Madin-Darby Canine Kidney (MDCK) cells were maintained at 37°C and 5% CO₂ in complete Dulbecco's modified Eagle Medium (DMEM; SIGMA). Virus stocks of the mouse-adapted influenza A/PR8/34 (H1N1) virus were prepared and titrated on MDCK cells as previously described (Barbosa et al., 2014). C. gattii (Cg) VGII, strain L27/01 (UFMG-CM-Y6141) was cultured on Sabouraud's Dextrose Agar (SDA) by 48 h at 37°C (Santos et al., 2014). Other Cryptococcus strains, such as, C. gattii R265 (VGII) and C. neoformans H99 (VNI) were also used for mice survival analysis.

Female C57BL/6 mice (6–8 weeks old) were obtained from the animal facilities of the Universidade Federal de Minas Gerais. All experimental procedures were carried out according to the standards of the Brazilian Society of Laboratory Animal Science/Brazilian College for Animal Experimentation (available at www.sbcal.org.br) and Brazilian Federal Law 11,794. The animal studies were approved by the Ethical Commission on Animals Use of the Universidade Federal de Minas Gerais (CEUA/UFMG, protocol n° 354/2015).

Six female C57BL/6 mice per group were anesthetized by intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). They were then inoculated intranasally (i.n.) with 20 μL of 1 × 10³ plaque-forming units (PFU)/animal of IAV diluted in PBS (0.1 LD⁵⁰) or mock infected with PBS (Barbosa et al., 2014) and/or inoculated intratracheally (i.t.) with 30 μL of 1 × 10⁴ colony-forming units (CFU)/animal of Cg or PBS (controls) (Ferreira et al., 2015). We evaluated two sequential co-infection protocols: (i) inoculation of IAV 10, 7, or 3 days before infection (d.b.i.) with Cg; (ii) Infection with IAV, 3, 7, or 10 days after infection (d.p.i.) with Cg. Behavior, weight and survival of infected animals were monitored daily (Figure 1A). After this step, we selected the inoculation of IAV 3 d.b.i. with Cg (IAV+Cg) for all the next analysis.

In another set of experiments, mice (n = 6 animals per group) infected with IAV alone or IAV+Cg were treated daily with oral Oseltamivir, 31 mg/kg/day (Oselt, Tamiflu®, Roche). Alternatively, mice infected with Cg alone or IAV+Cg were treated daily with intraperitoneal fluconazole, 10 mg/kg/day (FLZ, Sigma-Aldrich, St. Louis, MO). Additional co-infected animals were treated with both FLZ and Oselt. Treatment with Oselt was started at the time of infection with IAV, whereas treatment with FLZ was started at the time of infection with Cg. In summary, experimental groups included: Not-treated (NT) Cg, IAV, and IAV+Cg; IAV treated with Oselt, Cg treated with FLZ, IAV+Cg treated with Oselt, IAV+Cg treated with FLZ, and IAV+Cg treated with Oselt+FLZ (Figure 1A). All the experiments were performed at least twice to confirm the data and the results were always reproducible.

Mouse behavior and function was assessed longitudinally in the groups: non-infected (NI), Cg, IAV and IAV+Cg (3 d.b.i.) using the SHIRPA protocol (Figure 1A) for neurological diseases (Santos et al., 2014). These tests provide reliable information on murine cerebral dysfunction and their general status. Individual parameters evaluated were grouped into five functional categories: neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength, and reflex and sensory function. Individual parameters were summed up to determine a total score for each category (Santos et al., 2014).

After analysis of the survival curve, additional mice (n = 6 animals per group) were infected with Cg, IAV, or IAV+Cg or NI. The animals were anesthetized and euthanized by cervical dislocation 4 days post-infection with Cg (equivalent to 7 days infection by IAV). Lungs and brain were collected, homogenized in sterile PBS, plated onto SDA and incubated for 48 h at 37°C to determine the fungal burden, expressed as CFU per gram of tissue or milliliter of fluid (Costa et al., 2016). Likewise, viral loads in lung homogenates were assessed by titration in standard plaque assays on MDCK cells (Garcia et al., 2013). The viral titer was expressed as PFU per gram of tissue (Figure 1B).

