The BALB/c mice used were obtained from the Comisión Nacional de Energía Atómica (Buenos Aires, Argentina). Male TNFα-RKO (B6.129-Tnfrsf1a^tm1Mak), IL6KO (B6.129S2-Il6/^Jtm1Copf), and NLRP3 KO (B6.129S6-Nlrp3/J^tm1Bhk) mice were purchased from the Jackson Laboratory. Male C57BL/6J mice were from Universidad Nacional de La Plata (Argentina). All mice were inbred and housed according to institutional guidelines (28).

The T. cruzi infection protocols were performed as described (29). Briefly, the infection was maintained through intraperitoneal inoculations every 11 days. Then, blood-derived trypomastigotes were used to infect monolayers of Vero cells. After 7 days, supernatants were collected and stored at −80°C.

To obtain the BMDMs, the femur and tibia bones from different mice strains were flushed with cold RPMI 1640 containing 40 µg/mL gentamycin, following standard procedures (30). These recovered bone marrow progenitor cells were cultured in 100-mm bacteriologic plastic Petri dishes containing RPMI1640 supplemented with 10% FBS, 40 µg/mL gentamycin, 2 mM l-glutamine, and 30% L929-cell-conditioned medium (31) for 4 days. Then, bone marrow progenitors were supplemented with RPMI-LCM 30%. At day 6, BMDM were used as mentioned below, with a flow cytometric analysis of BMDM (Figure S1 in Supplementary Material) revealing that these cells were CD11b+ and F4/80+.

BALB/c mice underwent intraperitoneal infection as described previously (29), then PM were extracted and processed using several techniques. Non-infected animals were processed in parallel as control.

DMSO, LY294002, diphenyleneiodonium chloride (DPI), 1-methyl-dl-tryptophan, ATP, Griess reagent assay, DAPI, and phosphatase inhibitor (PhosphoStop) were obtained from Sigma-Aldrich (St. Louis, MO, USA); rapamycin and Fluorsave were purchased from Calbiochem (Darmstadt, Germany) and PP242 from Cayman Chemical (Ann Arbor, MI, USA); Recombinant mouse IFNγ, anti-F4/80+ (PE) and anti-CD11b+ (APC) FACS antibodies, recombinant mouse IL-4, ELISA Kits Assay IL-10, IL-12p70, TNFα, IL-6, and IL-1β were obtained from BioLegend (San Diego, CA, USA); LPS (from E. coli 0111:B4 strain) was from InvivoGen with DAF-FM, H2DCFDA, and MitoSOX TM Red probes and protein ladders being obtained from Thermo Fisher Scientifics (Waltham, MA, USA). Western blot (WB) antibodies anti-phospho-mTOR, anti-phospho-p70S6K, anti-phospho-4EBP1, anti-ATP-cytrate lyase, anti-β-actin, anti-NLRP3, and anti-IL-1β were acquired from Cell Signaling Technology (Danvers, MA, USA) and anti-Arginase I and anti-iNOS antibodies were obtained from Santa Cruz Biotechnology (Palo Alto, CA, USA). The Odyssey antibodies IRDye^® 680RD Donkey anti-Rabbit and IRDye^® 680RD Donkey anti-Mouse IgG were obtained from Li-Cor Biosciences (Lincoln, NE, USA). Finally, antibody anti-IgGh was from Biocientifica and protease inhibitor cocktail from Roche (Basilea, Switzerland).

Bone marrow-derived macrophage from Balb/c or C57BL6 mice at day 6 of differentiation were cultured for 3 h in RPMI-2%FBS for their adhesion. After that, cells were pretreated with DMSO as control; or pretreated with different inhibitors: 1-methyl-dl-tryptophan (1-MT, 100 µM, during 24 h, 37°C); diphenyleneiodonium chloride (DPI, 20 µM, during 3 h, 37°C), PP242 (40 or 80 nM, for 90 min., 37°C), LY294002 (10 or 50 nM, for 90 min.), rapamycin (50 or 100 nM, for 90 min.). After pretreatment, the BMDM were washed three times and then infected with T. cruzi (1:5, cell:parasites ratio). In addition, BMDM without inhibitor pretreatment were stimulated with LPS (1 µg/mL, as positive control of mTOR activation), LPS (1 µg/mL) + IFNγ (100 ng/mL), as positive control of M1 polarization, IL-4 (80 ng/mL), as positive control of M2 polarization, LPS (1 µg/mL) + ATP (5 mM) positive control of inflammasome. Then, cells and supernatants were collected for performing different techniques at 1, 3, 6, 12, 24, 48, and 72 p.i. The number of cells used for the different techniques was: 6 × 10⁶ cells/well for WB and ELISA, 3 × 10⁶ cells for Flow Cytometry, and 3 × 10⁵ cells for Immunofluorescence.

