Fungal strains

The wild type C. albicans strain (SC5314) used in this study was obtained from ATCC. Candida parapsilosis, Candida tropicalis, and Candida lusitaniae were obtained from Prof. Daniel Kornitzer (Israel Institute of Technology, Haifa, Israel). Candidalysin-mutant strains: ECE1 deletion strain (ece1Δ/Δ), ECE1-complemented strain (ece1Δ/Δ + ECE1), and C. albicans strain lacking only the candidalysin-encoding region in Ece1 (ece1Δ/Δ + ECE1 _Δ184–279) were produced as described in ref. ²⁶. Als mutant strains were als1Δ/Δ1467, als2Δ/Δ2757, als3Δ/Δ1843, als4Δ/Δ2034, als5Δ/Δ2373, als6Δ/Δ1420, als7Δ/Δ1429 and als9Δ/Δ2028, which are all derivatives of SC5314⁷. For all experiments, fungal cultures were inoculated overnight in Sabouraud dextrose (Sigma-Aldrich, Jerusalem, Israel) medium at 30 °C in aerobic conditions. The culture was then diluted 1:50 in a fresh secondary culture in Sabouraud dextrose medium for 2 h. Cells were then washed 3 times with ice cold PBS before their introduction into the experiment.

Systemic candidiasis mouse models

For the systemic candidiasis studies, 8-12-week-old males C57Bl/6 (Harlan, ENVIGO, UK), ΔdblGATA (backcrossed to the C57Bl/6 background for seven generations, a kind gift of Prof. Ariel Munitz, Department of Clinical Microbiology and Immunology, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel) and CD48^–/– mice (a kind gift of Prof. Arlene H. Sharpe Department of Microbiology and Immunobiology, Evergrande Center for Immunologic Diseases, Harvard Medical School, Boston, MA, USA) were injected via the lateral tail vein with either 5 × 10⁴ (for survival assay) or 5 × 10⁵ (fungal burden assay) C. albicans yeast for the determination of fungal burden respectively. C. albicans (strain SC5314 or als6Δ/Δ) was grown at 30 °C in yeast Sabouraud dextrose broth as described above. On the day of the experiment the fungal cells were washed 3 times in ice-cold PBS and taken to the animal facility on ice.

For fungal burden experiments, mice were sacrificed with 48 h post-infection and relevant organs were harvested. Organs were homogenized, filtered through a 70 μm strainer, serially diluted in ice-cold PBS and plated on Sabouraud dextrose agar plates (Sigma-Aldrich). The plates were incubated for 48 h in a stationary 30 °C incubator and once colonies were visible, they were counted.

For survival experiments, mice were weighed, clinically evaluated daily and euthanized once their initial weight was reduced by 20%.

For the depletion of eosinophils, WT and CD48^–/– mice were. injected i.p with 0.3 mg/100 µl of anti–IL-5 antibody in (TREK5; BioXCell) or with anti–horseradish peroxidase isotype control antibody (HRPN; BioXCell) at days −5, −3, and −1 before fungal infection.

All the animal experiments were performed according to Hebrew University ethical animal guidelines (MD-19-15990-5).

Human peripheral blood eosinophil purification

Human eosinophils were purified as previously described⁶⁶ from the peripheral blood of mildly atopic volunteers (blood eosinophil levels 5–10%), asymptomatic and therefore not taking any drug. Written informed consent was obtained according to the guidelines of the Hadassah-Hebrew University Human Experimentation Helsinki Committee (0410-14-HMO). Briefly, venous blood (150 ml) was collected in heparinized syringes and left to sediment in 6% dextran (Sigma-Aldrich). Leukocytes were centrifuged on Ficoll-Hypaque (density, 1.077; 25 min, 700 × g, 22 °C; Amersham Bioscience, Amersham, Buckinghamshire, UK). Neutrophils and contaminating lymphocytes were tagged in the granulocyte-enriched pellet with micromagnetic beads bound to anti-CD16 and anti-CD3 (Miltenyi Biotec, North Rhine-Westphalia, Germany). Eosinophils were purified by passing the cell suspension through a magnetic column (LS-MACS). Eosinophils were evaluated by flow cytometry using APC-anti-human CCR3 (Abcam, Cambridge, UK) and PE anti-human Siglec-8 (BioLegend, CA, USA) and collected at a purity of >98% (Kimura staining), with viability of >98% (trypan blue staining). Then, eosinophils were re-suspended (1 × 10⁶ cells/ml) in eosinophil medium consisting of RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS; HyClones; Logan, Utah, USA), penicillin-streptomycin solution (100 u/ml; Biological Industries, Beit Haemek, Israel), and GM-CSF (5 ng/ml; Peprotech, Rocky Hill, NJ, USA).

