All mice used in the experiments were on a C57BL/6 background. Wild-type (WT) C57BL/6-Kit^+/+ and MC-deficient C57BL/6-Kit^W/Kit^W−v mice were bred and housed at our facilities, and wild-type (WT) Cpa3-Cre/Mcl-1^+/+ and MC-deficient Cpa3-Cre/Mcl-1^fl/fl (Hello Kitty, HK) mice were also obtained from breeding colonies of the animal facilities of the Charite - Universitätsmedizin Berlin. All mice were kept under specific pathogen-free conditions, and all experiments were conducted according to institutional regulations.

The S. schenckii wild-type strain M-64 (ATCC MYA 4822) was a donation from Prof. Sandro Rogerio de Almeida's laboratory (Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, SP, Brazil). The Sporothrix albicans wild-type strain (S. albicans, ATCC® 201162^TM) was purchased from ATCC. Initially, the isolates were subcultured from conidia until complete differentiation into the yeast form. Yeast cells were grown in brain-heart infusion broth (BHI, HB8297-1, Hopebio, China) for 7 days at 37°C with constant rotary shaking at 150 cycles/min, in order to yield a high percentage of yeast conversion. Then, yeasts were harvested from the BHI culture by centrifugation, washed twice and adjusted to the required concentration in sterile phosphate buffered saline (PBS), pH 7.4, and stored at 2–8°C until use.

To study the role of MCs in skin infections with Sporothrix, we infected MC-deficient mice (HK) and WT littermates (C57BL/6) with S. schenckii subcutaneously by injecting 30 μl of PBS containing 3 × 10⁷ cells of S. schenckii yeast into the right foot pad, and PBS vehicle into the left foot pad as autologous control. Mice were then examined every 3 days during 5 weeks by evaluating the thickness, width, and length of foot inflammation with an electronic micrometer and the presence of skin ulceration or scarring in foot pad by photographing. The lesion volume was calculated (in mm³) as ellipsoids [(a/2×b/2×c/2)×4/3×π].

The fungal burden of infected skin sites of mice was measured by counting colony-forming units (CFU). Briefly, the organs were separated, weighed, and homogenized in sterile PBS with a tissue grinder. Samples (100 μL) of each homogenate were seeded on Petri dishes containing BHI agar and incubated at 37°C. The material was incubated for 7 days at room temperature, and the number of colonies formed on each plate (CFU) was counted, and the mean of viable fungi was then calculated for each group. The results were expressed as CFU/g tissue.

Tissue samples from infected mice were fixed in 10% formalin for 16 h, dehydrated in alcohol, and embedded in paraffin. Histological analyses of foot, spleen, lungs, liver, and lymph node were performed with a Zeiss Axioplan 2 Imaging microscope using a Zeiss AxioCam camera run by AxioVision Rel. 4.8 software. Giemsa stain was used to identify degranulated MCs.

For immunohistochemistry staining, sections were deparaffinized in xylene for 10 min and then rehydrated in graded alcohols and water. TNF staining was performed according to the manufacturer's instructions (DakoEnVision⁺ System- HRP Labeled PolymerAnti-Rabbit). Briefly, antigen retrieval was executed in citrate buffer (pH 6.0) in 95°C for 30 min. After blocking of non-specific binding with Block Dako for 10 min at room temperature, sections were incubated with primary antibodies against human and mouse TNF (ab9739, Abcam) in a humid chamber at 4°C overnight. Then sections were treated with H₂O₂ for 5 min at room temperature (RT). After three washes with TBS, sections were incubated with polymer secondary anti-rabbit secondary antibodies for 30 min, washed three times in TBS, and incubated with substrate AEC for 5 min. Slides were rinsed in TBS and counterstained with Mayer's hematoxylin (ab128990, Abcam; negative control = omission of the primary antibody). For IL-6 staining (Dako REAL™ Detection System, Alkaline Phosphatase/RED, Rabbit/Mouse, K5005), antigen retrieval was executed in citrate buffer (pH 6.0) in 95°Cfor 30 min. After blocking with avidin block, biotin block and 5% goat serum for 15 min each at room temperature, sections were incubated with primary antibodies against mouse IL-6 (sc-1265, Santa Cruz Biotechnology) and human IL-6 (sc-130326, Santa Cruz Biotechnology) in a humid chamber at 4°C overnight. After three washes with TBS, sections were incubated with biotinylated goat anti-rabbit secondary antibodies for 30 min, washed three times in TBS and incubated with ABC and substrate. Slides were rinsed in TBS and counterstained with Mayer's hematoxylin as described before. The expression of TNF and IL-6 was assessed by histomorphometry (Zeiss Axioplan 2 Imaging microscope using Zeiss AxioCam camera run by AxioVision Rel. 4.8 software).