For the assessment of intracellular NO, cytoplasmic ROS (cROS), and mtROS, BMDM from each experimental condition were collected at 4°C and washed with PBS 2% FBS. First, cells were stained with APC labeled anti-CD11b and with FITC labeled anti-F4/80 for 20 min at 4°C. After that, BMDM were incubated with 20 µM DAF-FM diacetate probe for 30 min at 37°C for NO detection; 20 µM H2DCFDA probe for 20 min at 37°C for cROS detection; or 5 µM of a MitoSOX TM Red probe 20 min at 37°C for mtROS detection. Finally, these cells were analyzed by FACS as described previously (29).

IL-10, IL-12p70, TNFα, IL-6, and IL-1β were measured in culture supernatants by ELISA sandwich following the manufacturer’s guidelines.

Cells were processed as previously described (29). Lysates were prepared by protein measurement using the Bradford micro-technique (32). Membranes were incubated overnight at 4°C with primary rabbit and mouse monoclonal antibodies anti-phospho-mTOR, anti-phospho-p70S6K, anti-phospho-4EBP1, anti-Arginase-I, anti-iNOS, anti-NLRP3, anti-IL-1β, anti-β-actin, or anti-ATP-cytrate lyase. Then, sheets were incubated with antibodies anti-Rabbit (IRDye^® 680RD Donkey anti-Rabbit) and anti-Mouse (IRDye^® 680RD Donkey anti-Mouse IgG) for 1 h, at room temperature and in darkness. An Odyssey CLx Infrared Imaging System (LI-COR, Inc.) was used to detect the bands.

Bone marrow-derived macrophages were treated as described in the Section “Inhibitors and Stimulus Treatments,” and non-internalized parasites were eliminated by performing washes with RPMI 24 h later. Parasite growth in BMDM was determined by counting the number of intracellular amastigotes using immunofluorescence assays as described (33). For nuclear staining, coverslips were incubated with DAPI, before being washed and incubated in mounting media FluorSave overnight. The slides were observed using an Olympus BX41 microscope (Olympus Corporation, Tokyo, Japan) and a Leica DMi8 microscope (Leica Microsystems). Images were processed with ImageJ software.

Nitric oxide levels were obtained by measuring those of the stable end product (nitrites) with Griess reagent assay (34). Absorbance at 540 nm was measured by the Bio Rad microplate reader and optical density was converted to micro molar of nitrites using a standard curve of sodium nitrite.

Arginase activity was measured in macrophage lysates as previously described (33, 35, 36). Cells were lysed with 100 µL of 1% Triton X-100 containing protease inhibitor cocktail and phosphatase inhibitor. Then after 30 min, lysates were prepared for protein measurement by using the Bradford micro-technique (32). The urea (produced by arginine hidrolysis) was measured at 540 nm. The results were expressed as micrograms of urea per microgram of protein.

Statistical analyses were performed using the Student’s t-test of GraphPad Prism software version 6.0 (GraphPad Software). Discrepancies with a value of p < 0.05 were considered significant.

It has been previously demonstrated that the invasion of trypomastigotes is reduced in HeLa cells treated with the mTOR inhibitor rapamycin (37). However, this has not yet been explored for mTOR function in macrophages during T. cruzi infection (38). Therefore, we infected BMDM with T. cruzi trypomastigotes and evaluated mTOR activation by WB through phosphorylation of its substrates 4EBP1 and P70S6K. The mTOR activation was observed at 6 and 24 h postinfection (Figure 1A), and in addition, we evaluated mTOR activation in peritoneal cells from mice at 15 days postinfection. The peritoneal cells from infected mice revealed an increase in 4EBP1 and P70S6K phosphorylation compared to control cells from uninfected mice (Figure 1B), with these results indicating that mTOR is an important pathway induced by the parasite in macrophages.

