Integrin αXβ2 Is a Leukocyte Receptor for Candida albicans and Is Essential for Protection against Fungal Infections

doi:10.4049/jimmunol.1200524

J Immunol. 2012 September 1; 189(5): 2468–2477.

Published online 2012 July 27. doi: 10.4049/jimmunol.1200524

PMCID: PMC3424400

Integrin α_Xβ₂ Is a Leukocyte Receptor for Candida albicans and Is Essential for Protection against Fungal Infections

Samir Jawhara,^* Elzbieta Pluskota,^* Dmitriy Verbovetskiy,^* Olena Skomorovska-Prokvolit,^† Edward F. Plow,^* and Dmitry A. Soloviev^*

^*Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic, Cleveland, OH 44195; and

^†Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, OH 44106

Corresponding author.

Address correspondence and reprint requests to Dr. Dmitry A. Soloviev, Cleveland Clinic, 9500 Euclid Avenue, Mail Code NB50, Cleveland, OH 44195. E-mail address: solovjd/at/ccf.org

Received February 10, 2012; Accepted June 28, 2012.

This article is distributed under The American Association of Immunologists, Inc., Reuse Terms and Conditions for Author Choice articles.

Abstract

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin α_Xβ₂ in the pathogenesis of fungal infection was assessed. Both purified α_Xβ₂ and α_Xβ₂-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by β-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of α_Mβ₂. Mice deficient in α_Xβ₂ were more prone to systemic infection with the LD₅₀ fungal inoculum decreasing 3-fold in α_Xβ₂-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 10⁴ cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of α_Xβ₂-deficient mice within 9 d. Organs taken from α_Xβ₂-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that α_Xβ₂ is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α_Xβ₂ to control fungal invasion.

Introduction

Candida albicans is a common opportunistic fungal pathogen. It is a dimorphic fungus existing as rounded yeast cells or as filamentous forms (1, 2). Although the yeast form can colonize mucosal membranes, it is thought that the filamentous form provides some protection to the microorganism against host defense systems, and the ability of C. albicans to rapidly and reversibly switch between yeast and filamentous morphologies is crucial to its pathogenicity (3–6). In recent years, Candida infections ranked as the fourth most common cause of nosocomial infections with immunocompromised patients being particularly susceptible (7, 8). Bloodstream fungal infections have an extremely high (30–70%, by different estimations) morbidity and mortality (8–11).

The innate immune system provides the principal protection against Candida infections. Polymorphonuclear leukocytes have been shown to be the primary components of the cellular immune defenses against Candida (12–14), and a protective role for macrophages in disseminated candidemia has also been suggested (13, 15, 16). The most prominent receptors on leukocytes used in fungal or microbial recognition are integrins of the β₂ subfamily (17, 18). This subfamily of leukocyte receptors is composed of four members that share a common β₂ subunit that associates noncovalently with one of four distinct but structurally homologous α subunits to form α_Mβ₂ (Mac-1, CD11b/CD18, and CR3), α_Lβ₂ (LFA-1 and CD11a/CD18), α_Xβ₂ (p150,95 CD11c/CD18 and CR4), and α_Dβ₂ (CD11d/CD18) (19–23). These cell surface receptors are expressed on monocytes, granulocytes, macrophages, and NK cells and have been implicated in diverse protective responses mediated by these cells, including phagocytosis, cell-mediated killing, chemotaxis, and cellular activation. Specifically, the β₂ integrins mediate migration of leukocytes to sites of infection and adhesion to microorganisms with subsequent phagocytosis or killing of many pathogens (12, 17, 24). Patients with leukocyte adhesion deficiency-1 (LAD-1), a rare hereditary disease that is characterized by low expression (mild LAD-1) or complete absence (severe) of all four of the β₂ integrins because of mutations in the ITGB2 (β₂) gene (25, 26), are highly susceptible to a wide range of bacterial and fungal infections (27, 28) [and the increased sensitivity of such patients to C. albicans infections has been discussed (29)]. Although other leukocyte pattern recognition receptors, which recognize fungal β-glucans (Dectin-1 and TLR2 (30, 31) and mannan-specific TLR4 (32)), also participate in fungal recognition and apparently are essential in leukocyte activation and notably in activation of β₂ integrins (33, 34), they do not directly facilitate leukocyte migration, adhesion, or phagocytosis.

