Caspofungin enhances antifungal immunity in human blood through transcriptional reprogramming

Abstract

Candida albicans is a leading cause of invasive candidiasis, a life-threatening infection with high mortality despite antifungal therapy. Echinocandins are first-line agents due to their fungicidal activity and low toxicity profile. Beyond direct killing, they may also modulate host immunity, e.g. through fungal cell wall remodeling. However, the systemic impact of echinocandins on human immune cells remains incompletely understood, particularly in the context of drug resistance. Using an ex vivo human whole-blood infection model, we assessed the effects of caspofungin during infection with echinocandin-susceptible (ECH^S/FKS^wt) and echinocandin-resistant (ECH^R/FKS^mut) C. albicans strains. Infection alone triggered a strong and conserved transcriptional immune program enriched for innate recognition and inflammatory pathways. Caspofungin treatment during infection with the susceptible strain selectively amplified these responses, engaging a sequential cascade of host defenses that linked pathogen recognition with innate effector functions and adaptive polarisation. In contrast, caspofungin induced only minimal transcriptional changes in resistant strain infections or in uninfected blood. These findings demonstrate that caspofungin efficacy arises not only from its fungicidal activity but also from amplification of host immunity, an effect primarily dependent on fungal susceptibility. This dual mode of action highlights a previously underappreciated role of echinocandins in shaping antifungal immunity and provides new mechanistic insight into drug-host–pathogen interactions during systemic candidiasis.

In human blood, caspofungin not only exerts fungicidal activity against Candida albicans but also potentiates host immune defenses, with both effects detectable only for susceptible strains.

Introduction

Candida albicans is a polymorphic opportunistic fungal pathogen that commonly resides as a commensal organism in the human gastrointestinal tract, oral cavity, and vaginal mucosa. Under conditions of immunosuppression or microbial dysbiosis, it can shift toward a pathogenic phenotype, causing superficial mucosal infections or, in severe cases, systemic candidiasis (Macias-Paz et al. 2023, Katsipoulaki et al. 2024, Schille et al. 2025), a life-threatening disease associated with high mortality rates despite the availability of antifungal treatment (Costa-de-Oliveira and Rodrigues 2020, Denning 2024).

Echinocandins, including caspofungin (CAS), anidulafungin, and micafungin, have become the preferred first-line treatment for invasive candidiasis due to their fungicidal activity against most Candida species, benign toxicity profile, and low potential for drug–drug interactions (Eschenauer et al. 2007, Costa-de-Oliveira and Rodrigues 2020, Cornely et al. 2025). Unlike azoles and polyenes, which target the fungal cell membrane, echinocandins specifically inhibit the synthesis of β-1,3-D-glucan that is an essential structural component of the cell wall in many pathogenic fungi. Inhibition of this pathway compromises cell wall integrity, making the fungus more susceptible to osmotic stress and ultimately leading to cell lysis in certain species, without harming mammalian cells (Letscher-Bru and Herbrecht 2003). Echinocandins have demonstrated strong antifungal activity in both in vitro and in vivo studies, including efficacy against Candida species resistant to azoles and amphotericin B, as well as fungistatic activity against Aspergillus species (Letscher-Bru and Herbrecht 2003, Maschmeyer and Glasmacher 2005).

Several studies have reported that, besides their direct antifungal activity, treatment with echinocandins such as CAS can modulate immune recognition of C. albicans and Aspergillus fumigatus both in vitro and in vivo. Mechanistically, this is explained by the increased surface exposure of β-1,3-D-glucan in the fungal cell wall, a potent pathogen-associated molecular pattern recognised by the C-type lectin receptor (CLR) dectin-1 (Wheeler and Fink 2006, Hohl et al. 2008, Wheeler et al. 2008, Walker and Munro 2020). This enhanced exposure promotes pattern recognition receptor (PRR) engagement and amplifies proinflammatory immune responses, suggesting that echinocandins indirectly influence host immunity through drug-induced remodeling of the fungal cell wall. In addition, CAS may exert immunomodulatory effects by acting directly on immune cells. At high concentrations, CAS suppresses zymosan-induced proinflammatory cytokine and chemokine release in THP-1 monocytes by inhibiting Syk-dependent signaling pathways (Itoh et al. 2021). Furthermore, CAS has been reported to alter the expression of immune-related genes in human monocytes during A. fumigatus infection (Henry et al. 2022). Thus, CAS may attenuate immune activation under specific stimulatory conditions. Collectively, these findings point to a dual immunomodulatory role for CAS: dampening inflammatory signaling when acting directly on innate immune cells, while enhancing pathogen recognition through cell wall alterations.

