All animal experiments were approved by and conducted in accordance with guidelines of the Animal Care and Use Committee of the Facultad de Ciencias Químicas, Universidad Nacional de Córdoba (Approval Number HCD 831/15). C57BL/6 (B6) was obtained from School of Veterinary, La Plata National University (La Plata, Argentina). All animals were housed in the Animal Facility of the Facultad de Ciencias Químicas, Universidad Nacional de Córdoba (OLAW Assurance number A5802-01). The Tulahuen strain of T. cruzi was used, which was maintained by weekly i.p. inoculations in mice.

Macrophages were cultured in complete medium: RPMI 1640 (Gibco, ThermoFisher Scientific) supplemented with 2 mM GlutaMAX (Gibco, ThermoFisher Scientific), 10% heat-inactivated fetal calf serum (Gibco, Thermo Fisher), and 50 µg/mL Gentamicin (Gibco, ThermoFisher Scientific). PBS was purchased from Gibco (ThermoFisher Scientific); bovine serum albumin and DMSO were obtained from Sigma-Aldrich.

Bone marrow-derived macrophages (BMM) were generated as described previously (41). Peritoneal macrophages (PM) were obtained as described previously (42). J774 murine macrophages and the human monocytic THP-1 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). THP-1 cell line was differentiated into macrophages by culture in complete medium supplemented with of 50 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 24 h followed by 24 h of incubation in complete medium.

Mo were incubated with LiCl (10 mM, #746460 Sigma-Aldrich), BIO (5 µM, #3194 TOCRIS), iCRT14 (50 µM, #4299, TOCRIS), CCT036477 (20 µM, #BML-WN114, ENZO), IWP-L6 (20 µM, #504819, Calbiochem), IFN-γ (10 ng/mL, Sigma-Aldrich) plus LPS (10 µg/mL, from Escherichia coli serotype 0111:B4, Sigma-Aldrich), 1-methyl-d-tryptophan (1-MT, #860646, Sigma-Aldrich) or complete medium before being infected with T. cruzi Tps (Mo:Tps = 1:3). Culture medium with PBS or DMSO was used as vehicle control for LiCl and 1-MT or iCRT14, CCT036477, and IWP-L6, respectively. MeBIO (5 µM, #3873, TOCRIS) was used as control of BIO.

The growth of parasites in Mos was evaluated by counting the intracellular amastigotes by immunofluorescence assay using serum of patient with chronic Chagas disease as described previously (8). The number of intracellular amastigotes was calculated by dividing intracellular parasites (n/200 cells) in differentially treated cultures by number of intracellular parasites (n/200 cells) in the corresponding vehicle-treated cultures, and expressed as relative units.

Spleen cells or BMM treated with RIPA buffer were analyzed by 10% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% milk the membranes were incubated with the following antibodies: anti-β-catenin (15B8, eBioscience), anti-p-Ser552-β-catenin (D8E11, Cell Signaling), anti-NFATc1 (7A6, Santa Cruz Biotechnology), anti-p-Thr286-CaMKII (D21E4, Cell Signaling), anti-p-Ser9-GSK-3β (37F11, Cell Signaling), anti-Wnt3a (ab19925-100 polyclonal, Abcam), anti-Wnt5a (ab72583 polyclonal, Abcam), anti-IDO antibody (Santa Cruz Biotechnology) followed by anti-rabbit or -mouse fluor-coupled secondary antibody. Anti-β-actin (mAbcam8226, Abcam) was used as loading control. The blots were revealed by incubation with corresponding IRD Fluor 800-labeled IgG or IRD Fluor 680-labeled IgG secondary antibody (LI-COR Inc., Lincoln, NE, USA) for 1 h at room temperature. After washing, the membranes were scanned with the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA) at a wavelength of 700–800 nm. Densitometric analysis was performed using ImageJ software.

The cells were fixed in 4% formaldehyde and perm with 10% TritonX-100 for 15 min, washed three times with 0.01 M PBS, and incubated with 5% bovine serum albumin (Sigma-Aldrich) for 1 h for blocking. The sections were then incubated overnight at 4°C with anti-β-catenin (15B8, eBioscience) or anti-NFATc1 (7A6, Santa Cruz Biotechnology) primary antibodies. Next, the cells were washed three times with 0.01 M PBS, incubated with secondary antibody Alexa Fluor 488-conjugated Goat anti-Mouse IgG (#A11001, Invitrogen) for 4 h and washed three times with 0.01 M PBS. For nuclear counterstaining, the cells were incubated with 4′,6-diamidino-2-phenylindone (Cell Signaling Technology, #4083) for 10 min, washed in 1× PBS, and the slides were set with FluorSave™ reagent (EMD Millipore). Expression and localization of beta-catenin were observed with an Olympus FV1200 laser scanning confocal microscope (Olympus Corporation) with fixed exposure time for all samples. Colocalization was quantified as a ratio of overlapping pixels to the total number of pixels by Threshold Mander’s coefficient and expressed as nuclear percentage (% nuclear). The preparations were visualized using confocal microscopy (Olympus FV1200), and the analyses of images were carried out using FIJI/ImageJ.

