Macrophage-derived CD36 + exosome subpopulations as novel biomarkers of Candida albicans infection

Invasive candidiasis (IC) is a notable healthcare-associated fungal infection, characterized by high morbidity, mortality, and substantial treatment costs. Candida albicans emerges as a principal pathogen in this context. Recent academic advancements have shed light on the critical role of exosomes in key biological processes, such as immune responses and antigen presentation. This burgeoning body of research underscores the potential of exosomes in the realm of medical diagnostics and therapeutics, particularly in relation to fungal infections like IC. The exploration of exosomal functions in the pathophysiology of IC not only enhances our understanding of the disease but also opens new avenues for innovative therapeutic interventions. In this investigation, we focus on exosomes (Exos) secreted by macrophages, both uninfected and those infected with C. albicans. Our objective is to extract and analyze these exosomes, delving into the nuances of their protein compositions and subgroups. To achieve this, we employ an innovative technique known as Proximity Barcoding Assay (PBA). This methodology is pivotal in our quest to identify novel biological targets, which could significantly enhance the diagnostic and therapeutic approaches for C. albicans infection. The comparative analysis of exosomal contents from these two distinct cellular states promises to yield insightful data, potentially leading to breakthroughs in understanding and treating this invasive fungal infection. In our study, we analyzed differentially expressed proteins in exosomes from macrophages and C. albicans -infected macrophages, focusing on proteins such as ACE2, CD36, CAV1, LAMP2, CD27, and MPO. We also examined exosome subpopulations, finding a dominant expression of MPO in the most prevalent subgroup, and a distinct expression of CD36 in cluster14. These findings are crucial for understanding the host response to C. albicans and may inform targeted diagnostic and therapeutic approaches. Our study leads us to infer that MPO and CD36 proteins may play roles in the immune escape mechanisms of C. albicans. Additionally, the CD36 exosome subpopulations, identified through our analysis, could serve as potential biomarkers and therapeutic targets for C. albicans infection. This insight opens new avenues for understanding the infection's pathology and developing targeted treatments.


Results
The principal findings of this investigation are comprehensively delineated in Table 1

Differentiation of Thp-1 cells into M0 macrophages detected by high content and q-PCR
The induction of THP-1 cells into M0-type macrophages was observed post-treatment with 75 ng/ml PMA for 72 h.Morphological changes, evident in Fig. 1A-D, included a transition from smaller, rounded suspended cells to larger, spindle-shaped, wall-adherent cells with multiple pseudopods and protrusions.An increase in cell volume correlating with stimulation time was noted, indicative of successful differentiation into M0-type macrophages.
The upregulation of CD11b, a surface marker for M0-type macrophages, was confirmed via q-PCR.Figure 1E shows a 15.24-fold increase in CD11b expression following 72 h of PMA treatment, further validating the differentiation of THP-1 cells into M0-type macrophages.

PBA detection of exosome surface proteins
PBA was utilized to evaluate individual EVs and their protein expressions in both sample groups (Fig. 4A).No significant differences were observed between the groups.Post-normalization using the TMM method, Fig. 4B,C illustrates the protein expression levels, highlighting the proteins with significant differential expression in the heatmap (Fig. 4D).Thirteen proteins were upregulated, and four downregulated in the MO + CA group, with notable increases in ACE2, CD36, CAV1, LAMP2, CD27, and MPO, and decreases in ITGA1, ICAM4, SDC1, and AIF1.Significant elevations in CD36 and MPO were observed in the MO + CA group compared to the M0 group (Figs.4E,F).FlowSOM analysis of single exosomes identified distinct subgroups based on exosomal protein characteristics.The distribution and proportion of all subgroups within two groups and each of their samples were visualized using t-SNE dimensionality reduction (Fig. 4G,H).The situation of proteins that are more highly expressed in each subgroup is depicted in Fig. 4I.Notably, Cluster14, although representing a minor proportion in the M0 group (0.96% as shown in Fig. 4G), was significantly increased to 2.61% in the M0 + CA group.Within Cluster14, 75.43% originated from the M0 + CA group.Cluster14 was then distinctly highlighted in the t-SNE plot (Fig. 4J) for an in-depth analysis of protein expression within this cluster, leading to the generation of a chart depicting the detection frequency of each protein in the subgroup (Fig. 4K).This analysis revealed a relatively higher expression of CD36.Consequently, we have designated Cluster14 as the CD36 subpopulation for further investigation.

