Eosinophil CD48 interactions with Candida albicans Als6 is protective in vitro and in mouse systemic candidiasis

Zaffran, Ilan; Gaur, Pratibha; Ofori, Prince; Charpak-Amikam, Yoav; Pahima, Hadas; Shasha, David; Osherov, Nir; Hube, Bernhard; Ben-Ami, Ronen; Naglik, Julian R.; Mandelboim, Ofer; Vitte, Joana; Levi-Schaffer, Francesca

doi:10.1038/s41467-025-64276-3

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Published: 21 October 2025

Eosinophil CD48 interactions with Candida albicans Als6 is protective in vitro and in mouse systemic candidiasis

Nature Communications volume 16, Article number: 9291 (2025) Cite this article

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Abstract

Eosinophils are innate immune cells with central roles in allergy, parasitic diseases and multiple inflammatory conditions. Moreover, their role in host-pathogen interactions has been well characterized. However, the role of eosinophils during fungal infection is poorly defined. In this study, we delineate the importance of eosinophils during C. albicans systemic infections. C. albicans is promptly phagocytosed by human eosinophils, but growing hyphae escape this mechanism by releasing the fungal toxin candidalysin, which causes eosinophil membrane damage and cell death. Concomitantly, eosinophil mediators, notably major basic protein 1 (MBP-1), released during cytolysis, inhibits C. albicans growth and viability. Moreover, systemic candidiasis in genetic (Δdbl/GATA) or anti–IL-5–mediated depletion of eosinophils results in increased fungal burden and decreased survival. We here identified CD48 as a major receptor of eosinophils and possibly of other immune cells involved in the recognition of C. albicans via agglutinin-like sequence 6 (Als6). CD48 is important for protection in a model of systemic candidiasis as shown in CD48^−/− mice and it binds clinical isolates of C. albicans. In conclusion, we have defined a protective role for eosinophils in vitro and in mouse C. albicans infections through CD48/Als6 host-pathogen interaction axis.

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Introduction

Worldwide, invasive fungal diseases (IFD) affect increasing numbers of individuals, both immunocompromised (i.e. HIV) and immunocompetent patients, leading to a staggering estimate of 1.5 million deaths annually due to underdiagnosis and lack of effective treatments¹. To foster research and intervention in this critical field, the World Health Organization (WHO) has established the first fungal pathogens priority list². Candida albicans (C. albicans) and Candida auris (C. auris) together with Aspergillus fumigatus (A. fumigatus) and Cryptococcus neoformans constitute the “critical priority group”, while two other Candida species (C. parapsilosis and C. tropicalis) are among the seven members of the “high priority group”². The commensal organism C. albicans, is a double-edged sword—contributing to both anti-fungal immunity³ and inflammatory responses. Additionally, its ability to switch between yeast and filamentous/ hyphae forms, which is linked to changes in virulence, highlights significant challenges in achieving accurate diagnosis and effective infection management.

In systemic candidiasis, innate immune cells such as neutrophils, macrophages, and natural killer (NK) cells are recruited to infected organs⁴ to clear the fungus⁵. C. albicans expresses mannoproteins that are typically recognized by innate immune cells via toll-like receptor 2 (TLR-2) and TLR-4. The dectin family receptors (dectin1/2/3) are also involved in the anti-fungal innate immunity by the recognition of β-glucans and α-mannans⁶. Moreover, C. albicans agglutinin-like sequence (Als) proteins were recently shown to bind the T cell immunoreceptor with Ig and ITIM domains (TIGIT) receptor and modulate the immune response of NK and T cells⁷.

Eosinophils are innate immune granulocytes, mainly known for their role in pathological conditions such as allergic diseases and parasitic infections⁸. Although eosinophil activation has been described mostly with allergenic fungi and therefore in the context of allergic diseases, a few studies indicate eosinophil function in fungal infections⁹. Recent data support a broader role for eosinophils in homeostasis^10,11 and in the immune surveillance against bacterial, viral, and fungal infections¹². Eosinophilia and candidiasis coexist in STAT3-loss of function hyper-IgE syndromes¹³, in pediatric IFD¹⁴, and in various other settings^15,16,17. Moreover, a clinical study found a significant association between absolute eosinophil count and serum (1,3)-β-D-glucan in patients with invasive fungal infections, comprising 10% with invasive candidiasis and 50% of these with C. albicans strains¹⁸, suggesting a possible role for eosinophils in systemic candidiasis.

Eosinophils express several receptors that sense and bind pathogens, including TLR-2, TLR-4¹⁹ and CD48²⁰. Upon activation, eosinophils release granular lytic mediators including eosinophils peroxidase (EPX), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and major basic protein 1 and 2 (MBP-1/2)²¹. Eosinophils also produce and release lipid membrane metabolites and an array of pre-formed and de novo synthesized pro-inflammatory cytokines and chemokines¹¹. Eosinophils can also phagocytose several pathogens²² and form both nuclear and mitochondrial eosinophil extracellular DNA traps (EETs)²³. Indeed, eosinophils were demonstrated more than 50 years ago to phagocytose C. albicans²⁴, and more recently also to release EETs²⁵ in response to this fungus.

In the present study, we show that recognition of C. albicans by human eosinophils leads to phagocytosis of the fungus. However, growing hyphae escape from the cells by secreting candidalysin, a recently described fungal toxin²⁶ that induces damage to the eosinophil membrane, resulting in eosinophil cytolysis. Notably, the release of MBP-1 during this lytic process affects fungal growth and reduces fungal viability. In addition, we identified CD48 as a receptor, expressed by eosinophils and other leukocytes as well, that interacts with Als6 on C. albicans, leading to inhibition of fungal growth in vitro and in vivo. Together, our results delineate a possible role for eosinophils in invasive systemic candidiasis.

