Trypanosoma cruzi Exploits Wnt Signaling Pathway to Promote Its Intracellular Replication in Macrophages

During the acute phase of Trypanosoma cruzi infection, macrophages can act as host cells for the parasites as well as effector cells in the early anti-parasitic immune response. Thus, the targeting of specific signaling pathways could modulate macrophages response to restrict parasite replication and instruct an appropriate adaptive response. Recently, it has become evident that Wnt signaling has immunomodulatory functions during inflammation and infection. Here, we tested the hypothesis that during T. cruzi infection, the activation of Wnt signaling pathway in macrophages plays a role in modulating the inflammatory/tolerogenic response and therefore regulating the control of parasite replication. In this report, we show that early after T. cruzi infection of bone marrow-derived macrophages (BMM), β-catenin was activated and Wnt3a, Wnt5a, and some Frizzled receptors as well as Wnt/β-catenin pathway’s target genes were upregulated, with Wnt proteins signaling sustaining the activation of Wnt/β-catenin pathway and then activating the Wnt/Ca+2 pathway. Wnt signaling pathway activation was critical to sustain the parasite’s replication in BMM; since the treatments with specific inhibitors of β-catenin transcriptional activation or Wnt proteins secretion limited the parasite replication. Mechanistically, inhibition of Wnt signaling pathway armed BMM to fight against T. cruzi by inducing the production of pro-inflammatory cytokines and indoleamine 2,3-dioxygenase activity and by downregulating arginase activity. Likewise, in vivo pharmacological inhibition of the Wnts’ interaction with its receptors controlled the parasite replication and improved the survival of lethally infected mice. It is well established that T. cruzi infection activates a plethora of signaling pathways that ultimately regulate immune mediators to determine the modulation of a defined set of effector functions in macrophages. In this study, we have revealed a new signaling pathway that is activated by the interaction between protozoan parasites and host innate immunity, establishing a new conceptual framework for the development of new therapies.

