C. mollis seed extract (10% w/v prepared in 0.15 M NaCl) was fractionated using ammonium sulfate (40–60% w/v) and the fraction obtained was submitted to affinity chromatography in a Sephadex G-75. The pCramoll preparation was bioselectively eluted with 0.3 M of D-glucose in 0.15 M NaCl, dialyzed against 0.15 M NaCl for 24 h and subsequently lyophilized (Correia and Coelho, 1995) and diluted in PBS to analyze protein concentration, as determined by the BCA kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Bone marrow cells were isolated as described previously (Weischenfeldt and Porse, 2008; de Souza et al., 2016). Femurs and tibias from mice were removed, disinfected by immersion in 70% ethanol and the ends of each bone were cut. Both bones were flushed with 5 mL of cold RPMI 1640 (HyClone, LGC Biotecnologia) using a 5-mL syringe and a 25-gauge needle. The cell suspension was centrifuged for 5 min at 1,200 rpm at 4°C and washed once with cold RPMI. Then, bone marrow cells were counted using a hemocytometer and the concentration was adjusted to 2 × 10⁶ cells/mL for plating on tissue culture–treated petri dishes in BMM medium (RPMI supplemented with 30% L929 growth conditioning media, 20% bovine fetal serum [Gibco], 2 mM glutamine [Sigma], 25 mM HEPES pH 72, 100 units/mL of penicillin-streptomycin [Life Technologies]). Fresh media were added every 48 h. Bone marrow–derived macrophages (BMDMs) were collected on day 7 and used for subsequent experiments. Under these conditions, macrophages/monocytes progenitors will proliferate and differentiate into a homogenous population of mature BMDMs, obtained >90% pure live macrophages (Zhang et al., 2008; Bouwman et al., 2014; Bhattacharya et al., 2015; Heung and Hohl, 2016). For all analyses, the cells were infected with L27/01 yeasts cells (growth on Sabouraud Dextrose Agar (SDA) medium, for 48 h at 35°C) opsonized with 10% murine serum in RMPI suspension (0.4 × 10⁶ cells/mL), at the proportion of 5 macrophages: 1 C. gattii, under cell treatment with 1 or 5 μM pCramoll and incubated for 3 or 24 h at 37°C under 5% CO₂.

The phagocytic index was performed using 24-well plates and a single coverslip 13 mm in diameter. After 3 h, these coverslips containing adherent cells were removed, stained with Giemsa and the index was calculated as the percentage of cells with internalized C. gattii 24 h post-infection (Santos et al., 2014). Images from the phagocytosis assay were obtain using a Nikon COOLPIX 4500 camera coupled to a Nikon ECLIPSE E200 microscope with 100 x of magnification and analyzed using the ImageJ Protocol (http://rsbweb.nih.gov/ij/); the cell area (average of 100 cells per coverslip) was measuring as described by Baviskar (2011).

The intracellular proliferation rate (IPR) assay was performed as previously described (Ma et al., 2009) with modifications. Non-internalized yeast cells in the supernatant were taken from the wells and the adherent phagocytes were washed with 200 μL PBS. These macrophages were lysed at 3 and 24 h with 200 μL of cold, sterile, distilled water and incubated for 30 min, then 100 μL was collected and plated on SDA medium, and the viable yeast cells were counted. The IPR was calculated as the quotient of the intracellular yeast cell numbers at 24 h (the point in time which featured the maximum intracellular yeast number) and 3 h.

The supernatant of phagocytosis assay was used for measurement of NO production using the Griess assay. The nitrite concentration was quantified by extrapolation from a sodium nitrite standard curve, determined at 540 nm with a microplate reader (Benchmark Plus, Bio-Rad, CA, US).

Macrophages/well in RPMI-1640 without phenol red (Sigma-Aldrich) were also infected with C. gatti (same conditions that phagocytosis assay), and the supernatant was used for ROS quantification using 2-7-dichlorofluorescein diacetate (DCFH-DA; Invitrogen, Life Technologies, Carlsbad, CA, USA) was used and the fluorescence was measured with a fluorometer (Synergy 2 SL Luminescence Microplate Reader; Biotek) the positive control was hydrogen peroxide at 10 μM (Ferreira et al., 2013).

The protocol of the animal studies was approved by the Comissão de Ética no Uso de Animais (CEUA) at the Universidade Federal de Minas Gerais (Protocol 310/2014). All mice were housed in clean cages, with food and water ad libitum. The controlled environment was set to a 12 h light/dark cycle at 23°C. Mice C57BL/6, 5–6 weeks old (n = 6/group) were anesthetized by intraperitoneal (i.p.) injection of ketamine hydrochloride (60 mg.kg⁻¹) and xylazine (10 mg.kg⁻¹) in PBS, and then inoculated by an intratracheal infection with 30 μL of 10⁴ CFU/animal of C. gattii, L27/01 strain. Intratracheal infection with the C. gattii L27/01 strain (GVII molecular type) has been previously shown in other works (Santos et al., 2014; Ferreira et al., 2015).

