Experimental sporotrichosis in a cyclophosphamide-induced immunosuppressed mice model

Experimental sporotrichosis in a cyclophosphamide-induced immunosuppressed mice model Abstract This report describes a model of host resistance for Sporothrix schenckii, an opportunistic fungi in immunosuppressed mice with cyclophosphamide (CY) to be used in studies of immunotoxicology and immunopharmacology. Two doses of CY were administered intraperitoneally: 200 mg/kg and a booster of 150 mg/kg at 9-day intervals. Three days after the first dose of CY the animals were infected subcutaneously with 1.8 × 108 yeast/ml (S. schenckii ATCC 16345). At 7 and 14 days post-infection, the animals were euthanized and analyzed the fungal load by unit forming colony count in the spleen and popliteal lymph nodes. The relative weight of thymus and spleen, splenic index, the frequency of T and B cells in spleen by flow cytometry, the hind paw inflammation index and cytokine (interleukin [IL]-17, IL-10, and interferon [IFN]-γ) profile were measured. Histopathological studies of the spleen and the hind paw were also assessed. The immunosuppression status was confirmed at the evaluated days by reduction of relative weight of thymus, reduction of the splenic white pulp, the population of B and T lymphocytes, and the cytokine profile in the treated mice with CY in comparison with nontreated groups, associated to higher fungal load in hind paw and spleen in the infected mice. The described model reveals an increasing in susceptibility to infection and severity when associated with immunosuppression. This model can serve as a reference for studies of S. schenckii host resistance in pharmaceutical and toxicological studies. Sporothrix schenckii, host resistance models, immunosuppression, cyclophosphamide Introduction Despite the existence of sophisticated molecular and genetic techniques to identify defects in resistance pathways at the molecular and message level, the host resistance assays using opportunist microorganisms remain the gold standard against which changes at the molecular or cellular level of immune function can be judged. This is due to resistance to infection, regardless of the actual pathogen, involves multiple pathways of effectors’ function to neutralize or eliminate pathogens.1 Isolated or multiple tests are even insufficient to evaluate the consequences of the immunosuppression for the risk of specific infections in several cases2. Sporotrichosis is a chronic fungal infection caused by different species of Sporothrix that are classified into five clades based on gene sequences of chitin synthase, β-tubulin and calmodulin: Sporothrix brasiliensis (Clade I); Sporothrix schenckii (Clade II); Sporothrix globosa (Clade III) Sporothrix mexicana (Clade IV) and Sporothrix pallida (formerly S. albicans) (Clade V).3 Sporotrichosis has been described on five continents, with a higher prevalence in tropical and subtropical regions.4,5 Infection generally occurs following traumatic inoculation with soil, plants, and organic matter contaminated with the fungus causing cutaneous and subcutaneous infection, leading to plaque formation as warts and lesions papules. Both manifestations can progress to ulcerative lesions. The main clinical manifestations of sporotrichosis are the lymphocutaneous forms, but the number of systemic and visceral cases has increased, particularly in immunodeficient patients.6 Zoonotic transmission has been increasing significantly, with a recent report of an epidemic outbreak with zoonotic transmission by infected cats in Brazil.7 Disseminated S schenckii infection usually occurs in the context of cellular immunodeficiency associated with: alcoholism, diabetes, sarcoidosis, tuberculosis, organ transplantation, malignancy, use of immunosuppressive agents, or AIDS.8–16 Nowadays, the influence of the environmental factors in the development of sporotrichosis is being studied.17,18 There have only been a few studies addressing the influence of the environment on the virulence of these pathogens. However, recent studies have demonstrated that adverse conditions in its natural habitats can trigger the expression of different virulence factors that confer survival advantages both in animal hosts and in the environment.17 Thus, having a host resistance models using S. schenckii as opportunistic fungi in immunosuppressed mice, will be a contribution to studies of sporotrichosis immunopathogenesis, the search of new prophylactic or therapeutic tools and evaluating the effects of xenobiotics on the susceptibility of sporotrichosis. Cyclophosphamide (CY) is an alkylating agent that can inhibit both the humoral and cellular immune responses. It is widely used as cytotoxic drug in the anticancer therapy and as immunosuppressive drug in autoimmune diseases and organ transplants, including prophylaxis of graft-versus-host disease (GVHD) after myeloablative allogeneic bone marrow transplantation.19–22 CY and Methotrexate (MTX) are the more frequently reported antineoplastic drugs associated to sporotrichosis. They are administrated in combined therapy joint to other immunosuppressive drug.23–27 In 1981, the susceptibility to sporotrichosis in a murine model of cyclophosphamide immunosuppression was described.28 However, no further description of S. schenckii as host resistance model in immunosuppressed mice, using quantitative immunological biomarkers were made. This study aimed to develop a murine model of host resistance to S. schenckii sensu stricto in immunosuppressed mice with CY as a tool for future studies of immunotoxicity and immunopharmacology in sporotrichosis. Methods Animals Male Balb/c mice, 5–7 week old at the time of inoculation, were purchased from “Centro Multidisciplinar para Investigação Biológica na Área da Ciência de Animais de Laboratório” (CEMIB), Universidade de Campinas (UNICAMP), São Paulo, Brazil. Animals were housed in individually ventilated cages in an ambient with controlled temperature and 12-h light/dark cycles. Water and food were offered ad libitum. All procedures were approved by the Institutional Ethics Committee (Protocol CEP/FCF/CAR no. 04/2014) and were in accordance with the National Institutes of Health Animal Care Guidelines. Microorganism and culture conditions S schenckii (ATCC® 16345™) isolated from a case of human lung infection (Baltimore, MD, USA), was kindly provided by Department of Microbiology, Materials laboratory references the Oswaldo Cruz Foundation, National Institute of Quality Control in Health, Rio de Janeiro, Brazil. Currently this isolated sustained in the Department of Clinical Analysis, Araraquara's School of Pharmaceutical Sciences, Universidade Estadual Paulista-UNESP, Júlio Mesquita Filho, in the filamentous phase amid MycoselTM at room temperature (±25°C). The yeast form is obtained in BHI (Brain Heart Infusion, Difco) at 37°C under constant stirring of 150 rpm / min for 5 days. Preparation of the heat-killed (HKss) S. schenckii yeast HKss cells were prepared from the same 5-day-old culture of the fungus in brain-heart infusion broth used for animal infection in each respective experiment.29 Yeast cells were separated from the supernatant by centrifugation at 200 g for 5 min at room temperature, washed twice with 8 ml of sterile phosphate-buffered saline pH 7.4 (PBS), and then ressuspended and adjusted to 2.5 × 108 yeast cells/ml in PBS, that was incubated for 1 h in a 60°C water bath and then stored at 2–8°C until use. As a control for the efficiency of the heat-killing process, 100 μl aliquots from each tube were inoculated on Mycosel agar plates and checked for colony-forming units (cfu) growth before use. HKss was used as a challenge in the esplenocyte culture for later cytokine quantification in the supernatant. A working suspension was obtained by making a 1/10 dilution of the stock suspension in Roswell Park Memorial Institute (RPMI) complete medium (defined as the base RPMI-1640 medium containing 20 μM of 2β-mercaptoethanol, 100 U/mL of penicillin and streptomycin, 2 mM of l-glutamine and 5% fetal calf serum). Infection standardization This kinetic study was performed to determinate the time when occurs the peak of the infection and the reduction of infection until the virtual fungal clearance. A yeast suspension was prepared in PBS, and each mouse was inoculated subcutaneously into the left hind footpad with 1.6 × 107 yeast cells in 0.02 ml of sterile PBS, as previously,30 with some modifications. Group of five animals were euthanized at the 0, 3, 7, 9, 11, 14, 21, 28, and 60 days after infection to determine of fungal load in spleen and lymph nodes by counting of cfu and the relative spleen weight (spleen weight divided by body weight). Additionally, a test of survival for 60 days was conducted. Immunosuppression model Immunosuppression of animals was induced with cyclophosphamide (CY). Animals received an initial dose of 200 mg/kg CY intraperitoneally in PBS, with maintenance doses of 150 mg/kg at 9 days, as described by Huyan and colleagues31, with some adaptations fixed in previous studies for immunosuppression optimization.32–34 Mice were infected 3 days after the first administration of CY.35 Control animals received only PBS, by the subcutaneous and intraperitoneal routes. The mice were euthanized on days 7 and 14 after