Despite the growing literature on host-fungal interactions, the specific impact of echinocandins such as CAS on immune signaling, particularly in the context of drug-induced cell wall remodeling, remains poorly understood. The situation is further complicated by the emergence of echinocandin resistance: clinical resistance can evolve during treatment and is almost exclusively linked to point mutations in the FKS genes encoding the echinocandin target β-1,3-D-glucan synthase (Perlin 2015, Barber et al. 2019, Aldejohann et al. 2021). These mutations significantly reduce drug-target affinity and thereby confer clinical resistance to echinocandins. Beyond resistance, they also induce structural alterations in the fungal cell wall, which may modify host–pathogen interactions by affecting recognition through PRRs on innate immune cells (Walker et al. 2013).

Given these complex and context-dependent effects, it remains unclear how the immune-modulating properties of CAS during C. albicans infection change patterns of systemic immune responses. To address this, we employed an ex vivo human whole-blood infection model (Hünniger et al. 2014) to assess CAS-driven transcriptional responses in immune cells following infection with echinocandin-susceptible (ECH^S/FKS^wt) and echinocandin-resistant (ECH^R/FKS^mut) C. albicans candidemia strains. RNA sequencing-based transcriptome analyses of host cells revealed that CAS treatment had no measurable transcriptional effect in uninfected whole blood, indicating that—unlike in isolated immune cells—CAS alone does not directly alter baseline immune gene expression in a complex medium like human blood. However, in the presence of infection, CAS induced substantial transcriptional changes in immune cells. Specifically, 570 differentially expressed genes (DEGs) were identified during ECH^S/FKS^wt infection with CAS compared to untreated controls, whereas such transcriptional changes were absent during ECH^R/FKS^mut infection. These results strongly suggest that the immunomodulatory effects of CAS are mediated predominantly by fungal strain-dependent cell wall alterations rather than by direct modulation of host immune cells, and are absent in infections caused by echinocandin resistant strains.

Material and methods

Ethics statement

Human peripheral blood was collected from healthy volunteers with written informed consent. This study was conducted in accordance with the Declaration of Helsinki and all protocols were approved by the Ethics Committee of the University Hospital Jena (permit number: 3639–12/12).

Strains and culture

Candida albicans candidemia isolates ECH^R/FKS^mut (SN-2021–0056), carrying a homozygous S645P mutation within the hot spot 1 region of the FKS1 gene, and ECH^S/FKS^wt (SN-2021–0283) were kindly provided by the German National Reference Center for Invasive Fungal Infection (NRZMyk). Species identification by MALDI-TOF and FKS1 sequencing were both performed by NRZMyk. Echinocandin susceptibility was confirmed by microdilution testing with the MICRONAUT-AM system (BRUKER), according to the manufacturer’s instructions (Table S1). For infection experiments, C. albicans strains were cultured overnight in a YPD medium (2% D-glucose, 1% peptone, 0.5% yeast extract, in water) at 30°C to stationary phase. Overnight cultures were diluted in a fresh YPD medium and incubated at 30°C until an OD₆₀₀ of 1.0 was reached. Cells were subsequently washed in HBSS, and cell numbers were determined using a Neubauer counting chamber.

Ex vivo whole-blood infection assay

The ex vivo whole-blood infection assay was performed as previously described (Hünniger et al. 2014), with minor modifications. Blood samples were collected from healthy donors in commercial hirudin-coated tubes (S-Monovette^® Hirudin, Sarstedt). On average, 10 mL of blood were collected per donor, and all samples were processed immediately after collection for use in the experiments. Whole blood was pre-treated with caspofungin (CAS, Sigma-Aldrich) at the indicated concentrations for 30 min, followed by the addition of C. albicans cells at a final concentration of 1 × 10⁶/ml. Samples were incubated at 37°C under constant rotation (5 rpm) for the indicated time points. Aliquots of whole blood were collected for RNA isolation. For plasma collection, aliquots were placed on ice immediately, centrifuged (10 min, 13 200 rpm, 4°C), and the resulting plasma was stored at −80°C until further analysis. Fungal survival was quantified by plating serial dilutions in triplicate on YPD agar and counting colony-forming units.

Quantification of secreted proteins

Cytokine and chemokine concentrations in plasma samples were determined using Luminex technology (Bio-Plex Pro Human Cytokine 27-plex Assay, Bio-Rad). Analyses were performed according to the manufacturer’s instructions.

RNA isolation

After 8 hours of infection, human RNA was stabilised in PAXgene Blood RNA tubes (PreAnalytiX) and extracted using the PAXgene Blood RNA Isolation Kit (PreAnalytiX) according to the manufacturer’s instructions. RNA concentration was measured with a NanoDrop One^C spectrophotometer (Thermo Fisher Scientific), and RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies).