Results

Eosinophils affect C. albicans growth in a Candida-induced eosinophil death mechanism

To determine the outcome of the interaction of eosinophils and C. albicans, human peripheral blood eosinophils were isolated and cultured with C. albicans in their yeast form at different multiplicities of infection (MOI) and time points. Eosinophil activation was assessed by measuring EPX release as a marker of degranulation. Significant levels of EPX were released after 3 h of incubation at a MOI of 1 (Fig. 1A), but not after 45 min (Fig. S1A). By observing trypan blue positive cells and measuring the release of lactate dehydrogenase (LDH) into the supernatants as a read-out for cell damage, we showed that C. albicans induced significant eosinophil death by 3 h at a MOI of 1 and after 20 h also at MOI of 0.1 (Fig. 1B, C). Nevertheless, eosinophil incubation with C. albicans significantly reduced the fungal viability by 60% as evaluated by colony-forming unit (CFU) count. Notably, this effect was observed after 20 h incubation (Fig. 1D) but not after 3 h (Fig. S1B). To assess whether eosinophil mediators released after interacting with C. albicans affect the viability of the fungus, supernatants of eosinophils incubated overnight alone or with C. albicans were added to fresh C. albicans cultures. A significant decrease in C. albicans CFU was observed only with the supernatants of eosinophils cultured with C. albicans, confirming that eosinophil released mediators affect C. albicans viability (Fig. 1E). Next, we assessed which eosinophil granule basic mediator was responsible for affecting C. albicans viability. We exposed C. albicans to several purified eosinophil granule proteins (EDN, MBP-1, EPX, ECP) at a concentration equivalent to the content found in 10⁵ eosinophils^21,27 (as used in the co-culture experiments described above) and found MBP-1 to be the most potent eosinophil basic protein responsible for the reduction of C. albicans viability, after 3 (Fig. S1C) and 20 h (Fig. 1F). Notably, C. albicans incubated with the four mediators combined reduced the CFU similarly to the eosinophils themselves (Fig. 1F).

To investigate the mechanism of eosinophil-induced C. albicans killing, host-fungal interaction was investigated by confocal fluorescence and scanning electron microscopy (SEM) analyses. Eosinophils phagocytosing C. albicans were observed 30 min after co-culture (Fig. 1G, H) together with EETs, that were visualized surrounding the yeast (Fig. 1H). Importantly, after 3 h of incubation (MOI 1), phagocytosed C. albicans was still capable of forming hyphae, thereby inducing damage to the eosinophils, resulting in cell death (Supplemental Movie 1). This correlated with the increase in LDH levels as described above (Fig. 1C). Therefore, we hypothesized that phagocytosis of C. albicans was the inducing mechanism of eosinophil mediator release as well as C. albicans death. To elucidate this, eosinophils were pre-treated with cytochalasin D, a well-characterized cytoskeleton inhibitor, and incubated with C. albicans. However, only a slight decrease in degranulation (Fig. 1I) and a minor increase in C. albicans viability (CFU; Fig. 1J) were detected in treated eosinophils, in comparison to untreated cells. Importantly, confocal microscopy showed that cytochalasin D treated eosinophils still interacted with C. albicans but did not phagocytose the fungus (Supplemental Movie 2).

Finally, we wondered if candidalysin, a fungal toxin secreted by C. albicans during hyphae formation, might be responsible for eosinophil activation and death. Therefore, we investigated the capacity of C. albicans mutants lacking candidalysin to induce eosinophil activation and death. Eosinophils were incubated with synthesized candidalysin, or mutant strains missing either the entire ECE1 gene (ece1Δ/Δ) or only the candidalysin-encoding sequence (ece1Δ/Δ + ECE1_Δ184–279), with the control ECE1 re-integrant strain (ece1Δ/Δ + ECE1)²⁶ or with the wild type (WT; SC3514) C. albicans strain. As shown by EPX levels (Fig. 1K), candidalysin triggered a dose-dependent degranulation of eosinophils. However, the candidalysin-deficient strains caused EPX released levels similar to those induced by C. albicans WT strain (Fig. 1K), suggesting that other mechanisms besides candidalysin may be involved in the degranulation of eosinophils. Moreover, eosinophils incubated with the wild type (WT) strain, or the different mutants showed a significant increase in LDH released after 20 h incubation. Interestingly only the highest candidalysin concentration (60 µM) induced a significant increase in LDH release (Fig. 1L).

Eosinophils decrease the fungal burden and improve survival in a murine model of systemic candidiasis

To investigate the role of eosinophils in systemic candidiasis in vivo, eosinophil deficient (ΔdblGATA) and WT mice were injected intravenously (i.v.) with 5 × 10⁵ viable C. albicans yeast and euthanized 2 days post-infection (dpi). Fungal burdens were then assessed in infected organs (kidneys, liver, and spleen) by CFU (Fig. 2A). A significantly higher fungal load was found in the kidneys of the eosinophil deficient mice but not in the liver and the spleen (Fig. 2B). Kidneys removed from C. albicans infected mice were enlarged and appeared visually more infected in ΔdblGATA mice as compared with WT mice (Fig. S1D). Staining of the kidneys with hematoxylin and eosin (H&E) revealed few intact eosinophils (Fig. S1E) and immunofluorescence staining of eosinophils by anti-Siglec-F showed mostly debris of cells surrounding the hyphae (Fig. S1E). Furthermore, levels of IL-6, a general inflammation marker, and of IFN-γ, a type 1 cytokine effective for anti-fungal defense, were assessed 2 dpi. ΔdblGATA mice presented significantly elevated levels of IL-6 and decreased levels of IFN-γ in serum (Fig. 2C) and homogenized kidneys (Fig. 2D) in comparison to WT mice. In addition, ΔdblGATA mice showed a significantly decreased survival rate compared to WT mice (Fig. 2F). Finally, we found that incubation of human eosinophils with C. albicans for 3 h induces the secretion of IFN-γ (Fig. 2G).