In addition, bronchoalveolar lavage fluid (BAL) was aspirated as previously described (Santos et al., 2013). Fungal colonies recovered onto SDA after 48 h at 37°C from the BAL of Cg and IAV+Cg mice were further used for morphometric analyzes and Zeta potential (ζ) measurements (Figure 1B) as described previously (Nosanchuk et al., 1999; Ferreira et al., 2015). Briefly, Cg cells were visualized after suspension in Indian ink with an optical microscope (Axioplan; Carl Zeiss); subsequently the capsule size and total cell diameter size (diameter plus capsule) of at least 100 cells was measured in ImageJ 1.40g software (National Institutes of Health (NHI), Bethesda, MD). Capsule size was expressed in μm and the total cell diameter size was expressed as a frequency (%) of enlarged cells (>10 μm) compared to the size of typical cryptococcal cell (5–10 μm), according to Okagaki et al. (2010). The Zeta potentials of the yeast cells were calculated using a Zeta potential analyzer (Zetasizer NanoZS90; Malvern, United Kingdom).

Brain and lungs were collected (n = 6 animals per group), processed and stained with hematoxylin-eosin (HE) (Figure 1B). Then, the samples were blinded examined under light microscopy at 200x magnification. Each of the following parameters were graded as 0 (absent), 1 (mild), 2 (moderate), 3 (intense), and 4 (severe): lung tissue damage; degeneration of the airways (bronchioles); congestion and edema of the alveolar septa; alveolar edema; bronchiolar inflammatory infiltrate; and alveolar septa inflammatory infiltrate. Results were expressed and summarized as the average of the combined scores for the distinct parameters evaluated.

For the better understanding of the increased susceptibility caused by IAV 3 d.b.i., six mice per group were infected with IAV or mock infected (NI), and were euthanized 3 days after infection (Figure 1C). MPO and NAG assays were performed on lung pieces (100 mg) (Costa et al., 2016), providing an indirect measurement of neutrophil and macrophage accumulation in lungs, respectively. The concentrations of IL-1β, IL-4, IL-6, IL-10, IFN-γ, TNF-α, and CXCL1 were measured by ELISA using commercially available antibodies from DuoSet Kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Fragments of lung tissue (100 mg) were homogenized with 1 ml of extraction buffer, prepared with phosphate buffered saline—PBS (pH 7.4) containing anti-proteases (0.1 mM phenylmethilsulfonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 KI aprotinin A, all purchased from Sigma-Aldrich) and 0.05% Tween 20. The expression of IFN-α4 and IFN-β genes was measured in lung tissue by qRT-PCR, using primers specific for murine samples (Costa et al., 2014). The primers used were: mIFNα4, 5′-CCA CAG CCC AGA GAG TGA CCA GC-3′ (forward) and 5′-AGG CCC TCT TGT TCC CGA GGT TA-3′ (reverse); mIFNβ, 5′-GAA AGG ACG AAC ATT CGG AAA T-3′ (forward) and 5′-CGT CAT CTC CAT AGG GAT CTT GA-3′ (reverse); and 18S ribosomal RNA, 5′-CGT TCC ACC AAC TAA GAA CG-3′ (forward) and 5′-CTC AAC ACG GGA AAC CTC AC-3′ (reverse).

Initially, phagocytosis was studied in vivo during co-infection by counting yeast cells internalized by phagocyte cells in BAL (Figure 1B). The phagocytic index was determined as the number of internalized yeast cells per 100 mononuclear cells and results were expressed as a percentage. Further, bone marrow-derived macrophages (BMDM) were isolated as described previously (Ribeiro et al., 2017). Briefly, bone marrow cells recovered from mice femurs and tibias were counted using a hemocytometer and the concentration adjusted to 2 × 10⁶ cells/mL for incubation in BMM medium (RPMI supplemented with 30% L929 growth conditioning media, 20% bovine fetal serum [Gibco], 2 mM glutamine [Sigma-Aldrich], 25 mM HEPES pH 7.2, 100 units/mL of penicillin-streptomycin [Life Technologies]). Fresh media were added every 48 h. BMDMs were collected on day 7 and used for subsequent experiments. Then, 2 × 10⁵ BMDM/mL were plated into 24-well plates for determining phagocytosis and Intracellular Proliferation Rate (IPR) and 96-well plates for detection of Reactive Oxygen Species (ROS) and peroxynitrite (PRN), and incubated overnight in RPMI supplemented with 10% fetal bovine serum at 37°C under 5% CO₂ (Figure 1D).