Initially, infected mice were intraperitoneally (i.p.) treated every 10 days with 1, 250, or 500 μg of lectin alone (the first dose was administered 1 day before intratracheal infection). For the group treated with the combination fluconazole (20 mg.kg⁻¹) and pCramoll at 1, 250, or 500 μg (the fluconazole was daily administered and every 10 days the association was used), or the mice were treated daily with fluconazole alone (20 mg.kg⁻¹) for survival monitoring. The untreated group (NT), and not infected group (NI) were inoculated with PBS. All animals were monitored twice daily for survival and behavior parameter analysis (SHIRPA protocol). The SHIRPA protocol analyzes the behavioral and functional assessment of neurological diseases (Rogers et al., 1997). The tasks are grouped into five functional categories: neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength, and reflex and sensory function (Santos et al., 2014; Costa et al., 2016). All mice were examined daily and the score for each functional category was calculated as the total of the evaluated parameters according to Lackner et al. (2006) and Pedroso et al. (2010) using the EpiData 3.1 software. The Table S1 describes all the parameters analyzed in the SHIRPA protocol.

Furthermore, other groups of mice were i.t. infected and treated with pCramoll at 1 μg per mouse (the concentration that provided the best results in the survival curve), combined or not with fluconazole to obtain lungs and brain at 15 or 35 days post-infection (d.p.i). The animals were euthanized by cervical dislocation under anesthesia. The lungs and brain were removed for determining the colony-forming units (CFU) as described previously (Santos et al., 2014). For this, the organs were homogenized with sterile PBS and plated in SDA medium. Specifically, 100 or 50 mg of lung tissue was homogenized with 1 or 0.5 mL of extraction solution of protein, containing PBS (pH 7.0) and anti-proteases (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, 20 Kallikrein inhibitor units of aprotinin A, all purchased from Sigma-Aldrich and 0.05% tween 20). The samples were centrifuged for 10 min at 3,000 × g, at 4°C and the supernatant was frozen at −20°C and utilized for cytokine analysis. The levels of IL-10 and IL-6 were determined using commercially available antibodies according to the manufacturer's instructions (R&B Systems, Minneapolis, MN, USA) and the levels of IFN-γ and IL-17A were measured by cytometric bead array (BD Biosciences, San Jose, CA, USA). Also, the bronchoalveolar lavage fluid (BAL) was obtained and centrifuged at 1,200 rpm for 5 min at 4°C. The cell pellet was suspended in 3% albumin solution (100 μl). A 10 ul aliquot of albumin solution containing the cells washed was diluted in 10 uL of Türck for cell count totals in a Neubauer chamber with an optical microscope at 40x magnification. For differential counts, smears were made in cellspin (CT-2000, CIENTEC), stained using the Giemsa method and cells were quantified by morphological criteria for the distinction of cellular types (mononuclear and polymorphonuclear) and the results were grouped according to the percent content of these two cells type (Maxeiner et al., 2007).

The infiltration of neutrophil in the lungs was indirectly measured by the assay of myeloperoxidase activity (MPO) according to Costa et al. (2016), by measuring the change in optical density (OD) using 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma-Aldrich). The absorbance reading was taken at 450 nm in a spectrophotometer. For N-acetylglucosaminidase activity (NAG), 2,24mMp-nitrophenyl-N-acetyl-β-D-glicosaminide (Sigma-Aldrich) was used and absorbance was determined at 400 nm (Baltazar et al., 2014).

The lungs were removed during necropsy and immediately fixed in buffered 10% formalin (v/v). The tissue was embedded in paraffin, and the sections were stained with hematoxylin and eosin (Sigma) and examined under light microscopy.

Statistical analysis of all data were performed using GraphPad Prism version 5.0 with p < 0.05 considered significant. The survival curve was plotted by Kaplan-Meier analysis and the results were analyzed using the log rank test, for behavior parameters the area under the curve was analyzed. Also, the results of the phagocytosis assay, IPR, measurement of ROS and NO production by macrophages, MPO and NAG activity, and quantification of cytokines were analyzed by analysis of variance (ANOVA) followed by Dunn's Multiple Comparison Test and the Student's t-test.

