Plasma gelsolin attenuates acute tissue inflammation and modulates monocyte responses in experimental candidemia

Highlights

• •pGSN attenuates acute pathological and inflammatory readouts in candidemia.
• •pGSN lowers blood fungal CFUs and acute kidney and lung pathology.
• •pGSN enhances monocyte uptake and lowers viable C. albicans recovery.
• •pGSN alters NO-, ROS-, and H₂S in human monocytes.

Abstract

Systemic Candida albicans (C. albicans) infections remain a significant cause of morbidity and mortality in immunocompromised patients, owing to both fungal proliferation and dysregulated host inflammation. Plasma gelsolin (pGSN), an actin-binding protein with immunomodulatory properties, has demonstrated protective effects in bacterial sepsis models, but its role in fungal infections remains unexplored. Here, we evaluated the impact of pGSN in an acute, 24-hour model of candidemia using athymic mice and investigated its effects on the antifungal function of primary human monocytes. Mice intravenously challenged with C. albicans developed increased 2-deoxyglucose-associated fluorescence, elevated whole-blood expression of inflammatory mediators, renal microabscesses, pulmonary pathology, and detectable circulating fungal cells. Subcutaneous administration of pGSN 12 h after infection reduced 2-deoxyglucose-associated signals in the kidneys and lungs, lowered inflammatory gene expression, decreased blood fungal CFUs, and reduced renal microabscess formation and pulmonary tissue injury. In primary human monocytes, pGSN increased fungal engagement and phagocytic uptake and generated a dose-dependent reduction in intracellular C. albicans survival. pGSN also modulated monocyte reactive radical production by increasing nitric oxide while reducing reactive oxygen species and hydrogen sulfide levels. Importantly, pGSN had no detectable direct candidacidal activity under cell-free conditions at the same concentrations, supporting a host-mediated effect. Collectively, these findings indicate that pGSN attenuates acute pathological and inflammatory responses while enhancing monocyte-mediated intracellular control of C. albicans. These results support further investigation of pGSN as a potential host-directed adjunct to antifungal therapy.

Graphical abstract

1. Introduction

Systemic candidiasis, most commonly caused by Candida albicans (C. albicans), remains a major cause of morbidity and mortality, particularly in immunocompromised patients and those receiving broad-spectrum antibiotics or invasive medical interventions (Pappas et al., 2018; Pfaller and Diekema, 2007). Despite the availability of antifungal therapies, mortality rates for candidemia can exceed 40%, especially in patients with delayed diagnosis or organ failure (Kullberg and Arendrup, 2015). The pathogenesis of systemic Candida infection is not solely attributed to fungal proliferation, but also to the host’s dysregulated immune response, which drives tissue damage and multiorgan dysfunction (Lionakis et al., 2023).

One of the major challenges in treating candidemia is the dual requirement for fungal clearance and control of inflammation. While antifungal drugs can suppress fungal growth, they do not address the collateral inflammatory damage induced by excessive cytokine release, immune cell infiltration, and microvascular dysfunction (Netea et al., 2015). Moreover, host immune responses vary across organs, with the kidneys exhibiting the most severe inflammation and tissue injury during fungal sepsis (Jae-Chen et al., 2015; Jawale and Biswas, 2021; Ngo et al., 2014). Therefore, there is a critical need for adjunctive therapies that can modulate the host immune response without compromising pathogen clearance.

Plasma gelsolin (pGSN) is a circulating actin-binding protein that plays a crucial role in maintaining immune homeostasis, scavenging extracellular actin, modulating Toll-like and scavenger receptor signaling, and preserving endothelial integrity (Bucki et al., 2005; Bucki et al., 2008; Lee et al., 2009; Suprewicz et al., 2022; Suprewicz et al., 2023). Under conditions of acute inflammation, including sepsis and trauma, pGSN levels drop significantly, impairing its protective functions (DiNubile, 2008). Restoring pGSN levels has been shown to mitigate inflammatory damage and improve survival in preclinical models of bacterial sepsis and acute lung injury (Cheng et al., 2017; DiNubile et al., 2020; Piktel et al., 2020; Zhi et al., 2024). Furthermore, pGSN has been shown to enhance scavenger receptor-dependent phagocytosis and killing of Candida auris by human neutrophils, suggesting that its effects may extend to other fungal pathogens (Suprewicz et al., 2023). However, the activity of pGSN during systemic C. albicans infection, including its effects on monocyte-mediated fungal uptake and intracellular survival, has not been established. Although pGSN has entered early clinical investigation in severe infectious and inflammatory conditions, its therapeutic efficacy remains to be determined (Catteeuw and DiNubile, 2021; DiNubile et al., 2022; Self et al., 2019).