RNA sequencing

Human mRNA sequencing was performed by Novogene (Novogene GmbH, Munich). RNA quality was assessed by RNA sample QC prior to library preparation. GlobinClear treatment was applied to remove globin mRNA, and libraries were prepared using a non-directional mRNA library preparation protocol. Sequencing was carried out on the Illumina NovaSeq X Plus platform, generating 150-bp paired-end reads (PE150). A minimum of 9 Gb of raw data was produced per sample, with sequencing quality reaching Q30 ≥ 85%. Raw sequencing data were subjected to standard quality control to remove adapter sequences, low-quality reads, and reads with excessive N content, resulting in high-quality clean data for downstream analyses.

RNA-seq data processing

Preprocessing of raw reads including quality control and gene abundance estimation was done with the GEO2RNaseq pipeline (v0.9.12) using the R scientific programming language (Seelbinder et al. 2019). Quality analysis was done with FastQC (v0.11.8) before and after trimming. Read-quality trimming was done with Trimmomatic (v0.36). Reads were rRNA-filtered using SortMeRNA (v2.1) with a single rRNA database combining all rRNA databases shipped with SortMeRNA. Reference annotation was created by extracting and combining exon features from corresponding annotation files. Reads were mapped against the human reference genome GRCh38.p14 using HiSat2 (v2.1.0, paired-end mode). Gene abundance estimation was done with featureCounts (v1.28.0) in paired-end mode with default parameters. MultiQC version 1.7 was finally used to summarise and assess the quality of the output of FastQC, Trimmomatic, HiSat, featureCounts, and SAMtools. The count matrix with gene abundance data without and with median-of-ratios normalisation were extracted (Anders and Huber 2010). The RNA sequencing data presented in this paper are available in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-16334.

Statistical analyses

Ex vivo whole-blood experiments

All ex vivo whole-blood infection experiments were performed with at least three independent replicates using peripheral blood from different donors. Data are presented as arithmetic means and standard deviation (SD). Statistical analyses were performed using two-sided unpaired t-test or two-way ANOVA with Šidák’s post-hoc pairwise comparisons and correction for multiple testing, as specified in the figure legends. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Transcriptional analyses

Given pronounced donor-impact on gene expression, we created a donor-sensitive design for differential gene expression analysis of condition-pairwise comparisons using DESeq2 (v1.48.1; design = ∼ donor + condition). Gene expression differences were considered significant if |log₂(fold change)| ≥ 0.5 and DESeq2 reported adjusted p < 0.05. The inclusion of donor as a covariate accounts for inter-individual baseline transcriptional differences, while the |log₂(FC)| ≥ 0.5 threshold ensures the detection of transcriptional changes corresponding to at least a 50% difference in expression between conditions, thereby capturing biologically meaningful yet moderate expression changes.

Gene set enrichment analysis for the sources GO biological process (GO: BP), KEGG, and Reactome (REAC) was accomplished using the g: profiler web frontend with Benjamini–Hochberg (FDR) correction for multiple test results and otherwise default parameter settings (https://biit.cs.ut.ee/gprofiler/). KEGG and REAC are both curated resources that provide pathway definitions based on distinct ontologies and nomenclatures. While KEGG primarily focuses on metabolic and signaling pathways, REAC additionally includes stress-induced and other regulatory pathways not represented in KEGG (Chowdhury and Sarkar 2015). We therefore considered the results from both analyses as complementary to achieve a broader and more comprehensive functional interpretation of the data. For KEGG, enriched pathways of the category Human Diseases (https://www.genome.jp/kegg/pathway.html#disease) were removed from subsequent analysis. For GO: BP, enriched pathways were filtered for all descendants of the GO: BP term “GO:0 006 955” (immune response). Expression overlay on KEGG pathway maps was done using the R package pathview (v1.48).

Results

Optimal CAS conditions distinguish resistant and susceptible C. albicans strains in human blood

CAS effects in human blood were assessed for C. albicans strains differing in echinocandin susceptibility to determine the optimal incubation time and drug concentration for subsequent experiments (Fig. 1). In a concentration-response assay performed over 4 h (Fig. 1A), the ECH^R/FKS^mut strain survived at levels comparable to the untreated control across all CAS concentrations tested, consistent with its resistant phenotype. In contrast, the ECH^S/FKS^wt strain showed a pronounced reduction in survival relative to the untreated control, with the largest differences observed at CAS concentrations ≥2 µg/ml, which is below achievable plasma levels (Würthwein et al. 2013). Of note, echinocandins are extensively bound by plasma proteins, reducing the free (pharmacologically active) fraction. Our whole-blood model naturally incorporates these plasma components. The pronounced reduction in survival observed for the ECH^S/FKS^wt strain at ≤2 µg/ml therefore likely reflects CAS activity under physiologically relevant protein-binding conditions. To further characterise the dynamics of CAS-mediated killing, we performed a time-course analysis using 2 µg/ml CAS (Fig. 1B). Survival of the ECH^R/FKS^mut strain declined similarly in treated and untreated samples, with no significant differences at any time point, whereas the ECH^S/FKS^wt strain showed a consistent reduction in survival compared to the untreated control at all tested time points, with the most pronounced drop occurring between 4 and 8 h post-treatment and suppression persisting through 16 h. The continuous decline of both ECH^R/FKS^mut and ECH^S/FKS^wt strains in untreated blood reflects immune-mediated killing, while in CAS-treated samples the ECH^S/FKS^wt strain exhibited an additional drug-specific reduction in survival that was absent in the ECH^R/FKS^mut background. Based on these results, a CAS concentration of 2 µg/ml and an incubation time of 8 h were chosen for all subsequent experiments.