To determine if mTOR activation is important for parasite survival, we performed experiments by targeting this pathway at different levels. First, BMDM were treated with the mTOR inhibitors rapamycin or PP242, or with Ly249002 (a PI3K inhibitor) or DMSO as control and incubated 90 min. Then, cells were washed and infected with T. cruzi trypomastigotes. The number of intracellular parasites was evaluated 72 h later by immunofluorescence. It was observed that pretreatment with rapamycin, PP242, and Ly294002 significantly reduced the number of parasites in BMDM (Figures 2A,C) and also in peritoneal infected cells compared to DMSO-treated and infected cells (Figure 2B). However, the number of parasites increased in IL-4 stimulated and infected cells, used as positive control of parasite replication (Figures 2A,B).

None of these treatments produced any cytotoxic effect on the cells according to cell viability measured by the release of LDH in the supernatants of these cultures (data not shown). The inhibitory effect of the drugs was not a consequence of its action on the trypomastigotes, since inhibitors were removed before incubation with parasites. In addition, it has been reported that pretreatment of parasites with rapamycin does not alter their infectivity in HeLa cells, even for high drug concentrations (37). Thus, our results indicate that the T. cruzi parasite activates the mTOR pathway in macrophages in order to promote its survival, since mTOR inhibitors control parasite replication.

It has been suggested that mTOR in macrophages enhances the expression of M2-associated cytokines (38–40). Moreover, the pharmacological inhibition of mTOR with rapamycin resulted in an inhibition of LPS induction of IL-10 mRNA and protein, but enhanced the pro-inflammatory cytokine TNFα production (41). Thus, to determine the role of mTOR in T. cruzi-induced cytokines and whether this could influence the survival of the parasite in macrophages, we performed experiments by incubating BMDM with rapamycin or DMSO as control for 90 min, then, cells were washed and infected with T. cruzi trypomastigotes. On evaluating IL-10 and IL-12 production 24 h later by ELISA, it was found that rapamycin pretreatment induced a switch in cytokine production in infected macrophages. Moreover, a rise in IL-12 but a reduction in IL-10 production were observed in rapamycin-pretreated and infected macrophages compared to DMSO control macrophages (Figures 3A,B). To achieve a better understanding about the mechanisms implicated in controlling the parasite in rapamycin-treated macrophages, we evaluated Arginase I and iNOS, which are hallmarks of the M1 or M2 activation profiles, respectively. BMDM were pretreated for 90 min with rapamycin or DMSO as control, after which, the cells were washed and infected with T. cruzi trypomastigotes and Arginase I and iNOS expression and activities were measured 24 h later. Arginase was slightly induced by the parasite whereas rapamycin pretreatment reduced its expression and activity (Figures 3C,D), correlating with a decreased IL-10 production (Figure 3A). Surprisingly iNOS expression was also reduced in rapamycin pretreated and infected macrophages (Figure 3E), as well as the nitrite production in culture supernatants (Figure 3F) and the frequency of NO producing cells (Figure 3G).

It has been previously shown that the ROS resulting from the respiratory burst have an important role in T. cruzi control (42–44), but ROS may be involved in cellular signaling and proliferation of this parasite (45). Nevertheless, on evaluating cROS production in rapamycin-treated and infected macrophages at different points after infection by FACS (using H2DCFDA, which measures cROS and mainly detects H₂O₂), we did not observe any differences in the frequency of the cROS producing macrophages between rapamycin-treated and infected macrophages and DMSO-treated and infected macrophages at 6 or 24 h p.i. (Figure 4A) or 24 h p.i. (Figure 4B). However macrophages stimulated with LPS plus IFN-γ, used as a positive control of ROS production, revealed a high frequency of cROS producing cells (Figure 4A).

On the other hand, the enzyme indoleamine 2,3-dioxigenase (IDO) of the tryptophan catabolism is involved in inhibiting intracellular pathogen replication (46, 47); moreover, IDO activity is induced by inflammatory cytokines and is essential for limiting the parasite’s reproduction in macrophages since T. cruzi amastigotes are sensitive to the l-kynurenine downstream metabolites (48). In order to examine the effect of IDO activity on the regulation of parasite growth in vitro in our experimental system, BMDM were cultured in the presence or absence of 1-methyl-d-tryptophan (1-MT), an IDO inhibitor, for 24 h, before being treated with rapamycin for 90 min and then infected with T. cruzi. After 24 h, the non-internalized parasites were eliminated through washes, and the intracellular parasites were counted by immunofluorescence 72 h later. IDO blockade with 1-MT was demonstrated to cause a strong stimulatory effect on intracellular parasite growth, as previously reported (48). However, 1-MT treatment did not reverse the effect of rapamycin (Figures 4B,C), indicating that IDO is not involved in limiting parasite growth in rapamycin-pretreated and infected macrophages.