In this study, we investigated the effects of recombinant human pGSN in an acute, 24-hour model of systemic C. albicans infection using athymic mice, thereby focusing primarily on innate immune responses in a T-cell-deficient host. We hypothesized that subcutaneous administration of pGSN would reduce acute inflammatory and pathological readouts without compromising antifungal host defense. We assessed whole-body and organ-specific 2-deoxyglucose-associated (2DG) fluorescence, body temperature, circulating fungal cells, tissue histopathology, and whole-blood inflammatory gene expression. In parallel, we examined pGSN-mediated changes in phagocytic engagement and uptake, intracellular fungal survival, and reactive radical production in primary human monocytes. Finally, we evaluated whether pGSN exerted direct candidacidal activity under cell-free conditions to distinguish direct fungal killing from host-cell-mediated effects.

2. Materials and methods

2.1. Plasma gelsolin

The recombinant human plasma gelsolin used in our study was expressed in E. coli cells and provided by BioAegis Therapeutics (North Brunswick, USA).

2.2. Inoculum preparation

The C. albicans reference strain (1408) was obtained from the Polish Collection of Microorganisms (Polish Academy of Sciences, Wroclaw, Poland) and stored in glycerol stocks at -80°C. For experimental use, the strain was plated on Sabouraud dextrose agar with chloramphenicol (Lab-Agar; Biomaxima, Lublin, Poland) and grown at 37°C. Yeast cells were counted using a hemocytometer and diluted to the desired concentration in sterile 0.9% NaCl for in vivo use or in cell culture medium for in vitro experiments.

2.3. Murine model of candidemia

Animal procedures were approved by the Local Ethics Committee for Animal Experimentation in Olsztyn, Poland (approval numbers 66/2020 and 12/2024). Female CAnN.Cg-Foxn1/CrL mice (10 weeks old; Charles River) were randomized into four experimental groups (Table 1). All mice were screened for bacterial, viral, and parasitic infections prior to the study (Alab Bioscience, Poland). Mice received an intravenous tail vein injection of C. albicans (10⁵ CFU/mL, 100 µL) or 0.9% NaCl, each combined with IRDye 800CW 2-deoxyglucose (10 nM, #926-08946, Li-Cor). At 12 h post-injection, animals were administered subcutaneously with either pGSN (10 mg/mL, 100 μL) or 0.9% NaCl. Whole-body near-infrared imaging was performed at 12, 16, 20 and 24 h post-infection. Surface body temperature measurements were taken every 4 h from the start of the experiment using a handheld infrared scanner. At 24 h, mice were sacrificed, and blood and organs were collected. Blood was drawn via cardiac puncture into EDTA tubes for analysis of fungal burden and gene expression. Organs were fixed in 10% formaldehyde and left for imaging and hematoxylin and eosin (H&E) staining for histopathological evaluation. All solutions were sterile-filtered using 0.2 μm syringe filters. A semi-quantitative analysis of H&E-stained sections of kidney pathology was quantified by counting microabscesses per 10 high-power fields (HPF), while lung inflammation was scored using a standardized ordinal scale (0–3) based on edema, inflammatory infiltrates, and structural disruption. Data are presented as mean ± SD according to scoring criteria: kidney microabscesses: discrete granulocytic inflammatory foci within renal tubules, counted across 10 randomly selected HPF per section. Lung inflammation score: 0 = no pathological changes; 1 = mild hyperemia or focal atelectasis; 2 = moderate edema, inflammatory infiltrates, alveolar wall thickening; 3 = severe edema, emphysema, widespread leukocyte infiltration.

Table 1.

Experimental setup for the systemic C. albicans infection model with plasma gelsolin intervention.

Group	N	IV injection (0 h)	SC injection (12 h)	Imaging of inflammation	Endpoint (24 h)
NaCl	7	100 μL NaCl (0.9%) + 2-DG 800CW (10 nM)	100 μL NaCl (0.9%)	At 12 h, 16 h, 20 h & 24 h	Blood & organ collection
Plasma gelsolin (pGSN)	6	100 μL NaCl (0.9%) + 2-DG 800CW (10 nM)	100 μL pGSN (10 mg/mL)
C. albicans	7	100 μL C. albicans (10⁵ CFU/mL) + 2-DG 800CW (10 nM)	100 μL NaCl (0.9%)
C. albicans + pGSN	7	100 μL C. albicans (10⁵ CFU/mL) + 2-DG 800CW (10 nM)	100 μL pGSN (10 mg/mL)

2.4. 2-deoxyglucose-associated fluorescence imaging

IRDye 800CW 2-deoxyglucose-associated fluorescence was used as an indicator of glucose-associated metabolic activity. At the indicated time points, animals were anesthetized with isoflurane and scanned using the Pearl Trilogy Imaging System (Li-Cor). After sacrifice, formalin-fixed organs were imaged ex vivo using the Odyssey CLx Imager (Li-Cor) to assess organ-specific fluorescence intensity.