Figure 1.

Survival of resistant and susceptible C. albicans in CAS-treated human blood. (A) Concentration-dependent survival after 4 h infection with increasing CAS doses (0–5 µg/ml). Survival values were normalised to the untreated control (0 µg/ml) of the corresponding strain and replicate (set to 100%, striped bars). (B) Time-dependent survival in untreated (−) or CAS-treated (+, 2 µg/ml) blood over 4–16 h. Survival was normalised to the initial inoculum (0 h) of the corresponding strain and replicate (set to 100%, striped bars). Bars in A and B represent mean ± SD from at least four independent experiments. Statistical significance was assessed by two-way ANOVA with Šídák’s multiple comparisons test, *p < 0.05, **p < 0.01. Legend: ECH^R/FKS^mut, echinocandin-resistant; ECH^S/FKS^wt, echinocandin-susceptible.

Candida albicans infection induces a conserved core immune transcriptional response independent of resistance or CAS treatment

To determine how antifungal treatment influences the host transcriptional programs, we analyzed the response of human blood to infection with either ECH^R/FKS^mut or ECH^S/FKS^wt  C. albicans, in the presence or absence of CAS (Fig. 2). Principal component analysis (PCA) demonstrated a clear separation between mock-infected and Candida-infected samples, irrespective of strain background, indicating that infection status was the primary driver of transcriptional variation (Fig. 2A). Within the infected groups, further stratification was observed for the ECH^S/FKS^wt strain, where CAS treatment led to distinct clustering of treated versus untreated samples per donor. In contrast, the ECH^R/FKS^mut strain displayed less pronounced separation under CAS exposure, suggesting that drug treatment exerted a stronger effect on the transcriptional profile during infection with the susceptible strain. This trend was reflected in the numbers of DEGs identified for each infection condition compared to the respective mock controls (Fig. 2B; Supplementary Data 1). While DEG numbers were high across all conditions (>1500 DEGs), CAS treatment caused a pronounced increase in blood infected with the ECH^S/FKS^wt strain relative to the untreated condition (1956 vs. 1511 DEGs, +29.4%), whereas only a modest rise was observed with the ECH^R/FKS^mut strain (2002 vs. 1807 DEGs, +10.8%). In all cases, upregulated genes outnumbered downregulated genes, indicating a predominantly activating effect on immune-related transcriptional programs. Venn diagram analysis revealed a large shared transcriptional signature, with 1129 DEGs (44.4%) common to all four infection conditions, representing a core host response to C. albicans independent of strain background or CAS treatment (Fig. 2C).

Figure 2.

Gene expression response of human blood to resistant versus susceptible C. albicans strains under CAS treatment. (A) PCA comparing transcriptomes of mock-infected blood with expression profiles of blood cells exposed to echinocandin-resistant (ECH^R/FKS^mut) or echinocandin-susceptible (ECH^S/FKS^wt) C. albicans strains in the absence (CAS⁻) or presence (2 µg/ml, CAS⁺) of CAS for 8 h. (B) Number of upregulated (striped) and downregulated (dotted) DEGs. DEGs were defined as genes with a log₂ fold change (log₂FC) ≥ 0.5 and an adjusted p-value < 0.05 compared to the corresponding mock-infected control. (C) Venn diagram summarising shared and condition-specific DEGs between the four infection conditions; 1129 DEGs were shared across all comparisons, defining the transcriptional core. (D) Significantly enriched categories (background ≤ 500, intersection ≥ 5, fraction > 0.1) among core DEGs shared across all infection conditions identified strong overrepresentation of immune-related categories from both KEGG and Reactome (REAC). The x-axis indicates the enrichment significance: −log₁₀(adjusted p-value), the point size reflects the fraction of genes overlapping with each term, and the color scale represents the FDR-adjusted p-value. (E) Cytokine quantification revealed elevated secretion across all infection conditions compared to the mock-infected control samples, consistent with the conserved immune activation program identified at the transcriptomic level. Bars represent mean ± SD from three independent experiments. Statistical significance was assessed by two-way ANOVA with Šídák’s multiple comparisons test, *p < 0.05, **p < 0.01, ****p < 0.0001. Legend: ECH^R/FKS^mut, echinocandin-resistant; ECH^S/FKS^wt, echinocandin-susceptible; untreated, CAS⁻; CAS-treated (2 µg/ml), CAS⁺.