CD48 binds C. albicans and regulates fungal growth

To investigate the mechanism involved in the interaction between human eosinophils and C. albicans, first we assessed the expression of TLR-2, TLR-4, and Dectin-1, which are involved in the innate immune response to C. albicans⁶. We also investigated CD48, an eosinophil surface protein that binds to bacterial surface molecules²⁰. In line with previous observations, CD48 but not TLR-2, TLR-4, or Dectin-1 was found to be expressed on freshly isolated eosinophils^19,28, (Fig. S2A). To evaluate whether C. albicans binds to CD48, we produced murine (mCD48-Fc) and human (hCD48-Fc) CD48 fusion proteins and used them to stain C. albicans yeast by flow cytometry (Fig. S2B). C. albicans effectively bound both human and mouse CD48 in comparison to the negative construct control (D1-Fc; Fig. 3A). Binding to hCD48-Fc and mCD48-Fc was further confirmed using plate-bound C. albicans (Fig. 3B) and visualized using immunofluorescence microscopy with both yeast (Fig. 3C) and hyphae (Fig. 3D).

Interestingly, other Candida species including C. lusitaniae and C. tropicalis, but not C. parapsilosis, also bound to CD48 (Fig. 3E), although to a far lesser degree than C. albicans.

To determine whether binding of C. albicans to CD48 could activate eosinophils, the degranulation of eosinophils induced by C. albicans preincubated with hCD48-Fc was measured. Under these conditions decreased EPX levels were observed in comparison to those released by eosinophils incubated with C. albicans alone (Fig. 3F). Moreover, eosinophil incubation with C. albicans significantly increased the phosphorylation of Lyn at 15 min, a signal transduction protein linked to CD48 (Fig. 3G), and slightly increased CD48 expression on eosinophils at 120 min (Fig. S2C).

To confirm the role of CD48 in C. albicans-eosinophil interactions, bone marrow derived eosinophils (BMEos) obtained from WT and CD48^–/– mice (Fig. S2D) were incubated with C. albicans (MOI 1) for 20 h and fungal viability was assessed. As with human eosinophils, WT BMEos co-cultured with C. albicans reduced significantly the fungal CFU, while no difference in CFU was found when the fungus was incubated with CD48^–/– BMEos (Fig. 3H), thus confirming a role for CD48 in C. albicans interactions with eosinophils.

Next, using CD48^–/– mice, we evaluated the involvement of CD48 in systemic candidiasis. CD48^–/– mice displayed a significant increase in fungal burden in the kidneys and a higher mortality rate than WT mice (Fig. 3I, K). Moreover, IL-6 but not IFN-γ levels were found to be significantly increased in the serum of the infected mice (Fig. 3J).

Antibody-mediated depletion of eosinophils increases the fungal burden of WT but not of CD48^–/– mice

To determine whether the CD48-dependent effect of fungal clearance is specific for eosinophils, we depleted eosinophils by injecting anti-IL-5 or rat IgG1 (as isotype control; Fig. S3A) in WT and CD48^–/– mice and assessed if this resulted in significant exacerbation of the infection. Similarly to ΔdblGATA mice, eosinophil depletion in WT mice led to a significantly higher fungal load in the kidneys (Fig. 4A). However, unlike WT mice, in which eosinophil depletion led to significantly higher fungal load, eosinophil depletion in CD48^–/– mice did not result in significant elevation in CFU in comparison to isotype control treated CD48^–/– mice, suggesting that CD48 expression on eosinophils is critical for their antifungal function. Notably, the fungal load in eosinophil-depleted CD48⁻/⁻ mice was significantly higher than in eosinophil-depleted WT mice, indicating that CD48 also plays an important role in antifungal defense through other CD48-expressing immune cells. Finally, IFN-γ levels in serum and kidney homogenates decreased following eosinophil depletion, confirming the production of IFN-γ by eosinophils (Fig. 4B, C).

Fig. 4: Antibody-mediated depletion of eosinophils increases the fungal burden in WT and CD48–/– mice. — **Fig. 4: Antibody-mediated depletion of eosinophils increases the fungal burden in WT and CD48^–/– mice.**

CD48 recognizes Als6 as a ligand on C. albicans

We next sought to identify the fungal ligand of CD48. S. cerevisiae α-agglutinin protein has sequence characteristics of immunoglobulin (Ig)-like protein and homology to human CD2 protein, a low-affinity ligand of CD48²⁹. Using a computational approach, we aligned the agglutinin sequences of S. cerevisiae and C. albicans with CD2 and detected 44% similarity between C. albicans agglutinin and CD2. Therefore, we assessed whether any of Als proteins could be CD48 ligand. For this purpose, we generated BW-hCD48 as reporter cells. BW (BW5147.3 cell line) cells were transfected with the extracellular domain of CD48 conjugated to mouse CD3ζ. Consequently, binding of CD48 led to the release of mouse interleukin-2 (mIL-2) detectable by enzyme-linked immunosorbent assay (ELISA)³⁰. C. albicans knock out strains for each of the ALS gene family (als1-9Δ/Δ) or WT (SC5314) strains were incubated with either BW-hCD48 or BW parental cells as a control (Fig. S4A) for 48 h, and mIL-2 levels in the supernatant were assessed as read outs of CD48 binding (Fig. 5A). As expected, BW parental cells did not release mIL-2 while BW-hCD48 cells incubated with WT C. albicans released mIL-2. Notably, incubation with als5Δ/Δ, als6Δ/Δ, and als7Δ/Δ strains showed significantly decreased levels of mIL-2, indicating that these Als proteins could also act as biologically relevant ligands for CD48 (Fig. 5B). To confirm this result, we assessed by ELISA the binding of hCD48-Fc to plate bound WT, als5Δ/Δ, als6Δ/Δ, and als7Δ/Δ C. albicans strains. As with BW system, WT but not als5Δ/Δ, als6Δ/Δ, and als7Δ/Δ was found to bind hCD48-Fc (Fig. 5C), confirming that Als5, Als6, and Als7 are ligands for CD48. Focusing on Als6 as the most compelling interactor, we confirmed the binding of CD48 to Als6 by a lack of staining of als6Δ/Δ with hCD48-Fc by flow cytometry (Fig. 5D).

