The murine monocyte-macrophage cell line RAW264.7 (obtained from Guangzhou Ye Shan Biotechnology Co.) was utilized in this study. The RAW264.7 cell line used in this study was derived from BALB/c mice, specifically isolated from peritoneal macrophages. Cells were cultured in DMEM supplemented with penicillin-streptomycin and maintained at 37°C in a 5% CO² atmosphere.

Fetal bovine serum (Hyclone, SH30087.01), DMEM-High Glucose Culture Medium (Hyclone, SH30022.01B), Penicillin-Streptomycin (Hyclone, SH30010), PBS Phosphate Buffered Solution (Hyclone, SH30256.01B). Sabouraud Dextrose Agar Plates (Guangdong Huan Kai Microbial Technology Co, CP0170). DHR123 (Sigma, D1054). Calcofluor White (Sigma, 18909-F). Dexamethasone (Source Leaf Biotech, S17003). Rapamycin (Cell Signaling, 9904S). Bafilomycin A1 (Selleck, S1413). Mouse IL-6 (Interleukin-6) ELISA Kit (Solarbio, SEKM-0007). Mouse TNF-α (Tumor Necrosis Factor-alpha) ELISA Kit (Solarbio, SEKM-0034). Anti-LC3B Antibody - Autophagosome Marker (Abcam, ab48394). Anti-mTOR (phospho S2448) Antibody [EPR426(2)] (Abcam, ab109268). p-mTOR Antibody (296.Ser 2481) (Santa Cruz, sc-293132). Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb (Cell Signaling Technology, 9234P). Phospho-p70 S6 Kinase (Ser371) Antibody (Cell Signaling Technology, 9208T). Phospho-Raptor (Ser792) (E4V6C) Rabbit mAb (Cell Signaling Technology, 89146). Phospho-S6 Ribosomal Protein (Ser240/244) (D68F8) XP^® Rabbit mAb (Cell Signaling Technology, 5364). Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP^® (Cell Signaling Technology, 4858). Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Cell Signaling Technology, 2855T). AO-EB Dual Staining Kit (Solarbio, CA1140). Gelatin (Sigma, G1393). Premo™ Autophagy Sensor (Thermo Fisher Scientific, P36235).

Candida glabrata was cultured in SDA (Sabouraud Dextrose Agar) medium and incubated at 37°C for 24 hours. Petri dishes were subsequently rinsed with sterile saline, and the liquid containing Candida glabrata was collected into centrifuge tubes. After centrifugation, the cells were ground, quantified using a haematocrit technical plate, and finally prepared into a suspension with a concentration of 10⁸ CFU (Colony-Forming Unit)/ml. The Candida glabrata suspensions were subjected to heat inactivation by incubating in a water bath at 56°C for 30 minutes. The inactivated Candida glabrata suspensions were stained with 0.1 mg/mL CFW (Calcofluor White) for 30 minutes at room temperature, washed twice with PBS, and utilized for fluorescence microscopy. For Candida glabrata spore preparation, spores were incubated with 30 µmol/L DHR123 (Dihydrorhodamine 123) at 37°C for 30 minutes, followed by centrifugation, washing with PBS, heat inactivation, and subsequent preparation for phagocytosis detection using flow cytometry.

Mouse monocyte-macrophage leukemia cells Raw264.7 (10⁵/ml) were co-cultured with Candida glabrata stained with CFW (10⁶CFU/ml) for 30 minutes, 1 hour, 2 hours, and 4 hours. At each time point, three randomly selected fields of view were captured, and the phagocytosis rate of Candida glabrata by macrophages was assessed. The percentage of cells phagocytosing more than three fungi was quantified under a 40x microscope. The phagocytosis rate was calculated as the number of macrophages phagocytosing Candida glabrata divided by the total number of macrophages, multiplied by 100%. The percentage of Candida glabrata with more than three fungi was determined as the number of macrophages phagocytosing three or more Candida glabrata divided by the total number of macrophages, multiplied by 100%.

Mouse monocyte-macrophage leukemia cells Raw264.7 (10⁵/ml) were co-cultured with DHR123-labeled Candida glabrata (10⁶ CFU/ml) for various time points (30 min, 1 h, 2 h, and 4 h). Gating based on FSC (Forward Scatter) and SSC (Side Scatter), phagocytosis was assessed, and 10,000 cells were collected per sample. The acquired data were analyzed using FlowJo to determine the proportion of cells positive for fluorescence and the Mean Fluorescence Intensity (MFI) for statistical evaluation.

Mouse monocyte macrophage leukemia cells Raw264.7 (10⁵/ml) were co-cultured with inactivated Candida glabrata (10⁶ CFU/ml) to generate the Candida suspension. Dexamethasone was introduced to the Candida suspension at a concentration of 200 ng/µl, followed by the addition of 100 µM rapamycin. To establish a baseline, Raw264.7 cells infected without Candida glabrata were designated as the control group (“C”). The experimental groups included Raw264.7 cells infected with Candida glabrata, labeled as the “Cg” group. Measurements were conducted at 6, 12, and 24 hours of co-cultivation. Another group, designated as “DEX+Cg”, consisted of Raw264.7 cells infected with Candida glabrata and pre-treated with dexamethasone, with measurements taken at the same time intervals. The third group, “DEX+Rapa+Cg”, involved Raw264.7 cells infected with Candida glabrata and pre-treated with dexamethasone and rapamycin, with measurements at 6, 12, and 24 hours of co-cultivation.