Discussion
Our data were indexed using bcl2fastq (Illumina) to convert the BCL file for each sample into fastq format and paired indexes.The complexTag, proteinTag, and moleculeTag were then extracted from the three fixed segments of each read.reads with only one count were excluded from the analysis.An in-house developed Perl script was then used to sequentially sort the tags based on complexTag, proteinTag, and moleculeTag.The number of exosomes with a particular protein combination was counted using an in-house developed R script.The applied t-SNE algorithm was implemented in the R package Rtsne with all parameters set to default values in our pipeline.
Recent studies increasingly underscore the significance of exosomes in both biophysiological and pathological processes, highlighting their evolving roles in diagnostics and therapeutics across a spectrum of diseases [20][21][22][23] .Furthermore, the exosomes secreted by host cells in response to fungal infections have garnered considerable attention in the scientific community, becoming a focal point of extensive research 24,25 .This growing body of www.nature.com/scientificreports/evidence reflects the expanding understanding of exosomal functions and their potential implications in disease management and treatment strategies.

MPO as a key factor in the immune evasion of C. albicans
In the context of increasing use of immunosuppressants, broad-spectrum antibiotics, and invasive procedures, the incidence of invasive fungal infections has risen significantly 26 .Among these, Candida species are predominant causative agents, with C. albicans being a common clinical species and a notable source of hospital-acquired  infections 27 Upon infection, C. albicans initiates immune responses through the recognition of its pathogenassociated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs).Endocytosis mediated by endothelial cells via N-cadherin plays a role in the internalization of C. albicans 28 , which can subsequently escape through vesicle phagocytosis or endothelial cell loss 29 .Then phagocytes, integral to the innate immune system, migrate from blood vessels to accumulate at sites of C. albicans infection 30 .This group primarily includes monocytes, macrophages, and neutrophils.Macrophages play a pivotal role in inhibiting and destroying C. albicans through mechanisms like nutrient deprivation, low pH environments, and oxidative stress 31 .Additionally, macrophages secrete immune factors that recruit more macrophages for the clearance of C. albicans 32 .However, C. albicans has developed various strategies to evade immune detection, diminish the effectiveness of antimicrobial responses, and escape from immune cells post-phagocytosis, though the specific mechanisms remain unclear 33 .Our study reveals that exosomes secreted by macrophages infected with C. albicans show a significant www.nature.com/scientificreports/increase in myeloperoxidase (MPO) expression compared to those from uninfected macrophages.MPO, a key enzyme secreted by neutrophils and macrophages, is linked to the severity and prognosis of various cardiovascular diseases 34 and is associated with NETosis, a form of cell death inducing the formation of extracellular traps (ETs) 35 .ETs, primarily identified in neutrophils as neutrophil extracellular traps (NETs), comprise components like histones, cathepsin G, neutrophil elastase, MPO, and others, crucial for pathogen capture and clearance 36 , are structures with bactericidal functions that occur by neutrophils stimulated by PMA, endotoxin, etc., and are known as neutrophil extracellular traps (NETs).The main components of ETs include histones (H1, H2A, H2B, H3, and H4), cathepsin G (CG), neutrophil elastase (NE), MPO, calcineurin, lactoferrin, and gelatinase 37 .These components are important for facilitating pathogen capture and clearance, with NE, MPO, and H3 being the major antimicrobial component proteins in NETs.The release of ETs is not limited to neutrophils but can also be released when other immune cells are exposed to different stimuli, including macrophages, mast cells, and others.In this paper, after THP-1 cells formed M0 macrophages after PMA stimulation, the exosomes produced by them expressed MPO, which may indicate that macrophages have macrophage extracellular traps (METs) released under PMA stimulation, while the exosomes produced by M0 macrophages after C. albicans infection expressed MPO was significantly increased, which may suggest that macrophages have increased release of METs under the dual stimulation of PMA and C. albicans.Bacterial and Fungal Infections Significantly Increased When MPO and NE Genes Were Knocked Out in Mice 38 , and it has been demonstrated that the formation of METs provides an immune escape route for C. albicans 39 .Thus, MPO emerges as a potential target for understanding and combating C. albicans immune escape mechanisms.