2.5. Fungal outgrowth in the blood and media

Whole-blood samples were serially diluted and plated on Sabouraud agar to quantify the fungal burden. Plates were incubated at 38°C for 48 h, after which colony counts were performed. In parallel, cell culture media containing C. albicans were exposed to pGSN at 50, 150, and 250 µg/mL, and to albumin (250 µg/mL), for 24 h, then plated as above to assess whether pGSN itself exhibits candidacidal activity.

2.6. RT-qPCR

Total RNA was extracted from 100 μL of blood using the Universal RNA Purification Kit (E3598-02; EURx, Gdansk, Poland) according to the manufacturer's instructions. RNA concentration and purity were assessed using a Qubit 4 fluorometer (Thermo Fisher Scientific). cDNA synthesis was performed with the iScript cDNA Synthesis Kit (1708891; Bio-Rad). qRT-PCR was carried out using 5 ng of cDNA and SsoAdvanced Universal SYBR Green Supermix (1725274; Bio-Rad) in a 20 μL reaction. Primers used are listed in Table S1. Reactions were run on the CFX Opus 96 Real-Time PCR Detection System (Bio-Rad) using the following program: 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. GAPDH served as the reference gene. Relative gene expression was calculated using the 2^-ΔΔCt method.

2.7. Human monocyte isolation

Peripheral blood was collected from healthy donors under the approval of the Bioethics Committee at the Medical University of Bialystok (APK.002.234.2021). Peripheral blood was obtained from three independent healthy, nonsmoking volunteers aged 25-35 years (2 male, 1 female) after written informed consent. Monocytes were isolated using the Dynabeads CD14+ positive isolation kit (11367D; Invitrogen) according to the manufacturer’s protocol. Cells were counted using a hemocytometer and resuspended in serum- and antimycotic-free RPMI 1640 medium (ATCC) to avoid interference from exogenous pGSN. Cells were maintained at 37°C with 5% CO₂.

2.8. Phagocytosis assay

Monocytes (1 × 10⁵ cells/well) were seeded in 96-well plates pre-coated with 0.01% poly-L-lysine (Sigma-Aldrich). After monocyte serum starvation for 2 h, calcofluor white-stained C. albicans (0.25 μg/mL for 30 min, RT) was added at a multiplicity of infection of 5 (MOI - a number of fungal cells per monocyte) with or without pGSN and albumin (250 µg/mL). After 2 h of incubation at 37°C, the cells were washed with PBS, fixed in 3.7% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 0.1% BSA for 30 min. Cells were stained with Alexa Fluor 647-conjugated wheat germ agglutinin (WGA; 5 μg/mL; W32466, Invitrogen) for 10 min, mounted in antifade medium (Abcam), and imaged using confocal microscopy (Leica DMI8). Phagocytic activity was assessed as (i) percentage of engaged cells (monocytes adhering to or internalizing fungi) and (ii) phagocytic index (fungal cells internalized per monocyte). At least eight randomly selected fields were analyzed per condition across four independent experiments.

2.9. Fungal intracellular survival assay

Monocytes were seeded in 24-well plates, serum-starved for 2 h, and then exposed to C. albicans at an MOI of 5 in the absence or presence of pGSN at 50, 100, or 250 µg/mL. Albumin at 250 µg/mL served as a protein control. After 2 h at 37°C and 5% CO₂, the culture supernatant was discarded, unbound fungi were washed with PBS, and adherent monocytes were collected using cell scrapers. Samples were transferred to microcentrifuge tubes and sonicated for 10 min to lyse the monocytes. Serial dilutions were plated on Sabouraud agar and incubated for 48 h at 38°C. Viable C. albicans was quantified by colony-forming unit enumeration and expressed as a percentage relative to the untreated condition without monocytes, with a sonication control.

2.10. Reactive species assessment

Monocytes (2 × 10⁵ cells/well) were plated in 96-well plates and serum-starved for 2 h. Cells were treated with C. albicans (MOI 5), pGSN, and their combination for 2 h, followed by incubation with redox-sensitive probes: DAF-2 (5 nM; nitric oxide), P3 (50 nM; hydrogen sulfide), and DCFH-DA (20 μM; reactive oxygen species) (Sigma-Aldrich). Fluorescence was recorded at 488/535 nm using a Varioskan Lux microplate reader (Thermo Fisher Scientific). Background fluorescence was subtracted, and data were normalized to untreated controls.

2.11. Statistical analysis

Statistical analyses were performed using OriginPro 2021. Individual animals or donors were considered biological replicates, and technical replicates were averaged before analysis. Normality was assessed using the Shapiro-Wilk test. Independent groups were compared using one-way ANOVA with Tukey’s post hoc test. Nonparametric data, including histopathology scores, were analyzed using the Kruskal-Wallis test. Blood CFUs were log₁₀-transformed, and RT-qPCR statistics were performed using ΔCt values. Data are presented as mean ± SD or median with interquartile range. Tests and replicate numbers are specified in the figure legends; p < 0.05 was considered significant.