infection (according to the previous results) for the analysis of immunosuppression. Spleens were aseptically removed; splenic length, width, and thickness were measured and used to calculate the splenic index, and weighted to evaluate the organ-to-body weight ratios (relative spleen weight). After that, the spleens were divided into two parts, one for histological analysis, the other for determine the number of cfu and to analyze the frequency of helper and cytotoxic T and B cells by flow cytometry. The thymus was also removed; weighed and the relative weight was measured. Preparation of total splenocytes A previously described, weighted spleen piece was macerated in 2 ml of sterile PBS and passed through a 100-μm cell strainer into a Petri dish. For red cell lysis, the resulting suspension was added with 6 ml of a 0.17 M ammonium chloride solution and then incubated on ice for 5 min. The splenocytes were then separated from the supernatant by centrifugation at 300 g for 5 min at 4°C, washed once with 3 ml of RPMI complete medium, and then ressuspended in 1 ml of the same medium. Cell concentration was determined by microscopy using the Trypan blue exclusion test and then the splenocytes were adjusted to 5 × 106 cells/ml in RPMI complete medium. Total splenocytes were used for determining distinct immune cell populations by flow cytometry and for ex vivo determination of cytokines. Flow cytometry of lymphocyte subpopulations To analyze the lymphocytes subpopulations, spleen cells were centrifugation and washing with PBS containing 1% bovine serum albumin (BSA) (Sigma), 1 × 106 of cells were stained with monoclonal antibodies anti-mouse: fluorescein isothiocyanate (FITC)-conjugated anti-CD3, allophycocyanin (APC)-conjugated anti-CD4, peridinin–chlorophyll–protein–cyanine 5.5 (PerCP Cy5.5)-conjugated anti-CD8, and phycoerythrin (PE)-conjugated anti-CD19 for analysis of frequency of this cells on spleen. Measurement of the ex vivo release of cytokines Cytokines interleukin (IL)-17, IL-10, and interferon (IFN)-γ were measured in the cell culture supernatant by enzyme-linked immunosorbent assay (ELISA; eBioscience) according to the manufacturer's instructions. Splenocytes obtained on the 14th day after infection, as described above, were cultured for 24 h at 37°C and 5% CO2 on flat bottom 48-well tissue culture plates in the presence of HKss, at a splenocyte-to-yeast ratio of 1:5. Final concentrations were 2.5 × 106 splenocytes/ml and 1.25 × 107 yeast cells/ml. Concanavalin A (0.25 μg/ml in RPMI complete medium) and RPMI alone were used as positive and negative controls, respectively. Fungal load in the spleen and popliteal lymph node The fungal load in the spleen and popliteal lymph node of the animals was determined by counting the number of cfu recovered such organs after maceration. For this, aliquots of the organs were diluted appropriately and plated on agar MycoselTM, incubated at 37°C and checked after 3 and 7 days for the count of cfu/g of organ. Hind paw inflammation index The swelling of hind paws was measured once a week, by measuring the thickness of the hind paws from a lateral view using a digital caliper (Mitutoyo, Japan). The swelling index was calculated for hind paws by the mean difference between the injected and the contra lateral paws. Spleen and footpad histopathology After euthanasia, the spleen and footpads of the mice were removed aseptically and immediately submerged in 4% formaldehyde for histological analysis. Hematoxylin-eosin (H&E) stain was used to evaluate the decrease in the cellularity of the spleen white pulp in mice treated or not with CY and the Grocott-Gomori methenamine-silver nitrate stain was used to observe the presence of the fungi in the tissue. Analysis was made by optical microscopy (NIKON Eclipse TS100, Tokyo, Japan), and photos were captured by VD480 OPTMEDICAL software. Statistical analysis Statistical analysis was performed in GraphPad Prism ver. 6.01, by using one- or two-way analysis of variance (ANOVA) with Tukey or Sidak's multiple comparisons test, respectively, as indicated. Confidence interval was set at 95% for all tests. Groups of three or four animals were used in each experiment. Results Infection standardization During the 60 days of the study, no mice mortality was recorded. As shown in Figure 1A, the days indicated, mice were euthanized, and both spleens and lymph nodes were removed for the evaluation of systemic fungal load. The spleens were previously weighted. Figure 1. View largeDownload slide (A) Kinetics infection. The cfu spleen and popliteal lymph node of infected mice subcutaneously with S. schenckii. (B) Relative spleen weight (spleen weight/animal weight) of infected mice subcutaneously with S. schenckii. The data represent the mean ± SD of four animals per day. Figure 1. View largeDownload slide (A) Kinetics infection. The cfu spleen and popliteal lymph node of infected mice subcutaneously with S. schenckii. (B) Relative spleen weight (spleen weight/animal weight) of infected mice subcutaneously with S. schenckii. The data represent the mean ± SD of four animals per day. The results show that local infection observed by the cfu in popliteal lymph node reached the peak on the 7th day post-infection and regressed gradually until the almost fungal clearance by most animals when completed 60 days post-infection. Moreover, the infection becomes systemic since the first 3 days post-infection with cfu in the spleen. Notably, the relative spleen weight increased at the beginning of the infection and, with the passage of days, it decreased along with the fungal load (Fig. 1B). With this result, we selected the 7th and the 14th days for fungal load and immunological evaluation in further studies. Fungal load in spleen and local lymph node The fungal load in the spleen was similar between both groups infected (previously immunosuppressed or not), but at day 14 was observed a cfu reduction in the non-immunosuppressed group, while the immunosuppressed group still maintained high concentrations fungal colonization in the organ. In contrast, in the local lymph node there were not observed changes in the fungal load at 7th and 14th days in the same group, but the immunosuppressed mice showed higher fungal colonization (Fig. 2). Figure 2. View largeDownload slide The cfu obtained from (A) spleen and (B) popliteal lymph node of infected and immunosuppressed or only infected mice, 7 and 14 days after infection footpad with S. schenckii. The data represent the mean ± SD of four animals per day. *P < .05; **P < .01. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide The cfu obtained from (A) spleen and (B) popliteal lymph node of infected and immunosuppressed or only infected mice, 7 and 14 days after infection footpad with S. schenckii. The data represent the mean ± SD of four animals per day. *P < .05; **P < .01. This Figure is reproduced in color in the online version of Medical Mycology. Relative weights and splenic index The mice did not exhibit any differences in weight between the experimental groups (Fig. Supl. 1). However, the relative weight of spleen had a significant increase in immunosuppressed and infected mice when compared to all other groups both at 7th and 14th days. Moreover, the relative weight of the spleen in mice both treated with CY and infected separately was higher than the control group treated with PBS (Fig. 3A). On the other hand, the relative weight of thymus just showed a significant increase in immunosuppressed infected and non-infected mice when compared to the other groups (Fig. 3C). Thus, the infection does not alter thymus weight when compared to control and, in fact, CY-induced immunosuppression reduced thymus weight whether or not infection was present. The higher spleen index was observed in the group immunosuppressed and infected mice. However, groups only immunosuppressed or only infected, also had statistical difference in splenic index when compared to the control group on the studied days (Fig. 3B). Figure 3. View largeDownload slide Relative (A) spleen and (C) thymus weight (organ weight ÷ animal weight), splenic index (length × width × thickness) (B) immunosuppressed mice (or not), 7 and 14 days after infection with S. schenckii (or not). The data represent the mean ± SD of four animals. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group at each point corresponding unless otherwise indicated. Relative thymus weight was significantly reduced in mice receiving cyclophosphamide infected or not, especially in the 14-day post-infection with a significant difference between these groups. This Figure is reproduced in color in the online version of Medical Mycology. Figure 3. View largeDownload slide Relative (A) spleen and (C) thymus weight (organ weight ÷ animal weight), splenic index (length × width × thickness) (B) immunosuppressed mice (or not), 7 and 14 days after infection with S. schenckii (or not). The data represent the mean ± SD of four animals. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group at each point corresponding unless otherwise indicated. Relative thymus weight was significantly reduced in mice receiving cyclophosphamide infected or not, especially in the 14-day post-infection with a significant difference between these groups. This Figure is reproduced in color in the online version of Medical Mycology. Flow cytometry of lymphocyte subpopulations from the spleen The populations of lymphocytes in the spleen of the animals were defined as helper (CD3+CD4+) T cells, cytotoxic (CD3+CD8+) T cells and B (CD19+) cells. Figure 4 shows the results expressed as the mean ± SD of the frequencies (%) of cell populations obtained from each group on days indicated. Statistical analysis showed that the frequency of helper and cytotoxic T cells as well as B cells in the spleen of mice receiving CY infected or not was significantly lower than control animals or infected without CY. This significant difference existed in both the 7th day and on the 14th day post-infection. Furthermore, it is possible to observe the maintenance of reduced frequency of T and B lymphocytes in the spleen of the animals to the 14th day post-infection certainly due to the booster dose. Interestingly, in this model, a reduction in both subpopulations of T cells was observed at 7th and 14th days in the nontreated and infected group, but B cells were elevated at 7th day in comparison with the control group without both infection and treatment. Figure 4. View largeDownload slide Frequency (%) of CD3+CD4+ and CD3+CD8+ T lymphocytes and B lymphocytes CD19+ in samples of immunosuppressed mice (or not), 7 and 14 days after infection with S. schenckii (or not). The graphs represent the mean values of each group (4 animals) by day examined. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group (spleen). This Figure is reproduced in color in the online version of Medical Mycology. Figure 4. View largeDownload slide Frequency (%) of CD3+CD4+ and CD3+CD8+ T lymphocytes and B lymphocytes CD19+ in samples of immunosuppressed mice (or not), 7 and 14 days after infection with S. schenckii (or not). The graphs represent the mean values of each group (4 animals) by day examined. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group (spleen). This Figure is reproduced in color in the online version of Medical Mycology. Cytokine production When the group infected was compared with the group treated with CY and infected (CY+ fungi), it was possible to observe a reduction in the production of all cytokines. However, the anti-inflammatory cytokine IL-10, was the least affected with the immunosuppressive therapy and the infection (Fig. 5). Figure 5. View largeDownload slide Measurements of IFN-γ, IL-17, and IL-10 in spleen samples of immunosuppressed mice (or not), at 14 days after infection with S. schenckii (or not). The graphs represent the mean values of each group (n = 4) by day examined. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group (spleen). This Figure is reproduced in color in the online version of Medical Mycology. Figure 5. View largeDownload slide Measurements of IFN-γ, IL-17, and IL-10 in spleen samples of immunosuppressed mice (or not), at 14 days after infection with S. schenckii (or not). The graphs represent the mean values of each group (n = 4) by day examined. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group (spleen). This Figure is reproduced in color in the online version of Medical Mycology. Histological analysis of spleen Figure 6 depicts histological sections from spleens of the animals immunosuppressed or not, 7th and 14th days, after infection with S. schenckii or not. The spleens of mice receiving only PBS presents its natural morphology in both the 7th and the 14th day post-infection, with obvious germinal centers and the normal relation of cellularity of white pulp (WP) containing lymphocytes and immune cells and red pulp (RP). Already in the spleen of immunosuppressed mice (infected and noninfected) it is possible to observe a marginal zone disorganization and absence of germinal centers, with decreased cellularity of white pulp. On the 7th day post-infection this characteristic seems to be more evident. At the 7th day, an infiltration of the red pulp with myeloid cells, owing to extramedullary hematopoiesis (EMH), was observed in the immunosuppressed group. The myeloid cells infiltration was minor in the infected and immunosuppressed group and had very reduced at the 14th day of the study, being only observed in the peripherical areas of the red pulp. Figure 6. View largeDownload slide Histology of the spleen on the 7th and 14th days after infection in control (PBS), immunosuppressed with cyclophosphamide (CY), infected (Fungi) or infected and immunosuppressed mice (CY+Fungi). On indicated days, the mice were euthanized and had their spleens removed, processed, and H&E stained. EMH, Extramedular hematopoiesis; RP, Red pulp; WP, White pulp. This Figure is reproduced in color in the online version of Medical Mycology. Figure 6. View largeDownload slide Histology of the spleen on the 7th and 14th days after infection in control (PBS), immunosuppressed with cyclophosphamide (CY), infected (Fungi) or infected and immunosuppressed mice (CY+Fungi). On indicated days, the mice were euthanized and had their spleens removed, processed, and H&E stained. EMH, Extramedular hematopoiesis; RP, Red pulp; WP, White pulp. This Figure is reproduced in color in the online version of Medical Mycology. The spleen of infected animals presented only natural morphology, with the presence of germinal centers. However, the spleen of immunocompromised and infected animals has a morphology similar to the spleen of immunosuppressed animals and uninfected, with differences between the 7th and 14th days post-infection. On the 7th day, the germinal centers appear to try to retake its natural structure, organized due to the presence of the fungus. However, on day 14, with already reduced fungal load, it is seen again decreased cellularity of the white pulp of the spleen and an absence of germinal centers. Hind paw inflammation index and histological fungal infiltration As depicted in Figure 7, infected and immunocompetent mice exhibited the highest inflammation index. Despite the hind paw inflammation index in the CY+fungi group was lower than the infected group, the fungal colonization in the immunosuppressed group was higher. In histological slices, the paw of control infected mice showed only isolated yeasts, while the paw of CY+fungi mice showed yeast agglomerates. Figure 7. View largeDownload slide Hind paw inflammation (A) and fungal infiltration index (B) in control (PBS), immunosuppressed with cyclophosphamide (CY), infected (Fungi) or infected and immunosuppressed mice (CY+Fungi). (C) Representative images of the hind paw inflammation and microscopic fields of fungal (Sporothrix schenckii) infiltration (Gomori-Grocott methenamine silver stain) (100×). This Figure is reproduced in color in the online version of Medical Mycology. Figure 7. View largeDownload slide Hind paw inflammation (A) and fungal infiltration index (B) in control (PBS), immunosuppressed with cyclophosphamide (CY), infected (Fungi) or infected and immunosuppressed mice (CY+Fungi). (C) Representative images of the hind paw inflammation and microscopic fields of fungal (Sporothrix schenckii) infiltration (Gomori-Grocott methenamine silver stain) (100×). This Figure is reproduced in color in the online version of Medical Mycology. Discussion For the achievement of an efficient host defense against pathogenic fungi, an adequate function of innate and adaptive immune response is necessary.35 In the case of sporotrichosis, the causal agent is considered opportunistic fungi in immunossupressed and the host immune response determines the clinical forms of this disease.33 Different studies performed of our group using murine models of S. schenckii infection, evidenced the role of the innate and adaptative immune response in controlling the fungus at different moments of the infectious process.36–44 At present, there is great interest in developing therapeutic and prophylactic alternatives based in immunomodulation for the control of this disease.45–49 In this context, the development of a S. schenckii host resistance model in immunosuppressed mice would be extremely useful and necessary for conducting successful studies of experimental infections of this opportunistic fungus, including immunotoxicological and immunopharmacological assessment. Immunosuppressive drugs have often been used to obtain a state of immunodeficiency in mice.32–34,50 In our study, mice were immunosuppressed using CY owing to the wide use of this drug as an immunosuppressor in different animal models for fungal diseases.28,50 Thus, a reasonable selection of CY dose, time, and frequency of administration are critical for the successful establishment of CY-induced immunosuppression animal models, resulting in effective inhibition of immune function and survival of the animals at long-term.31 Among the effects caused by CY highlights the induction of myelosuppression, since this drug interferes in the proliferation and differentiation of bone marrow cells.51,52 This happened because CY, a nitrogen mustard alkylating agent, binds to DNA and disrupts cell replicatin.53 These changes are associated with a marked leukopenia and neutropenia and increased susceptibility to infections.54,55 To optimize our experimental model of infection with S. schenckii, mice were inoculated subcutaneously with 20 μl of a suspension of 8 × 108/ml of