Cytokines were measured in culture supernatants of BMM 24 h post-infection (pi) by capture ELISA using antibodies and protocols suggested by the manufacturer (eBioscience). The cytokine concentration was expressed as index of cytokine release (Index) obtained by dividing the cytokine release of inhibitors/activators-treated BMM by the cytokine release of vehicle-treated BMM.

RNA was extracted from BMM by the Trizol reagent (Invitrogen, ThermoFisher Scientific) and reverse-transcribed into cDNA by using Revert Aid First Strand cDNA Synthesis (Fermentas). Transcripts were quantified by real-time quantitative PCR on an StepOnePlus™ Real-Time System (ThermoFisher Scientific) using SYBR Green (ThermoFisher Scientific) with the following primers (all primers listed in the 5′ to 3′ orientation): Wnt3a TTC TTA CTT GAG GGC GGA GA (forward) and CTG TCG GGT CAA GAG AGG AG (reverse); Wnt5a GCA GGA CTT TCT CAA GGA CA (forward) and CCC TGC CAA AGA CAG AAG TA (reverse); Ctnnb1 AGC CGA GAT GGC CCA GAA T (forward) and AAG GGC AAG GTT TCG AAT CAA (reverse); Ccnd1 AGT GCG TGC AGA AGG AGA TT (forward) and CAC AAC TTC TCG GCA GTC AA (reverse); Axin1 CTG GGC TTG TAT CCC ACT GT (forward) and ACC AAG CTG GTG GCT AGA GA (reverse); Wisp1 CTG GAC AGA AAA GGG CAT GT (forward) and AGG AAG GAG GGG AAA TCT CA (reverse); Fzd4 CTG CAG CAT GCC TAA TGA GA (forward) and CGT CTG CCT AGA TGC AAT CA (reverse); Fzd6 TCC GAC GCT TGA AGA AAA CT (forward) and CAA CCC CAG GTC CTC AAG TA (reverse); Fzd8 GCA GCA TGT TCG CTA TGA AA (forward) and AGT AGC CTG CTA TGG CCT CA (reverse); Fzd9 AGA GCC TGT GCT ACC GAA AA (forward) and CAA GGA GGG AGC AAC CAT AA (reverse); Actb CGC CAC CAG TTC GCC ATG GA (forward) and TAC AGC CCG GGG AGC ATC GT (reverse). Relative expression was normalized to β-actin (Actb) and expressed as mRNA relative levels. The cycling conditions included a hot start at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Specificity was verified by melting curve analysis and agarose gel electrophoresis.

Arginase and IDO assays were performed as described previously (8, 43) with cell lysates that had been cultured in different conditions as indicated in the figure legends.

The production of NO was measured indirectly by assaying nitrites in the culture supernatant using the Griess reaction as described previously (8).

Lactate dehydrogenase release was measured in the supernatant of BMM using LDH colorimetric assay kit (Wiener Lab) following the manufacturer’s protocol and expressed as international units (I/U).

Groups of 6 mice (6–8 weeks old) maintained under standard conditions were infected with 5,000 bloodstream T. cruzi Tps by the i.p. route. On days 5, 8, 11, and 14 pi, mice were treated with IWP-L6 (7.5 mg/kg/dose) by the i.p. route. We resuspended IWP-L6 in DMSO (10 mg/mL) and then injected diluted in PBS, with this vehicle also being employed as a negative control. The levels of parasitemia were monitored every 2 days as described earlier, and the number of deaths was recorded daily. Determination of tissue parasitism was assessed in heart and liver obtained from 180-day-infected IWP-L6 or control mice as described previously (7).

Statistical analyses were performed using Student’s t-test, and two-way ANOVA followed by Bonferroni’s post-test for comparing more than two groups. The Kaplan–Meier analysis test was used for comparing survival of control and treated mice. Percent survival at each time was also compared by using the log-rank and Gehan–Breslow–Wilcoxon tests. The results were considered significantly different when P < 0.05.