Of the β₂ integrins, α_Mβ₂ has been specifically implicated in the recognition of C. albicans. Polymorphonuclear leukocytes and NK cells use α_Mβ₂ to adhere only to the filamentous form but not to the yeast form of C. albicans (35, 36). C. albicans pH-regulated Ag 1 (Pra1) (37), also known as fibrinogen binding protein 1 (38) or C. albicans 58-kDa mannoprotein (39), was identified as the major ligand of α_Mβ₂ among C. albicans proteins (40). Pra1p is a mannoprotein (1, 41) and is expressed on the surface of the hyphae but not on the yeast form of C. albicans (3, 41). Expression of Pra1p is strongly pH dependent and is also regulated by nutrition and certain other fungal genes (37, 41, 42). Disruption of the PRA1 gene protects the fungus against leukocyte killing in vitro and in vivo, impedes the innate immune response to infection, and increases overall fungal virulence and organ invasion in vivo (29, 43).

Although mutations in α_M subunits have been previously described (44), it appears that the clinical manifestations of selective loss of α_Mβ₂ are less severe than when all four β₂-integrins are absent, which suggests that α_Mβ₂ may share at least part of its surveillance functions with other β₂ integrins, most likely with the integrin α_Xβ₂ (19, 45). Integrin α_Xβ₂ is present on the surface of all leukocyte subsets that express α_Mβ₂, with the exception of dendritic cells, which have CD11c (α_X) as a major surface marker. These integrins are ~70% identical and also share a number of ligands, most notably fibrinogen (46), ICAM-1 (47), and iC3b, a component of the complement system (48). However, the functions of α_Xβ₂ are less well studied, and its functions in innate immunity are still unclear. It was shown that α_Xβ₂ is involved in macrophage-mediated phagocytosis of Mycobacterium tuberculosis (49) and Mycobacterium leprae (50) and may play a role in the development of gastric ulcers in chronic Helicobacter pylori infection (51).

The present study was undertaken to determine the role and significance of the α_Xβ₂ in C. albicans pathogenicity and the effects of its elimination on host defense in vivo, using α_Xβ₂-deficient mice in a model of systemic murine candidiasis.

Materials and Methods

C. albicans strains

C. albicans strain SC5314 was used in most in vitro and in vivo experiments. In some experiments, the Pra1-depleted strain CAMB5-18 (pra1::hisG/pra1::hisG iro1-ura3Δ/IRO1-URA3) was also used. This strain was derived from the strain CAMB435 by reversion of the iro1-ura3 deletion as previously described (52) and was characterized by us previously (29). All strains were routinely maintained on Difco Sabouraud Dextrose Agar (SDA) plates (BD Biosciences, Sparks, MD).

Animals

α_X-Knockout (KO) mice (Δα_Xβ₂) were provided by Dr. C. M. Ballantyne (Baylor College of Medicine, Houston, TX). This mouse line was generated in parallel with other β₂-KO lines (53). All these β₂-KO murine lines have been used in a number of studies in comparison with the Δα_M mice (54–56), and there are no separate publications only characterizing the Δα_X mice. In our laboratory, these mice were backcrossed for more than 12 generations into a C57BL/J6 background. Before experiments, the genotypes of all mice were confirmed by PCR of blood DNA samples. Age-matched C57BL/J6 mice, obtained from The Jackson Laboratory (Bar Harbor, ME), were used as controls (wild-type [WT] mice). All protocols involving mice were approved by the Institutional Animal Care and Use Committee in accordance with the Public Health Service policy, the Health Research Extension Act (PL99-158), and the Cleveland Clinic policy. All experiments on mice involving C. albicans infections were carried out in a BSL2 facility. The mice were maintained during experiments on a 12-h alternating light/dark cycle and supplied with food (diet number 2918; Harlan Teklad, Madison, WI) and sterilized water ad libitum.