3. Results

3.1. pGSN reduces acute 2DG-associated fluorescence and circulating fungal burden

To investigate the therapeutic potential of pGSN in systemic C. albicans infection, we established a murine model of candidemia using athymic nude mice (CAnN.Cg-Foxn1<nu>/CrL), which lack functional T cells and therefore allow us to study mostly innate immunity, which is often imbalanced in immunocompromised patients at highest risk for invasive fungal disease. Mice were randomized into four experimental groups receiving either C. albicans or NaCl intravenously, with or without subcutaneous pGSN treatment 12 h post-infection (Table 1 and Fig. 1A).

Fig. 1.

Plasma gelsolin diminished the inflammatory response caused in a murine model of candidemia. (A) Schematic overview of the experimental workflow. Female CAnN.Cg-Foxn1<nu>/CrL mice (10 weeks old) were intravenously injected with Candida albicans (10⁵ CFU/mL) or NaCl, along with IRDye 800CW 2-deoxyglucose (10 nM). At 12 h post-injection, animals received a subcutaneous dose of either pGSN (10 mg/mL) or NaCl. Mice were imaged at 12, 16, 20, and 24 h and sacrificed at 24 h for sample collection. (B) Representative whole-body near-infrared images of mice from each treatment group acquired at 12, 16, 20, and 24 h post-injection of fungal cells. The color scale indicates the relative intensity of the inflammatory signal. (C) Surface body temperature of mice over the 24-hour course, showing elevated temperature in C. albicans-infected animals and mitigation in the pGSN-treated group. (D) Fungal burden (log CFU) in blood at 24 h post-infection. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s test (C) or an unpaired Student’s t-test (D). n = 6-7 per group.

To monitor changes in glucose-associated metabolic activity during infection, mice were co-injected with IRDye 800CW 2-deoxyglucose (2DG-800CW), a near-infrared glucose analog that accumulates in metabolically active, inflamed tissues (Piktel et al., 2020). Serial imaging over a 12-hour period revealed a progressive increase in whole-body fluorescence in C. albicans-infected animals, with a notable signal localized to the abdominal region, corresponding to the kidneys. In contrast, pGSN-treated animals displayed a visibly reduced 2DG-associated fluorescence throughout the imaging time course (Fig. 1B).

In parallel, we measured surface body temperature to assess systemic inflammation. C. albicans infection induced a significant rise in temperature compared to NaCl controls, whereas pGSN treatment decreased this response, maintaining values closer to baseline (Fig. 1C).

To determine whether pGSN affected circulating fungal burden, blood CFUs were quantified at 24 h. Mice receiving pGSN exhibited a marked reduction in colony-forming units (CFU) in blood compared to untreated C. albicans-infected animals (Fig. 1D), indicating pGSN's ability to reduce systemic inflammation and decrease fungal burden.

3.2. pGSN is associated with reduced kidney and lung pathology during acute candidemia

To assess the distribution and severity of inflammation at the organ level, we performed ex vivo imaging of dissected organs 24 h post-infection. In C. albicans-infected animals, the strongest fluorescence signal of 2DG was observed in the liver, lungs, and kidneys (Fig. 2A). Quantitative analysis of fluorescence intensities across all individual animals confirmed these trends (Figs. 2B and S2). pGSN-treated C. albicans-infected mice showed significantly lower 2DG fluorescence in the lungs and kidneys compared to untreated infected mice. At the same time, pGSN caused a less pronounced decrease in 2DG in the liver, spleen, or heart of C. albicans-infected mice. As expected, a high baseline 2DG signal was also detected in the liver and kidneys across all groups, including the NaCl and pGSN controls, due to the physiological metabolism of 2DG, as well as the clearance and excretion of the fluorescent probe (Huang et al., 2022; Linder et al., 2011; Marshall et al., 2010). This background signal was consistent among animals (Fig. S1). These findings indicate that pGSN diminished inflammation at key target sites of fungal dissemination.

Fig. 2.

Plasma gelsolin reduces organ-specific inflammatory burden in systemic candidiasis. (A) Ex vivo near-infrared fluorescence imaging of isolated organs (spleen, liver, kidneys, lungs, and heart) collected at 24 h post-intravenous injection of C. albicans or NaCl. Mice received either plasma gelsolin (pGSN) or NaCl subcutaneously at 12 h. Representative fluorescence images show elevated glucose uptake in the liver, kidneys, and lungs of infected animals, indicating tissue inflammation. (B) Heatmap showing log₂-transformed mean fluorescence intensities of 2-deoxyglucose in each organ in each animal. Each column represents a single animal; n = 6-7 per group.

To evaluate the extent of organ damage during candidemia, formalin-fixed lungs, kidneys, and liver were processed for hematoxylin and eosin (H&E) staining and assessed by a pathologist. Consistent with the ex vivo imaging data, C. albicans infection induced changes in the kidneys and lungs, while the liver remained largely unaffected (Table 2).

