All studies involving mice were approved by the animal research ethics committee at UFU (Comitê de Ética na Utilização de Animais da Universidade Federal de Uberlândia—CEUA/UFU), under protocol number 109/16. All procedures including housing and welfare were carried out in accordance with the recommendations in the Guiding Principles for Biomedical Research Involving Animals of the International Council for Laboratory Animal Science (ICLAS), countersigned by the Conselho Nacional de Controle de Experimentação Animal (CONCEA; Brazilian National Consul for the Control of Animal Experimentation) in its E-book (http://www.mct.gov.br/upd_blob/0238/238271.pdf). The UFU animal facility (Centro de Bioterismo e Experimentação Animal—CBEA/UFU) is accredited by the CONCEA (CIAEP: 01.0105.2014) and Comissão Técnica Nacional de Biossegurança (CTNBio, Brazilian National Commission on Biosecurity; CQB: 163/02).

For the experiments done in this work, we have used wild-type (WT) mice on a C57BL/6 background, along with genetically deficient littermates in Dectin-1 (Dectin-1^−/−), and NADPH oxidase isoform 2 (NOX2^−/−), with 6–8 weeks old. All mice were bred and maintained at CBEA/UFU, in groups of maximum five animals inside each isolator cages, with light/dark cycle of 12 h, food, and water ad libitum.

Neospora caninum tachyzoites (Nc-1 isolate) and T. gondii tachyzoites (RH, Pru, and Me49 strains) were maintained by continuous passages in a diploid-immortalized cell line derived from cervical cancer (HeLa; CCL-2, ATCC, Manassas, VA, USA). Briefly, HeLa cells were cultured in RPMI-1640 medium (Thermo Scientific Inc., Waltham MA, USA) supplemented with 25mM HEPES, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated calf fetal serum [fetal calf serum (FCS); Thermo Scientific] in an incubator under controlled temperature and atmosphere (37°C, 5% CO₂, 95% relative humidity; Thermo Scientific). After HeLa infection with N. caninum, these cells were cultured in RPMI-1640 medium without FCS or, after infection with different strains of T. gondii, with 2% FCS. Extracellular parasites were washed twice (720 × g, 10 min, 4°C) with phosphate-buffered saline (PBS, 0.01M, pH 7.2), and the resulting pellet was resuspended in RPMI. Finally, the parasites were suspended in RPMI-1640 medium, and numbers of viable tachyzoites were determined by Trypan blue exclusion (Sigma-Aldrich, St. Louis, MO, USA) in a hemocytometer (43).

In order to obtain tissue cysts of the type II T. gondii Me49 strain, we maintained mice chronically infected with sub-lethal doses of the parasite. After 30–45 days of infection, the brains were removed, homogenized, and washed in sterile PBS (pH 7.2) at 1,000 × g for 10 min. Tissue cysts were then counted under light microscopy for the experimental infection protocols. For in vivo assays involving survival and parasite burden, mice were infected through oral route (gavage) with 20 and 5 cysts, respectively (44, 45).

Bone marrow-derived macrophages were generated from bone marrow stem cells of WT and Dectin-1^−/− mice, as previously described (46). Briefly, stem cells were cultured on 10 cm-diameter polystyrene plates, for 7 days in RPMI-1640 medium, containing HEPES 15 mM, 2 g of sodium bicarbonate/L, 1 mM l-glutamine, supplemented with 20% heat-inactivated FCS and 30% cell-conditioned medium, obtained from the supernatant of confluent L929 cells (LCCM). Differentiated BMDMs were removed from the substrate by vigorous pipetting of ice-cold PBS. Cells were counted and used in the proportion of 1 × 10⁶/mL for the experiments, in 96-well plates.

Bone marrow-derived dendritic cells (BMDCs) were generated as previously cited (24). Briefly, bone marrow stem cells were cultured on 10 cm-diameter polystyrene plates in RPMI-1640 medium supplemented with 10% FCS and murine granulocyte-macrophage colony-stimulating factor (25 ng/mL; BD Biosciences, San Jose, CA, USA). On day 3, supernatant was gently removed and replaced with the same volume of supplemented medium. On day 6, non-adherent cells were removed and placed in 96-well plates (1 × 10⁶ cells/mL) prior to stimulation.