CD36 subpopulations as a potential biomarker for C. albicans Infection
CD36, a widely expressed scavenger receptor on various immune and non-immune cells, mediates numerous biological processes including inflammation, angiogenesis, atherosclerosis, and innate immunity 40,41 .Acting as a pattern recognition receptor (PRR) on macrophages, CD36 is involved in the phagocytosis and elimination of pathogens 42 .Additionally, CD36 recognizes endogenous ligands such as thrombospondin-1 (TSP-1), amyloid, advanced glycation end-products (AGEs), and advanced oxidation protein products (AOPPs) 43 .These ligands, indicative of cellular oxidative stress and lipid or protein denaturation, upon binding to CD36, trigger several pathophysiological responses, including inflammation and intracellular lipid accumulation 44,45 .Given its multifunctionality, CD36 is a promising biomarker for numerous diseases.
In the realm of gene expression, CD36 can be modulated by various environmental stimuli, transcription, post-translational modifications, or translocation to the plasma membrane 41 .Our study revealed a significant differential expression of CD36 in exosomal subpopulations between two groups, with a notably high expression level of CD36.This observation indicates that CD36 subpopulations may serve as viable biomarkers for the early detection of C. albicans infection, a hypothesis that merits further investigation and validation through future studies utilizing animal models.Moreover, CD36 may be implicated in another form of immune escape by C. albicans, known as cellular pyroptosis.Following phagocytosis by macrophages, C. albicans transforms within phagosomes from yeast to mycelium morphology.When mycelium growth within the phagosomes exceeds the phagosomal membrane's capacity, the membrane ruptures, activating the host's Nlrp3 inflammasome 46 .The structural interaction between receptor proteins and their ligands serves as a bridge, connecting receptor and effector proteins, which induces the activation of the inflammatory protease caspase-1.Caspase-1 cleaves gasdermin D, which then binds to phospholipoproteins on the cell membrane, forming pores and releasing a significant amount of pro-inflammatory factors.This cascade results in cell lysis and pyroptosis 39 .Notably, in atherosclerosis 47 , CD36 has been identified to play a dual role in both the initiation and activation of Nlrp3 inflammasomes, a hallmark of cellular pyroptosis.While the precise mechanism remains to be fully elucidated, it is plausible that CD36 also plays a crucial role in mediating immune responses following C. albicans infection in macrophages.Consequently, CD36 emerges not only as a promising biomarker for C. albicans infection but also as a potential target for therapeutic intervention in fungal infections and associated inflammatory responses.

Conclusion
In conclusion, employing the PBA approach enabled us to analyze millions of individual exosomes per sample, facilitating the screening for biomarkers of C. albicans infection at the level of individual exosomes.Our findings suggest that Myeloperoxidase (MPO) may play a role in the immune escape mechanisms of C. albicans, and that CD36 holds potential as a biomarker for detecting such infections.We observed that C. albicans infection alters the surface protein profiles of exosomes in both study groups.These alterations could represent a compensatory mechanism that promotes inflammatory and immune responses, thereby enriching our understanding of the immune mechanisms involved in C. albicans infections.Furthermore, to validate these early diagnostic markers and therapeutic targets, and to confirm the functional roles of MPO and CD36, multicenter studies involving both animal models and patient populations are essential.Future research should focus on elucidating the molecular mechanisms of MPO and CD36, which will deepen our understanding of their roles in C. albicans infections.

Materials and methods
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to.No ethical approval was required as the research in this article related to micro-organisms.
The flowchart of this paper is shown in Fig. 5. www.nature.com/scientificreports/frozen storage tubes, were inoculated into 5 ml of YPD liquid medium in a clean room biosafety cabinet.The culture was maintained at 37 °C on a shaker at 150 rpm overnight to ensure the strain reached the logarithmic growth phase.The culture was then centrifuged at 1500 rpm for 10 min; the supernatant was discarded, and the cells were washed twice with PBS.A 0.9% sodium chloride solution was used to adjust the bacterial solution concentration to 2 × 10 8 CFU/mL 48 .

Cell culture and induction
Frozen THP-1 cell lines were resuscitated in T300 culture flasks at 37 °C in a humid environment containing 5% CO 2 using RPMI-1640 medium (containing 1% double antibody, 10% fetal bovine serum, and 0.05 mM β-mercaptoethanol) 49 .Upon reaching a cell density of 8 × 10 5 cells/ml, the cells were centrifuged at 1000 rpm for 5 min, and the medium was replaced with fresh THP-1 complete medium.Cells were then distributed into 6-well plates at 2 mL per well, and Phorbol-12-Myristate-13-Acetate (PMA) was added to achieve a concentration of 75 ng/ml 50 .Cells were incubated at various time points (0 h, 24 h, 48 h, 72 h) for high internal culture and imaging.

q-PCR to detect Thp-1 cell differentiation
RNA was extracted using TRIzol reagent, and the total RNA content was measured using a nucleic acid quantifier.Reverse transcription was performed following the instructions of the Reverse Transcription Kit (Item No.7E652J2, Novozymes).The q-PCR reaction conditions were set at 37 °C for 45 min, followed by 85 °C for 5 s.The reaction products were preserved for subsequent experiments.q-PCR was performed according to the Realtime PCR Master Mix Kit (SYBR Green Realtime PCR Master Kit, Toyobo), with GAPDH as the internal reference.The primer sequences 51 are shown in Table 2:

C. albicans stain
A 1 mL sample of C. albicans (2 × 10 8 CFU/ml) was centrifuged and the supernatant discarded; the cells were washed twice with wash solution and then centrifuged (500 g, 5 min, 4 °C).The cells were resuspended in 1 mL of wash solution and added to pHrodo Deep Red stain 52 , shaken until dissolved, and incubated at 22.5 °C for 2 h.After adding 1 mL of DB medium to resuscitate the fungus, the cells were centrifuged again to discard the supernatant.The cells were washed once with RPMI-1640, centrifuged, and then resuspended in 1 mL of RPMI-1640, adjusting the bacterial concentration accordingly.

Highly visceral record of infection
Stained C. albicans was uniformly added to the THP-1 cell culture medium at a ratio of 1 fungus per macrophage, and the cells were placed under high internalization for observation of phagocytosis.

Exosome purification and characterization
In this study, research subjects were allocated into two distinct groups for analytical purposes: the negative control group consisted of subjects with macrophage-derived exosomes in a non-infected state; conversely, the   www.nature.com/scientificreports/positive control group comprised subjects exhibiting macrophage-derived exosomes subsequent to infection with C. albicans.THP-1 cells were cultured in T300 flasks until reaching a density of 8 × 10 5 cells/ml.Cells were then centrifuged at 1000 rpm for 5 min, and the medium was replaced with fresh THP-1 complete medium supplemented with 75 ng/ml PMA.The cells were allowed to differentiate and adhere to the flask walls for 72 h at 37 °C in a 5% CO 2 atmosphere.Post-differentiation, the medium was discarded, and cells were washed and replenished with RPMI-1640 medium.Half of the flasks (T300 × 3) continued incubation for 48 h, after which the supernatant was collected.The remaining flasks were treated with a uniform C. albicans suspension and incubated for 2 h at 37 °C, 5% CO 2 .Post-incubation, the flasks were washed thrice with sterile PBS to remove external C. albicans.Serum-free 1640 medium was then added, and the supernatant was collected after a further 6 h incubation.The collected supernatant was centrifuged at 3000 g for 10 min, and the resulting supernatant was stored on ice (or at − 80 °C if not used immediately, then thawed at 37 °C and placed on ice).
Exosome extraction was performed through differential centrifugation: The sample was first melted at 37 °C and then centrifuged at 2000 g for 30 min at 4 °C.The supernatant was transferred to a new tube and further centrifuged at 10,000 g for 45 min at 4 °C to remove larger vesicles.It was then filtered through a 0.45 μm filter membrane.The filtrate was ultracentrifuged at 100,000 g for 70 min at 4 °C.The pellet was resuspended in 10 mL of pre-cooled PBS and ultracentrifuged again under the same conditions.Finally, the pellet was resuspended in 300μL of pre-cooled PBS for storage at − 80 °C.
Transmission electron microscopy (TEM) was used to observe the exosome samples 53 : 10 μL of the exosome suspension was placed on a copper grid, allowed to settle for 1 min, and excess liquid was removed.After drying at room temperature for several minutes, the samples were observed under TEM at 100 kV.
Particle tracking analysis (NTA) of exosome samples 54 : frozen samples were taken, thawed in a 25 °C-water bath and placed on ice.Exosome samples were then diluted with 1 × PBS for direct NTA assay.
Western Blotting was performed to detect exosomal surface markers 51 : RIPA buffer (Beyotime, China), supplemented with protease and phosphatase inhibitors (Selleck, China), was used for protein extraction.Protein concentrations were determined using a BCA kit (Beyotime, China).Following protein blotting, detection and imaging were performed using a gel imager (Pinghao, Beijing, China) and chemiluminescent horseradish peroxidase substrate BrightTM ECL (Beyotime, China).To enhance specificity and clarity of detection, WB membranes were carefully trimmed to remove non-targeted areas before incubation with specific antibodies.This procedure was aimed at minimizing non-specific signals and focusing on the proteins of interest.