To investigate common functional patterns in our gene expression profiles, we performed gene set enrichment using both the KEGG and REAC pathway databases by overrepresentation analysis. Applied to the core gene set, functional enrichment revealed a strong overrepresentation of immune-related pathways (Fig. 2D, Supplementary Data 2). KEGG analysis highlighted cytokine-cytokine receptor interaction, NOD-like receptor signaling, Toll-like receptor (TLR) signaling, and JAK-STAT signaling, underscoring central roles for cytokine-mediated communication and pathogen recognition. REAC pathway analysis further confirmed significant enrichment of immune system processes, with more specific modules such as neutrophil degranulation as well as interleukin and interferon signaling. Together, these findings demonstrate that C. albicans infection triggers a conserved host transcriptional program characterised by activation of innate immune and inflammatory defense mechanisms, irrespective of antifungal resistance or drug exposure. Consistent with this, cytokine quantification revealed similarly elevated secretion levels across all infection settings compared to the respective mock-infected controls, with increases in IL-1β, IL-12p70, IFN-γ, MIP-1α, and TNF-α, mirroring the conserved immune activation profile observed in the transcriptomic core (Fig. 2E). These cytokines and chemokines are primarily released by monocytes and NK cells during the early innate immune response (Hünniger et al. 2014, Kämmer et al. 2020), likely preceding CAS-induced effects on the fungus and associated host adaptations.

CAS selectively amplifies host immune transcriptional responses to susceptible C. albicans

We next examined how CAS treatment modulates the host immune transcriptional response by performing direct pairwise analyses within each condition (Mock, ECH^R/FKS^mut, and ECH^S/FKS^wt), comparing samples with versus without CAS exposure. Differential gene expression analysis revealed a striking difference between conditions (Fig. 3A and B, Supplementary Data 1). CAS treatment of mock-infected blood resulted in only a very limited number of DEGs (9 up- and 7 downregulated). A similarly weak response was observed in blood challenged with the ECH^R/FKS^mut strain, where CAS induced 18 DEGs (17 up- and 1 downregulated). Thus, exposure of host cells to the drug alone or in the absence of fungal susceptibility exerts minimal impact on host transcriptional programs. In sharp contrast, infection with the ECH^S/FKS^wt strain elicited a pronounced host response to CAS compared to the no-drug condition, with 570 genes significantly altered during drug exposure. These comprised 502 upregulated and 68 downregulated transcripts, indicating that the response was largely driven by transcriptional activation. These findings suggest that antifungal efficacy in the susceptible strain context not only reduces fungal viability but also amplifies host immune transcriptional programs.

Figure 3.

CAS selectively amplifies immune transcriptional programs during infection with the susceptible strain. (A) Number of upregulated (striped) and downregulated (dotted) DEGs between CAS-treated (CAS⁺) and untreated (CAS⁻) whole blood within each condition (Mock, ECH^R/FKS^mut, ECH^S/FKS^wt). DEGs were defined as genes with a log₂ fold change (log₂FC) ≥ 0.5 and an adjusted p-value < 0.05. (B) Venn diagram depicting overlap of DEGs across the three conditions. (C–D) KEGG and Reactome (REAC) enrichment of the 570 DEGs from ECH^S/FKS^wt CAS⁺ vs. ECH^S/FKS^wt CAS⁻ comparison highlight immune and inflammatory pathways. Shown are significantly enriched categories (background ≤ 500, intersection ≥ 5, fraction > 0.05). The x-axis indicates the enrichment significance: −log₁₀(adjusted p-value), the point size reflects the fraction of genes overlapping with each term, and the color scale represents the FDR-adjusted p-value. (E) Heatmap of representative DEGs mapping to interferon-α/β signaling and cytokine–cytokine receptor interactions identified as enriched in response to CAS treatment in ECH^S/FKS^wt-infected blood compared to no-drug condition (log₂FC ≥ 0.5, adjusted p-value < 0.05).