Table 2.

Pathological changes in major organs 24 h after the initiation of treatment.

Group	Lungs	Kidneys	Liver
NaCl	mild hyperaemia, reduced or no atelectasis	no visible changes/mild hyperaemia	no visible changes/mild hyperaemia
C. albicans	edema, emphysema, hyperaemia, predominance of peripheral atelectasis, numerous hemosiderin-laden macrophages, single granulocytes	inflammatory foci characterized by numerous abscesses localized within tubules, significant hyperaemia, inflammatory infiltrates of granulocytes, presence of fungi in microabscesses, no changes in renal calyces, all changes confined to the parenchyma
C. albicans + pGSN	no granulocytes, mild atelectasis, mild hyperaemia	few microabscesses containing single fungal cells
pGSN	mild hyperaemia, reduced or no atelectasis	no visible changes/mild hyperaemia

Kidney sections from infected mice revealed extensive inflammatory foci characterized by tubular microabscesses, marked granulocytic infiltration, and the presence of fungal cells within abscesses (Fig. 3, Fig. 4). These lesions were primarily confined to the renal parenchyma, with no apparent involvement of the renal calyces. In contrast, mice treated with pGSN exhibited only sparse microabscesses, rarely containing single fungal cells, and lesser granulocytic infiltration. In the lungs, Candida-infected animals showed signs of pulmonary injury, including edema, emphysema, peripheral atelectasis, and the presence of hemosiderin-laden macrophages, indicative of tissue damage and hemorrhage (Fig. 3, Fig. 4). These pathological changes were notably reduced in animals receiving pGSN, which exhibited only mild hyperemia and minimal structural disruption. Semi-quantitative scoring of histological sections (Table S2) demonstrated significantly fewer renal microabscesses and reduced pulmonary inflammation in pGSN-treated animals, supporting the qualitative pathology findings shown in Fig. 3, Fig. 4. Liver tissue from all groups showed minimal to no pathological alterations. Neither uninfected mice treated with pGSN nor vehicle-treated controls exhibited histological abnormalities, indicating that recombinant pGSN does not induce baseline inflammation or hepatic damage. Collectively, these findings confirm that at the 24-hour endpoint, pGSN-treated infected mice exhibited fewer renal microabscesses and lower pulmonary pathology scores than untreated infected mice.

Fig. 3.

Histopathological analysis of kidney, lung, and liver tissue reveals tissue-specific inflammation and the protective effect of plasma gelsolin. Representative hematoxylin and eosin (H&E) stained sections of kidney, lung, and liver harvested at 24 h post-treatment from all experimental groups. In C. albicans-infected animals, kidneys show prominent inflammatory foci with numerous microabscesses (indicated by green stars), while lungs exhibit emphysematous changes and thickened alveolar walls. No major pathology is observed in the liver. Co-administration of plasma gelsolin (pGSN) with C. albicans reduces inflammation and largely prevents abscess formation. Scale bars = 200 μm.

Fig. 4.

Plasma gelsolin reduces microabscess formation in the kidneys and alleviates lung pathology during systemic C. albicans infection. High-magnification hematoxylin and eosin (H&E) stained images of kidney and lung tissue collected at 24 h post-intravenous injection of C. albicans, with or without plasma gelsolin (pGSN) treatment. In the kidneys of infected animals, multiple tubular microabscesses containing fungal cells (red arrows) are characterized by granulocytic infiltration (green arrows). Co-administration of pGSN markedly reduces the number and extent of abscesses. (B) Lung sections from infected mice reveal alveolar collapse, hyperemia, and leukocyte infiltration. Blue arrows indicate hemosiderin-rich macrophages. These inflammatory changes are mitigated in animals treated with pGSN. Scale bars = 50 μm.

3.3. pGSN reduces inflammatory gene expression in whole blood

To assess the systemic immune response to C. albicans infection and the effect of pGSN treatment, we analyzed mRNA levels of inflammatory cytokines using quantitative reverse transcription PCR (qRT-PCR) in RNA isolated from whole blood collected 24 h after infection. C. albicans-infected animals showed a robust upregulation of several pro-inflammatory genes, including IL-1β, IL-6, TNF-α, and TGF-β (Figs. 5A–D, respectively). Treatment with pGSN reduced the whole-blood transcript abundance of these genes, suggesting a systemic anti-inflammatory effect. These observations are consistent with the attenuation of both body temperature and organ-specific inflammation shown in Fig. 1, Fig. 3. No significant between-group differences were detected for IL-10 or IFN-γ (Figs. 5E and F). These data indicate that pGSN modulates systemic inflammation at the transcriptional level during candidemia by suppressing pro-inflammatory gene expression.

Fig. 5.