We next sought to determine the functional relevance of CD48-Als6 binding in the antifungal activity of eosinophils. Therefore, C. albicans als6Δ/Δ was incubated with either CD48^–/– or WT BMEos. In both cases, similar CFUs were observed (Fig. 5E), contrasting with our previous observation with the WT C. albicans strain, where WT but not CD48^–/– BMEos showed a significant reduction of the fungi viability (Fig. 3H). Finally, to evaluate the relevance of these findings in vivo, we injected als6Δ/Δ C. albicans strain into WT and CD48^–/– mice and evaluated the fungal load in the kidneys, the serum IL-6, and the IFN-γ levels in the kidneys 2 dpi. In contrast to data obtained with WT C. albicans strain where a significant increase in fungal load, serum IL-6 and IFNγ was found in CD48^–/– mice, these parameters were all similar in WT and CD48^–/– mice infected with als6Δ/Δ C. albicans (Fig. 5F, G, H).

Next, we sought to determine whether CD48 might play a general role in eosinophil-fungal interactions. We collected 34 clinical isolates of C. albicans from a cohort of patients with invasive candidiasis, and- using BW-hCD48 and BW cells we evaluated their CD48 binding capacity. We found that 55% of the clinical isolates bound significantly to BW-hCD48 cells as compared to BW-hCD48 cultured in medium alone (Fig. 5I), while incubation of BW parental cells with the same strains did not show any binding (Fig. S4B). To note, most clinical strains displayed similar or even higher binding levels than the WT strain, highlighting the potential clinical relevance of CD48.

Discussion

The role of eosinophils in allergic diseases, including those caused by fungal allergens, is well defined. However, eosinophil contribution in fungal infections, and particularly in candidiasis, remains unclear. A clinical study demonstrated that absolute eosinophil count is significantly associated with serum (1,3)-β-D-Glucan in patients with invasive fungal infections¹⁸, implying a possible role for eosinophils. Eosinophil depletion by biological drugs (mepolizumab, benralizumab) for treatment of severe eosinophilic asthma or other eosinophil-linked diseases (hypereosinophilic syndrome, eosinophilic esophagitis, etc.) is being increasingly employed. However, post market surveillance (pharmacovigilance) of these drugs highlights the increased risk of infections for patients^31,32,33,34.

In the current study, we show a protective role of eosinophils against C. albicans in vitro and during systemic candidiasis in vivo. Using human peripheral blood eosinophils, we found that eosinophils were activated by C. albicans and inhibited fungal growth via a complex mechanism. C. albicans is phagocytosed by eosinophils, but the growing hyphae within the eosinophils and the subsequent secretion of candidalysin, allow the fungi to escape and to cause eosinophil degranulation, cytokine production (IFN-γ) and cytolysis. This dual killing mechanism has also been demonstrated in human macrophages after C. albicans phagocytosis³⁵ and in eosinophils in response to A. alternata²⁸. Importantly, another study also reported that stimulating human eosinophils with GM-CSF, IL-1, IL-3, and IL-5 augmented phagocytosis of C. albicans, but this paradoxically did not result in increased killing of the organism³⁶ and in line with our findings confirms that C. albicans may escape from eosinophils phagocytosis.

The killing of C. albicans appears predominantly mediated by the release of the eosinophil basic granule proteins, mainly MBP-1. MBP-1 is a strongly basic protein (pI = 11.4)²¹ and is active against a wide range of microorganisms, including bacteria, helminths, and fungi³⁷. The cationic property of MBP-1 affects the charge of surface membranes, resulting in their permeabilization and disruption, as shown with Staphylococcus. aureus (S. aureus) and E. coli³⁸. In another study, it was reported that recombinant RNase3/ECP displays anti-C. albicans properties due to its basic characteristics^39,40. Together, this suggests that strong positively charged compounds influence C. albicans growth/viability, even though C. albicans can tolerate a broad range of pH (from 2 to 10)⁴¹.

In our study eosinophil death appears to be mediated by hyphal growth and candidalysin secretion. Candidalysin directly permeabilizes host cell membranes, driving cell damage²⁶, and is a key driver of inflammasome-independent cytolysis⁴². Candidalysin also induces immune responses in host cells including macrophages⁴², epithelial cells⁴³, and mast cells⁴⁴. In this study, we demonstrate that candidalysin induced eosinophil degranulation while its capacity to induce eosinophil death was limited only with the highest candidalysin concentration used (60 µM). Notably, eosinophils co-cultured with candidalysin-deficient strains displayed similar degranulation and killing as that induced by WT strain, suggesting that other mechanisms, for example those induced by growing hyphae, may contribute to eosinophil degranulation and cytolysis.

Moreover, SEM analysis after co-culture revealed EETs surrounding C. albicans. EETs formation in response to C. albicans, E-coli, S. aureus²⁵, and A. fumigatus⁴⁵ is a well-known phenomenon that occurs upon cytolytic eosinophil death⁴⁶. Therefore, EET formation may be an additional contributor to anti-Candida activity and eosinophil death.

Using eosinophil deficient ΔdblGATA mice we demonstrate that eosinophils play an important anti-fungal role in a systemic candidiasis model. GATA-1 mutation causes not only eosinophil deficiency but also an impairment of hematopoietic stem and progenitor cells⁴⁷, together with a concomitant reduction in IL-4 production, mast cell numbers, and IgE mediated-activation⁴⁸. These features together with the absence of eosinophils might influence the outcome of systemic candidiasis in the ΔdblGATA mice. We therefore performed another model in which eosinophils were specifically depleted by injection of anti-IL-5 antibody. In both models, eosinophils deficient mice showed a higher fungal load in the kidneys together with increased IL-6 levels in serum, decreased IFN-γ in serum and homogenates kidneys and increased mortality. Importantly, the decreased IFN-γ levels found in both eosinophil deficient mice models, together with the release of this cytokine by human eosinophils in response to C. albicans in vitro, suggests that eosinophils may also affect the immune response to decrease the fungal burden. Notably, eosinophil staining by H&E and anti-Siglec-F in the infected kidneys revealed few intact eosinophils and mostly eosinophil debris as described by the Splendore–Hoeppli phenomenon, that is characterized by microorganisms (fungi, bacteria and parasites) surrounded by eosinophilic debris and material^49,50. To note, this phenomenon was also described in microabscesses and granulomas throughout the body of patients with systemic candidiasis⁵¹. Moreover, the hyphal form was predominantly observed in infected kidneys, while the yeast form was predominantly observed in the liver and spleen⁴², consistent, with our in vitro data showing that eosinophil anti-fungal activity took place after hyphae formation.