Femoral and tibial bone marrow from 4 to 6-week old C57BL/6 mice was cultured in DMEM plus recombinant stem cell factor (SCF, 20 ng/mL, Biolegend) and recombinant interleukin-3 (IL-3, 20 ng/mL, Biolegend) containing medium for 4–8 weeks to generate populations of bone marrow-derived cultured mast cells (BMCMCs) that were >95% pure as assessed by flow cytometry counting CD117 and FceRI double positive cells.

BMCMCs (10⁶ in 50 μl 0.9% NaCl) were injected intradermally and mice were used for experiments, together with sex- and age-matched MC-deficient Kit^W/Kit^W−v and Kit⁺/⁺ mice, 4 wk after adoptive transfer. Reconstitution of cutaneous MC populations was confirmed by histomorphometric analyses of paraffin-embedded, Giemsa-stained sections of injected skin (Supplementary Figure 8).

Assessment of degranulation by flow cytometry, Wild-type BMCMCs were incubated with different concentrations of S. schenkii yeasts, medium alone as a negative control, or PMA/ionomycin as a positive control, respectively. After 0.5 h of incubation with S. schenkii yeasts, BMCMCs were stained with APC anti-mouse CD63 antibody (143906, Biolegend). APC Rat IgG2a, κ- antibody (400512, Biolegend) was used as isotype control. Cells were analyzed with a Coulter Epics XL Flow cytometer or a Coulter FC 500 ANALYZER (Beckman Coulter). The relevant data were obtained and analyzed using FlowJo software, version 7.6.

BMCMCs (5 × 10⁶) were incubated with or without yeasts (9.4 × 10⁷, 4.7 × 10⁷, or 2.35 × 10⁷) of S. schenkii or S. albicans at 37°C in supplemented RPMI 1640 medium under an atmosphere of 5% CO₂ for 24 h. Cell-free supernatants were collected and stored at −80°C until assayed for TNF and IL-6. Then, we used ELISA kits (Biolegend) to determine the levels of TNF, IL-6, IL-1β, IL-10, and MCP-1 in cell culture media according to the manufacturer's instructions. Serum levels of TNF and IL-6 in sporotrichosis patients were determined using human TNF or IL-6 enzyme-linked immunosorbent assay kits, respectively (R&D Systems). The results were expressed in pg/ml.

Ethical approval from the Ethics Committee of Department of Dermatology, First Hospital, Peking University (Beijing, China) was obtained prior to the study. A total of 15 sporotrichosis patients in Han Chinese population (n = 15, male: 6, female: 9; aged 30–50 years, n = 4, mean 43.75; aged 50–70 years, n = 7, mean 61;aged 70-90 years, n = 4, mean 78;) were included after informed consent (Table 1). The definitive diagnosis of sporotrichosis was established by isolation of the fungus from skin specimens (exudate, scales, and skin biopsy), which were sent to the Laboratory of Mycology. The definitive diagnosis was made by isolation of Sprothrix species as previously described. The control group consisted of 15 plastic surgery-derived skin samples of healthy individuals.

All results are presented as means ± SEM unless specified otherwise. The two-tailed t-test and one-way or two-way ANOVA were used to determine significance between two groups or multiple groups. A P-value ≤ 0.05 was considered to reflect statistical significance.