Trypanosoma cruzi-infected B6 mice have great difficulty in controlling inflammatory response, resulting in the premature death of these animals by liver failure. Interestingly, we have observed that B6, unlike BALB/c mice, did not expand the population of Treg cells in parallel with the large expansion undergone by the T cell compartment resulting in an increased ratio of T effector/Treg cells (not shown). These results suggest that, as observed in patients with severe chronic Chagas disease cardiomyopathy (12, 44), the fatal outcome in B6 mice may be linked to an unbalanced inflammatory response. For that, although in T. cruzi-infected B6 mice the main target organ of pathological inflammatory response is the liver, and not the heart as in infected human patients, we performed the experiments in B6 mice to evaluate both parasite replication and inflammatory pathology.

To study whether experimental infection with T. cruzi induces the production of Wnt pathway proteins, the expression of the most common inflammation-linked Wnt proteins such as Wnt3a and Wnt5a, and β-catenin was evaluated in spleen cells from infected B6 mice at different times pi. Figure 1 shows that, as T. cruzi infection progresses, the expression of Wnt3a, Wnt5a, and β-catenin is positively regulated in spleen cells. These results suggest that T. cruzi recognition induces in spleen cells the expression of Wnt proteins that could signal for the activation of the Wnt/β catenin canonical pathway, taking into account the accumulation of β-catenin.

To evaluate whether T. cruzi experimental infection induces the expression of proteins involved in Wnt signaling in macrophages, BMM were in vitro infected with T. cruzi Tps and the expression of Wnt3a and Wnt5a transcripts and proteins determined by q-PCR and Western blot. Figures 2A,B show that in vitro infection of BMM with T. cruzi induced the expression of Wnt5a and Wnt3a transcripts and proteins. The evaluation by means of q-PCR of the transcripts corresponding to Fzd receptors that most strongly interact with Wnt3a and Wnt5a, such as Fzd4, Fzd6, Fzd8, and Fzd9 (45), revealed that as early as 5 min pi, there is an increase in the transcription of these genes (Figure 2C), suggesting that after the recognition of T. cruzi by innate immune receptors start the transcription of Wnt proteins that could signal through Fzd receptors to activate the Wnt signaling pathways.

To evaluate whether T. cruzi infection activates β-catenin in BMM, the kinetics of expression of β-catenin (mRNA and protein) and its translocation to the nucleus were evaluated in in vitro-infected BMM at different times pi. At very short times after the infection (5 min), a significant increase in the transcription of the gene coding for β-catenin (Ctnnb1) was detected (Figure 3A), while the protein expression and translocation to the nucleus began at 2 h pi and reached the maximum between 8 and 12 h pi (Figures 3A,B). Moreover, the upregulation of Wnt/β-catenin pathway-target genes transcription, such as Wisp1, Axin1, and Ccnd1, correlates with β-catenin accumulation and nucleus translocation (Figure 3C). In addition, accumulation of β-catenin was observed in PM from B6-infected mice obtained at 24 h pi (Figure 3D). Taken together, our results showed that in vitro and in vivo T. cruzi infection was associated with significant activation of the genes and proteins involved in the canonical Wnt signaling pathway in macrophages.

Next, we tested whether T. cruzi infection is also able to activate Wnt/Ca⁺² pathway by analyzing the expression of CaMKII phosphorylated at Thr286 [phosphorylated CAMKII (p-CAMKII)] and the activation and nuclear translocation of NFATc1. As shown in Figure 4A, T. cruzi infection led to an increase in the phosphorylation of CaMKII at Thr286 from 12 h pi. We also detected an induced NFATc1 activation after 18 h pi as measured by faster migrating, dephosphorylated, active NFATc1 bands on Western blot (arrows; Figure 4B) and the increased expression and nuclear translocation of NFATc1 detected by immunofluorescence (Figure 4C). Interestingly, the upregulation of p-CaMKII and activation of NFATc1 were dependent on the secretion of Wnt proteins; since the use of IWP-L6, a porcupine inhibitor that is the O-acetyltransferase membrane involved in the palmitoylation of Wnt proteins, a critical modification for its secretion, was able to inhibit both p-CaMKII upregulation and NFATc1 activation (Figures 4D,E). Treatment of BMM with the calcium ionophore ionomycin was used as positive control of Ca⁺² pathway activation (46). Moreover, we were unable to detect NFATc1 upregulation or translocation to the nucleus in PM obtained at 24 h pi (Figure S1 in Supplementary Material), even though it’s upregulation was evident in PM obtained from infected mice at 18 days pi (Figure S2 in Supplementary Material).