Plasma gelsolin attenuates systemic inflammatory gene expression in whole blood during candidemia. pGSN treatment reduced C. albicans-driven elevated expression of several inflammatory mediators, including (A) IL-1β, (B) IL-6, (C) TNF-α, and (D) TGF-β. No statistically significant change was recorded for (E) IL-10 and (F) IFN-γ. Expression levels were normalized to housekeeping genes and are shown relative to the NaCl-only group. Data are presented as mean ± SD. Each dot represents one animal (n = 6-7). Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s test, for all pairwise comparisons. P values are shown where significant (p ≤ 0.05); non-significant comparisons (p > 0.05) are not labeled.

3.4. pGSN enhances monocyte uptake and control of C. albicans

To investigate the immunomodulatory effects of pGSN on innate immune function in a human context, we examined the interaction between C. albicans and primary human monocytes. Peripheral blood monocytes were selected due to their abundance and clinical relevance during infections.

Using fluorescence and confocal microscopy, we analyzed the uptake of C. albicans by monocytes in the presence or absence of pGSN. Cells were stained with wheat-germ agglutinin (WGA) to define monocyte membranes, while calcofluor white distinguished fungal cells. High-resolution 3D-stack imaging revealed that pGSN-treated monocytes internalize fungal cells, as visualized by the presence of engulfed Candida within the monocyte cytoplasm (Figs. 6A and Ai).

Fig. 6.

Plasma gelsolin enhances the phagocytic activity of human monocytes against C. albicans. (A) Representative confocal microscopy images of monocytes (magenta) isolated from healthy donors, exposed to C. albicans. Internalized fungal cells (cyan) are visible within the monocyte cytoplasm, while extracellular yeasts are green. (Ai) 3D-confocal stack image showing a monocyte with an internalized Candida cell within the cytoplasm. (Aii) Confocal stack showing a monocyte attached to the extracellular fungal cell. (B) Quantification of the percentage of monocytes engaged in phagocytosis. (C) Phagocytic index calculated as the average number of C. albicans cells internalized per monocyte. (D) Intracellular survival of C. albicans following phagocytosis by monocytes in the presence or absence of pGSN. Data represent mean ± SD from n = 3-4 independent experiments. Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s test, for all pairwise comparisons. P values are shown where significant (p ≤ 0.05); non-significant comparisons (p > 0.05) are not labeled.

Quantification of monocyte-fungus engagement showed a significant increase in the percentage of monocytes associated with or internalizing C. albicans following pGSN treatment (Figs. 6Aii, B, and S3). The phagocytic index, defined as the number of internalized fungal cells per monocyte, was also significantly elevated in the presence of pGSN (Fig. 6. C), indicating enhanced fungal uptake.

To determine whether increased uptake was accompanied by improved intracellular control, we quantified the number of viable C. albicans recovered from monocytes after phagocytosis. pGSN treatment produced a dose-dependent reduction in intracellular fungal survival at concentrations of 50, 100, and 250 µg/mL (Fig. 6D), consistent with enhanced monocyte-mediated fungal killing. Importantly, pGSN did not affect C. albicans viability in a cell-free killing assay at the same concentrations (Fig. S4), excluding a detectable direct candidacidal effect under the conditions tested.

Together, these findings demonstrate that pGSN enhances both the phagocytic uptake and control of C. albicans by human monocytes. The absence of direct candidacidal activity suggests that these effects are mediated through modulation of monocyte antifungal function and may contribute to the lower circulating fungal burden observed in pGSN-treated mice.

3.5. pGSN alters reactive-species-associated signals in human monocytes during fungal challenge

To characterize the effect of pGSN on monocyte redox responses, primary human monocytes were exposed to C. albicans in the absence or presence of pGSN at 50, 100, or 250 µg/mL.C. albicans increased NO-, ROS-, and H₂S-associated fluorescence compared with untreated (NaCl) monocytes (Figs. 7A–C). pGSN differentially altered these responses, NO-associated fluorescence increased most prominently at 250 µg/mL, whereas C. albicans-induced ROS- and H₂S-associated signals were reduced across the tested pGSN concentrations. In monocytes not exposed to C. albicans, pGSN modestly increased NO-associated fluorescence but did not produce comparable increases in ROS- or H₂S-associated signals (Fig. S5). These findings show that pGSN differentially modifies reactive-species-associated fluorescence during fungal challenge, the signaling pathways responsible for these changes and their contribution to monocyte-mediated fungal killing remain to be established.

Fig. 7.

Plasma gelsolin modulates monocyte reactive radical production in response to fungal challenge. Monocytes isolated from healthy human donors were incubated with C. albicans in the presence or absence of plasma gelsolin (pGSN). Production of (A) nitric oxide, (B) reactive oxygen species, and (C) hydrogen sulfide was measured using fluorescent probes. Data are presented as mean ± SD from n = 3 independent experiments. Statistical significance was determined by one-way ANOVA with post-hoc Tukey’s test for all pairwise comparisons. P values are shown where significant (p ≤ 0.05); non-significant comparisons (p > 0.05) are not labeled.