Previously, the pCramoll toxicity analysis in Caenorhabditis elegans model was performed complementarily. Non-toxicity was verified in these worms (data not shown) and confirms the non-cytotoxicity of the pCramoll concentration used in this study and published previously by da Silva et al. (2014). Subsequently, we investigated the antifungal activity of pCramoll against C. gattii by screening and determining the minimum inhibitory concentration (MIC). Neither of the concentrations tested (0.93–120 μg/mL) were able to inhibit fungal growth (data not shown). Indeed, no interaction between pCramoll and fluconazole was obtained by the checkerboard test (data not shown).

pCramoll at 1 μM increased the phagocytic index after 3 h of incubation (Figure 1A), compared with the untreated group. At the same time, the levels of ROS were increased in the presence of pCramoll at 1 and 5 μM, both for infected and uninfected macrophages (Figure 1B), especially with pCramoll at 1 μM with significant increase in the production of nitrosative species (Figure 1C). This increase in ROS levels was associated with a reduced IPR (Figure 1D). Another interesting observation was the presence of macrophage aggregates, vacuoles and increased expansion of the cells (area) in the presence of increasing concentration of pCramoll (Figure 1E). Bone marrow-derived macrophages stimulated with pCramoll at 1 and 5 μM showed, 3 h post-incubation, 0.080 and 0.197 μm² of area, respectively, while at 24 h: 0.214 and 0.483 μm², respectively. There was an increase of up to 5 × in cell area compared with untreated macrophages infected with C. gattii after 3 h and to 7 × after 24 h of incubation (Figure 1F).

An increase in survival of animals treated with pCramoll was observed. The untreated group (NT) showed a median survival (MS) of 20 days (Figure 2A), while the MS for groups treated with the lectin at 500, 250, and 1 μg, were 30.5 (p < 0.05; data not shown), 30 (p < 0.01; data not shown) and 29.5 (p < 0.01) days (Figure 2A), respectively, an increase of 50% (approximately) for all doses tested, and an independent dose response. pCramoll at 500, 250, and 1 μg was also tested in uninfected animals and no change in the survival/behavior of animals compared to NI mice was found (data not shown). When combined with fluconazole, the influence of lectins on survival was dose dependent. The group treated with fluconazole alone showed an MS of 46 days, while the groups where pCramoll was combined with the antifungal demonstrated a MS of 51 days for 500 μg of pCramoll (data not shown), 58 days for 250 μg of pCramoll (data not shown) and 62 days for 1 μg of pCramoll (p < 0.05; Figure 2A). Based on these results, the dose of 1 μg of pCramoll combined or not with fluconazole was used in the further experiments. Indeed, the cellular polysaccharide extracted from strain L27/01 (PSC) of C. gattii was associated with pCramoll and fluconazole, and were tested in our model. The administration of PSC only hurried the death of infected animals (MS of 17 days), compared to the NT group (20 days). The same was observed where PSC was associated with pCramoll and fluconazole (MS of 43 days), compared to the FCZ-treated group (MS of 46 days), where the animals had previously succumbed (data not shown).

The behavior analysis of basic functions such as sensory-reflex (Figure 2B), muscle tone and strength (Figure 2C) demonstrated improved behavior by day 20 post-infection when animals were treated with pCramoll, fluconazole and the combination compared to the untreated group (NT). Moreover, the combination is significantly more efficient than fluconazole alone (p < 0.05). A similar behavioral profile was observed for parameters involved in the neuropsychiatric state (Figure 2D) and body weight (Figure 2E), which improved by day 22 day post-infection (p < 0.05) for all the treated groups, but with better performance for groups treated with the combination (p < 0.05). There was no difference between the treated and untreated groups for motor behavior (Figure 2F). Finally, as shown in Figure 2G, autonomic function was only improved when the combination was used (p < 0.05).

All the treatments reduced the fungal burden in the lungs after 15 dpi. At 35 days post infection, fluconazole combined with pCramoll was better than fluconazole alone in reducing pulmonary fungal burden (p < 0.05; Figure 3A). In brain tissue, only the use of fluconazole reduced the fungal burden after 15 dpi (Figure 3B), however, the combination (fluconazole + pCramoll) was able to reduce cerebral fungal burden significantly when compared with fluconazole alone after 35 dpi (p < 0.05). Histological analysis of lung tissue from untreated infected mice at 15 dpi showed numerous yeasts in the pulmonary parenchyma, mild perivascular infiltrate and alveolar thickening. Moderate to intense perivascular inflammation was observed in lung tissue from pCramoll, and pCramoll + fluconazole groups. Lungs from treated groups showed less yeast in alveolar space than the untreated group (Figure 3C).

Regarding the inflammatory infiltrate in the bronchoalveolar lavage fluid (Figure 4A), we verified a predominance of mononuclear cells for all groups except the one treated with the combination, for which there was a balance between mononuclear and polymorphonuclear cells, at both 15 and 35 dpi (Figure 4B). Additionally, MPO (Figure 4C) and NAG (Figure 4D) activities were performed in order to confirm the presence of neutrophils and macrophages, respectively, where the combination demonstrated higher activities of both enzymes at 15 dpi.

The levels of IFN-γ (Figure 4E) were higher for the group treated with the combination at 35 dpi. The levels of IL-6 (Figure 4F) and IL-10 (Figure 4G) were augmented in the NT group. An increase in IL-17A (Figure 4H) was verified for the combination and for fluconazole alone at 15 dpi.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CPT and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.