Pathway enrichment analyses were used to define the biological functions associated with these 570 DEGs (Fig. 3C and D, Supplementary Data 3). KEGG highlighted immune pathways central to pathogen recognition, with strong enrichment for TLR signaling, phagosome maturation, and antigen presentation. In parallel, cytokine-mediated responses were represented by cytokine-cytokine receptor interaction, Th17 cell differentiation, JAK-STAT signaling, and related inflammatory programs (Fig. 3C). A complementary REAC analysis confirmed and extended this pattern, revealing broad activation of immune regulatory processes, with top categories comprising neutrophil degranulation, interferon α/β and γ signaling, and interleukin signaling (Fig. 3D). Taken together, these results show that CAS treatment during infection with the ECH^S/FKS^wt strain amplifies host immune pathways at multiple levels. Notably, many enriched categories overlapped with those identified in the functional enrichment of the core gene set (Fig. 2D), indicating that CAS reinforces pre-existing host defense programs in the susceptible strain context.

To further dissect the contribution of individual genes driving pathway enrichment, we examined the expression profiles of DEGs mapping to interferon α/β signaling and cytokine-cytokine receptor interactions (Fig. 3E). These categories represent central regulators of antifungal immunity and showed marked amplification in CAS-treated blood infected with the ECH^S/FKS^wt strain. Under these condition, interferon-stimulated genes such as IRF7, ISG15, OASL, IFI6, IFIT2, and IFIT3 were strongly induced respective to the untreated condition. In parallel, chemokine genes, including CXCL9, CXCL10, CXCL11, CXCL16, CCL7, and CCL8, were robustly upregulated, consistent with enhanced cytokine-mediated signaling. In contrast, mock-infected blood samples, with or without CAS, displayed little to no induction of these immune effectors. These findings demonstrate that the observed transcriptional activation is specific to CAS treatment in the context of infection with the susceptible strain, aligning with KEGG and REAC enrichment results.

To determine whether CAS-dependent transcriptional amplification results in downstream effector activity, we quantified cytokine secretion after 16 h of infection (Fig. 4). Consistent with the REAC enrichment of interferon α/β, interferon γ, and IL-10 signaling pathways (Fig. 3D), CAS treatment during infection with the ECH^S/FKS^wt strain significantly increased secretion of IFN-α, IFN-γ, and IL-10 compared with the no-drug condition, whereas cytokine levels remained unchanged in the presence of the resistant strain. Notably, at 8 h post-infection, IFN-γ secretion already showed an upward trend in the ECH^S/FKS^wt strain upon CAS treatment (CAS⁺/CAS⁻: 119 ± 4.2%), although this difference did not reach statistical significance, while no such trend was observed for the resistant strain (99.6 ± 25.6%) (Fig. 2E). A significant difference became evident at 16 h, in line with the CAS-induced transcriptional changes. These results provide functional confirmation that CAS enhances cytokine and interferon responses only when the fungus is susceptible to the drug.

Figure 4.

CAS enhances cytokine secretion during infection with the susceptible strain. Whole blood was infected with ECH^R/FKS^mut or ECH^S/FKS^wt  C. albicans for 16 h in the presence (2 µg/ml, CAS⁺) or absence (CAS⁻) of CAS. Concentrations of IL-10, IFN-α, and IFN-γ were measured in plasma from four independent experiments and normalised to the untreated condition to account for donor variability, and are expressed as % CAS⁺/CAS⁻. Data represent mean ± SD. Statistical significance was assessed by two-tailed, unpaired t-test, *p < 0.05, ***p < 0.001.

Overall, these data demonstrate that CAS enhances host antifungal immunity in a strain-dependent manner, coupling effective fungal clearance with reinforced host defense pathways during infection with the susceptible strain.

CAS augments host immunity across sequential antifungal pathways

To place these transcriptional changes into a pathway context, we next mapped expressed genes from whole blood infected with the ECH^S/FKS^wt  C. albicans strain in the presence versus absence of CAS onto KEGG pathways central to antifungal defense (Figs 5–6). Within the TLR pathway map, we observed transcriptional upregulation of receptor genes (CD14, TLR1, TLR2, TLR4) as well as the adaptor molecule gene MYD88 and the downstream signaling kinase gene IRAK1 (Fig. 5A). Similarly, the CLR pathway showed elevated expression of receptor genes such as Mincle and MCL, known contributors to fungal recognition, together with the adaptor molecule gene Syk (Fig. 5B). These PRR pathways were linked to central downstream signaling cascades, including NF-κB and JAK-STAT (Figs S1 and S2), thereby directing transcriptional programs that regulate cytokine and chemokine expression.

Figure 5.

KEGG pathview maps for TLR signaling pathway (A) and CLR signaling pathway (B). Gene expression changes in whole blood during ECH^S/FKS^wt infection are displayed as log₂(fold-change) for CAS⁺ versus CAS⁻ conditions.

Figure 6.