In a lung infection model caused by intranasal inoculation of A. fumigatus, ΔdblGATA mice showed a higher fungal burden in the lungs⁵², similar to our data in the kidneys.

The decrease in eosinophil blood counts in WT mice infected with C. albicans and their absence in the kidney at 18 h post infection may indicate a role for these granulocytes in systemic candidiasis. Conversely, it is possible to hypothesize that the association of eosinophil plasma membrane markers of activation and the declining blood eosinophil counts are indicative of an eosinophil cytolytic process. A similar phenomenon was described in several infectious diseases, including bacterial infections⁵³ and COVID-19⁵⁴. Nevertheless, there is still a lack of evidence from human patients treated with biological drugs for depletion of eosinophils. It is possible to hypothesize that eosinophils and their effect in systemic candidiasis may be redundant in comparison to “professional” immune cells such as neutrophils, macrophages, or T cells. However, several case studies reveal that patients with candida infection and neutrophil impairment due to caspase recruitment domain-containing protein 9 (CARD9) deficiency showed an increased eosinophil count in blood and cerebrospinal fluid (CSF)^55,56,57, suggesting a potential role for eosinophils.

Next, we report here on the role of CD48 in the interaction with C. albicans. CD48 is a glycosylphosphatidylinositol (GPI) anchored activatory receptor expressed by several myeloid and lymphoid cells and recognizes bacteria such as E. coli²⁰ and Mycobacterium tuberculosis⁵⁸. CD48 also plays a prominent role in the binding and activation of eosinophils by S. aureus²⁰. Here, we demonstrated that CD48 binds C. albicans yeast and hyphae and that blocking the CD48-C. albicans axis, by incubation of C. albicans with hCD48-Fc, reduced eosinophil activation. Moreover, CD48^–/– BMEos co-cultured with C. albicans could not induce reduction in C. albicans viability unlike WT BMEos.

CD48 plays a significant role in antifungal immunity in vivo, as evidenced by the increased kidney fungal burden, elevated serum IL-6 levels, and higher mortality observed in CD48^–/– mice in models of systemic candidiasis. However, since CD48 is expressed on multiple leukocyte subsets, its antifungal function cannot be attributed solely to eosinophils. To explore this further, we depleted eosinophils in both CD48^–/– and WT mice and compared their fungal burdens. Our results demonstrated that while CD48 is essential for eosinophil-mediated antifungal activity, its role extends beyond eosinophils. Overall, these findings identify CD48 as an antifungal receptor that contributes to host defense across various CD48-expressing immune cells.

The biological and clinical relevance of the CD48-C. albicans interaction was also assessed in this study. We report here two other Candida species including C. lusitaniae and C. tropicalis, but not C. parapsilosis to bind CD48. Furthermore 19 out of the 34 systemic C. albicans clinical isolates that were assessed significantly bound CD48.

C. albicans infections often exacerbate inflammatory diseases such as IBD⁵⁹, asthma⁶⁰ and COPD⁶¹. Zhang et al.⁶² suggested that the ability of certain fungi to exacerbate asthma symptoms is dependent on their immunomodulatory functions and independent of their ability to act as sensitizing allergens. Based on our results, we might assume that C. albicans affect the inflammatory milieu in the lungs and exacerbate disease via CD48, which we previously reported as being a pivotal eosinophil receptor in allergic eosinophilic airway inflammation^63,64.

The Als family is cell surface glycoproteins required for adhesion of C. albicans to host cells, including endothelial, vaginal and buccal epithelial cells⁶⁵. Here, we identified Als5, Als6, and Als7 as potential ligands of CD48. Focusing on Als6, we found that its interaction with CD48 was required to induce eosinophil mediated C. albicans killing in vitro and an anti-fungal activity in vivo. In contrast with the data observed with the WT C. albicans, CD48^–/– mice injected with als6Δ/Δ mutant showed comparable kidney fungal burdens and IL-6 serum levels as WT mice. This indicates that Als6-CD48 interaction is crucial for a protective anti-fungal response.

Importantly, it was previously reported that C. albicans binds the TIGIT receptor via Als6, Als7 and Als9, inhibiting NK and T cell activity⁷. This shows that the Als proteins may function as adhesion molecules as well as immune modulators by binding activatory and inhibitory receptors.

In conclusion, we demonstrate that eosinophils, by a distinct mechanism, (Fig. 6) are important in host protection against C. albicans infection, and that the CD48-Als6 axis is a key driver of the anti-fungal immune cell response.

Methods

Fungal strains

The wild type C. albicans strain (SC5314) used in this study was obtained from ATCC. Candida parapsilosis, Candida tropicalis, and Candida lusitaniae were obtained from Prof. Daniel Kornitzer (Israel Institute of Technology, Haifa, Israel). Candidalysin-mutant strains: ECE1 deletion strain (ece1Δ/Δ), ECE1-complemented strain (ece1Δ/Δ + ECE1), and C. albicans strain lacking only the candidalysin-encoding region in Ece1 (ece1Δ/Δ + ECE1 _Δ184–279) were produced as described in ref. ²⁶. Als mutant strains were als1Δ/Δ1467, als2Δ/Δ2757, als3Δ/Δ1843, als4Δ/Δ2034, als5Δ/Δ2373, als6Δ/Δ1420, als7Δ/Δ1429 and als9Δ/Δ2028, which are all derivatives of SC5314⁷. For all experiments, fungal cultures were inoculated overnight in Sabouraud dextrose (Sigma-Aldrich, Jerusalem, Israel) medium at 30 °C in aerobic conditions. The culture was then diluted 1:50 in a fresh secondary culture in Sabouraud dextrose medium for 2 h. Cells were then washed 3 times with ice cold PBS before their introduction into the experiment.