MC-deficient HK (Hello Kitty) mice and WT (Wild type) mice both developed typical skin sporotrichosis lesions with ulceration and scarring after subcutaneous inoculation with S. schenckii (Figure 1A). However, WT mice infected with S. schenckii developed markedly larger skin lesions compared with HK mice (Figures 1A,B). Differences were most pronounced on day 4 (260 ± 35 vs. 199 ± 42.1 mm³, p = 0.03) and on day 7 post-infection (280 ± 46 vs. 211 ± 12 mm³, p = 0.02), with lesion volumes decreased some 25% in HK mice vs. WT mice (Figure 1B). WT mice also exhibited increased lesional fungal burden, more than double as compared to HK mice on day 7 (Figure 1C; 49 ± 13 vs. 20 ± 6 CFU/mm³, p = 0.002). No inflammation or spores were found in the lungs, spleens, or livers of any of the mice (Supplementary Figure 6A). WT and HK mice showed no spores and similar levels of immune cell infiltration in the draining lymph nodes of sites of infection (Supplementary Figure 6B).

MC-competent WT mice also developed larger sporotrichosis lesions as compared to MC-deficient C57BL/6-Kit^W/Kit^W−v mice infected with S. schenckii (Figure 1D). Reconstitution of skin MCs by local adoptive transfer to C57BL/6-Kit^W/Kit^W−v mice 4 weeks prior to infection resulted in skin lesions comparable to those of WT mice (Figure 1D). In WT mice, infection with S. schenckii resulted in 2-fold increased numbers of MCs at sites of infection, but not non-infected skin sites (Supplementary Figures 1A,B).

To investigate how MCs contribute to S. schenckii infections, we tested if MCs degranulate in response to S. schenckii, in vitro and in situ. MCs did not degranulate in vitro as assessed by β-hexosaminidase release and CD63 surface expression by flow cytometry in BMCMCs (Figures 2A,B and Supplementary Figure 2A). Also, MCs did not exhibit signs of degranulation in response to S. schenckii in situ, as assessed by quantitative histomorphometric analyses of infected skin sites (Supplementary Figure 1C). In contrast, MCs dose-dependently released substantial amounts of the early response cytokines IL-6, TNF, IL-10, and IL-1β, but not MCP-1, in response to S. schenckii (Figures 2C–F and Supplementary Figure 2B). MC-deficient mice showed markedly reduced expression of TNF and IL-6 at sites of S. schenckii infection as compared to WT mice (Figure 3). In addition, in WT mice, most cutaneous MCs at sites of S. schenckii infection were positive for IL-6 and/or TNF, and most IL-6/TNF positive cells were MCs (Figures 4, 5 and Supplementary Figure 7A).

To assess the relevance of MC cytokine production in sporotrichosis, we used S. schenckii and the less virulent Sporothrix species S. albicans. We first compared skin responses of mice and then the release of cytokines of MCs exposed to these two Sporothrix species. Mice infected with S. schenckii developed markedly larger skin lesions than mice infected with S. albicans (Figure 6A, 175 ± 38 vs. 112 ± 45 mm³ at day 7, p = 0.02). In addition, S. schenckii-infected mice, but not S. albicans-infected mice developed necrosis, with scabbing and ulcerations at sites of infection (Supplementary Figure 3).

When compared for their effects on MC cytokine release, S. schenkii induced more than 11-fold and 8-fold higher release of IL-6 and TNF, respectively, as compared to S. albicans (Figures 6B,C), whereas IL-10 and IL-1ß release were similar (Supplementary Figures 4A,B). Immunohistochemical analyses showed that TNF and IL-6 were also increased in the skin lesions of S.schenckii-infected mice as compared to S. albicans-infected mice (Figure 6D and Supplementary Figure 5A).

Patients with sporotrichosis showed markedly higher serum levels of TNF and IL-6 than healthy controls (HC; Figure 7; TNF-α: 53 ± 24 vs. 6 ± 10 pg/ml, P < 0.001; IL-6: 80 ± 42 vs. 25 ± 10 pg/ml, P < 0.001). Serum levels of TNF and IL-6 were highest in patients of disseminated type sporotrichosis, followed by patients with lymphocutaneous sporotrichosis, and lowest in fixed cutaneous sporotrichosis patients (Figures 7A,B). Expression levels of TNF and IL-6 in the lesional skin of sporotrichosis patients were markedly higher as compared to healthy control skin (Figure 7C and Supplementary Figure 5B). Some cutaneous IL-6/TNF-positive cells were MCs, and most MCs were IL-6/TNF-positive (Figures 7C, 8 and Supplementary Figure 7B).

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.