To this aim, cells were inoculated with IAV (M.O.I. = 1) 2 h before (IAV+Cg) infection with Cg (5 cells:1 yeast) or with Cg or IAV alone (Figure 1D). The phagocytic index post 3 h inoculation with Cg was determined as described above. For the IPR assay, cell culture supernatants were removed and non-internalized and adherent yeast cells were removed by two washes with PBS. BMDMs then were lysed with 200 μL of sterile distilled water for 30 min at 37°C, and 50 μL of this suspension were plated on SDA for CFU determination. The IPR was calculated as the quotient of the CFU/mL at 24 and 3 h post Cg inoculation (Ma et al., 2009; Ribeiro et al., 2017).

The above experiments were also performed using RPMI-1640 without phenol red (Sigma-Aldrich), followed by incubation with 2′,7′-dichlorofluoresceindiacetate (DCFH-DA; Invitrogen, Life Technologies, Carlsbad, CA, USA) or dihydrorhodamine-123 (DHR-123; Invitrogen) for ROS and PRN measurements, respectively. Fluorescence was assessed 3 h post-inoculation of Cg using a fluorometer (Synergy 2 SL Luminescence Microplate Reader; Biotek) with excitation and emission wavelengths of 485/530 nm. The data are expressed as arbitrary units of fluorescence (Ferreira et al., 2013). The supernatant from the phagocytosis assay was used to quantify nitric oxide (NO) production using the Griess assay. The nitrite concentration was determined by extrapolation from a sodium nitrite standard curve, read at 540 nm with a microplate reader. For all these tests, we also have groups pre-incubated overnight with IFN-γ (50 U/mL).

All statistical analyzes were performed using GraphPad Prism, version 5.00, for Windows (GraphPad Software, San Diego, CA, USA) with p < 0.05 considered to be significant. Kaplan-Meier survival curves were generated and analyzed using the log rank test. SHIRPA data were analyzed using the area under the curve, followed by analysis of variance (ANOVA) and a Tukey test. The histopathology analysis, phagocytosis and IPR assay, ROS, PRN, and NO measurements were analyzed by ANOVA followed by Tukey test to compare different groups. The results of CFU/g, PFU/g, capsule size, zeta potential, MPO and NAG activities, cytokines and chemokines levels were analyzed by the non-parametric Friedman test, used to compare two groups. All the tests were performed at least twice and the results were always reproducible.

Initially we demonstrate that a sublethal dose of influenza virus alters the survival of mice infected with C. gattii. Survival studies reveal that infection with Cg alone causes 100% mortality by 35 days of infection (Figures 2A,B). Mice inoculated with IAV alone, lost substantial weight up to day 6 (Figure 2C), but survived the infection (Figure 2A). Notably, infection with IAV 3 days before Cg resulted in faster and higher weight lost and reduced survival of infected mice (death within 8 days) (p < 0.05) compared with mice infected with Cg (Figures 2A,C). Identical results were obtained using C. gattii R265 and C. neoformans H99 strains (Supplementary Figure 1). In contrast, when we compare Figures 2A,B, we observed that mice infected with IAV 3 days after Cg did not present difference in survival compared to animals infected only by Cg (Figure 2B); otherwise mice infected with IAV 3 days before Cg has a marked increase in the rate of mortality. In the same way, there was no difference in survival of mice infected with IAV 10 or 7 days prior to inoculation with Cg (Supplementary Figure 2). Similarly, there was no difference in survival of mice when IAV was inoculated 7 or 10 days after Cg (Supplementary Figure 2).

Our previous study showed that the IAV load in lungs peaked at about the fourth day after infection and that IAV was cleared by day 10 (Barbosa et al., 2014). Therefore, our data strongly suggested a link between high viral replication and increased susceptibility to Cg during acute influenza infections. To test this hypothesis, we treated mice daily with oseltamivir and/or fluconazole. Remarkably, we verified that treatment with oseltamivir delayed the mortality in co-infected mice (Figure 2D). While treatment with the antifungal fluconazole improved survival of mice infected with Cg, it did not improve survival in co-infected mice (Figure 2E). However, combined treatment (Oselt+FLZ) significantly improved survival in co-infected mice (Figure 2F).

Application of the SHIRPA protocol showed that infection with IAV resulted in undetectable cerebral impairment while infection with Cg did cause cerebral dysfunction. Moreover, morbidity in co-infected mice was increased compared with all other groups, showing a severe decline (p < 0.0005) in each of the five functional categories evaluated (Figures 3A–E).