During the acute phase of Trypanosoma cruzi infection, macrophages can act as host cells for the parasites as well as effector cells in the early anti-parasitic immune response. Thus, the targeting of specific signaling pathways could modulate macrophages response to restrict parasite replication and instruct an appropriate adaptive response. Recently, it has become evident that Wnt signaling has immunomodulatory functions during inflammation and infection. Here, we tested the hypothesis that during T. cruzi infection, the activation of Wnt signaling pathway in macrophages plays a role in modulating the inflammatory/tolerogenic response and therefore regulating the control of parasite replication. In this report, we show that early after T. cruzi infection of bone marrow-derived macrophages (BMM), β-catenin was activated and Wnt3a, Wnt5a, and some Frizzled receptors as well as Wnt/β-catenin pathway's target genes were upregulated, with Wnt proteins signaling sustaining the activation of Wnt/β-catenin pathway and then activating the Wnt/Ca +2 pathway. Wnt signaling pathway activation was critical to sustain the parasite's replication in BMM; since the treatments with specific inhibitors of β-catenin transcriptional activation or Wnt proteins secretion limited the parasite replication. Mechanistically, inhibition of Wnt signaling pathway armed BMM to fight against T. cruzi by inducing the production of pro-inflammatory cytokines and indoleamine 2,3-dioxygenase activity and by downregulating arginase activity. Likewise, in vivo pharmacological inhibition of the Wnts' interaction with its receptors controlled the parasite replication and improved the survival of lethally infected mice. It is well established that T. cruzi infection activates a plethora of signaling pathways that ultimately regulate immune mediators to determine the modulation of a defined set of effector functions in macrophages. In this study, we have revealed a new signaling pathway that is activated by the interaction between protozoan parasites and host innate immunity, establishing a new conceptual framework for the development of new therapies.
Keywords: Trypanosoma cruzi infection, Wnt proteins, beta-catenin, macrophages, cytokines, indoleamine 2,3-dioxygenase Wnt Signaling Promotes T. cruzi Replication Frontiers in Immunology | www.frontiersin.org April 2018 | Volume 9 | Article 859 inTrODUcTiOn Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, represents a major cause of heart disease and cardiovascularrelated deaths in endemic areas and causes a significant economic burden on the affected countries. Approximately 8 million people are infected with T. cruzi in Central and South America, and at least 120 million are at risk of infection (1). Currently, there are no vaccines available to prevent Chagas disease, and treatment options are limited to anti-parasitic drugs that are expensive, not well tolerated, and effective only during short periods of the acute phase (2). The entry of metacyclic trypomastigotes (Tps) of T. cruzi into the mammalian host initiates the invasion by these parasites of several host cell types where they are converted into amastigotes, which are the replicative form of this parasite (3). During the acute phase of the infection macrophages represent an important target of Trypanosoma cruzi and therefore, these cells are central for the control of this pathogen. Within macrophages, the replication of T. cruzi can be either inhibited or favored leading to the dissemination of the parasite (4). Thus, it has been reported that during the early phase of infection, the control T. cruzi parasitism is dependent on macrophage activation through toll-like receptors (TLRs) and their subsequent activation by pro-inflammatory cytokines. Activated macrophages upregulate inducible nitric oxide synthase (iNOS) and indoleamine 2,3-dioxygenase (IDO) enzymes, leading to the production of nitric oxide (NO) and kynurenines, with both being important effector molecules that induce death of the amastigotes (5-9). The protective mechanisms of cell-mediated immunity (Th1 cells) are required for the resistance during this infection; nevertheless, unbalanced Th1 cells can also orchestrate a deleterious response that can cause tissue damage and fibrosis, since high levels of NO, IFN-γ, and tumor necrosis factor (TNF) have been associated with the pathogenesis of chronic Chagas disease (10)(11)(12)(13)(14). Therefore, a better understanding of the cellular and molecular mechanisms that orchestrate the different signals that promote the effective macrophage activation (able to restrict parasite replication) followed by its opportune contraction to prevent immunopathology is mandatory to design improved therapeutic strategies.