Cells

Human epithelial kidney 293 (HEK293) cells expressing α_Xβ₂ (HEK293/α_Xβ₂ cells) were prepared as described previously (57–59). Briefly, human full-length α_X and β₂ cDNAs were blunt-end cloned into TOPO-pCDNA 3.1(+) expression vector (Life Technologies, Carlsbad, CA). Plasmids were sequenced to ensure appropriate insert orientation and cotransfected into HEK293 cells using the LipofectAMINE Plus reagent (Life Technologies). To prepare control mock-transfected cells, the vector alone was used. Transfected cells were selected using neomycin sulfate (Life Technologies), and cells expressing the integrin were detected and sorted by flow cytometry (FACS) using a FACStar cell sorter (BD Biosciences) and anti-human CD18 mAb (clone IB4). The sorted cells were subcloned, and α_Xβ₂ expression was characterized by FACS. The cell lines obtained were routinely grown in a monolayer in DMEM/F12 medium, supplemented with 10% FBS (all from BioWhittaker). For experiments, the cells were harvested using enzyme-free Cell Dissociation buffer (Life Technologies), washed with HBSS, and resuspended in HBSS containing 100 mM HEPES (pH 7.4), 5 mM CaCl₂, and 5 mM MgCl₂ (HBSS/HEPES). Monocytes were isolated from human donor blood using the Pan Monocyte Isolation Kit (Miltenyi Biotec) following the manufacturer’s instructions.

All studies involving human blood cells were performed in accord with protocols and policies approved by the Institutional Review Board at Cleveland Clinic and with the Helsinki Declaration of 1975 as revised in 2000.

Abs and inhibitors

mAbs used in this study were as follows: 44a (anti-human α_M I-domain, IgG1), OKM1 (anti-human α_M lectin domain, IgG2b), IB4 (anti-human β₂, IgG2a). The hybridoma cell lines producing these mAbs were obtained from the American Type Culture Collection and adapted to growing in serum-free media in CELLine Bioreactor Flasks (Integra Biosciences, Hudson, NH) in the Cleveland Clinic Hybridoma Core. The mAbs were purified from conditioned media using recombinant protein G columns (Life Technologies).

The mAb clone 3.9 (anti-human α_X, IgG1) and clone N418 (anti-mouse α_X) were purchased from BioLegend (San Diego, CA), and mAb clone YW62.3 (anti-mouse CD45) was obtained from Serotec (Raleigh, NC).

Baker yeast β-glucan, mannan, and echistatin (60, 61) were purchased from Sigma-Aldrich (St. Louis, MO). The recombinant hookworm neutrophil inhibitory factor (NIF) was prepared as described previously (58).

Tissue section preparation and assaying

Brains and livers from experimental mice were snap-frozen in OCT. Brain sections (6 μm thick) were processed for immunohistochemical staining with the following Abs: hamster anti-mouse CD11c (BioLegend), followed by biotinylated anti-hamster Ab and streptavidin Alexa Fluor 568. CD45 was stained using rat anti-mouse CD45 mAb (BD Biosciences) and rabbit–anti-rat Ab conjugated to Alexa Fluor 488. The slides were mounted using Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). To visualize the extent of the fungal cell invasion and proliferation, the brain and liver sections were stained with periodic acid–Schiff stain. The images were observed using a Leica DMR microscope equipped with ×10/0.4 and ×20/0.5 NA objective lenses (Leica Microsystems, Wetzlar, Germany) and photographed with a Qimaging Retiga ExiFas camera (Qimaging, Burnaby, British Columbia, Canada) using ImagePro 5.1 software (Media Cybernetics, Silver Spring, MD).The images were processed with Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA).