yeast. After that, they were euthanized in different intervals for monitoring the fungal load in the draining lymph node (in this case, the popliteal lymph node in the left rear paw) and in the spleen, to evaluate the evolution from local to infection systemic infection. Also, the relative spleen weight was evaluated. The results of the infection kinetics showed that both local and systemic infection reached its peak by day 7 post-infection and regressed gradually. Even at 60 days post-infection all mice survived, in spite of the high fungal dose used. We decided to evaluate in the following studies on days 7 and 14, coinciding with the maximal fungal load and around half of the decreasing phase. The protocol of immunosuppression adopted was based on the data reported by Huyan and colleagues31, which showed that high doses of CY (150–200 mg/kg) are able to decrease the frequency of T helper cells (CD3+CD4+) and cytotoxic (CD3+CD8+) and B cells (CD19+), inhibiting the innate immunity and adaptive animal. Still, the authors show that on the 10th day post-CY, the percentage of T and B lymphocytes begins to gradually increase, but less than half the percentage basal and only return to normal levels in 30–60 days. In addition, Salva and colleagues have reported that a single dose CY (150 mg/kg) induced a significant decrease in the number of blood leukocytes at day 1 post-CY decreasing until the 3rd day. On day 3 there was an increase in the number of leukocytes, which achieved normal values after 8 days. Between days 9 and 11 post-CY, these values were higher than normal, returning to baseline after 12 days.33 From these data, we decided to administer a booster for the maintenance of immunosuppression animals. The day of infection adopted was based on work reported by Czink and colleagues50, which described that mice were infected with Staphylococcus aureus, Candida albicans, and Plasmodium berghei 3 days after administration of the first dose of CY. In our study, we observed a significant decrease in lymphocyte populations in the spleen of immunosuppressed animals, both on the 7th day and the 14th post-infection. The maintenance of the reduced frequency of T and B lymphocytes in the spleen of these animals to the 14th day demonstrates the need and efficiency of the booster dose. Interestingly, infected mice without CY treatment exhibited a significant reduction in the T-cell populations, while the B cells were elevated in comparison with the control group. In this way, there are evidences that S. schenckii is able to induce transitory T-cell unresponsiveness associated with endogenous nitric oxide.56 This transitory immunosuppression seems to be a mechanism for immune evasion and different mechanisms still insufficient studied can be involved. The reduced count in T cells matched with the relative weight of the thymus showing the immunosuppression status. On the other hand, is possible that the fungal load influenced the relative weight of the spleen since immunosuppressed mice had a significant higher relative weight of the spleen associated with the colonization of the fungi in this organ. Concordant effects were observed in the spleen histological analysis, where the mice receiving only PBS presents its natural morphology, on the 7th and the 14th day post-infection, with germinal centers and the normal relation of cellularity of white pulp and red pulp. In the spleen of immunosuppressed mice, there was a disorganization of the marginal zone with the absence of germinal centers, and consequently decreased cellularity of white pulp. Interestingly, it is observed areas of extramedullary granulopoiesis within the red pulp, as expression of the severe medular insufficiency due to the immunosuppression, as described by other studies.57,58 However, in infected mice treated with CY, a reduction of areas with splenic granulopoiesis was observed, possibly owing to the migration of granulocytes toward the other areas of fungal infiltration. At the 14th day, there was still a marked reduction of the white pulp in the immunosuppressed mice. The immunosuppression induced by CY favored the fungal load in the local lymph nodes and in the spleen. In the site of fungal inoculation was observed a mild reduction of the inflammatory response associated with a higher fungal colonization compared with the immunocompetent mice. This reduction of the local inflammation was also associated with a reduction in the cytokine production, mainly IFN-γ and IL-17 in the immunosuppressed mice. It is suggested that this reduction in the cytokine production observed in our study can be due to the reduction in the T-cell populations and inhibition of protein synthesis caused by a high dose cyclophosphamide.59,60 In summary, the described murine model reveals an increasing susceptibility to infection and severity of the fungal infection associated to immunosuppression by CY, so it can serve as a reference in immunotoxicological evaluation using S. schenckii for host resistance assays and in immunopharmacology studies evaluating drug candidates and vaccines for prevention and treatment of sporotrichosis in immunosuppressed host. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgements Financial support was provided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) with the following Grants: Postdoctoral fellowship Proc. 2014/00914-5; Regular Research Proc. 2015/04023-0. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Luebke RW . Nematodes as host resistance models for detection of immunotoxicity . Methods . 2007 ; 41 : 38 – 47 . Google Scholar CrossRef Search ADS PubMed 2. Burleson GR , Burleson FG . Testing human biologicals in animal host resistance models . J Immunotoxicol . 2008 ; 5 : 23 – 31 . Google Scholar CrossRef Search ADS PubMed 3. Sasaki AA , Fernandes GF , Rodrigues AM et al. Chromosomal polymorphism in the Sporothrix schenckii complex . PLoS One . 2014 ; 9 : e86819 . Google Scholar CrossRef Search ADS PubMed 4. Carlos IZ , Batista-Duharte A . Sporotrichosis: An emergent disease . In: Carlos IZ , ed. 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Comparison of virulence of different Sporothrix schenckii clinical isolates using experimental murine model . Med Mycol . 2007 ; 45 : 721 – 729 . Google Scholar CrossRef Search ADS PubMed 31. Huyan XH , Lin YP , Gao T , Chen RY , Fan YM . Immunosuppressive effect of cyclophosphamide on white blood cells and lymphocyte subpopulations from peripheral blood of Balb/c mice . Int Immunopharmacol . 2011 ; 11 : 1293 – 1297 . Google Scholar CrossRef Search ADS PubMed 32. Wang H , Wang M , Chen J et al. A polysaccharide from Strongylocentrotus nudus eggs protects against myelosuppression and immunosuppression in cyclophosphamide-treated mice . Int Immunopharmacol . 2011 ; 11 : 1946 – 1953 . Google Scholar CrossRef Search ADS PubMed 33. Salva S , Marranzino G , Villena J , Agüero G , Alvarez S . Probiotic Lactobacillus strains protect against myelosuppression and immunosuppression in cyclophosphamide-treated mice . Int Immunopharmacol . 2014 ; 22 : 209 – 221 . 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Mycopathologia . 2006 ; 161 : 11 – 19 . Google Scholar CrossRef Search ADS PubMed 38. Maia DC , Gonçalves AC , Ferreira LS et al. Response of cytokines and hydrogen peroxide to Sporothrix schenckii exoantigen in systemic experimental infection . Mycopathologia . 2016 ; 181 : 207 – 215 . Google Scholar CrossRef Search ADS PubMed 39. Negrini T , de C , Ferreira LS , Alegranci P et al. Role of TLR-2 and fungal surface antigens on innate immune response against Sporothrix schenckii . Immunol Invest . 2013 ; 42 : 36 – 48 . Google Scholar CrossRef Search ADS PubMed 40. Sassá MF , Saturi AE , Souza LF , Ribeiro LC , Sgarbi DB , Carlos IZ . Response of macrophage Toll-like receptor 4 to a Sporothrix schenckii lipid extract during experimental sporotrichosis . Immunology . 2009 ; 128 : 301 – 309 . Google Scholar CrossRef Search ADS PubMed 41. Sassá MF , Ferreira LS , de Abreu Ribeiro LC , Carlos IZ . Immune response against Sporothrix schenckii in TLR-4-deficient mice . 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Google Scholar CrossRef Search ADS PubMed 45. de Almeida JR , Kaihami GH , Jannuzzi GP , de Almeida SR . Therapeutic vaccine using a monoclonal antibody against a 70-kDa glycoprotein in mice infected with highly virulent Sporothrix schenckii and Sporothrix brasiliensis . Med Mycol . 2015 ; 53 : 42 – 50 . Google Scholar CrossRef Search ADS PubMed 46. Oliveira AH , de Oliveira GG , Carnevale Neto F , Portuondo DF , Batista-Duharte A , Carlos IZ . Anti-inflammatory activity of Vismia guianensis (Aubl.) Pers. extracts and antifungal activity against Sporothrix schenckii . J Ethnopharmacol. 2017 ; 195 : 266 – 274 . Google Scholar CrossRef Search ADS PubMed 47. Batista-Duharte A , Lastre M , Romeu B et al. Antifungal and immunomodulatory activity of a novel cochleate for amphotericin B delivery against Sporothrix schenckii . Int Immunopharmacol . 2016 ; 40 : 277 – 287 . Google Scholar CrossRef Search ADS PubMed 48. Portuondo DL , Batista-Duharte A , Ferreira LS et al. A cell wall protein-based vaccine candidate induce protective immune response against Sporothrix schenckii infection . Immunobiology . 