The Wnt pathway is an evolutionarily highly conserved signaling system that plays a critical role in cellular development, motility, polarization, survival, and proliferation (15, 16). In the last years, emerging studies have highlighted that the Wnt signaling pathway, particularly in dendritic cells (DC) and macrophages, plays a major role in regulating tolerance versus immunity. Members of the Wnt family are lipid-modified glycoproteins secreted by different cell types that bind to G-protein-coupled receptors of the Frizzled (Fzd) family and different coreceptors to activate a signaling cascade involved in complex mechanisms of gene regulation. In mice and human, 19 ligands secreted glycoproteins of the Wnt family, 10 seven-transmembrane receptors of the Fzd family, as well as several coreceptors or alternative receptors are known (16,17). Depending on the composition of the Wnt-Fzd-coreceptor complex and the cellular context, Wnt proteins can initiate at least three different intracellular signaling cascades that regulate many cellular events: the Wnt/β-catenin called canonical pathway, the Wnt/Planar cell polarity pathway, and the Wnt/Ca +2 pathway (18,19). In resting state, cytosolic/nuclear β-catenin is maintained at a very low level through rapid turnover of free β-catenin. This turnover is executed through a multi-protein complex, termed the β-catenin destruction complex, integrated by AXIN1/2, adenomatous polyposis coli, casein kinase I-alpha, and glycogen synthase kinase 3 beta (GSK-3β). GSK-3β sequentially phosphorylates β-catenin with β-catenin hyperphosphorylated subjected to ubiquitination and proteasomal degradation. Activation of Wnt/β-catenin signaling pathway results in the inhibition of the activity of GSK-3β, which leads to the accumulation of β-catenin in the cytoplasm and its translocation to the nucleus, where it interacts with T-cell factor/ lymphoid enhancer factor family members and regulates the transcription of several target genes (16,17). While canonical Wnt signaling has been extensively studied from the view point of innate and adaptive immune response, the non-canonical Wnt/ Ca +2 signaling cascade has been less focused on. The binding of Wnt ligand to cognate Fzd receptor leads to the activation of calcium/ calmodulin-dependent kinase II (CaMKII) and protein kinase C (PKC). Both CaMKII and PKC activate many nuclear transcription factors as NF-κB and CREB. Similarly, calcium ions can activate widely expressed protein phosphatase calcineurin that can activate cytoplasmic protein nuclear factor associated with T cells (NFAT) via dephosphorylation. This event results in accumulation, nuclear translocation and transcription of several NFAT target genes, including pro-inflammatory genes in lymphocytes (20).
Human and murine DC and macrophages express various receptors and Wnt proteins and are susceptible to both canonical and non-canonical signaling, and recently, several studies have emphasized the important role of DC and macrophages Wnt/βcatenin pathway in regulating inflammatory responses to microbial infections (21,22). In DC, the activation of canonical and Wnt/Ca +2 pathways is associated with the development of a tolerogenic profile, secretion of anti-inflammatory cytokines, induction of Treg cells, and the inhibition of the adaptive immune response (23)(24)(25)(26)(27)(28). However, Wnt pathways activation in macrophages may have both pro-and anti-inflammatory consequences, and different Wnt proteins may effectively cross-regulate each other in macrophages [reviewed in Ref. (21,29)]. Wnt proteins and Fzd receptors are induced by STAT3-, TLR2-, TLR4-, and NF-κB-mediated signaling and, in addition to direct activation of β-catenin by Wnt proteins, TLR-mediated signals can also directly activate β-catenin (21,26,28,(30)(31)(32)(33)(34)(35)(36)(37). Considering that TLR2 and TLR4 are involved in the recognition of different T. cruzi components (38)(39)(40), here we tested the hypothesis that during T. cruzi infection, the activation of Wnt signaling pathway in macrophages plays a role modulating the inflammatory/tolerogenic response and therefore regulating the control of intracellular parasite replication.