Systemic candidiasis mouse models

For the systemic candidiasis studies, 8-12-week-old males C57Bl/6 (Harlan, ENVIGO, UK), ΔdblGATA (backcrossed to the C57Bl/6 background for seven generations, a kind gift of Prof. Ariel Munitz, Department of Clinical Microbiology and Immunology, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel) and CD48^–/– mice (a kind gift of Prof. Arlene H. Sharpe Department of Microbiology and Immunobiology, Evergrande Center for Immunologic Diseases, Harvard Medical School, Boston, MA, USA) were injected via the lateral tail vein with either 5 × 10⁴ (for survival assay) or 5 × 10⁵ (fungal burden assay) C. albicans yeast for the determination of fungal burden respectively. C. albicans (strain SC5314 or als6Δ/Δ) was grown at 30 °C in yeast Sabouraud dextrose broth as described above. On the day of the experiment the fungal cells were washed 3 times in ice-cold PBS and taken to the animal facility on ice.

For fungal burden experiments, mice were sacrificed with 48 h post-infection and relevant organs were harvested. Organs were homogenized, filtered through a 70 μm strainer, serially diluted in ice-cold PBS and plated on Sabouraud dextrose agar plates (Sigma-Aldrich). The plates were incubated for 48 h in a stationary 30 °C incubator and once colonies were visible, they were counted.

For survival experiments, mice were weighed, clinically evaluated daily and euthanized once their initial weight was reduced by 20%.

For the depletion of eosinophils, WT and CD48^–/– mice were. injected i.p with 0.3 mg/100 µl of anti–IL-5 antibody in (TREK5; BioXCell) or with anti–horseradish peroxidase isotype control antibody (HRPN; BioXCell) at days −5, −3, and −1 before fungal infection.

All the animal experiments were performed according to Hebrew University ethical animal guidelines (MD-19-15990-5).

Human peripheral blood eosinophil purification

Human eosinophils were purified as previously described⁶⁶ from the peripheral blood of mildly atopic volunteers (blood eosinophil levels 5–10%), asymptomatic and therefore not taking any drug. Written informed consent was obtained according to the guidelines of the Hadassah-Hebrew University Human Experimentation Helsinki Committee (0410-14-HMO). Briefly, venous blood (150 ml) was collected in heparinized syringes and left to sediment in 6% dextran (Sigma-Aldrich). Leukocytes were centrifuged on Ficoll-Hypaque (density, 1.077; 25 min, 700 × g, 22 °C; Amersham Bioscience, Amersham, Buckinghamshire, UK). Neutrophils and contaminating lymphocytes were tagged in the granulocyte-enriched pellet with micromagnetic beads bound to anti-CD16 and anti-CD3 (Miltenyi Biotec, North Rhine-Westphalia, Germany). Eosinophils were purified by passing the cell suspension through a magnetic column (LS-MACS). Eosinophils were evaluated by flow cytometry using APC-anti-human CCR3 (Abcam, Cambridge, UK) and PE anti-human Siglec-8 (BioLegend, CA, USA) and collected at a purity of >98% (Kimura staining), with viability of >98% (trypan blue staining). Then, eosinophils were re-suspended (1 × 10⁶ cells/ml) in eosinophil medium consisting of RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS; HyClones; Logan, Utah, USA), penicillin-streptomycin solution (100 u/ml; Biological Industries, Beit Haemek, Israel), and GM-CSF (5 ng/ml; Peprotech, Rocky Hill, NJ, USA).

Eosinophil viability and degranulation assay

For viability and degranulation assays, eosinophils (10⁵ cells/well) were incubated at 37 °C, 5% CO₂ for 1, 3, and 20 h in eosinophil medium with C. albicans at MOI 0.1, and 1 or with commercially synthesized candidalysin peptide toxin (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK; >98% pure; Peptide Protein Research Ltd, Hampshire, UK). Blocking of CD48 was assessed by pre-incubation of C. albicans with D1-Fc or hCD48-Fc (5 µg/ml) for 40 min on ice. Yeast was then washed with phosphate buffer saline (PBS) prior to the co-culture. After the indicated time points, viable cell count was determined by trypan blue exclusion test using a Neubauer chamber cell, and supernatants were collected to evaluate the levels of EPX and LDH. EPX levels were measured using a standardized colorimetric assay with purified human EPX (a kind gift of Prof. Gerald J. Gleich, University of Utah Health Sciences Center, Salt Lake City, UT, USA) and freshly prepared peroxidase substrate solution containing o-phenylenediamine dihydrochloride (OPD; Thermo-Fisher Scientific; Kyriat Shmona, Israel) as previously described⁶⁷. LDH levels were determined using Lactate dehydrogenase assay kit (Abcam).

Generation of human and mouse CD48-Fc

Fusion proteins were generated as previously described⁵⁰. In brief, the sequence encoding the extracellular part of human CD48 was amplified by PCR using the 5ʹ primer cccACCGGT GCCGCCACC (including AgeI restriction site) and the 3ʹ primer CATTCTTGGCCTGTTACTTACGGATCCccc (including BamHI restriction site). This PCR fragment was cloned into an expression vector containing a mutated Fc portion of human IgG1 (CSI-Ig Puro plasmid). The same protocol was performed for the generation of the mouse CD48-Fc (mCD48-Fc). All proteins were generated in 293 T cells and were purified on a protein G column (HiTrap TM protein G HP, GE Health Care, Chicago, IL, USA).