On the third day of infection, peritoneal cells from WT mice infected with 1 × 10⁷ Nc-1 tachyzoites were collected and stained for phenotypic characterization and Dectin-1 expression. Briefly, mice were euthanized, and their peritoneal cavities were washed with ice-cold PBS. The suspension was then centrifuged at 400 × g, at 4°C, for 10 min. The cell pellet was resuspended in PBS with 5% of normal rabbit serum, at room temperature, for 15 min, prior to incubation with the appropriate antibodies: anti-CD11b-APC-Cy7, anti-CD11c-FITC, anti-MHCII-PE, anti-CD11c-V450, anti-CD19-APC-Cy7, anti-CD3-Pacific Blue, anti-CD49b-APC (BD Biosciences), and anti-Dectin-1-PE (R&D Systems, Minneapolis, MN, USA). Cells were incubated with primary antibodies conjugated to the different fluorochromes for another 30 min, at room temperature. After washing, cells were suspended in PBS with 3% formaldehyde, read by flow cytometry (FACSCantoII, BD Biosciences), and analyzed using dedicated software (FlowJo X, Tree Star Inc., Ashland, OR, USA).

Reactive oxygen species and NO production by peritoneal cells after 3 days of infection were determined through fluorescent probes [CellROX Green Reagent (Thermo Scientific) and DAF-2 DA (Sigma-Aldrich), respectively], according to the manufacturers’ instructions. Briefly, peritoneal cells obtained from infected mice were incubated with each probe for 30 min, at 37°C, followed by four washing cycles with PBS. The stained cell suspension was read by flow cytometry (FACSCantoII, BD Biosciences) and analyzed by dedicated software (FlowJo, Tree Star). Additionally, in vitro experiments were also undertaken, in order to determine ROS production in BMDMs generated from WT and Dectin-1^−/− mice. Briefly, BMDMs (10⁶ cells/mL) were infected with live Nc-1 tachyzoites (0.5:1, parasite:cell ratio) for 1 h, at 37°C, 5% CO₂, pretreated or not with laminarin (LAM) (500 μg/mL) for 3 h. After incubation, the supernatant was removed, and the cells were incubated with the probe for 30 min, at 37°C, followed by six washing cycles with PBS. The reaction was read at a plate reader (M2e, Molecular Devices, Sunnyvale, CA, USA) under 488 nm emission and 525 nm excitation.

IL-12p40 production was analyzed in the peritoneal fluids and spleen homogenates of naïve and infected mice, as well as the supernatants harvested from cultured BMDCs and BMDMs. The samples were collected and immediately stored at −70°C until being assayed according to the manufactures’ instructions (Opteia set, BD Biosciences).

In vivo LAM (Sigma-Aldrich) treatment was performed during seven consecutive days. After the fourth day of treatment, mice were challenged with non-lethal (1 × 10⁶) or lethal (1 × 10⁷) doses of N. caninum tachyzoites, according to the aim of each assay. The dose of LAM used was 1 mg/kg/day, by intraperitoneal route. For survival analysis, mice were followed twice a day, until the 30th day of infection, for clinical symptoms compatible with the infection. After advancement in morbidity scores and before the occurrence of natural death induced by the experimental challenge, mice were euthanized to avoid excessive suffering.

For acute parasitism analysis, we stained the parasites with fluorescent reactive ester dyes, to allow the identification of parasite-associated cells as previously described (43). Briefly, approximately 3 × 10⁷ tachyzoites/mL was pre-stained with CFDA-SE (5 μM/mL; CFSE; Thermo Scientific). After 10 min at 37°C, the tachyzoites were washed with 10 mL of RPMI-1640 with 10% FCS and centrifuged at 800 × g, for 10 min, at 4°C. Viable tachyzoites were determined with the Trypan blue exclusion test and used to infect mice, BMDCs and BMDMs. For in vivo assays, mice were intraperitonally infected with 1 × 10⁷ CFSE⁺ Nc-1 tachyzoites, and the peritoneal cells were extracted on the third day of infection for analysis of CFSE-associated cells gated within the monocyte region, determined by gating under FSC-A and SSC-A parameters. In vitro, BMDMs were infected with CFSE⁺ Nc-1 tachyzoites (MOI 1) and analyzed after 24 h of infection. The parasitism was given as percent or intensity of CFSE⁺ cells after being read in a flow cytometer (FACSCantoII, BD) and analyzed by dedicated software (FlowJo, Tree Star).

