Healthy donors were informed and gave their written consent to participate in the present study. The study protocol was reviewed and approved by the Ethics Committee of Lille University Hospital. The study was conducted according to the principles expressed in the Declaration of Helsinki.

Whole blood was collected from healthy donors. Platelets were isolated by differential centrifugation and then washed in modified Tyrode's buffer (2.7 mM KCl, 3.3 mM Na₂HPO₄, 137 mM NaCl, 1.2 mM NaHCO₃, 1 mg/ml bovine serum albumin, 3.8 mM HEPES, 5 mM glucose) (Byzova and Plow, 1997).

C. albicans strain SC5314 was used in this study and maintained at 4°C in yeast peptone dextrose broth (YPD; 1% yeast extract, 2% peptone, 2% dextrose). To prepare the yeast suspension, C. albicans cells were cultured in Sabouraud dextrose broth (Sigma-Aldrich, St. Quentin Fallavier, France) for 24 h at 37°C in a rotary shaker. C. albicans cells were harvested by centrifugation. After washing several times with phosphate-buffered saline (PBS), C. albicans cells were resuspended in PBS.

C. albicans cell pellets were washed twice in PBS. Chitin was extracted from C. albicans yeast cells as described previously (Charlet et al., 2018b). Briefly, 20 mL of 10% KOH was added to the C. albicans cell pellet. This procedure was repeated twice and the pellet was autoclaved at 120°C for 2 h. After washing with distilled water, the supernatant was removed and 50% acetic acid and 50% hydrogen peroxide were added to the pellet, which was then autoclaved at 120°C for 2 h. After washing with distilled water, the pellet was centrifuged and the chitin fraction was lyophilized. Nuclear magnetic resonance (NMR) analysis was performed to confirm the nature of the chitin. Intact chitin purified from C. albicans was added to deuterated hexafluoroisopropanol (Euriso-Top) at 70°C until dissolved. All experiments were performed using a Bruker Avance 600 MHz (13.1 T) spectrometer with Bruker standard pulse programs. MALDI-TOF mass spectra were acquired on a Voyager Elite DE-STR mass spectrometer (Perspective Biosystems, Framingham, MA). A mixture of matrix (1 μL containing 10 mg/mL DHB and 5% acetonitrile) and 1 μL of sample was deposited on a MALDI plate. Small and large soluble chitin fragments were detected by MALDI-TOF mass spectrometry. A BiCinchoninic acid assay was employed to determine the chitin concentration extracted from C. albicans (Le Devedec et al., 2008). The standard range for the N-acetyl-glucosamine (GlcNAc) control was 0.1–5 mg/mL.

Neutrophils were prepared from the peripheral blood of healthy donors by gradient centrifugation, according to the protocol of Pluskota et al. (2003). Fresh platelet poor plasma (200 μL) was added to each well of a 96-well plate and incubated for 2 h at 37°C (Clark et al., 2007). After several washes, a suspension of 10⁶ neutrophils in 200 μL of RPMI medium containing 1 mM Mg²⁺/Ca²⁺ was added to each well of a 96-well plate coated with platelets. Non-adherent cells were removed and the wells were washed with Hank's balanced salt solution (HBSS) (Bouaouina et al., 2004). To assess platelet adhesion to C. albicans, 200 μL of RPMI medium containing 10⁵ C. albicans cells was added to each well of a 96-well plate. This plate was then incubated at 37°C to allow C. albicans to adhere to the bottom of the plate. Thrombin-stimulated platelets (10⁶ cells/mL) were labeled with calcein and then pre-treated for 30 min with chitin (20–200 μmol/L) incubated at 37°C for 20 min (Clark et al., 2007). After several washes, the percentage platelet adhesion to C. albicans or neutrophils was examined using a fluorometer (FLUOstar® Omega; BMG Labtech, Champigny sur Marne, France). To examine platelet adhesion to C. albicans by confocal microscopy, washed human platelets were stained with calcein and C. albicans cells were labeled with 5B2 monoclonal antibody (1:2,000 dilution) (Vancraeyneste et al., 2016). Slides were examined by confocal microscopy (Zeiss LSM710). For THP-1 cell culture, THP-1 cells were incubated in RPMI 1640 medium (Gibco by Life Technologies™, France) supplemented with 10% fetal bovine serum, 50 IU/mL penicillin and 50 IU/mL streptomycin. THP-1 cells were differentiated into macrophages in the presence of phorbol-12-myristate 13-acetate (100 nM; Sigma-Aldrich, St. Quentin Fallavier, France) at 37°C and 5% CO₂.