We have shown that rapamycin pretreatment induced an increase in IL-12 and a corresponding reduction in IL-10 production in infected macrophages (Figures 3A,B). It has been reported that rapamycin differentially modulates both pro- and anti-inflammatory cytokine production in macrophages in response to LPS such as TNF-α, IL-6, and IL-1β, among others (24, 25, 49). BMDM were pretreated for 90 min with rapamycin or DMSO as control. Then, cells were washed and infected with T. cruzi trypomastigotes and cytokine production was evaluated at different time points after infection. mTOR inhibition by rapamycin in BMDM infected with T. cruzi led to a strong upregulation of IL-12 production (Figures 3B and 5B). At the same time, the anti-inflammatory cytokine IL-10 was remarkably suppressed (Figures 3A and 5A), whereas, importantly, mTOR inhibition increased the production of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β (Figures 5C–E respectively). These results indicate that mTOR inhibition alters the cytokine balance of macrophages toward a pro-inflammatory phenotype upon T. cruzi infection.

It has been shown that NLRP3 is involved in the antiparasitic response against T. cruzi, but the mechanisms involved are known to depend on the experimental design employed (50). Here, we evaluated NLRP3 expression and IL-1β production in rapamycin pretreated and infected macrophages. Briefly, BMDM were pretreated for 90 min with rapamycin or DMSO as control and cells were washed and infected with T. cruzi trypomastigotes. Cell lysates and supernatants were collected 6 h p.i., and NLRP3 expression and IL-1β production were measured by wild type. We observed that mTOR inhibition by rapamycin in T. cruzi-infected BMDM led to a potent upregulation of NLRP3 and an increase in IL-lβ production (Figure 6A), with BMDM stimulated with ATP plus LPS serving as a positive control of inflammasome activation.

Then, to study the relevance of NLRP3 activation during mTOR inhibition in infected macrophages, we obtained BMDM from NLRP3 KO or wild-type (WT) mice and pretreated them with rapamycin or DMSO for 90 min. Then, cells were infected and T. cruzi intracellular replication was measured by immunofluorescence. In agreement with Figure 2 and Figure S2 in Supplementary Material, a reduction in parasite growth in rapamycin pretreated and infected BMDM from WT was observed. However, rapamycin pretreated and infected BMDM from NLRP3 KO showed a strong increase in parasite replication, which was even more robust than that observed in IL-4 stimulated BMDM used as control (Figure 6B). Representative images from DMSO or rapamycin treated and infected WT BMDM (Figures 6C) and NLRP3 KO BMDM are shown in Figure 6D. In addition, in rapamycin pretreated and infected BMDM from TNFα-R KO and IL-6 KO mice there was an increase in parasite replication compared with rapamycin-pretreated and infected BMDM from WT mice. However, this effect was less evident than in the BMDM from NLRP3 KO mice (Figure 6B and Figure S3 in Supplementary Material).

It has been previously shown that mitochondria participate in inflammasome activation (51). The NLRP3 inflammasome is the best characterized among various inflammasome complexes, with its activation depending on different stress signals, including ROS production promoted by mitochondrial dysfunction (51). Thus, we evaluated mtROS production in rapamycin treated and infected BMDM at different points after infection using MitoSOX, which measures mtROS and mainly detects superoxide radical. We found a significant increase in mtROS production in rapamycin-pretreated and infected BMDM, with increased levels of mtROS being observed in ATP plus LPS-stimulated BMDM, used as positive control (Figure 7A). Then, to evaluate whether mtROS is relevant for controlling parasite replication, BMDM from WT mice were pretreated with rapamycin, DPI (NADPH oxidase inhibitor), rapamycin + DPI, or DMSO before being infected, and the parasite load was studied by immunofluorescence. It was observed that BMDM incubated with rapamycin plus DPI had a significantly higher parasite load compared to rapamycin-pretreated BMDM (Figure 7B), with a representative image from DMSO, rapamycin, DPI, and rapamycin plus DPI treated and infected BMDM being shown in Figure 7C. Additionally, we have observed that DPI inhibits mtROS production induced by rapamycin pretreatment and T. cruzi infection, Figure S4 in Supplementary Material. This may indicate that mtROS would participate in the control of T. cruzi replication. These findings strongly suggest that mTOR inhibition during T. cruzi infection in BMDM induces inflammasome NLRP3 activation and mtROS production, which controls parasite survival.