2016 ; 221 : 300 – 309 . Google Scholar CrossRef Search ADS PubMed 49. Manente FA , Ferreira LS , Quinello C et al. Assessment of imiquimod, alone or in association with itraconazole, as an alternative for sporotrichosis therapy . Cytokine . 2016 ; 87 : 87 – 88 . 50. Czink I , Koncz Á , Molnár S , Hernádi S , Hernádi F . Neutropenia induced by cyclophosphamide. Mouse model . Pharmacol Res . 1992 ; 25 : 296 – 297 . Google Scholar CrossRef Search ADS 51. Guest L , Uetrecht J . Drugs toxic to the bone marrow that target stromal cells . Immunopharmacology . 2000 ; 46 : 103 – 112 . Google Scholar CrossRef Search ADS PubMed 52. Pass GJ , Carrie D , Boylan M et al. Role of hepatic cytochrome p450s in the pharmacokinetics and toxicity of cyclophosphamide: Studies with the hepatic cytochrome p450 reductase null mouse . Cancer Res . 2005 ; 65 : 4211 – 4217 . Google Scholar CrossRef Search ADS PubMed 53. Okuda DT . Immunosuppressive treatments in multiple sclerosis . Handb Clin Neurol . 2014 ; 122 : 503 – 511 . Google Scholar CrossRef Search ADS PubMed 54. Jang SE , Joh EH , Lee HY et al. Lactobacillus plantarum HY7712 ameliorates cyclophosphamide-induced immunosuppression in mice . J Microbiol Biotechnol . 2013 ; 23 : 414 – 421 . Google Scholar CrossRef Search ADS PubMed 55. Buisman AM , Van Zwet TL , Langermans JA , Geertsma MF , Leenen PJ , van Furth R . Different effect of granulocyte colony-stimulating factor or bacterial infection on bone-marrow cells of cyclophosphamide-treated or irradiated mice . Immunology . 1999 ; 97 : 601 – 610 . Google Scholar CrossRef Search ADS PubMed 56. Fernandes KS , Neto EH , Brito MM , Silva JS , Cunha FQ , Barja-Fidalgo C . Detrimental role of endogenous nitric oxide in host defence against Sporothrix schenckii . Immunology . 2008 ; 123 : 469 – 479 . Google Scholar CrossRef Search ADS PubMed 57. Joyce RA , Hartmann O , Chervenick PA . Splenic granulopoiesis in mice following administration of cyclophosphamide . Cancer Res . 1979 ; 39 : 215 – 218 . Google Scholar PubMed 58. Wang Y , Meng Q , Qiao H , Jiang H , Sun X . Role of the spleen in cyclophosphamide-induced hematosuppression and extramedullary hematopoiesis in mice . Arch Med Res . 2009 ; 40 : 249 – 255 . Google Scholar CrossRef Search ADS PubMed 59. Short RD , Rao KS , Gibson JE . The in vivo biosynthesis of DNA, RNA, and proteins by mouse embryos after a teratogenic dose of cyclophosphamide . Teratology . 1972 ; 6 : 129 – 137 . Google Scholar CrossRef Search ADS PubMed 60. Xu X , Zhang X . Effects of cyclophosphamide on immune system and gut microbiota in mice . Microbiol Res . 2015 ; 171 : 97 – 106 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Experimental sporotrichosis in a cyclophosphamide-induced immunosuppressed mice model

Medical Mycology , Volume 56 (6) – Aug 1, 2018

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Taylor & Francis
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© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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1369-3786
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1460-2709
D.O.I.
10.1093/mmy/myx098
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Abstract

Abstract This report describes a model of host resistance for Sporothrix schenckii, an opportunistic fungi in immunosuppressed mice with cyclophosphamide (CY) to be used in studies of immunotoxicology and immunopharmacology. Two doses of CY were administered intraperitoneally: 200 mg/kg and a booster of 150 mg/kg at 9-day intervals. Three days after the first dose of CY the animals were infected subcutaneously with 1.8 × 108 yeast/ml (S. schenckii ATCC 16345). At 7 and 14 days post-infection, the animals were euthanized and analyzed the fungal load by unit forming colony count in the spleen and popliteal lymph nodes. The relative weight of thymus and spleen, splenic index, the frequency of T and B cells in spleen by flow cytometry, the hind paw inflammation index and cytokine (interleukin [IL]-17, IL-10, and interferon [IFN]-γ) profile were measured. Histopathological studies of the spleen and the hind paw were also assessed. The immunosuppression status was confirmed at the evaluated days by reduction of relative weight of thymus, reduction of the splenic white pulp, the population of B and T lymphocytes, and the cytokine profile in the treated mice with CY in comparison with nontreated groups, associated to higher fungal load in hind paw and spleen in the infected mice. The described model reveals an increasing in susceptibility to infection and severity when associated with immunosuppression. This model can serve as a reference for studies of S. schenckii host resistance in pharmaceutical and toxicological studies. Sporothrix schenckii, host resistance models, immunosuppression, cyclophosphamide Introduction Despite the existence of sophisticated molecular and genetic techniques to identify defects in resistance pathways at the molecular and message level, the host resistance assays using opportunist microorganisms remain the gold standard against which changes at the molecular or cellular level of immune function can be judged. This is due to resistance to infection, regardless of the actual pathogen, involves multiple pathways of effectors’ function to neutralize or eliminate pathogens.1 Isolated or multiple tests are even insufficient to evaluate the consequences of the immunosuppression for the risk of specific infections in several cases2. Sporotrichosis is a chronic fungal infection caused by different species of Sporothrix that are classified into five clades based on gene sequences of chitin synthase, β-tubulin and calmodulin: Sporothrix brasiliensis (Clade I); Sporothrix schenckii (Clade II); Sporothrix globosa (Clade III) Sporothrix mexicana (Clade IV) and Sporothrix pallida (formerly S. albicans) (Clade V).3 Sporotrichosis has been described on five continents, with a higher prevalence in tropical and subtropical regions.4,5 Infection generally occurs following traumatic inoculation with soil, plants, and organic matter contaminated with the fungus causing cutaneous and subcutaneous infection, leading to plaque formation as warts and lesions papules. Both manifestations can progress to ulcerative lesions. The main clinical manifestations of sporotrichosis are the lymphocutaneous forms, but the number of systemic and visceral cases has increased, particularly in immunodeficient patients.6 Zoonotic transmission has been increasing significantly, with a recent report of an epidemic outbreak with zoonotic transmission by infected cats in Brazil.7 Disseminated S schenckii infection usually occurs in the context of cellular immunodeficiency associated with: alcoholism, diabetes, sarcoidosis, tuberculosis, organ transplantation, malignancy, use of immunosuppressive agents, or AIDS.8–16 Nowadays, the influence of the environmental factors in the development of sporotrichosis is being studied.17,18 There have only been a few studies addressing the influence of the environment on the virulence of these pathogens. However, recent studies have demonstrated that adverse conditions in its natural habitats can trigger the expression of different virulence factors that confer survival advantages both in animal hosts and in the environment.17 Thus, having a host resistance models using S. schenckii as opportunistic fungi in immunosuppressed mice, will be a contribution to studies of sporotrichosis immunopathogenesis, the search of new prophylactic or therapeutic tools and evaluating the effects of xenobiotics on the susceptibility of sporotrichosis. Cyclophosphamide (CY) is an alkylating agent that can inhibit both the humoral and cellular immune responses. It is widely used as cytotoxic drug in the anticancer therapy and as immunosuppressive drug in autoimmune diseases and organ transplants, including prophylaxis of graft-versus-host disease (GVHD) after myeloablative allogeneic bone marrow transplantation.19–22 CY and Methotrexate (MTX) are the more frequently reported antineoplastic drugs associated to sporotrichosis. They are administrated in combined therapy joint to other immunosuppressive drug.23–27 In 1981, the susceptibility to sporotrichosis in a murine model of cyclophosphamide immunosuppression was described.28 However, no further description of S. schenckii as host resistance model in immunosuppressed mice, using quantitative immunological biomarkers were made. This study aimed to develop a murine model of host resistance to S. schenckii sensu stricto in immunosuppressed mice with CY as a tool for future studies of immunotoxicity and immunopharmacology in sporotrichosis. Methods Animals Male Balb/c mice, 5–7 week old at the time of inoculation, were purchased from “Centro Multidisciplinar para Investigação Biológica na Área da Ciência de Animais de Laboratório” (CEMIB), Universidade de Campinas (UNICAMP), São Paulo, Brazil. Animals were housed in individually ventilated cages in an ambient with controlled temperature and 12-h light/dark cycles. Water and food were offered ad libitum. All procedures were approved by the Institutional Ethics Committee (Protocol CEP/FCF/CAR no. 04/2014) and were in accordance with the National Institutes of Health Animal Care Guidelines. Microorganism and culture conditions S schenckii (ATCC® 16345™) isolated from a case of human lung infection (Baltimore, MD, USA), was kindly provided by Department of Microbiology, Materials laboratory references the Oswaldo Cruz Foundation, National Institute of Quality Control in Health, Rio de Janeiro, Brazil. Currently this isolated sustained in the Department of Clinical Analysis, Araraquara's School of Pharmaceutical Sciences, Universidade Estadual Paulista-UNESP, Júlio Mesquita Filho, in the filamentous phase amid MycoselTM at room temperature (±25°C). The yeast form is obtained in BHI (Brain Heart Infusion, Difco) at 37°C under constant stirring of 150 rpm / min for 5 days. Preparation of the heat-killed (HKss) S. schenckii yeast HKss cells were prepared from the same 5-day-old culture of the fungus in brain-heart infusion broth used for animal infection in each respective experiment.29 Yeast cells were separated from the supernatant by centrifugation at 200 g for 5 min at room temperature, washed twice with 8 ml of sterile phosphate-buffered saline pH 7.4 (PBS), and then ressuspended and adjusted to 2.5 × 108 yeast cells/ml in PBS, that was incubated for 1 h in a 60°C water bath and then stored at 2–8°C until use. As a control for the efficiency of the heat-killing process, 100 μl aliquots from each tube were inoculated on Mycosel agar plates and checked for colony-forming units (cfu) growth before use. HKss was used as a challenge in the esplenocyte culture for later cytokine quantification in the supernatant. A working suspension was obtained by making a 1/10 dilution of the stock suspension in Roswell Park Memorial Institute (RPMI) complete medium (defined as the base RPMI-1640 medium containing 20 μM of 2β-mercaptoethanol, 100 U/mL of penicillin and streptomycin, 2 mM of l-glutamine and 5% fetal calf serum). Infection standardization This kinetic study was performed to determinate the time when occurs the peak of the infection and the reduction of infection until the virtual fungal clearance. A yeast suspension was prepared in PBS, and each mouse was inoculated subcutaneously into the left hind footpad with 1.6 × 107 yeast cells in 0.02 ml of sterile PBS, as previously,30 with some modifications. Group of five animals were euthanized at the 0, 3, 7, 9, 11, 14, 21, 28, and 60 days after infection to determine of fungal load in spleen and lymph nodes by counting of cfu and the relative spleen weight (spleen weight divided by body weight). Additionally, a test of survival for 60 days was conducted. Immunosuppression model Immunosuppression of animals was induced with cyclophosphamide (CY). Animals received an initial dose of 200 mg/kg CY intraperitoneally in PBS, with maintenance doses of 150 mg/kg at 9 days, as described by Huyan and colleagues31, with some adaptations fixed in previous studies for immunosuppression optimization.32–34 Mice were infected 3 days after the first administration of CY.35 Control animals received only PBS, by the subcutaneous and intraperitoneal routes. The mice were euthanized on days 7 and 14 after infection (according to the previous results) for the analysis of immunosuppression. Spleens were aseptically removed; splenic length, width, and thickness were measured and used to calculate the splenic index, and weighted to evaluate the organ-to-body weight ratios (relative spleen weight). After that, the spleens were divided into two parts, one for histological analysis, the other for determine the number of cfu and to analyze the frequency of helper and cytotoxic T and B cells by flow cytometry. The thymus was also removed; weighed and the relative weight was measured. Preparation of total splenocytes A previously described, weighted spleen piece was macerated in 2 ml of sterile PBS and passed through a 100-μm cell strainer into a Petri dish. For red cell lysis, the resulting suspension was added with 6 ml of a 0.17 M ammonium chloride solution and then incubated on ice for 5 min. The splenocytes were then separated from the supernatant by centrifugation at 300 g for 5 min at 4°C, washed once with 3 ml of RPMI complete medium, and then ressuspended in 1 ml of the same medium. Cell concentration was determined by microscopy using the Trypan blue exclusion test and then the splenocytes were adjusted to 5 × 106 cells/ml in RPMI complete medium. Total splenocytes were used for determining distinct immune cell populations by flow cytometry and for ex vivo determination of cytokines. Flow cytometry of lymphocyte subpopulations To analyze the lymphocytes subpopulations, spleen cells were centrifugation and washing with PBS containing 1% bovine serum albumin (BSA) (Sigma), 1 × 106 of cells were stained with monoclonal antibodies anti-mouse: fluorescein isothiocyanate (FITC)-conjugated anti-CD3, allophycocyanin (APC)-conjugated anti-CD4, peridinin–chlorophyll–protein–cyanine 5.5 (PerCP Cy5.5)-conjugated anti-CD8, and phycoerythrin (PE)-conjugated anti-CD19 for analysis of frequency of this cells on spleen. Measurement of the ex vivo release of cytokines Cytokines interleukin (IL)-17, IL-10, and interferon (IFN)-γ were measured in the cell culture supernatant by enzyme-linked immunosorbent assay (ELISA; eBioscience) according to the manufacturer's instructions. Splenocytes obtained on the 14th day after infection, as described above, were cultured for 24 h at 37°C and 5% CO2 on flat bottom 48-well tissue culture plates in the presence of HKss, at a splenocyte-to-yeast ratio of 1:5. Final concentrations were 2.5 × 106 splenocytes/ml and 1.25 × 107 yeast cells/ml. Concanavalin A (0.25 μg/ml in RPMI complete medium) and RPMI alone were used as positive and negative controls, respectively. Fungal load in the spleen and popliteal lymph node The fungal load in the spleen and popliteal lymph node of the animals was determined by counting the number of cfu recovered such organs after maceration. For this, aliquots of the organs were diluted appropriately and plated on agar MycoselTM, incubated at 37°C and checked after 3 and 7 days for the count of cfu/g of organ. Hind paw inflammation index The swelling of hind paws was measured once a week, by measuring the thickness of the hind paws from a lateral view using a digital caliper (Mitutoyo, Japan). The swelling index was calculated for hind paws by the mean difference between the injected and the contra lateral paws. Spleen and footpad histopathology After euthanasia, the spleen and footpads of the mice were removed aseptically and immediately submerged in 4% formaldehyde for histological analysis. Hematoxylin-eosin (H&E) stain was used to evaluate the decrease in the cellularity of the spleen white pulp in mice treated or not with CY and the Grocott-Gomori methenamine-silver nitrate stain was used to observe the presence of the fungi in the tissue. Analysis was made by optical microscopy (NIKON Eclipse TS100, Tokyo, Japan), and photos were captured by VD480 OPTMEDICAL software. Statistical analysis Statistical analysis was performed in GraphPad Prism ver. 6.01, by using one- or two-way analysis of variance (ANOVA) with Tukey or Sidak's multiple comparisons test, respectively, as indicated. Confidence interval was set at 95% for all tests. Groups of three or four animals were used in each experiment. Results Infection standardization During the 60 days of the study, no mice mortality was recorded. As shown in Figure 1A, the days indicated, mice were euthanized, and both spleens and lymph nodes were removed for the evaluation of systemic fungal load. The spleens were previously weighted. Figure 1. View largeDownload slide (A) Kinetics infection. The cfu spleen and popliteal lymph node of infected mice subcutaneously with S. schenckii. (B) Relative spleen weight (spleen weight/animal weight) of infected mice subcutaneously with S. schenckii. The data represent the mean ± SD of four animals per day. Figure 1. View largeDownload slide (A) Kinetics infection. The cfu spleen and popliteal lymph node of infected mice subcutaneously with S. schenckii. (B) Relative spleen weight (spleen weight/animal weight) of infected mice subcutaneously with S. schenckii. The data represent the mean ± SD of four animals per day. The results show that local infection observed by the cfu in popliteal lymph node reached the peak on the 7th day post-infection and regressed gradually until the almost fungal clearance by most animals when completed 60 days post-infection. Moreover, the infection becomes systemic since the first 3 days post-infection with cfu in the spleen. Notably, the relative spleen weight increased at the beginning of the infection and, with the passage of days, it decreased along with the fungal load (Fig. 1B). With this result, we selected the 7th and the 14th days for fungal load and immunological evaluation in further studies. Fungal load in spleen and local lymph node The fungal load in the spleen was similar between both groups infected (previously immunosuppressed or not), but at day 14 was observed a cfu reduction in the non-immunosuppressed group, while the immunosuppressed group still maintained high concentrations fungal colonization in the organ. In contrast, in the local lymph node there were not observed changes in the fungal load at 7th and 14th days in the same group, but the immunosuppressed mice showed higher fungal colonization (Fig. 2). Figure 2. View largeDownload slide The cfu obtained from (A) spleen and (B) popliteal lymph node of infected and immunosuppressed or only infected mice, 7 and 14 days after infection footpad with S. schenckii. The data represent the mean ± SD of four animals per day. *P < .05; **P < .01. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide The cfu obtained from (A) spleen and (B) popliteal lymph node of infected and immunosuppressed or only infected mice, 7 and 14 days after infection footpad with S. schenckii. The data represent the mean ± SD of four animals per day. *P < .05; **P < .01. This Figure is reproduced in color in the online version of Medical Mycology. Relative weights and splenic index The mice did not exhibit any differences in weight between the experimental groups (Fig. Supl. 1). However, the relative weight of spleen had a significant increase in immunosuppressed and infected mice when compared to all other groups both at 7th and 14th days. Moreover, the relative weight of the spleen in mice both treated with CY and infected separately was higher than the control group treated with PBS (Fig. 3A). On the other hand, the relative weight of thymus just showed a significant increase in immunosuppressed infected and non-infected mice when compared to the other groups (Fig. 3C). Thus, the infection does not alter thymus weight when compared to control and, in fact, CY-induced immunosuppression reduced thymus weight whether or not infection was present. The higher spleen index was observed in the group immunosuppressed and infected mice. However, groups only immunosuppressed or only infected, also had statistical difference in splenic index when compared to the control group on the studied days (Fig. 3B). Figure 3. View largeDownload slide Relative (A) spleen and (C) thymus weight (organ weight ÷ animal weight), splenic index (length × width × thickness) (B) immunosuppressed mice (or not), 7 and 14 days after infection with S. schenckii (or not). The data represent the mean ± SD of four animals. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group at each point corresponding unless otherwise indicated. Relative thymus weight was significantly reduced in mice receiving cyclophosphamide infected or not, especially in the 14-day post-infection with a significant difference between these groups. This Figure is reproduced in color in the online version of Medical Mycology. Figure 3. View largeDownload slide Relative (A) spleen and (C) thymus weight (organ weight ÷ animal weight), splenic index (length × width × thickness) (B) immunosuppressed mice (or not), 7 and 14 days after infection with S. schenckii (or not). The data represent the mean ± SD of four animals. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group at each point corresponding unless otherwise indicated. Relative thymus weight was significantly reduced in mice receiving cyclophosphamide infected or not, especially in the 14-day post-infection with a significant difference between these groups. This Figure is reproduced in color in the online version of Medical Mycology. Flow cytometry of lymphocyte subpopulations from the spleen The populations of lymphocytes in the spleen of the animals were defined as helper (CD3+CD4+) T cells, cytotoxic (CD3+CD8+) T cells and B (CD19+) cells. Figure 4 shows the results expressed as the mean ± SD of the frequencies (%) of cell populations obtained from each group on days indicated. Statistical analysis showed that the frequency of helper and cytotoxic T cells as well as B cells in the spleen of mice receiving CY infected or not was significantly lower than control animals or infected without CY. This significant difference existed in both the 7th day and on the 14th day post-infection. Furthermore, it is possible to observe the maintenance of reduced frequency of T and B lymphocytes in the spleen of the animals to the 14th day post-infection certainly due to the booster dose. Interestingly, in this model, a reduction in both subpopulations of T cells was observed at 7th and 14th days in the nontreated and infected group, but B cells were elevated at 7th day in comparison with the control group without both infection and treatment. Figure 4. View largeDownload slide Frequency (%) of CD3+CD4+ and CD3+CD8+ T lymphocytes and B lymphocytes CD19+ in samples of immunosuppressed mice (or not), 7 and 14 days after infection with S. schenckii (or not). The graphs represent the mean values of each group (4 animals) by day examined. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group (spleen). This Figure is reproduced in color in the online version of Medical Mycology. Figure 4. View largeDownload slide Frequency (%) of CD3+CD4+ and CD3+CD8+ T lymphocytes and B lymphocytes CD19+ in samples of immunosuppressed mice (or not), 7 and 14 days after infection with S. schenckii (or not). The graphs represent the mean values of each group (4 animals) by day examined. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group (spleen). This Figure is reproduced in color in the online version of Medical Mycology. Cytokine production When the group infected was compared with the group treated with CY and infected (CY+ fungi), it was possible to observe a reduction in the production of all cytokines. However, the anti-inflammatory cytokine IL-10, was the least affected with the immunosuppressive therapy and the infection (Fig. 5). Figure 5. View largeDownload slide Measurements of IFN-γ, IL-17, and IL-10 in spleen samples of immunosuppressed mice (or not), at 14 days after infection with S. schenckii (or not). The graphs represent the mean values of each group (n = 4) by day examined. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group (spleen). This Figure is reproduced in color in the online version of Medical Mycology. Figure 5. View largeDownload slide Measurements of IFN-γ, IL-17, and IL-10 in spleen samples of immunosuppressed mice (or not), at 14 days after infection with S. schenckii (or not). The graphs represent the mean values of each group (n = 4) by day examined. *P < .05; **P < .01; ***P < .001; ****P < .0001. All groups showed significantly greater difference when compared to the PBS group (spleen). This Figure is reproduced in color in the online version of Medical Mycology. Histological analysis of spleen Figure 6 depicts histological sections from spleens of the animals immunosuppressed or not, 7th and 14th days, after infection with S. schenckii or not. The spleens of mice receiving only PBS presents its natural morphology in both the 7th and the 14th day post-infection, with obvious germinal centers and the normal relation of cellularity of white pulp (WP) containing lymphocytes and immune cells and red pulp (RP). Already in the spleen of immunosuppressed mice (infected and noninfected) it is possible to observe a marginal zone disorganization and absence of germinal centers, with decreased cellularity of white pulp. On the 7th day post-infection this characteristic seems to be more evident. At the 7th day, an infiltration of the red pulp with myeloid cells, owing to extramedullary hematopoiesis (EMH), was observed in the immunosuppressed group. The myeloid cells infiltration was minor in the infected and immunosuppressed group and had very reduced at the 14th day of the study, being only observed in the peripherical areas of the red pulp. Figure 6. View largeDownload slide Histology of the spleen on the 7th and 14th days after infection in control (PBS), immunosuppressed with cyclophosphamide (CY), infected (Fungi) or infected and immunosuppressed mice (CY+Fungi). On indicated days, the mice were euthanized and had their spleens removed, processed, and H&E stained. EMH, Extramedular hematopoiesis; RP, Red pulp; WP, White pulp. This Figure is reproduced in color in the online version of Medical Mycology. Figure 6. View largeDownload slide Histology of the spleen on the 7th and 14th days after infection in control (PBS), immunosuppressed with cyclophosphamide (CY), infected (Fungi) or infected and immunosuppressed mice (CY+Fungi). On indicated days, the mice were euthanized and had their spleens removed, processed, and H&E stained. EMH, Extramedular hematopoiesis; RP, Red pulp; WP, White pulp. This Figure is reproduced in color in the online version of Medical Mycology. The spleen of infected animals presented only natural morphology, with the presence of germinal centers. However, the spleen of immunocompromised and infected animals has a morphology similar to the spleen of immunosuppressed animals and uninfected, with differences between the 7th and 14th days post-infection. On the 7th day, the germinal centers appear to try to retake its natural structure, organized due to the presence of the fungus. However, on day 14, with already reduced fungal load, it is seen again decreased cellularity of the white pulp of the spleen and an absence of germinal centers. Hind paw inflammation index and histological fungal infiltration As depicted in Figure 7, infected and immunocompetent mice exhibited the highest inflammation index. Despite the hind paw inflammation index in the CY+fungi group was lower than the infected group, the fungal colonization in the immunosuppressed group was higher. In histological slices, the paw of control infected mice showed only isolated yeasts, while the paw of CY+fungi mice showed yeast agglomerates. Figure 