Generation of WT and CD48^–/– BMEos

Bone marrow cells obtained from femurs of CD48^–/– and WT mice were differentiated as previously described⁶⁸. On days 10–12, eosinophil purity was examined by flow cytometry. Eosinophils were recognized by their expression of characteristic cell surface markers using PE-anti-mouse Siglec-F (BD Biosciences, NJ, USA) and APC-anti-mouse CCR3 (BioLegend). CD48 expression was also evaluated by flow cytometry using FITC-anti-mouse CD48 or hamster IgG isotype control (eBioscience, CA, USA).

Determination of fungal colony forming unit (CFU) in vitro

To evaluate C. albicans viability. human and murine eosinophils (1 × 10⁵ cells/well) were co-cultured for 3 and 20 h with C. albicans at MOI 0.1 or 1. To assess the effect of eosinophil mediators, C. albicans (1 × 10⁵ yeast/well) was incubated with purified eosinophil basic mediators, each at the concentration present in 1 × 10⁵ eosinophils^21,27. Cells were then collected and diluted in PBS by serial dilutions and plated onto Sabouraud agar plates, incubated at 30 °C for 48 h, and CFU numbers were counted.

Flow cytometry

For the staining of cell surface receptors, cells were harvested, centrifuged at 250 × g for mammalian cells and 3000 × g for fungal cells, washed twice in flow cytometry buffer (PBS, 0.1% bovine serum albumin (BSA)), and counted using a hemocytometer. Cells (1 × 10⁵ cells/well) were plated into a 96 U-bottom plate and resuspended in blocking buffer (flow cytometry buffer supplemented with 5% goat serum, Biological Industries) for 15 min on ice. Human eosinophils were incubated with the following primary antibodies: anti-human CD48 (BioLegend), anti-human Dectin-1 (Abcam), anti-human TLR2 (BioLegend), anti-human TLR4 (BioLegend), anti-phospho-Lyn (Cell signaling, Danvers, MA USA) or the appropriate isotype control (mouse IgG1 or mouse IgG2a; all at 2.5 μg/ml) for 40 min on ice. Cells were then washed again and incubated with the secondary antibody Fluorescein (FITC)-goat anti-mouse IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 30 min on ice in the dark. C. albicans staining was assessed using in-house murine and human CD48-Fc or D1-Fc (5 µg/ml), followed by FITC-goat anti-human IgG (1:2000 in FC buffer; Jackson ImmunoResearch). Afterward, the cells were washed twice, resuspended in 200 μl flow cytometry buffer, and analyzed. Samples were acquired by Cytoflex flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo (FlowJo X 10.0.7r2, Tree Star, Ashland, OR, USA) software.

Immunofluorescence microscopy

C. albicans yeast/hyphae were grown as described above and washed twice in ice cold PBS. Hyphal cells were grown overnight at 37 °C with RPMI-1640 supplemented with 10% FCS (Sigma-Aldrich) and 1% penicillin-streptomycin solution (100 U/ml; Biological Industries). Cells (2 × 10⁵) were incubated in the presence of FITC (0.1 mg/ml, diluted in PBS) for 30 min, washed twice in ice-cold PBS (3000 G, 4 °C, 5 min) and blocked using PBS/5% BSA for 2 h at 4 °C. Next, the cells were washed again in PBS followed by incubation with hCD48-Fc or the negative control protein D1-Fc for 2 h at 4 °C. Unbound protein was then washed with ice cold PBS and incubated with the secondary antibody PE-anti-human IgG (1:500; Jackson ImmunoResearch) for 2 h at 4 °C and then washed with PBS. Finally, cells were mounted with DAPI mounting media and visualized using an Olympus Fluoview FV1000 confocal microscope.

Generation of BW-hCD48 cells

Reporter cells for hCD48 (BW-hCD48) were generated as previously described⁶⁹. The inserts for the expression vectors encoding CD48 and zeta chain were generated by PCR using the following primers:

1-CD48 forward: cccAAGCTT GCCACC ATGTGCTCCAGAGGTTGGGA

2-CD48 reverse containing additional sequences complementary to zeta chain forward: TTCCATCTAGCAAGTAGCAGAG GGACCGGGCCAGGGTACAGGG

3-Zeta chain forward containing additional sequences complementary to CD48 reverse CCCTGTACCCTGGCCCGGTCC CTCTGCTACTTGCTAGATGGAA

4-Zeta chain reverse gggCTCGAG TTAGCGAGGGGCCAGGGTCTGC.

The full construct was then introduced into a plasmid with an antibiotic resistance (Puromycin) and inserted into BW5147.3 cell line (BW) using electroporation. Cells were cultured in RPMI-1640 supplemented with 10% FCS, penicillin-streptomycin solution (100 u/ml; Biological Industries) and gentamycin (5 µg/ml, G418; Sigma-Aldrich) for selection. Before each experiment, cells were tested by flow cytometry for CD48 expression as described above.

BW reporter assay

WT and C. albicans mutant strains: als1Δ/Δ1467, als2Δ/Δ 2757, als3Δ/Δ 1843, als4Δ/Δ 2034, als5Δ/Δ 2373, als6Δ/Δ 1420, als7Δ/Δ 1429, als9Δ/Δ 2028 and the C. albicans clinical strains were grown as described above. The fungal cells (5 × 10⁴ yeast/well) were first incubated in a 96 flat-bottom plate for 2 h at 37 °C to adhere to the plate. Then, BW or BW-hCD48 cells were added (MOI 1) in a final volume of 200 μl RPMI-1640 growth media as described above and for an additional 48 h (37 °C; 5% CO₂). The medium was supplemented with fluconazole (5 µg/ml) to prevent hyphae formation. After 48 h cells were centrifuged and supernatants were collected for measurement of released mIL-2 by ELISA.