Chronic parasitism was determined in brain tissue from mice after 30 days of infection with N. caninum or T. gondii by real-time quantitative polymerase chain reaction as described elsewhere (47). Primer pairs designed for the Nc-5 region (Np6/Np21) of N. caninum (sense: 3′-GCTGAACACCGTATGTCGTAAA-5′; antisense: 3′-AGAGGAATGCCACATAGAAGC-5′) and T. gondii’s B1 gene (sense: 3′-GCTCCTCCAGCCGTCTTG-5′; antisense: 3′-TCCTCACCCTCGCCTTCAT) were used in assays based on SYBR green detection system (Promega Co., Madison, WI, USA). DNA extraction was performed from 20 mg of murine brain tissues using a commercial kit (Wizard SV Genomic DNA kit, Promega) according to the manufacturer’s instructions. DNA concentrations were determined by UV spectrophotometry (260 nm; Nanodrop Lite, Thermo Scientific) and adjusted to 200 ng/μL with sterile DNAse free water. Assays to determine N. caninum and T. gondii parasite loads were performed through real-time PCR (StepOnePlus, Thermo Scientific) and calculated by interpolation from a standard curve with DNA equivalents extracted from tachyzoites included in each run. Brain tissue from naïve mice was analyzed in parallel as negative control.

Brain tissue sections obtained from mice after 30 days of infection were stained with hematoxylin and eosin and examined by light microscopy to detect tissue damage. Inflammatory scores were represented as arbitrary units: 0–1, mild; 1–2, moderate; 2–3, severe; and >3, very severe; according to previously described protocol (48). Brain tissue samples from naïve mice served as negative controls.

Statistical significance of the results was checked using non-parametric one-way analysis of variance followed by Bonferroni’s post hoc test (for three or more groups) comparing all pairs of columns, or two-tailed Student’s t-test (for two groups). Differences in survival rates between groups were compared using Kaplan–Meier survival analysis, through a log-rank Mantel–Cox test. In all measurements, a p value < 0.05 was deemed as statistically significant. Analysis was performed with the aid of commercial software (GraphPad Prism, San Diego, CA, USA).

Dectin-1 is an innate receptor related until now with β-glucan recognition. Its role as a PRR has been described in infections by fungal, bacteria, and some protozoa. With that intent, our initial goal was to evaluate the role of this receptor in mice survival and parasite burden during lethal infection protocol (DL100) with N. caninum tachyzoites. Dectin-1^−/− mice were more resistant to infection (p = 0.0090), since 50% of the genetically deficient mice survived the DL100 challenge (Figure 1A). Our next step was to evaluate if Dectin-1 also interfered with the acute and chronic parasitism, during a non-lethal infection protocol. Dectin-1^−/− mice presented a decreased number of parasitized (CFSE⁺) cells in the peritoneal cavity during the acute phase (p < 0.01, Figure 1B), and lower concentration of parasite genomic DNA during chronic infections (p = 0.0002, Figure 1C), demonstrating that the presence of functional Dectin-1 contributes to the susceptibility of mice to the infection by N. caninum.