Washed human platelets (10⁸ cells/mL) were pre-treated with chitin (20 μmol/L) for 30 min in HBSS and then treated with thrombin (0.2 U/mL; Sigma-Aldrich, St. Quentin Fallavier, France) to promote platelet aggregation (Cambi et al., 2008). Platelet aggregation was assessed continuously after thrombin addition for 5–10 min using an aggregometer (Labor Biomedical Technologies Gmbh, LABITEC APACT 4004).

Ca²⁺ measurements were performed as described previously (Hussain and Mahaut-Smith, 1999). Briefly, 5 μM fura-2 acetoxymethyl ester (Invitrogen, France) and 0.2 μg/mL Pluronic F-127 were added to washed platelets pre-treated with different concentrations of chitin (20, 50, 100, or 200 μg/mL) in HBSS without Ca²⁺ at 37°C for 30 min. Platelets were then washed once and resuspended in HBSS. After platelet activation with thrombin (0.05 U/mL), the Ca²⁺ response was measured using a fluorometer (FLUOstar® Omega; BMG Labtech, Champigny sur Marne, France). The ratio of absorbance at 340/380 nm was converted into Ca²⁺ concentration (Ca²⁺).

This was performed in RPMI medium (300 μL) containing 5 × 10⁵ platelets/mL; RPMI medium does not induce platelet activation (Speth et al., 2013). Platelets were pre-treated with chitin at different concentrations (20–200 μmol/L) for 2 min and then activated with thrombin (0.05 U/mL). The activation or expression of platelet receptors was assessed using specific or isotype antibodies linked to fluorochromes (anti-human/mouse CD62P P-selectin, anti-human PAC-1, anti-mouse PE IgG1k, Brilliant Violet 421 anti-human CD41; Ozyme, France). After incubation for 30 min in the dark, platelets were washed twice with 300 μl PBS. The platelets were resuspended in 300 μL PBS. Specific isotype controls (mouse IgG1, κ Isotype Control Biolegend®, and mouse IgG1, κ Isotype Control BD Parmagen™) were used in each flow cytometry experiment. The expression/activation of platelet receptors was determined by flow cytometry (BD LSRFortesa® X20). Unstimulated or stimulated platelets with thrombin (0.05 U/mL) were used as controls for each experiment. The data obtained were analyzed using Kaluza software®. To examine the expression of platelet receptors by confocal microscopy, washed human platelets were labeled with TLR monoclonal antibodies (Vancraeyneste et al., 2016). Specific slides (wells 6.7 mm; Thermo Scientific, France) were used in this experiment and the coverslips were examined by confocal microscopy (Zeiss LSM710, Zeiss Airyscan SR mode x6311.4).

A Nucleospin RNA/Protein kit (Macherey-Nagel, France) was used to isolate total RNA and proteins from platelets. Quantification of RNA in each sample was performed by spectrophotometry (Nanodrop; Nyxor Biotech, France). Reverse transcription of mRNA was performed in a final volume of 20 μL containing 1 μg total RNA (high capacity cDNA RT kit; Applied Biosystems). cDNA synthesis was performed according to the High Capacity DNA Reverse Transcription (RT) protocol, using Master Mix (Applied Biosystems). SYBR green dye intensity was determined using one-step software (Jawhara et al., 2008). All results were normalized to the reference gene, GAPDH. For Western blot analysis, proteins were recovered in lysis buffer (Tris/HCl 50 mM; Triton-X100, 1%; SDS 0.1%; EDTA 5 mM; NaCl 150 mM). The protein content of each sample was estimated using a BiCinchoninic acid protein assay (Pierce) and adjusted to the same protein concentration prior to analysis by SDS-PAGE on a 10% acrylamide gel [18]. Proteins are transfered onto a nitrocellulose membrane (iBlot 2; Thermofisher Scientific). Membranes were blocked with TBS-BSA 3% for 1 h and then incubated overnight with monoclonal anti-TLR (dilution 1:250) (TLR4 Biotechne® TLR8 GeneTex®). Horseradish peroxidase-labeled secondary antibody (1:5,000 dilution) (Southern Biotech) were used to detect monoclonal antibodies.

Statistical analysis was performed using XLSTAT and Prism 4.0 (GraphPad). Data were analyzed using the Mann-Whitney U test to compare pairs of groups. The results are expressed as the mean ± SD of individual experimental groups. Differences were considered significant when the P-value was < 0.05.