KEGG pathview maps for phagosome pathway (A) and antigen processing and presentation pathway (B). Gene expression changes in whole blood during ECH^S/FKS^wt infection are displayed as log₂(fold-change) for CAS⁺ versus CAS⁻ conditions.

This upstream activation was also mirrored in the phagosome pathway (Fig. 6A), which further displayed increased expression of multiple components governing phagosome maturation. These included genes encoding the early endosomal regulator Rab5, the sorting protein Hrs, v-ATPase subunits that drive intraphagosomal acidification, lysosomal membrane protein LAMP, and degradative cathepsins. In parallel, upregulation of the NOX2 NADPH oxidase complex (CYBA/p22phox, NCF1/p47phox, NCF2/p67phox, NCF4/p40phox) pointed to enhanced capacity for reactive oxygen species production. Together, these changes suggest that CAS exposure in the presence of susceptible C. albicans induced a coordinated transcriptional program to support fungal killing.

Consistently, the antigen processing and presentation pathway displayed upregulation of components of both the MHC class I and class II routes (Fig. 6B). These included the ER chaperone CALR and the peptide transporter TAP1/2, lysosomal processing factors (GILT, CTSB), and central MHC components (MHCI, MHCII, Ii/CLIP, HLA-DM). These changes support enhanced antigen presentation to CD8⁺ and CD4⁺ T cells as well as modulation of NK-cell activity, thereby linking innate recognition and phagocytosis to downstream activation of adaptive immunity. In line with this, Gene Ontology Biological Process (GO: BP) enrichment analysis highlighted immune differentiation programs, including polarisation toward Th1/Th17 lineages, among the top categories (Fig. S3, Supplementary Data 3), supporting a shift toward adaptive immune polarisation.

Taken together, the pathview analyses demonstrate that CAS amplifies host defenses during whole-blood infections with the ECH^S/FKS^wt strain, reinforcing immune activation from pathogen recognition through intracellular killing to antigen presentation and adaptive programming. These findings indicate that CAS efficacy derives not only from direct fungicidal activity against susceptible C. albicans but also from coordinated amplification of host immunity across multiple layers.

Discussion

In this study, we adapted our ex vivo human whole-blood infection model combined with transcriptomic profiling to dissect the immunomodulatory properties of CAS during infection with C. albicans strains differing in echinocandin susceptibility. Our study confirms previous indications that CAS not only exerts direct antifungal activity against C. albicans but also amplifies host immune responses (Wheeler and Fink 2006, Hohl et al. 2008, Wheeler et al. 2008, Walker and Munro 2020). However, both effects occur only in the context of the drug-susceptible strain. CAS treatment alone or during infection with the ECH^R/FKS^mut strain induced little to no host transcriptional change compared to the untreated controls, whereas CAS exposure during ECH^S/FKS^wt strain infection led to the induction of more than 500 DEGs. These findings demonstrate that the observed immunomodulation arises from drug-pathogen interactions rather than direct stimulation of immune cells.

Independent of CAS exposure, both ECH^R/FKS^mut and ECH^S/FKS^wt strains triggered a conserved transcriptional immune signature characterised by innate immune activation, cytokine-mediated signaling, and effector functions such as neutrophil degranulation, demonstrating that the innate immune system mounts a robust and stereotyped defense during bloodstream infection. However, CAS markedly amplified these host programs across multiple immune layers when used against the susceptible strain, including PRR signaling, phagosome maturation, antigen processing, and the initiation of adaptive immune polarisation. Notably, interferon-stimulated genes (e.g. IRF7, ISG15, OASL, IFI6) and genes expressing chemokines (e.g. CXCL9, CXCL10, CXCL11) were strongly induced, reflecting enhanced immune communication and leukocyte recruitment. The upregulation of genes involved in antigen processing and presentation, together with the enrichment of diverse immune differentiation programs under CAS treatment suggests that drug efficacy may not only enhance innate immunity but also facilitate bridging to adaptive immunity, potentially leading to a more effective and long-lasting immune response against infection. In contrast, in resistant-strain infections, CAS neither reduced fungal viability nor amplified immune transcriptional programs, showing that immune modulation depends on drug effectiveness. Consistent with this, cytokine quantification revealed that CAS treatment increased secretion of IFN-α, IFN-γ, and IL-10 during infection with the susceptible strain but not the resistant strain. This functional validation supports the conclusion that CAS-driven transcriptomic changes translate into measurable effector responses when the fungus is susceptible to the drug. Together, these results highlight that CAS not only acts directly against the fungus but also strengthens host immunity.