In our study, Raw264.7 cells were co-cultured with DHR123-labeled Candida glabrata at a ratio of 1:10. The untreated cell group served as the control, denoted as “1:10Cg”. Raw264.7 cells were seeded at a density of 1.5×10⁵/dish and pretreated for 24 hours under different conditions: the control group (“1:10Cg”), and experimental groups: “10DEX1d+1:10Cg”, “100DEX1d+1:10Cg”, and “200DEX1d+1:10Cg”, with dexamethasone concentrations of 10ng/µl, 100ng/µl, and 200ng/µl, respectively. Additionally, Raw264.7 cells were pretreated with 100 µmol/L rapamycin for 12 hours and then co-cultured with Candida glabrata. This group of cells was labeled as “Rapa 12h+1:10Cg”. Macrophage phagocytosis of Candida glabrata was investigated at different time points (0.5h, 1h, 2h, and 4h) during co-culture.

Levels of IL-6 and TNF-α in the supernatant from Raw264.7 macrophage cell cultures and Candida suspension were assessed using ELISA (Solarbio, SEKM-0034) and ELISA (Solarbio, SEKM-0007). The Candida suspension was co-treated for 6h, 12h, and 24h, respectively. Direct supernatant (100 µL) and diluted samples (20-50 times) were subjected to analysis on ELISA plates. Cytokine concentrations were determined in accordance with the manufacturer’s instructions.

Protein extraction from the Candida suspension was performed using Radio-Immunoprecipitation Assay (RIPA) lysate. The protein concentration was determined, and the collected protein samples were supplemented with an appropriate amount of concentrated Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Protein Sampling Buffer. The mixture was heat-treated in a boiling water bath for 3-5 minutes to achieve complete protein denaturation. After cooling to room temperature, the protein samples were directly loaded into the SDS-PAGE wells. Separated proteins from the gel were transferred onto a membrane (Polyvinylidene difluoride - PVDF) via electroblotting. Non-specific binding sites on the membrane were blocked by incubating it with a blocking solution. The membrane was then incubated with a primary antibody specific to the target protein. Following this, the membrane was washed to remove unbound primary antibodies. Subsequently, the membrane was incubated with a secondary antibody conjugated to an enzyme, and unbound secondary antibodies were washed away. Protein detection was achieved using BeyoECL Plus reagents. The resulting film was scanned or photographed and analyzed by Quantity One for determining the molecular weight and net optical density values of the target bands.

A working solution of Acridine Orange (AO) and Ethidium Bromide (EB) was prepared by mixing the two dyes in a ratio of 1:1. After treatment, Raw264.7 cells were harvested and washed with PBS. The AO/EB working solution was then added to the cell pellet, and the cells were incubated for 2-5 minutes at room temperature in the dark. Stained cells were visualized under a fluorescence microscope using appropriate filter sets for AO (green fluorescence) and EB (red fluorescence). Digital images of the stained cells were captured using a fluorescence microscope equipped with a camera. The percentage of viable cells was quantified based on the intensity and morphology of the fluorescence signals.

Add 2 μL of Premo™ LC3B-GFP Kit Autophagy Sensor (Component A) medium per 10,000 Raw264.7 cells and incubate the cells at 37°C for 16 hours. The different stages of autophagy were monitored using the Premo™ Autophagy Sensor GFP-LC3B Kit (Thermo, P36235) following the manufacturer’s instructions.

Utilizing laser confocal microscopy, we conducted a comprehensive examination of macrophage fluorescence, emphasizing both quantification and spatial distribution subsequent to transfection with GFP-LC3B baculovirus. Moreover, the visualization of macrophage viability was achieved through the observation of live and dead cells stained with Acridine Orange and Ethidium Bromide (AO/EB) using laser confocal microscopy.

Statistical analysis was conducted using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA). ELISA, AO/EB staining, Western blot, and macrophage phagocytosis of Candida glabrata were assessed, with data presented as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was employed to compare data among three or more groups. Differences were considered statistically significant when the p-value was less than 0.05.

Under Candida glabrata stimulation, the secretion of TNF-α and IL-6 by macrophages exhibited a time-dependent increase, demonstrating statistical significance (P<0.05, Figures 1A, B ). This indicates that Candida glabrata induces the secretion of pro-inflammatory factors TNF-α and IL-6 by macrophages. To explore the impact of glucocorticoids on intrinsic immunity during Candida glabrata phagocytosis by macrophages, cells were pre-treated with the glucocorticoid dexamethasone. The co-culture of macrophages with Candida glabrata for 24 hours revealed a significant inhibition of TNF-α and IL-6 expression levels by glucocorticoids (P<0.05, Figures 1A, B ). This suggests that glucocorticoids suppress intrinsic immunity during Candida glabrata infection. To investigate whether glucocorticoids inhibit intrinsic immunity by influencing macrophage autophagy, macrophages were pre-treated with the autophagy inducer rapamycin. Under the influence of glucocorticoids, rapamycin further decreased the expression levels of IL-6 and TNF-α when macrophages were co-cultured with Candida glabrata for 24 hours (P<0.05, Figures 1A, B ).