To evaluate the disease caused by Influenza-Cryptococcus co-infection, mice were infected with IAV 3 days before infection by Cg (IAV+Cg) or infected only by Cg; IAV or non-infected (NI). Four days (7 days of protocol) post-infection with Cg, these animals were ethically euthanized for disease profile investigation. We found no significant differences (p > 0.05) in the viral (Figure 4A) and fungal burdens (Figure 4B) in lungs of mice infected with IAV or Cg alone, respectively, compared with co-infected mice. However, fungal loads in the brain were significantly higher in co-infected mice than in mice infected with Cg alone (Figure 4D). In addition, histopathological scores were higher in lungs of mice infected with IAV or co-infected when compared with those infected with Cg alone (Figure 4C). Histopathological findings in the groups infected with IAV alone or co-infected showed that the virus directly damages the lung structure, including the mild airway degeneration, congestion and edema of the alveolar septa and increased bronchial and septal inflammation (Figure 4E). The presence of the fungus is evidenced by arrow in the Cg group (amplified box, Figure 4E).

Considering the higher transmigration of Cg to CNS, we verified if this fact could be associated with the fungal adaptation (morphological modifications of Cg during co-infection) to the host in the presence of virus. For this, colonies recovered from BAL of mice co-infected or infected only by Cg were submitted to capsule size and zeta potential determination. Our data demonstrated an increased capsule size (Figure 5A) and increased electronegativity (Figure 5B) in the fungal cells recovered from BAL of mice infected with IAV+Cg, when compared with those infected with Cg alone and cultured fungi (Cg control). Figure 5C demonstrates a phagocyte (arrow head) attempting to engulf an enlarged encapsulated Cg cell (thick arrow, Figure 5C). In the same way, our results for the total cell diameter size showed the higher proportion of enlarged cells (>10 μm) in the co-infected group (35.9%), while mice infected only by Cg present only 18.0% of enlarged cells (Figure 5D).

Given the severe histopathological alterations caused by IAV infection, we investigated the host response against IAV that may also be responsible for the increased susceptibility to secondary C. gattii infection. Further, to determine the inflammatory profile of mice at the time they would be infected with Cg, mice were infected only by IAV (1 × 10³ PFU) or NI and they were ethically euthanized and lungs collected 3 days after IAV infection. The expression of the genes of type 1 interferons, i.e., interferon alpha (IFN-α4) and beta (IFN-β) were significantly increased (p < 0.0005) in the IAV infection (Figures 6A,B, respectively). Interestingly, the levels of interferon gamma (IFN-γ) were reduced (p < 0.005) in comparison with non-infected mice (Figure 6C). Additionally, the levels of tumor necrosis factor-alpha (TNF-α) were not altered by IAV (Figure 6D). We verified increased levels of CXCL-1 (Figure 6E), interleukin 1-β (IL-1β) (Figure 6F), and IL-6 (Figure 6G). Moreover, the levels of cytokines IL-4 (Figure 6H) and IL-10 (Figure 6I) were lower than those in NI mice. Here we used MPO as an indirect measurement of neutrophil accumulation, since MPO is the most abundant pro-inflammatory enzyme stored in the azurophilic granules of neutrophilic granulocytes (Pulli et al., 2013). In the same way, an estimate of macrophage infiltration is given by the level of NAG which is present in high specific activity in activated macrophages (Bailey, 1988). The activities of the enzymes MPO (Figure 6J) and NAG (Figure 6K) were significantly (p < 0.05) increased in lungs of mice infected with IAV compared with NI mice, suggesting that neutrophils and macrophages, respectively, had been recruited to the site of infection.

In order to assess the influence of IAV infection in Cg phagocytosis, we performed the in vivo and in vitro phagocytosis assays. The phagocytic index of Cg, as assessed using mononuclear cells of BAL aspirated from IAV+Cg-co-infected mice, was reduced in comparison with those infected with Cg alone (Figure 7A). These data were confirmed in the in vitro assay using BMDM (Figure 7B). Furthermore, the IPR assay revealed an increased ability of Cg to grow inside BMDMs that had been virus-infected 2 h prior to the addition of Cg (IAV+Cg) (Figure 7C). In addition, challenge with IAV or IAV+Cg reduced the ability of macrophages to produce ROS compared to cells infected with Cg (Figure 7D), but not PRN (Figure 7E) and NO (Figure 7F). Treatment with IFN-γ improved the oxidative burst activity of macrophages and decreased the IPR for BMDM challenged with Cg alone. However, in co-infected BMDMs, IAV impaired the fungicide activity of macrophages independently of the increase in ROS, PRN, and NO production induced by IFN-γ.

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.