Since Dectin-1^−/− mice presented increased resistance to N. caninum, our next step was to evaluate if the expression of the receptor was altered during the acute phase of the infection. For that purpose, WT mice were infected with non-lethal doses of N. caninum tachyzoites and evaluated for Dectin-1 expression in different phenotypes of peritoneal cells after 3 days of infection. The most pronounced differences in Dectin-1 expression were observed in antigen-presenting cells (dendritic cells and macrophages), which are the primary inducers of appropriated adaptive immune responses. Resident dendritic cells (CD11b⁺CD11c⁺ cells, gated in the monocyte region) in the peritoneal cavity of naïve mice showed constitutive expression of Dectin-1 in its surface (Dectin-1⁺ DCs = 97.6%). However, during acute infection, two distinct populations were clearly observed (CD11b⁺CD11c⁺Dectin-1⁻ and CD11b⁺CD11c⁺Dectin-1⁺), where the expression of Dectin-1 in positive cells was considerably increased (Figure 2A). The same pattern of events was observed for macrophages (CD11b⁺CD11c⁻ cells, gated in the monocyte region). While a homogenous constitutive expression of the receptor was observed in resident cells obtained from naïve mice, macrophages obtained during acute infection showed polarized cell phenotypes: an increased proportion of Dectin-1⁻ macrophages versus Dectin-1⁺ cells with high expression of the receptor (Figure 2A). No relevant differences in Dectin-1 expression were observed in polymorphonuclear cells and lymphocytes (NK, T, and B; data not shown).

Given the differences observed in Dectin-1 expression induced by the infection in antigen-presenting cells, our next question was if the presence of the functional receptor altered IL-12p40 production, an important proinflammatory mediator related to the control of parasite replication during the acute phase of the infection. With that intent, WT and Dectin-1^−/− mice were infected with non-lethal doses of N. caninum tachyzoites for the assessment of IL-12p40 production after 3 days of infection. Our results showed increased levels of this cytokines in the peritoneal fluid (p < 0.001, Figure 2B) and spleen homogenates (p < 0.001, Figure 2C), when compared to WT mice.

Due to the previous observations, and taken that antigen-presenting cells are crucial for the induction of proper Th1-biased immune responses against N. caninum, we then aimed to evaluate whether Dectin-1 would directly affect the ability of dendritic cells and macrophages to increase the production of IL-12p40, a crucial molecule during antigen presentation in N. caninum infection. For that, we undertook experiments, which exposed BMDCs and BMDMs, obtained from WT and Dectin-1^−/− mice, to live N. caninum tachyzoites in vitro. In that context, we observed that Dectin-1^−/− BMDC (Figure 2D) and BMDM (Figure 2E) presented augmented production of IL-12p40 (p < 0.001), compared to WT cells after incubation with N. caninum live tachyzoites for 24 h.

Taken together, these data suggest that N. caninum tampers with Dectin-1 expression in antigen-presenting cells, negatively regulating the production of crucial molecules for the mounting of effective immune responses against the infection.

Once we observed that the absence of Dectin-1 is associated with increased resistance of the host against N. caninum infection, due to an increment of IL-12p40 linked to the infection control, we next aimed to assess whether the use of LAM, a competitive inhibitor of Dectin-1, would be suitable to be employed in therapeutic measures against the parasite. Treatment with LAM rescued 50% of the challenged animals with DL100 (p = 0.0415), similar to the result obtained using Dectin-1^−/− mice (Figure 3A). In addition, after 3 days of infection, mice challenged with N. caninum and treated with LAM presented increased levels of IL-12p40 in the peritoneal fluid (p = 0.0159, Figure 3B) associated with decreased parasitism in peritoneal monocytic cells (p = 0.0110, Figure 3C) during the acute phase of a non-lethal infection protocol. During the chronic phase of the infection, this phenotype of increased resistance was maintained in mice treated with LAM, which presented a reduced concentration of parasite genomic DNA in the central nervous system (p = 0.0097, Figure 3D), associated with a decreased inflammatory score (p = 0.0095, Figure 3E).