The viability of macrophages significantly decreased (P<0.01, Figures 1C , 2 ) after 24 hours of co-culture with Candida glabrata. This observation aligns with previous findings, indicating a notable surge in IL-6 and TNFα secretion by macrophages at the 24-hour co-culture mark compared to earlier time points ( Figures 1A, B ). In contrast, when macrophages were co-cultured with Candida glabrata for 6 and 12 hours, treatment with glucocorticoids resulted in a remarkable increase in macrophage viability (P<0.01, Figures 1C , 2 ). Furthermore, under the influence of glucocorticoids, rapamycin treatment significantly enhanced the macrophage viability (P<0.01, Figures 1C , 2 ).

Excessive production of proinflammatory factors has been associated with macrophage apoptosis. Both rapamycin and glucocorticoids are known for their robust immunosuppressive properties. Therefore, it is postulated that glucocorticoids and rapamycin function to inhibit macrophage apoptosis during the phagocytosis of Candida glabrata.

In the context of glucocorticosteroid influence on macrophages, the phagocytosis rate of Candida glabrata by macrophages and the proportion of macrophages engulfing more than three yeast cells significantly increased at 4h compared to 0.5h, 1h, and 2h (P<0.05, Figure 3B ). The number of Candida glabrata phagocytosed by macrophages demonstrated a time-dependent escalation ( Figure 3 ). Dexamethasone treatment hindered the phagocytosis of Candida glabrata by macrophages (P<0.05, Figures 3–5), but the phagocytosis rate was not distinct under varying glucocorticosteroid concentrations ( Figure 3A ). The autophagy inducer rapamycin had no substantial impact on the phagocytosis of Candida glabrata by macrophages ( Figure 3 ). For co-culture durations of 0.5h and 1h, rapamycin marginally inhibited the phagocytosis, with no discernible difference ( Figure 3 ). However, for 2h and 4h co-culture, rapamycin enhanced the phagocytosis, yet without significant differentiation ( Figure 3 ).

Dexamethasone exhibited a significant inhibitory effect on the phagocytosis of Candida glabrata by macrophages. Interestingly, the impact of the autophagy inducer rapamycin on phagocytosis displayed a dual nature, showing both inhibitory and promotional effects on Candida glabrata phagocytosis by macrophages ( Figure 3 ). Consequently, further investigation is warranted to elucidate the specific interactions between dexamethasone and autophagy in this context.

During cellular autophagy, the transition of the LC3BI (LC3 cytoplasmic phenotype) to a LC3BII (LC3 membrane phenotype) occurs. This process, impeded by the BAF-A1 (lysosomal inhibitor bafilomycin-A1) at the final step of autophagy, leads to the LC3BII/LC3BI or LC3BII/GAPDH(Glyceraldehyde 3-phosphate dehydrogenase) ratio, serving as an indicator of autophagy levels. The ratio positively correlates with the extent of autophagy. LC3B predominantly localizes to the cell membrane of macrophages( Figure 6A ). Under Candida glabrata stimulation, dexamethasone significantly reduced the accumulation of LC3BII in macrophages, indicating the inhibition of macrophage autophagy(P<0.01, Figures 6 , 7 ). These differences were statistically significant after co-cultivation of Candida glabrata with macrophages for 6h, 12h, and 24h (P<0.01, Figures 6 , 7 ). Additionally, the autophagy inducer rapamycin enhanced the accumulation level of LC3BII in macrophages (P<0.05, Figures 6, 7), highlighting its potential to promote macrophage autophagy.

In our preceding experiments, we established that glucocorticoids hinder macrophage phagocytosis of Candida glabrata by suppressing macrophage autophagy. To delve deeper into the mechanistic link between glucocorticoids and cellular autophagy regulation through the MTOR pathway, we investigated alterations in the MTOR pathway within each macrophage group. As pivotal serine/threonine kinases, the MTOR pathway serves as a principal regulator of cellular metabolism, and its inhibition is known to induce autophagy.

Upon co-culturing Candida glabrata with macrophages, we observed that Candida glabrata triggered the activation of the MTOR pathway ( Figure 8 ). However, this activation diminished over time. Conversely, when Candida glabrata and macrophages were co-cultured and subjected to glucocorticoid stimulation, the inhibitory impact of glucocorticoids on the MTOR pathway showed a time-dependent reduction, and by 24 hours, glucocorticoids exhibited an activating effect on the MTOR pathway ( Figure 8 ). Furthermore, under glucocorticoid stimulation, co-culturing Candida glabrata with macrophages and applying rapamycin resulted in the inhibition of the MTOR pathway ( Figure 8 ).