4. Discussion

This study provides evidence that pGSN modifies acute host responses during systemic C. albicans infection. In an athymic mouse model, pGSN administration was associated with lower 2DG-associated fluorescence in the kidneys and lungs, reduced whole-blood expression of inflammatory mediators, lower circulating fungal CFUs, and decreased renal and pulmonary pathology at the 24-hour endpoint. In primary human monocytes, pGSN increased fungal engagement and phagocytic uptake while reducing the recovery of viable monocyte-associated C. albicans. The absence of direct candidacidal activity in cell-free conditions suggests that the reduction in viable fungi observed during monocyte coculture reflects a host-cell-mediated effect rather than direct fungal killing by pGSN. pGSN also differentially altered NO-, ROS-, and H₂S-associated signals during fungal challenge. Collectively, these findings support an acute immunomodulatory effect of pGSN that combines attenuation of pathological inflammatory readouts with enhancement of selected monocyte antifungal functions.

The IRDye 800CW 2-deoxyglucose signal used in this study should be interpreted as an indicator of glucose-associated metabolic activity rather than a specific measurement of inflammation. Increased uptake can occur in metabolically active immune cells and injured tissues but may also be influenced by physiological metabolism, probe distribution, and organ-specific clearance (Huang et al., 2022; Linder et al., 2011; Marshall et al., 2010). Accordingly, the imaging findings were interpreted together with histopathological changes, surface-temperature measurements, and whole-blood inflammatory gene expression. pGSN reduced IL-1β, IL-6, TNF-α, and TGF-β transcript abundance, consistent with attenuation of the acute transcriptional response to infection. However, circulating cytokine protein concentrations were not measured, and changes in whole-blood transcript abundance may reflect both transcriptional regulation and differences in circulating leukocyte populations. Protein-level cytokine measurements and immune-cell phenotyping will therefore be required to more fully characterize the systemic inflammatory response.

In this study, the 24-hour endpoint was selected to capture the acute innate immune phase of disseminated C. albicans infection, during which inflammatory signaling, cytokine production, and neutrophil infiltration reach maximal activity (Lionakis et al., 2011; Lionakis et al., 2023; Ngo et al., 2014). Longer observation periods in immunodeficient mice often lead to severe fungal dissemination and tissue necrosis, limiting the interpretability of immune modulation. Future time-course studies in immunocompetent models will help define the temporal dynamics of pGSN activity during prolonged infection (Chin et al., 2014; Zhu et al., 2023). The in vivo dose of 10 mg/mL and in vitro concentrations of 50-250 µg/mL were chosen based on previously established, non-toxic therapeutic ranges that restore physiological pGSN levels and exert immunomodulatory effects in sepsis and inflammatory models (Bhat et al., 2024; DiNubile et al., 2020; Piktel et al., 2020). In our study, neither histological nor morphological evidence of toxicity was observed, supporting the safety and biological relevance of these concentrations. We administered pGSN subcutaneously, a route that offers practical advantages over intravenous delivery, especially in clinical settings. Subcutaneous injection enables sustained release and absorption, potentially prolonging the bioavailability of therapeutic proteins, such as pGSN (Bittner et al., 2018). This delivery method is minimally invasive and easy to administer outside hospital settings. It has already been successfully tested in clinical trials involving critically ill patients (e.g., COVID-19), supporting its feasibility for broader therapeutic use (Malekzadeh et al., 2020). While antifungal agents such as echinocandins and azoles directly target the fungal cell wall or membrane, they do not address the excessive inflammatory response that contributes to sepsis-related organ failure (Ghannoum and Rice, 1999). Furthermore, antifungal resistance poses a significant threat, limiting therapeutic options (Fisher et al., 2022). Our findings support the idea that pGSN, which has been evaluated for safety and efficacy in patients with infectious diseases (DiNubile et al., 2022; Self et al., 2019), could be deployed as an adjunctive, host-directed therapy in combination with standard-of-care antifungals.

The observed renal pathology is consistent with the established organ tropism of C. albicans in intravenous mouse models, in which the kidney is the principal site of progressive fungal accumulation and inflammatory injury (Jawale and Biswas, 2021). Untreated infected mice developed numerous renal microabscesses containing fungal elements and granulocytic infiltrates, whereas pGSN-treated mice exhibited fewer microabscesses at 24 h. Lung pathology, although not associated with visible fungal cells, was characterized by emphysema, atelectasis, and the presence of hemosiderin-rich macrophages and also reduced following pGSN treatment. The absence of visible fungal cells suggests that pulmonary injury may partly reflect a secondary inflammatory response; however, direct fungal involvement cannot be excluded by histopathology alone (Moldoveanu et al., 2009). Similarly, the lower blood CFU count cannot be interpreted as evidence of reduced fungal burden within individual organs or complete fungal clearance. The conclusions regarding tissue pathology are therefore restricted to the kidneys, lungs, and liver examined in this study. Future studies may need to include other organs, such as the brain, to assess the potential for cerebral dissemination in experimental candidiasis. This should also be done over longer periods, beyond 24 h, to evaluate the long-term safety and protective effects of pGSN as a standalone intervention and combined with standard antifungal agents.