Based on these findings, we next sought to shed some light into the molecular mechanism rendering protection against N. caninum infection, upon blockage of Dectin-1. During infections, it is well described that different effector molecules are related to the elimination of intracellular pathogens, with ROS and NO as hallmarks of this process (38, 49–52). Thus, we evaluated if LAM treatment interfered with the production of these free radicals during in vivo experiments with non-lethal infections with live tachyzoites. We observed that LAM significantly increased ROS expression in monocytic peritoneal cells (p = 0.0273) but maintained stable percentage of NO⁺ cells after 3 days of infection (Figure 4A). We further confirmed this phenomenon with an in vitro experiment (Figure 4B), where we demonstrate that the treatment of WT BMDMs with LAM does significantly increase ROS production (p = 0.008) during infection with live tachyzoites, similar to the levels produced by Dectin-1^−/− BMDMs. On the other hand, and as expected, LAM did not produce an additive effect in ROS produced by Dectin-1^−/− BMDMs (p = 0.751). To evaluate if ROS, which has not yet been described during N. caninum infection, is related to parasite control, we used WT and genetically deficient mice in NOX2^−/− to evaluate their ability to control acute parasite replication using CFSE-stained parasites. As expected, we observed that NOX2^−/− mice failed to control parasite replication due to the increased expression of CFSE detected in the peritoneal cells gated in the monocyte region (Figure 4C), demonstrating that ROS is critical for the control of N. caninum replication.

To elucidate whether LAM would be able to control parasite replication in a ROS-independent manner, which would indicate that the molecular mechanism behind protection upon Dectin-1 blockage would be performed by distinct molecules, WT and NOX2^−/− mice were treated with LAM and infected to evaluate the monocytic peritoneal parasitism with CFSE⁺ tachyzoites. Interestingly, while LAM-treated WT mice presented a decreased peritoneal monocytic parasite burden; the intensity of CFSE⁺ cells in the peritoneal cavity of NOX2^−/− remained practically unaltered upon treatment (Figure 4D). Finally, we aimed to correlate if Dectin-1 mediated inhibition of ROS production during N. caninum infection would also affect IL-12p40 production in the infected mice. For that, WT and NOX2^−/− mice were treated with LAM and infected for 3 days for cytokine measurement in the peritoneal fluid. As already demonstrated, WT mice produced IL-12p40 upon infection, which was significantly increased in LAM-treated mice (p = 0.0188). Surprisingly, NOX2^−/− mice were not able to respond to the infectious stimulus with the production of the referred cytokine, which was not even partially reverted by LAM (Figure 4E).

In that context, our results suggest that LAM treatment is efficient to control the pathogenesis of the infection in the proposed models, through a mechanism regulated by the increase in ROS production and consequent upregulation of IL-12p40 during the acute phase of the infection.

Although closely related, N. caninum and T. gondii present marked biological distinctions, as its surface carbohydrate contents (15). We then aimed to answer whether the absence of Dectin-1 would protect mice against T. gondii infection, in the same fashion as seen for N. caninum. For this, a set of in vivo experiments using different clonal T. gondii strains, doses and routes of infection were carried out in WT and Dectin-1^−/− mice. Overall, no differences were noted in susceptibility/resistance of the animals submitted to the infectious protocols.

Initially, survival assays were undertaken. WT and Dectin-1^−/− mice infected by intraperitoneal route with 1 × 10² tachyzoites of RH strain (type I) succumbed, in a similar fashion, to the challenge, with 100% lethality of the different groups until the 10th day postinfection (Figure 5A). Dectin-1^−/− mice were also checked for increased survival against type II strains. WT and Dectin-1 mice were challenged with Pru strain tachyzoites (1 × 10⁴), by intraperitoneal route, with full lethality being observed in both groups after 7 days of infection (Figure 5B).

We also evaluated if the infection protocol was affecting the experimental observations. For that, we adopted Me49 strain (type II) to test distinct infectious doses and routes. WT and Dectin-1^−/− mice were then infected with 1 × 10³ or 1 × 10⁵ tachyzoites, by intraperitoneal route, and both groups showed similar survival curves, with all animals succumbing to the infection until the 12th day (Figures 5C,D). To evaluate whether the route of the infection would induce differential resistance mediated by Dectin-1, WT and Dectin-1^−/− mice were infected by the oral route, with 30 cysts of Me49 strain for survival analysis, or 5 cysts for the evaluation of chronic parasitism in the brain. Once more, our results did not point out differences in survival rates among the groups (Figure 5E), nor in chronic phase parasitism (Figure 5F).

Thus, unlike of the observed for N. caninum, these sets of in vivo experiments with T. gondii demonstrate that Dectin-1 is differentially regulated in infections caused by the two closely related parasites.