7. View largeDownload slide Hind paw inflammation (A) and fungal infiltration index (B) in control (PBS), immunosuppressed with cyclophosphamide (CY), infected (Fungi) or infected and immunosuppressed mice (CY+Fungi). (C) Representative images of the hind paw inflammation and microscopic fields of fungal (Sporothrix schenckii) infiltration (Gomori-Grocott methenamine silver stain) (100×). This Figure is reproduced in color in the online version of Medical Mycology. Figure 7. View largeDownload slide Hind paw inflammation (A) and fungal infiltration index (B) in control (PBS), immunosuppressed with cyclophosphamide (CY), infected (Fungi) or infected and immunosuppressed mice (CY+Fungi). (C) Representative images of the hind paw inflammation and microscopic fields of fungal (Sporothrix schenckii) infiltration (Gomori-Grocott methenamine silver stain) (100×). This Figure is reproduced in color in the online version of Medical Mycology. Discussion For the achievement of an efficient host defense against pathogenic fungi, an adequate function of innate and adaptive immune response is necessary.35 In the case of sporotrichosis, the causal agent is considered opportunistic fungi in immunossupressed and the host immune response determines the clinical forms of this disease.33 Different studies performed of our group using murine models of S. schenckii infection, evidenced the role of the innate and adaptative immune response in controlling the fungus at different moments of the infectious process.36–44 At present, there is great interest in developing therapeutic and prophylactic alternatives based in immunomodulation for the control of this disease.45–49 In this context, the development of a S. schenckii host resistance model in immunosuppressed mice would be extremely useful and necessary for conducting successful studies of experimental infections of this opportunistic fungus, including immunotoxicological and immunopharmacological assessment. Immunosuppressive drugs have often been used to obtain a state of immunodeficiency in mice.32–34,50 In our study, mice were immunosuppressed using CY owing to the wide use of this drug as an immunosuppressor in different animal models for fungal diseases.28,50 Thus, a reasonable selection of CY dose, time, and frequency of administration are critical for the successful establishment of CY-induced immunosuppression animal models, resulting in effective inhibition of immune function and survival of the animals at long-term.31 Among the effects caused by CY highlights the induction of myelosuppression, since this drug interferes in the proliferation and differentiation of bone marrow cells.51,52 This happened because CY, a nitrogen mustard alkylating agent, binds to DNA and disrupts cell replicatin.53 These changes are associated with a marked leukopenia and neutropenia and increased susceptibility to infections.54,55 To optimize our experimental model of infection with S. schenckii, mice were inoculated subcutaneously with 20 μl of a suspension of 8 × 108/ml of yeast. After that, they were euthanized in different intervals for monitoring the fungal load in the draining lymph node (in this case, the popliteal lymph node in the left rear paw) and in the spleen, to evaluate the evolution from local to infection systemic infection. Also, the relative spleen weight was evaluated. The results of the infection kinetics showed that both local and systemic infection reached its peak by day 7 post-infection and regressed gradually. Even at 60 days post-infection all mice survived, in spite of the high fungal dose used. We decided to evaluate in the following studies on days 7 and 14, coinciding with the maximal fungal load and around half of the decreasing phase. The protocol of immunosuppression adopted was based on the data reported by Huyan and colleagues31, which showed that high doses of CY (150–200 mg/kg) are able to decrease the frequency of T helper cells (CD3+CD4+) and cytotoxic (CD3+CD8+) and B cells (CD19+), inhibiting the innate immunity and adaptive animal. Still, the authors show that on the 10th day post-CY, the percentage of T and B lymphocytes begins to gradually increase, but less than half the percentage basal and only return to normal levels in 30–60 days. In addition, Salva and colleagues have reported that a single dose CY (150 mg/kg) induced a significant decrease in the number of blood leukocytes at day 1 post-CY decreasing until the 3rd day. On day 3 there was an increase in the number of leukocytes, which achieved normal values after 8 days. Between days 9 and 11 post-CY, these values were higher than normal, returning to baseline after 12 days.33 From these data, we decided to administer a booster for the maintenance of immunosuppression animals. The day of infection adopted was based on work reported by Czink and colleagues50, which described that mice were infected with Staphylococcus aureus, Candida albicans, and Plasmodium berghei 3 days after administration of the first dose of CY. In our study, we observed a significant decrease in lymphocyte populations in the spleen of immunosuppressed animals, both on the 7th day and the 14th post-infection. The maintenance of the reduced frequency of T and B lymphocytes in the spleen of these animals to the 14th day demonstrates the need and efficiency of the booster dose. Interestingly, infected mice without CY treatment exhibited a significant reduction in the T-cell populations, while the B cells were elevated in comparison with the control group. In this way, there are evidences that S. schenckii is able to induce transitory T-cell unresponsiveness associated with endogenous nitric oxide.56 This transitory immunosuppression seems to be a mechanism for immune evasion and different mechanisms still insufficient studied can be involved. The reduced count in T cells matched with the relative weight of the thymus showing the immunosuppression status. On the other hand, is possible that the fungal load influenced the relative weight of the spleen since immunosuppressed mice had a significant higher relative weight of the spleen associated with the colonization of the fungi in this organ. Concordant effects were observed in the spleen histological analysis, where the mice receiving only PBS presents its natural morphology, on the 7th and the 14th day post-infection, with germinal centers and the normal relation of cellularity of white pulp and red pulp. In the spleen of immunosuppressed mice, there was a disorganization of the marginal zone with the absence of germinal centers, and consequently decreased cellularity of white pulp. Interestingly, it is observed areas of extramedullary granulopoiesis within the red pulp, as expression of the severe medular insufficiency due to the immunosuppression, as described by other studies.57,58 However, in infected mice treated with CY, a reduction of areas with splenic granulopoiesis was observed, possibly owing to the migration of granulocytes toward the other areas of fungal infiltration. At the 14th day, there was still a marked reduction of the white pulp in the immunosuppressed mice. The immunosuppression induced by CY favored the fungal load in the local lymph nodes and in the spleen. In the site of fungal inoculation was observed a mild reduction of the inflammatory response associated with a higher fungal colonization compared with the immunocompetent mice. This reduction of the local inflammation was also associated with a reduction in the cytokine production, mainly IFN-γ and IL-17 in the immunosuppressed mice. It is suggested that this reduction in the cytokine production observed in our study can be due to the reduction in the T-cell populations and inhibition of protein synthesis caused by a high dose cyclophosphamide.59,60 In summary, the described murine model reveals an increasing susceptibility to infection and severity of the fungal infection associated to immunosuppression by CY, so it can serve as a reference in immunotoxicological evaluation using S. schenckii for host resistance assays and in immunopharmacology studies evaluating drug candidates and vaccines for prevention and treatment of sporotrichosis in immunosuppressed host. Supplementary material Supplementary data are available at MMYCOL online. Acknowledgements Financial support was provided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) with the following Grants: Postdoctoral fellowship Proc. 2014/00914-5; Regular Research Proc. 2015/04023-0. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Luebke RW . Nematodes as host resistance models for detection of immunotoxicity . Methods . 2007 ; 41 : 38 – 47 . Google Scholar CrossRef Search ADS PubMed 2. Burleson GR , Burleson FG . Testing human biologicals in animal host resistance models . J Immunotoxicol . 2008 ; 5 : 23 – 31 . Google Scholar CrossRef Search ADS PubMed 3. Sasaki AA , Fernandes GF , Rodrigues AM et al. Chromosomal polymorphism in the Sporothrix schenckii complex . PLoS One . 2014 ; 9 : e86819 . Google Scholar CrossRef Search ADS PubMed 4. Carlos IZ , Batista-Duharte A . Sporotrichosis: An emergent disease . In: Carlos IZ , ed. 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Medical MycologyOxford University Press

Published: Aug 1, 2018

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