Mice and Parasites
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 reagents 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.

cells
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. In Vitro Trypanocidal activity assay

In Vitro infections and Treatments
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.

β-catenin and nFaTc1 immunofluorescence
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.

Measurement of cytokine Production
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/activatorstreated BMM by the cytokine release of vehicle-treated BMM.

arginase and iDO activity
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.

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

iWP-l6 Treatment
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 analysis
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.

T. cruzi infection Promotes Wnt signaling Pathway activation in spleen cells
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.

T. cruzi infection induces expression of Wnt Proteins and Fzd receptors in Macrophages
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.

Pathway in Macrophages
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 Nuclear staining was detected with 4′,6-diamidino-2phenylindone (DAPI), and the levels of expression of β-catenin (% of total area) and the threshold Mander's colocalization (% nuclear) coefficients calculated using FIJI/ImageJ program as described in Section "Materials and Methods." Green, β-catenin; red, DAPI (*P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001). 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).

The inhibition of Wnt/β-catenin signaling Pathway limits the replication of T. cruzi in Macrophages
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/βcatenin 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.
In Vivo inhibition of Wnt signaling improves the resistance to T. cruzi infection  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).

DiscUssiOn
Given that during the acute phase of T. cruzi infection macrophages can act as host cells for the parasites as well as effector cells in the early anti-parasitic immune response, the targeting of specific signaling pathways in macrophages could modulate its response  Figure 5B (*P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001). to restrict parasite replication and instruct an appropriate adaptive immune response. In recent years, it has become apparent that Wnt signaling pathway, known for its essential participation in embryonic development and tissue homeostasis, exerts immunomodulatory functions during inflammation and infection. In this study, we have demonstrated that early after the recognition of T. cruzi by innate immune receptors in BMM, β-catenin was activated and Wnt3a, Wnt5a, some Fzd receptors as well as target genes of Wnt/β-catenin pathway as Axin1, Wisp1, and Ccnd1 were upregulated. Subsequently to canonical pathway activation, Wnt/Ca +2 pathway was activated; since we have demonstrated an Wnt proteins-dependent upregulated of p-CaMKII-Thr286 and activated NFATc1 expression after infection. Many studies have demonstrated that T. cruzi utilize the host Ca +2 signaling to establish the infection (50) and several mechanisms have been proposed to explain the intracellular Ca +2 influx that occurs during T. cruzi infection (51). In addition, Kayama et al. (52) have reported that NFATc1 is activated in response to T. cruzi infection in a TLR-independent manner, but the critical molecules and signaling pathways that lead to NFATc1 activation during T. cruzi infection have not yet been identified. Wnt3a and Wnt5a are more commonly associated with canonical and non-canonical Wnt signaling, respectively (19). However, Wnt5a can also activate discrete β-catenin signaling (53,54), and recent reports have suggested that the activity of Wnt ligands and their binding to Fzd receptors depend on the cellular context; therefore, Wnt and Fzd proteins cannot be rigorously subdivided according to the pathways they induce (55). Thus, considering that we found that T. cruzi infectioninduced β-catenin, p-CaMKII-Thr286, and activated NFATc1 upregulation were suppressed by IWP-L6 treatment, these results suggest that both Wnt3a and Wnt5a proteins, and others Wnt proteins not evaluated in this paper, could be the responsible for the activation and maintenance of both canonical and noncanonical signaling pathways during T. cruzi infection which allows the parasite to spread in the host.
Resistance to T. cruzi infection has been associated with the capacity of NK cells and T lymphocytes to generate IFN-γ which can, in turn, activate macrophages to kill the obligate intracellular amastigote form of T. cruzi. The trypanocidal activity of pro-inflammatory cytokines-activated macrophages is mediated at least by the upregulation of the enzymes iNOS and IDO which lead to the production of NO and kynurenines, respectively (5-9, 56). On the other hand, susceptibility to infection is associated with the production of IL-10 and transforming growth factor beta (TGF-β) (5,56). Here, we have demonstrated that while the activation of Wnt/β-catenin pathway did not promote the intracellular parasite replication, the treatments of macrophages with specific inhibitors of β-catenin transcriptional activity or the inhibition of Wnt proteins secretion were able to inhibit the parasite replication by modifying macrophages activity. Inhibition of Wnt signaling pathway enhanced production of the pro-inflammatory cytokines IFN-γ, IL-12, TNF and IL-6 and suppressed production of TGF-β, results that are in agreement  with previous reports showing that the activation of the canonical pathway in macrophages and DC controls the inflammatory response (28). In addition, these treatments induced downregulation of arginase activity, an enzyme that counteracts iNOS activity, but failed to upregulate iNOS activity, suggesting that the resulting treated-macrophages do not fully fit in the classically activated/inflammatory macrophage phenotype. Interestingly, and despite the fact that there is a close relationship between the activation of the Wnt/β-catenin pathway and the induction of IDO and vice versa (27,57,58), in our experimental model the inhibition of β-catenin-induced transcription or Wnt proteins secretion upregulated IDO expression and activity. In addition, IDO activity proved to be critical for the control of T. cruzi replication in BMM, as denoted by the recovery of T. cruzi replication observed in cultures where the inhibitors were combined with 1-MT. Expression of IDO and activation of β-catenin within macrophages and DC under tolerogenic conditions are particularly important mechanisms that limits inflammation within the gastrointestinal tract and tumor cell microenvironment (28,59). The IDO promoter has been shown to exhibit LEF-1 binding sites, and kynurenine and quinolinic acid, produced by IDO activity, can activate the Wnt/β-catenin pathway (60,61). However, as IDO gene expression is induced not only in tolerogenic conditions but also by IFNs and TNF during inflammatory conditions (62,63), in our experimental settings the upregulation of IDO could be induced by the milieu of pro-inflammatory cytokines generated in inhibitors-treated T. cruzi-infected macrophages. Thus, our results suggest that the anti-T. cruzi activity of inhibitor-treated macrophages is due to the production of pro-inflammatory cytokines-inducible antimicrobial molecules, with IDO being one of the most important.
In summary, in this study, we have revealed a new signaling pathway that is activated by the interaction between protozoan parasites and host innate immunity. In this context, it is well founded that T. cruzi infection activates a plethora of signaling pathways that ultimately regulate the immune mediators to determine the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.

eThics sTaTeMenT
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). acKnOWleDgMenTs XV, LA, and CI thank Consejo Nacional de Investigaciones Científicas y Técnicas from Argentina for the fellowships granted.