is further upregulated in infected mice fed with high-fat diet [17]. Rapamycin treatment in the context of T. cruzi infection has only been demonstrated in vitro, showing decreased parasite proliferation [18]. In Leishmania major, in vivo treatment using rapamycin reduced cutaneous inflammatory lesions in mice [16].…”

Early Trypanosoma cruzi Infection Triggers mTORC1-Mediated Respiration Increase and Mitochondrial Biogenesis in Human Primary Cardiomyocytes

“…Different works found the activation of AKT in T. cruzi infected cells ( Chuenkova et al, 2001 ; Woolsey et al, 2003 ), and perturbations in the host mTORC1 pathway were also described in T. cruzi infected HeLa ( Caradonna et al, 2013 ) and macrophages ( Rojas Marquez et al, 2018 ) cells. The PI3K/AKT pathway, can lead to the phosphorylation of the serine/threonine mTOR kinase which then can form the mammalian target of rapamycin complex 1 (mTORC1).…”

Efeito da rapamicina na infecção de cardiomiócitos humanos por Trypanosoma cruzi

“…Destacado com a seta está um parasita no VP.De maneira diferente do que foi mostrado por Libisch (61), em nosso modelo após utilizar o inibidor de mTORC1, a Rapamicina, há diminuição na replicação parasitária (Figura 9). Dados similares foram encontrados utilizando macrófagos, onde a via foi ativada após a infecção e sua inibição causou prejuízo na replicação parasitária bem como mudança no perfil inflamatório dessas células(60). A importância de mTOR na relação parasita/hospedeiro já foi descrita utilizando modelos de infecção por Leishmania, onde a inibição da autofagia que é gerada de maneira dependente e também independente de mTOR, se mostra importante na sobrevivência e replicação parasitária(54,55).…”