Our results align with prior studies reporting strong interferon and cytokine activation during C. albicans infection (Smeekens et al. 2013, Niemiec et al. 2017). Previous work with human monocytes suggested that CAS can directly reprogram immune responses, in some cases suppressing cytokine and chemokine release or broadly altering immune-related gene expression (Itoh et al. 2021, Henry et al. 2022). However, these studies used supratherapeutic drug concentrations or simplified cell culture systems. In contrast, our whole-blood data indicate that, under clinically relevant conditions (Kurland et al. 2022, Meng et al. 2025), CAS does not directly modulate host immune cell gene expression. Instead, CAS indirectly enhances immune responses by acting on the fungus, particularly during infection with the susceptible strain. In this context, the observed immune transcriptional programs are most likely driven by highly transcriptionally active innate immune cells, such as monocytes. This apparent discrepancy likely reflects differences in experimental systems: isolated-cell assays can uncover direct off-target effects, whereas whole blood preserves the natural milieu of cellular interactions, plasma proteins, complement, and cytokines (Nguyen et al. 2007, Duggan et al. 2015, Kurland et al. 2022), thereby more closely mimicking the in vivo environment and providing greater translational relevance.

The mechanism by which CAS amplifies immune transcription remains to be directly demonstrated in this study. A plausible explanation, supported by prior in vitro work, is that inhibition of β-1,3-glucan synthesis by CAS in susceptible strains not only compromises viability but also triggers structural alterations in the cell wall, increasing exposure of immunostimulatory epitopes such as β-1,3-glucan (Wheeler and Fink 2006, Hohl et al. 2008, Wheeler et al. 2008, Walker and Munro 2020). This unmasking has been shown to enhance dectin-1-mediated recognition and cytokine induction. Our transcriptional data are consistent with this model, showing amplified activation of multiple immune pathways (e.g. TLR, CLR and NF-κB signaling pathway) under CAS treatment. In contrast, infection with the resistant strain carrying an FKS1 mutation did not trigger comparable amplification of host transcriptional responses in the presence of CAS. One possible explanation is that the unmasking of immunostimulatory epitopes may be attenuated or counterbalanced by compensatory cell wall remodeling, such as increased chitin deposition, a mechanism previously linked to echinocandin resistance in vivo (Lee et al. 2012). These compositional changes can also alter immune-cell recognition. In C. albicans, elevated chitin levels, as observed in fks1 mutants, have been associated with reduced inflammatory responses and attenuated virulence, consistent with dampened host activation. Moreover, ultrapurified C. albicans chitin can attenuate immune recognition by blocking dectin-1-dependent sensing and lowering cytokine production (Mora-Montes et al. 2011). Taken together, antifungal-induced remodeling, encompassing altered β-1,3-glucan exposure and chitin accumulation, may either enhance or suppress host recognition and defense. Thus, CAS-driven changes in fungal cell wall architecture likely modulate immune responses, although direct evidence of such remodeling in our system will require future experimental validation. Several limitations should be acknowledged. First, the ex vivo whole-blood model captures early immune responses but does not replicate the complexity of in vivo infection, including tissue-specific microenvironments and long-term immune dynamics. Second, investigation of additional clinical isolates will be necessary to assess the generalisability of our findings. Finally, it remains to be determined whether the immunomodulatory effects mediated by CAS also extend to other echinocandins or antifungal drug classes.

In conclusion, our findings demonstrate that CAS efficacy against susceptible C. albicans extends beyond direct fungicidal activity to amplify host immune responses at multiple levels, whereas resistance abolishes both effects. These results highlight the dual role of antifungal therapy as pathogen-targeting and immunomodulatory. Future studies combining whole-blood and isolated-cell approaches, with functional and cell wall-focused analyses, will be useful to elucidate the mechanisms by which antifungal therapy shapes host immunity.

Author contributions

Conceptualization: K.H., O.K.; Formal analysis: S.S.; Funding acquisition: O.K., G.P.; Investigation: A.S.; Methodology: A.S., K.H., S.S.; Project administration: G.P., K.H., O.K.; Resources: A.M.A, G.P., O.K., R.M.; Software: S.S.; Supervision: G.P., K.H., O.K.; Visualization: A.S., K.H., S.S.; Writing—original draft: A.S., K.H.; Writing—review & editing: A.M.A, G.P., O.K., R.M, S.S..

Conflicts of interest

We have no conflicts of interest to declare. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funding

The study was supported by the Deutsche Forschungsgemeinschaft (DFG) within the Collaborative Research Center CRC TR124 FungiNet “Pathogenic fungi and their human host: Networks of interaction,” DFG project number 210879364 (C3 to O.K. and INF to G.P.), and by the German Federal Ministry of Education and Research (BMBF) through the CompLS—Computational Life Sciences program in the framework of the project MuMoSim (grant 031L0291B, O.K.). The work of the NRZMyk is supported by the Robert Koch Institute from funds provided by the German Federal Ministry of Health (grant 1369–240, O.K.).