Eosinophil viability and degranulation assay

For viability and degranulation assays, eosinophils (10⁵ cells/well) were incubated at 37 °C, 5% CO₂ for 1, 3, and 20 h in eosinophil medium with C. albicans at MOI 0.1, and 1 or with commercially synthesized candidalysin peptide toxin (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK; >98% pure; Peptide Protein Research Ltd, Hampshire, UK). Blocking of CD48 was assessed by pre-incubation of C. albicans with D1-Fc or hCD48-Fc (5 µg/ml) for 40 min on ice. Yeast was then washed with phosphate buffer saline (PBS) prior to the co-culture. After the indicated time points, viable cell count was determined by trypan blue exclusion test using a Neubauer chamber cell, and supernatants were collected to evaluate the levels of EPX and LDH. EPX levels were measured using a standardized colorimetric assay with purified human EPX (a kind gift of Prof. Gerald J. Gleich, University of Utah Health Sciences Center, Salt Lake City, UT, USA) and freshly prepared peroxidase substrate solution containing o-phenylenediamine dihydrochloride (OPD; Thermo-Fisher Scientific; Kyriat Shmona, Israel) as previously described⁶⁷. LDH levels were determined using Lactate dehydrogenase assay kit (Abcam).

Generation of human and mouse CD48-Fc

Fusion proteins were generated as previously described⁵⁰. In brief, the sequence encoding the extracellular part of human CD48 was amplified by PCR using the 5ʹ primer cccACCGGT GCCGCCACC (including AgeI restriction site) and the 3ʹ primer CATTCTTGGCCTGTTACTTACGGATCCccc (including BamHI restriction site). This PCR fragment was cloned into an expression vector containing a mutated Fc portion of human IgG1 (CSI-Ig Puro plasmid). The same protocol was performed for the generation of the mouse CD48-Fc (mCD48-Fc). All proteins were generated in 293 T cells and were purified on a protein G column (HiTrap TM protein G HP, GE Health Care, Chicago, IL, USA).

Generation of WT and CD48^–/– BMEos

Bone marrow cells obtained from femurs of CD48^–/– and WT mice were differentiated as previously described⁶⁸. On days 10–12, eosinophil purity was examined by flow cytometry. Eosinophils were recognized by their expression of characteristic cell surface markers using PE-anti-mouse Siglec-F (BD Biosciences, NJ, USA) and APC-anti-mouse CCR3 (BioLegend). CD48 expression was also evaluated by flow cytometry using FITC-anti-mouse CD48 or hamster IgG isotype control (eBioscience, CA, USA).

Determination of fungal colony forming unit (CFU) in vitro

To evaluate C. albicans viability. human and murine eosinophils (1 × 10⁵ cells/well) were co-cultured for 3 and 20 h with C. albicans at MOI 0.1 or 1. To assess the effect of eosinophil mediators, C. albicans (1 × 10⁵ yeast/well) was incubated with purified eosinophil basic mediators, each at the concentration present in 1 × 10⁵ eosinophils^21,27. Cells were then collected and diluted in PBS by serial dilutions and plated onto Sabouraud agar plates, incubated at 30 °C for 48 h, and CFU numbers were counted.

Flow cytometry

For the staining of cell surface receptors, cells were harvested, centrifuged at 250 × g for mammalian cells and 3000 × g for fungal cells, washed twice in flow cytometry buffer (PBS, 0.1% bovine serum albumin (BSA)), and counted using a hemocytometer. Cells (1 × 10⁵ cells/well) were plated into a 96 U-bottom plate and resuspended in blocking buffer (flow cytometry buffer supplemented with 5% goat serum, Biological Industries) for 15 min on ice. Human eosinophils were incubated with the following primary antibodies: anti-human CD48 (BioLegend), anti-human Dectin-1 (Abcam), anti-human TLR2 (BioLegend), anti-human TLR4 (BioLegend), anti-phospho-Lyn (Cell signaling, Danvers, MA USA) or the appropriate isotype control (mouse IgG1 or mouse IgG2a; all at 2.5 μg/ml) for 40 min on ice. Cells were then washed again and incubated with the secondary antibody Fluorescein (FITC)-goat anti-mouse IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 30 min on ice in the dark. C. albicans staining was assessed using in-house murine and human CD48-Fc or D1-Fc (5 µg/ml), followed by FITC-goat anti-human IgG (1:2000 in FC buffer; Jackson ImmunoResearch). Afterward, the cells were washed twice, resuspended in 200 μl flow cytometry buffer, and analyzed. Samples were acquired by Cytoflex flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo (FlowJo X 10.0.7r2, Tree Star, Ashland, OR, USA) software.

Immunofluorescence microscopy

C. albicans yeast/hyphae were grown as described above and washed twice in ice cold PBS. Hyphal cells were grown overnight at 37 °C with RPMI-1640 supplemented with 10% FCS (Sigma-Aldrich) and 1% penicillin-streptomycin solution (100 U/ml; Biological Industries). Cells (2 × 10⁵) were incubated in the presence of FITC (0.1 mg/ml, diluted in PBS) for 30 min, washed twice in ice-cold PBS (3000 G, 4 °C, 5 min) and blocked using PBS/5% BSA for 2 h at 4 °C. Next, the cells were washed again in PBS followed by incubation with hCD48-Fc or the negative control protein D1-Fc for 2 h at 4 °C. Unbound protein was then washed with ice cold PBS and incubated with the secondary antibody PE-anti-human IgG (1:500; Jackson ImmunoResearch) for 2 h at 4 °C and then washed with PBS. Finally, cells were mounted with DAPI mounting media and visualized using an Olympus Fluoview FV1000 confocal microscope.