PBA treatment of exosomal samples and high-throughput sequencing
The exosomes secreted by macrophages and those post-C.albicans infection were analyzed using PBA 18 .This involved the use of antibodies labeled with DNA probes containing a unique protein tag for protein identification, a molecular tag for repeat assays, and a universal binding site for subsequent processes.EV capture was performed using 96-well plates coated with Cholera Toxin subunit B (CTB) [55][56][57][58] .Antibody-tagged oligonucleotides on the same EV were assigned a unique EV tag.DNA sequences comprising EV tag-protein tag-molecular tag barcodes were prepared into sequencing libraries.Sequencing was conducted using the DNBSEQ-T7 platform (UWI, Shenzhen, China) or NovaSeq S4 (Illumina, USA).Raw sequencing data were converted from bcl to fastq files using MegaBolt (MGI) or bcl2fastq (Illumina).
Exosome proteomic data analysis and statistical analysis [59][60][61] Sequencing reads were analyzed using EVisualizer® decoding software (version 1.0, Secretech, Shenzhen, China) to generate EV ID-protein expression datasets as PBA raw data, and then analyzed for protein expression, combinations, and EV subgroups.To analyze the differentially expressed proteins (DEP) and differentially expressed protein combinations (DEPC), the following statistical analyses were performed, and the normality of the data was checked using the Shapiro-Wilk test.Homogeneity of the data was checked using F-test or Bartlett's test.When comparing two sets of data, the t-test was used if the data were normally distributed with a chi-squared variance.If the data are not normally distributed, the Mann-Whitney U test is used.Welch't test is used for data that is normally distributed but not chi-square.When comparing more than two groups (> 2 variables or categories), we chose the ANOVA test for normally distributed and chi-squared data sets.Significantly different expressions were analyzed by Duncan's test.If the data did not obey normal distribution or variance chi-square, the nonparametric test Kruskal-Wallis test was used instead.Significantly different expressions were analyzed using the paired Wilcoxon rank sum test.We utilized the Benjamini-Hochberg (BH) method to adjust p values.To generate EV subpopulations, the unsupervised FlowSOM 62 algorithm is used.Distributed Stochastic Neighborhood Embedding (t-SNE) and Uniform Flow Approximation and Projection (UMAP) methods 63 can be used to map the EV subpopulations.EV analysis based on the generated EV subpopulations is performed in the interactive interface of the software EVisualizer® online viewer (version 1.0, Secretech, Shenzhen, China).

The strength and limitations of the present study
Exosomes, as a major hot topic at present, have been relatively little studied in the field of fungi, this paper seizes this breakthrough point and explores the immune role of macrophage-derived exosomes in fungal infections before and after Candida albicans infection; In this study, the proteomic resolution of single exosomes was carried out by this technology of PBA, which changed the dilemma of traditional exosome technology limited to total protein and total nucleic acid detection, and improved the precision of exosome protein detection to the level of single molecule, and realized the study of heterogeneity of exosomes with high precision and high sensitivity.The limitations of this study are that the inhibition of Candida albicans morphological transformation by macrophage-derived exosomes and the screening of possible biomarkers were explored and discussed www.nature.com/scientificreports/based on the in vitro level, and future multicenter studies in animal models as well as in patient populations are needed to validate some of the early diagnostic markers and therapeutic targets and to confirm the functional roles of the above proteins.

Figure 3 .
Figure 3. Characterization of two groups of exosomes.(A) TEM images of M0 group exosomes.Scale bar = 100 nm, (B) TEM images of exosomes in M0 + CA group.Scale bar = 100 nm.(C) NTA analysis of the size and distribution of exosomes in group M0.(D) NTA analysis of the size and distribution of exosomes in M0 + CA group.(E) Expression of the exosome marker TSG101 and no expression of the negative marker protein Calnexin were detected by western blot Supplementary Information.

Figure 4 .Figure 4 .Figure 4 .
Figure 4. Identification and characterization of exosome subpopulations.(A) Number of exosomes detected, number of proteins, and number of proteins per exosome for both sets of samples.(B, C) Protein expression on the surface of exosomes in each sample.(D) Proteins differentially expressed by exosomes in the M0 and MO + CA groups.(E) CD36 expression was higher in the M0 + CA group, **P < 0.01.(F) MPO expression was higher in the M0 + CA group, *P < 0.05.(G) Distribution and proportion of exosome subpopulations in the M0 and MO + CA groups.(H) Distribution and proportion of exosome subpopulations in each sample.(I) Protein expression of each exosome subpopulation.(J) Distribution and proportion of cluster14 in the M0 and MO + CA groups, respectively.(K) Protein expression of cluster14.

Figure 5 .
Figure 5. Flow chart of this study.

Table 1 .
The principal findings of this investigation.
FindingsSignificantly increasedMPOin infected versus uninfected macrophages.< br > Elevated METs release under PMA and C. albicans stimulation Notably higher CD36 levels in exosomal subpopulations during C. albicans infection Clinical Implications MPO as a novel target for elucidating C. albicans immune evasion strategies CD36 subpopulations as promising early biomarkers for C. albicans infection; potential therapeutic targets in fungal infections