Platelets can bind directly to different pathogens through different surface glycoproteins. In order to evaluate the effect of chitin on the C. albicans-platelet adhesion process, we used an increasing concentration of fungal chitin ranging from 10 to 200 μg/mL.

These different chitin concentrations were used in the present study as there as there is no routine clinical test to measure the concentration of circulating chitin in the blood of patients with candidaemia.

Increasing concentrations of fungal chitin were associated with a decrease in percentage platelet adhesion to C. albicans (Figure 1). This decrease was significant for chitin concentrations ≥20 μg/mL. Activated platelets are crucial for the activation of neutrophils and in neutrophil-mediated inflammatory responses. The impact of chitin on platelet-neutrophil interactions was assessed. Platelets pre-treated with chitin (50, 100, or 200 μg/mL) showed a significant decrease in adhesion to neutrophils (Figure 1).

In order to determine the effect of chitin on platelet aggregation involving α_IIbβ₃ integrin, platelets were pre-exposed to different chitin concentrations (50, 100, or 200 μg/mL) for 30 min and then stimulated with 0.2 U/mL thrombin (Figure 2A). A decrease in the percentage of platelet aggregation was observed in the presence of fungal chitin with a dose-dependent effect. There was a significant decrease in aggregation in the presence of chitin at a concentration of 100 or 200 μg/mL (Figure 2B).

The effect of chitin on platelet activation was determined using fura-2, which exhibits an increase in fluorescence intensity on binding to intracellular Ca²⁺. The concentration of intracellular calcium in platelets stimulated with thrombin (0.05 U/mL) was higher than that in unstimulated platelets (Figure 2C). Pre-treatment of platelets with chitin decreased the concentration of intracellular Ca²⁺ (Figure 2C). P-selectin expression, which is a crucial marker of platelet activation, was also investigated. Platelets were labeled with a specific CD41 marker by targeting the 2b chain of the alpha integrin of the GPIIb-IIIa complex. In the presence of thrombin, the majority of the CD41⁺ platelet population expressed P-selectin while platelets pre-treated with chitin showed a significant decrease in platelet activation proportional to the fungal chitin concentration, with a greater effect of chitin at a concentration of 200 μg/mL suggesting that chitin has a dose-dependent effect (Figure 3). In parallel, the activation of integrin α_IIbβ₃ was assessed in thrombin-stimulated platelets pretreated with chitin at a concentration of 20 μg/mL (Supplementary Data). A significant decrease in integrin α_IIbβ₃ activation was observed in thrombin-stimulated platelets when compared to those untreated with chitin. These data corroborate the aggregation assay showing that chitin pre-treatment reduces platelet aggregation.

Using PCR, we also investigated the expression of different TLRs that are known to sense many PAMP molecules, including chitin, promoting the host defense against pathogens (Figure 4). Western blot was then performed to confirm the expression of these receptors. A histone marker was used to exclude the presence of leukocytes in the platelet isolate. The human macrophage-differentiated THP-1 cell line was employed as a control. Platelet isolation was histone-free, indicating that the platelet preparation was not contaminated with leukocytes. In terms of mRNA expression levels for TLR2, TLR4, and TLR9, no significant difference was observed between platelets pre-treated with chitin (50 μg/mL) and untreated platelets (Figure 4). In contrast, TLR8 mRNA was highly expressed in thrombin-activated platelets pre-treated with chitin compared to that in untreated platelets, and this expression was concentration-dependent, indicating that chitin and thrombin stimulation modulate TLR8 mRNA expression.

The mRNA data correlated with those of Western blot analysis showing that TLR4 expression is independent of chitin exposure while high expression levels of TLR8 were detected in thrombin-activated platelets pre-treated with increasing concentrations of chitin (Figure 5). We performed quantification of Western Blot band intensity corresponding to platelets alone (CTL–), platelets in the presence of thrombin (CTL+) and thrombin-stimulated platelets pre-treated with chitin at different concentrations, using ImageJ bundled with 64-bit Java 1.8.0-112. The intensity of each band was normalized to that of beta-actin. We observed an increase in TLR8 band intensity with an increase in chitin concentration (Supplementary Data). In parallel, we examined the expression of TLR8 and TLR9 platelet receptors by confocal microscopy. In contrast to TLR9, TLR8 expression was only detected in thrombin-activated platelets pre-treated with chitin (Figure 5B).

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.