Generation of BW-hCD48 cells

Reporter cells for hCD48 (BW-hCD48) were generated as previously described⁶⁹. The inserts for the expression vectors encoding CD48 and zeta chain were generated by PCR using the following primers:

1-CD48 forward: cccAAGCTT GCCACC ATGTGCTCCAGAGGTTGGGA

2-CD48 reverse containing additional sequences complementary to zeta chain forward: TTCCATCTAGCAAGTAGCAGAG GGACCGGGCCAGGGTACAGGG

3-Zeta chain forward containing additional sequences complementary to CD48 reverse CCCTGTACCCTGGCCCGGTCC CTCTGCTACTTGCTAGATGGAA

4-Zeta chain reverse gggCTCGAG TTAGCGAGGGGCCAGGGTCTGC.

The full construct was then introduced into a plasmid with an antibiotic resistance (Puromycin) and inserted into BW5147.3 cell line (BW) using electroporation. Cells were cultured in RPMI-1640 supplemented with 10% FCS, penicillin-streptomycin solution (100 u/ml; Biological Industries) and gentamycin (5 µg/ml, G418; Sigma-Aldrich) for selection. Before each experiment, cells were tested by flow cytometry for CD48 expression as described above.

BW reporter assay

WT and C. albicans mutant strains: als1Δ/Δ1467, als2Δ/Δ 2757, als3Δ/Δ 1843, als4Δ/Δ 2034, als5Δ/Δ 2373, als6Δ/Δ 1420, als7Δ/Δ 1429, als9Δ/Δ 2028 and the C. albicans clinical strains were grown as described above. The fungal cells (5 × 10⁴ yeast/well) were first incubated in a 96 flat-bottom plate for 2 h at 37 °C to adhere to the plate. Then, BW or BW-hCD48 cells were added (MOI 1) in a final volume of 200 μl RPMI-1640 growth media as described above and for an additional 48 h (37 °C; 5% CO₂). The medium was supplemented with fluconazole (5 µg/ml) to prevent hyphae formation. After 48 h cells were centrifuged and supernatants were collected for measurement of released mIL-2 by ELISA.

ELISA assays

Cytokine levels in the serum or in homogenized kidneys of mice infected with C. albicans were determined 2 dpi by commercial kits for IFN-γ (Peprotech, Rehovot, Israel), IL-6 (ELISA MAX™ Deluxe Set Mouse IL-6, BioLegend) according to the supplier’s instructions.

mIL-2 levels in the supernatants from BW and BW-CD48 cells were determined by in-house ELISA. ELISA plates were initially coated with anti-mouse-IL-2 (0.1 μg/100 µl PBS; BioLegend) for 2 h, 37 °C. and blocked with PBS/1% BSA for an additional 2 h at room temperature. Supernatants were then added to the plates and incubated overnight at 4 °C. The plates were then washed 4 times with PBST (PBS/0.05% tween20) and a detection antibody biotinylated anti-mouse-IL-2 (0.1 μg/100 µl PBS; BioLegend) from a different clone than the coating antibody was added for 1 h at room temperature. Next the plates were washed 6 times with PBST, incubated for 30 min with Streptavidin-HRP (1:1000; Jackson ImmunoResearch) and again washed 6 times using PBST. Finally, the plates were developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (SouthernBiotech) and read at 650 nm using an ELISA reader (BIO-TEK, Winooski, VT, USA).

Quantification and statistical analysis

If not indicated otherwise, experiments were always performed in triplicates and repeated at least three times. Due to strong eosinophil donor variability, data from experiments with human eosinophils are shown as representative of three or more independent experiments performed with different donors. Results are expressed as mean ± SD or mean ± SEM and statistical tests were performed using Prism (GraphPad Prism 10.5.0.730) software. The unpaired two-tailed Student’s t test was used to compare differences between two groups. One-way ANOVA with Tukey’s test for multiple comparisons was used to evaluate experiments containing more than two groups. Survival curves were analyzed by Log-rank (Mantel Cox) test. P values less than 0.05 were considered significant for all analyses.

Ethics statement

Peripheral blood eosinophils were obtained according to the guidelines of the Hadassah-Hebrew University Human Experimentation Helsinki Committee (0410-14-HMO). All work involving human patients was approved by the Tel Aviv Sourasky Medical Center Institutional Ethics Committee (approval number 0729-16). The need for informed consent was waived given the retrospective observational nature of this study.

Mice experiments were performed according to the guidelines of the Hebrew University ethics committee for animal studies (MD-19-15990-5).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.