ELISA assays

Cytokine levels in the serum or in homogenized kidneys of mice infected with C. albicans were determined 2 dpi by commercial kits for IFN-γ (Peprotech, Rehovot, Israel), IL-6 (ELISA MAX™ Deluxe Set Mouse IL-6, BioLegend) according to the supplier’s instructions.

mIL-2 levels in the supernatants from BW and BW-CD48 cells were determined by in-house ELISA. ELISA plates were initially coated with anti-mouse-IL-2 (0.1 μg/100 µl PBS; BioLegend) for 2 h, 37 °C. and blocked with PBS/1% BSA for an additional 2 h at room temperature. Supernatants were then added to the plates and incubated overnight at 4 °C. The plates were then washed 4 times with PBST (PBS/0.05% tween20) and a detection antibody biotinylated anti-mouse-IL-2 (0.1 μg/100 µl PBS; BioLegend) from a different clone than the coating antibody was added for 1 h at room temperature. Next the plates were washed 6 times with PBST, incubated for 30 min with Streptavidin-HRP (1:1000; Jackson ImmunoResearch) and again washed 6 times using PBST. Finally, the plates were developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (SouthernBiotech) and read at 650 nm using an ELISA reader (BIO-TEK, Winooski, VT, USA).

Quantification and statistical analysis

If not indicated otherwise, experiments were always performed in triplicates and repeated at least three times. Due to strong eosinophil donor variability, data from experiments with human eosinophils are shown as representative of three or more independent experiments performed with different donors. Results are expressed as mean ± SD or mean ± SEM and statistical tests were performed using Prism (GraphPad Prism 10.5.0.730) software. The unpaired two-tailed Student’s t test was used to compare differences between two groups. One-way ANOVA with Tukey’s test for multiple comparisons was used to evaluate experiments containing more than two groups. Survival curves were analyzed by Log-rank (Mantel Cox) test. P values less than 0.05 were considered significant for all analyses.

Ethics statement

Peripheral blood eosinophils were obtained according to the guidelines of the Hadassah-Hebrew University Human Experimentation Helsinki Committee (0410-14-HMO). All work involving human patients was approved by the Tel Aviv Sourasky Medical Center Institutional Ethics Committee (approval number 0729-16). The need for informed consent was waived given the retrospective observational nature of this study.

Mice experiments were performed according to the guidelines of the Hebrew University ethics committee for animal studies (MD-19-15990-5).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All the reagents used, and data supporting the findings of this study are available within the article and in the Supplementary Table 1, or available from the corresponding author upon request. Source data files are provided with this paper. Source data are provided with this paper.

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Acknowledgements

This study was supported by the Israel Science Foundation (442/18), Aimwell Charitable Trust (UK), and the Emalie Gutterman Memorial Endowment Fund to F.L.S.; and by the Israeli Scholarship Education Foundation (ISEF) to I.Z. J.R.N. was supported by the Wellcome Trust (214229_Z_18_Z). J.V. was a Jacob and Lena Joels Memorial Foundation Visiting Professor at the HUJI. We thank Prof Ariel Munitz (Tel Aviv University) for his valuable comments on mice experiments and Dr. Eduard Berenshtein from the electron microscopy core facility of the Hebrew University for his assistance with scanning electron microscopy.

Author information

These authors contributed equally: Ilan Zaffran, Pratibha Gaur.

Authors and Affiliations

Pharmacology and Experimental Therapeutics Unit, School of Pharmacy, Institute for Drug Research, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel
Ilan Zaffran, Pratibha Gaur, Prince Ofori, Hadas Pahima, Joana Vitte & Francesca Levi-Schaffer
The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research Israel-Canada (IMRIC), Hebrew University Medical School, Jerusalem, Israel
Yoav Charpak-Amikam & Ofer Mandelboim
Infectious Diseases Unit, Tel Aviv Sourasky Medical Center; Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
David Shasha & Ronen Ben-Ami
Department of Clinical Microbiology and Immunology Gray School of Medical Sciences, Tel-Aviv University, Tel-Aviv, Israel
Nir Osherov
Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knoell Institute, Jena, Germany
Bernhard Hube
Institute of Microbiology, Friedrich Schiller University, Jena, Germany
Bernhard Hube
Centre for Host-Microbiome Interactions, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, London, UK
Julian R. Naglik
INSERM UMR-S 1250 P3CELL and Laboratory of Immunology, Faculty of Medicine; University Hospital of Reims, University of Reims, Champagne-Ardenne, Reims, France
Joana Vitte
Montpellier University, Institut Desbrest d’Épidémiologie et de Santé Publique, Institut National de la Sante et de la Recherche Medicale, UMR UA 11, Montpellier, France
Joana Vitte

Authors

Ilan Zaffran
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Pratibha Gaur
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Prince Ofori
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Yoav Charpak-Amikam
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Hadas Pahima
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David Shasha
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Nir Osherov
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Bernhard Hube
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Ronen Ben-Ami
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Julian R. Naglik
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Ofer Mandelboim
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Francesca Levi-Schaffer
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Contributions

I.Z., P.G., and P.O. performed the experiments, analyzed data, and wrote the manuscript; Y.C.A., B.H., J.R.N., R.B.A., N.O., and O.M. assisted with methodology; H.P., D.S., B.H., J.R.N., and R.B.A. provided reagents and fungal strains; J.V. edited the manuscript; Y.C.A., B.H., J.R.N., and O.M. reviewed the manuscript; F.L.S. designed the research, provided overall supervision, and edited the manuscript.

Corresponding author

Correspondence to Francesca Levi-Schaffer.

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Competing interests

J.V. reports speaker and consultancy fees and travel support in the past 5 years from Astra Zeneca, HpVac, Novartis, L’Oréal, Sanofi, Stallergènes-Greer, and Thermo Fisher Scientific. The other authors have no financial competing interests.

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Zaffran, I., Gaur, P., Ofori, P. et al. Eosinophil CD48 interactions with Candida albicans Als6 is protective in vitro and in mouse systemic candidiasis. Nat Commun 16, 9291 (2025). https://doi.org/10.1038/s41467-025-64276-3

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Received: 04 July 2023
Accepted: 15 September 2025
Published: 21 October 2025
Version of record: 21 October 2025
DOI: https://doi.org/10.1038/s41467-025-64276-3