The human monocyte experiments strengthen the interpretation that pGSN can enhance innate antifungal function. pGSN increased both the proportion of monocytes engaged with C. albicans and the number of fungal cells internalized per monocyte. In parallel, pGSN reduced viable fungal recovery following monocyte coculture in a dose-dependent manner. Because pGSN did not affect C. albicans viability in the absence of monocytes at the same concentrations, these findings are consistent with enhanced monocyte-mediated fungal control rather than direct candidacidal activity. They also extend previous observations that pGSN enhances scavenger receptor-dependent phagocytosis and killing of Candida auris by human neutrophils (Suprewicz et al., 2023). Nevertheless, the current CFU assay measures viable monocyte-associated fungi after washing and host-cell lysis and does not fully separate the contributions of increased uptake and subsequent intracellular killing. Kinetic experiments quantifying viable intracellular fungi immediately after uptake and at later time points would more precisely define the effect of pGSN on intracellular fungal survival. Moreover, the relatively small donor cohort and exclusive use of monocytes limit generalization to other innate immune populations, particularly neutrophils, which have a central role in systemic candidiasis. pGSN also altered reactive-species-associated signals in C. albicans-challenged monocytes. NO-associated fluorescence increased most prominently at the highest pGSN concentration, whereas ROS- and H₂S-associated signals were reduced. NO, ROS, and H₂S can exert antimicrobial or tissue-damaging effects depending on their magnitude, timing, cellular source, and intracellular localization (Adhikari et al., 2007; Park and Lee, 2025). Therefore, the present measurements do not establish that the observed redox profile is inherently protective or directly responsible for enhanced fungal killing. Previous studies have linked pGSN to scavenger receptor-mediated phagocytosis, NOS3 activity, and modulation of NF-κB-dependent inflammatory responses in other cellular and infectious contexts (Bucki et al., 2008; Suprewicz et al., 2022; Suprewicz et al., 2023; Yang et al., 2015). However, receptor dependency, NF-κB activation, nitric oxide synthase activity, inflammasome signaling, and downstream transcriptional programs were not examined here. The association between altered reactive-species signals and enhanced monocyte antifungal activity should consequently be considered hypothesis-generating rather than mechanistic.

The use of athymic mice enabled assessment of an innate-dominant response in a host lacking functional T cells. This model is relevant to selected immunocompromised settings but does not capture the full complexity of candidemia in immunocompetent hosts or in patients with other forms of immunosuppression (Bays et al., 2024; Gao et al., 2022; Vidya Vijayan et al., 2017). Adaptive responses, including T-cell-mediated coordination of antifungal immunity, could alter fungal dissemination, inflammatory pathology, and the response to pGSN. In addition, the 24-hour endpoint was selected to capture the acute phase of infection but precludes conclusions about survival, sustained fungal control, organ recovery, adaptive immune responses, or delayed toxicity. The present findings should therefore be interpreted as an acute proof of concept rather than evidence of durable therapeutic efficacy.

5. Conclusion

This study provides evidence that pGSN attenuates acute pathological and inflammatory readouts while enhancing monocyte-mediated antifungal activity during C. albicans infection. In the mouse model, pGSN treatment was associated with lower organ-specific 2DG-associated fluorescence, reduced inflammatory gene expression, decreased circulating fungal CFUs, and reduced renal and pulmonary pathology. In primary human monocytes, pGSN increased fungal uptake and reduced viable monocyte-associated C. albicans without exerting direct candidacidal activity under cell-free conditions, supporting a host-cell-mediated effect. pGSN also altered monocyte reactive-species-associated signals, although the underlying pathways remain unresolved.

Because the study used an acute 24-hour, T-cell-deficient model and did not assess organ-specific fungal burden, survival, or combination treatment with antifungal agents, the findings should be considered proof of concept. Nevertheless, they support further evaluation of pGSN as a potential host-directed adjunct to standard antifungal therapy in longer-term, mechanistic, and immunocompetent models of systemic candidiasis.

CRediT authorship contribution statement

Łukasz Suprewicz: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Visualization, Writing – original draft, Writing – review & editing, Project administration, Funding acquisition. Alicja Walewska: Methodology, Investigation, Visualization, Writing – review & editing. Andrzej Namiot: Formal analysis, Visualization, Writing – review & editing. Sylwia Deptuła-Chmielewska: Methodology, Investigation, Data curation, Writing – review & editing. Paul A. Janmey: Conceptualization, Resources, Supervision, Writing – review & editing. Robert Bucki: Conceptualization, Methodology, Resources, Supervision, Project administration, Funding acquisition, Writing – review & editing.

Declaration of competing interest

The authors declare no conflict of interest.