To evaluate the role of the Wnt/β-catenin pathway activation in the control of T. cruzi intracellular replication, BMM were pretreated with LiCl or BIO, both inhibitors of GSK-3β activity which mimics activation of Wnt/β catenin signaling, or specific inhibitors of β-catenin-responsive transcription such as iCRT14 and CCT036477 for 24 h before being infected. Then, the intracellular amastigotes were counted by IF assay, with the results shown in Figures 5A,B. Treatment of BMM with IFN-γ plus LPS was used to activate BMM to control T. cruzi replication as described previously (8). β-Catenin-responsive transcription blockade with iCRT14 and CCT036477 was shown to result in a strong inhibitory effect on intracellular parasite growth. Similar results were obtained by inhibiting the secretion of Wnt proteins with IWP-L6 (Figures 5A,B). The inhibition of parasite replication observed by β-catenin transcription blockade was even more significant than those observed after the activation of BMM with IFN-γ plus LPS, and these results were not due to drugs-induced cytotoxicity, as revealed by using LDH release assay to determine cell membrane integrity (Figure 5C). In addition, the treatment with IWP-L6 inhibited both β-catenin accumulation and nucleus translocation at 12 h pi (Figure 5D). Similar results were obtained when the drugs were added to the culture at the same time as the parasites (not shown). Likewise, inhibition of Wnt/β-catenin pathway or Wnt protein secretion in human monocyte-derived THP-1 macrophages and J774 cell line (macrophages derived from BALB/c mice) resulted in the suppression of intracellular parasite replication (Figure S3 in Supplementary Material). On the other hand, treatments with β-catenin activators (LiCl and BIO) were unable to significantly increase the intracellular parasite growth (Figures 5A,B).

Then, we studied whether the inhibition of canonical Wnt signaling regulates macrophages function by modulating the secretion of pro-inflammatory and anti-inflammatory cytokines. Figure 6A shows that inhibition of Wnt/β-catenin signaling induced the secretion of the pro-inflammatory cytokines IFN-γ, IL-12, IL-6, and TNF while inhibited the production of TGF-β (Figure 6A; Table S1 in Supplementary Material), an effect that correlates with the control of parasite replication observed in iCRT14-, CCT036477-, or IWP-L6-treated BMM (Figures 5A,B). In addition, canonical signaling activation using LiCl or BIO induced a slightly upregulation of TGF-β while only LiCl increased IL-10 production by T. cruzi-infected BMM (Figure 6A).

Nitric oxide production is counteracted by the expression of arginase, an enzyme that competes with iNOS for l-arginine that leads to l-ornithine and urea production (47). T. cruzi antigens can upregulate arginase activity with this type of activation profile associated with the ability to promote the intracellular growth of T. cruzi (48). Interestingly, although the pharmacological inhibition of Wnt/β-catenin pathway did not affect NO production (Figure 6B), the treatments with CCT036477 and IWP-L6 significantly downregulated arginase activity in infected BMM (Figure 6C).

Because IDO activity is also critical for the control of T. cruzi amastigote growth in macrophages (7–9), we analyzed the effect of the inhibition of Wnt/β-catenin signaling on the activity and expression of IDO and their role in the intracellular parasite replication control. The inhibition of both β-catenin-responsive gene transcription as well as Wnt secretion induced upregulation of IDO expression and activity in infected BMM (Figures 6C,D). Remarkably, the inhibitory effect of intracellular amastigote growth induced by pharmacological inhibitors of the Wnt/β-catenin pathway was reversed when IDO activity was blocked using 1-MT (Figure 6E). In addition, as it was previously demonstrated, 1-MT exacerbated the intracellular parasite replication in untreated BMM (Figure 6E). Taken together, these results demonstrated that pharmacological inhibition of Wnt/β-catenin pathway activates macrophages to fight against T. cruzi.

Next, we evaluated whether the inhibition of Wnt proteins secretion in vivo would result in the control of the parasite load. Five days after T. cruzi infection with a lethal dose 50 (DL50) of Tps, the mice were treated every 3 days with IWP-L6 (Figure 7A). The dose of IWP-L6 used has been previously assayed to examine the effect of Wnt proteins in an experimental model of cancer (49). As expected, in vivo IWP-L6 treatment could maintain inhibited both Wnt/β-catenin as well as Wnt/Ca⁺² signaling pathways until 18 days pi, as denoted by the lack of β-catenin or NFATc1 accumulation in PM from treated versus control mice (Figure S2 in Supplementary Material). Mice infected with T. cruzi and injected with vehicle presented high levels of parasitemia, causing death of ~50% of mice between 18 and 30 days pi (Figure 7B). By contrast, although only four doses of IWP-L6 were given, 100% of treated mice survived to acute infection and displayed lower parasitemia than control mice during the acute phase of the infection (Figure 7C). In addition, IWP-L6-treated mice showed significantly lower parasite load in liver and heart than control mice during the chronic phase of the disease (Figure 7D).