C. albicans staining with soluble α_Xβ₂

Recombinant α_Xβ₂ was isolated from HEK293/α_Xβ₂ cells with the method previously used by us to purify α_Mβ₂ (40, 59). Briefly, 10 g cells was harvested, washed, and lysed with 1% Triton X-100 in TBS containing protease inhibitor mixture for mammalian cells (Sigma-Aldrich). The cell lysate was clarified by centrifugation, diluted with TBS containing CaCl₂ and MgCl₂ and loaded onto a column of immobilized IB4 (anti-β₂). To prepare the immunoadsorbent, purified IB4 mAbs were coupled to cyanogen bromide-activated Sepharose 4B (GE Biosciences, Piscataway, NJ) following the manufacturer’s protocol to final concentrations of 2.2–2.8 mg immobilized proteins per 1 ml swollen gel. After washing with TBS containing 10 mM octyl-β-d-glucopyranoside (OG; Calbiochem, San Diego, CA) and CaCl₂/MgCl₂, bound protein was eluted with three column volumes of 20 mM sodium acetate buffer (pH 4.2), containing OG and Ca/Mg²⁺. Immediately after elution, 100 μl 1 M HEPES-NaOH (pH 8) was added to each 1 ml of the column eluate to neutralize the acidic pH. Protein fractions were pooled and dialyzed against HEPES-NaCl, OG, and Ca/Mg²⁺. The proteins were biotinylated using Sulfo-NHS-LC-Biotin (Pierce), according to the manufacturer’s protocol. To visualize α_Xβ₂–C. albicans interaction, C. albicans strain SC5314 was allowed to germinate in RPMI 1640 medium for 2 h and then purified. Biotinylated α_Xβ₂ was added to obtain a 1 μg/ml concentration, and the samples were incubated for 1 h at 37°C. After incubation, the fungi were washed with Dulbecco's PBS (D-PBS) and incubated with FITC–streptavidin conjugate for 30 min at room temperature. Subsequently, the fungi were again washed with D-PBS. Blankophor, the β-glucan, and chitin-specific dye were added, and the mixture was incubated for an additional 30 min (62). Finally, the fungi were washed with D-PBS and analyzed by fluorescence microscopy (Leica Microsystems) at a magnification of ×800.

Cell adhesion assays

Cell adhesion assays were performed as described previously (40, 63). Briefly, to determine cell adhesion to fungal hyphae, 48-well Costar tissue culture plates (Corning, Corning, NY) were precoated with polyvinylpyrrolidone (PVP; Sigma-Aldrich) and washed with HBSS, and aliquots of 5 × 10⁵ C. albicans yeast were added and incubated overnight at 37°C to germinate. For adhesion to C. albicans yeast, the fungi were incubated in YNB broth to prevent germination. After incubation, the supernatant was removed and adherent fungi were carefully washed with HBSS. A total of 10⁵ PMA-activated peripheral blood human monocytes or HEK293/α_Xβ₂ cells were added in HBSS/HEPES and assay plates were incubated at 37°C for 1 h. Control wells were coated with PVP only. Each experimental point was in triplicate. Subsequently, plates were washed, and the number of adherent cells in each well was quantified using the CyQUANT Cell Proliferation Assay kit (Life Technologies) as described previously (40, 63). For inhibition assays, before addition to the plate wells, the HEK293/α_Xβ₂ cells or isolated monocytes were preincubated with 10–20 μg/ml selected Abs, 1 mM β-glucan, 1 mM mannan, or 5 μM echistatin for 10 min at room temperature. Data from cell adhesion and migration (see below) are presented as percentage (mean ± SE) of total cells (to which was assigned the value of 100%) and represent the results of three independent experiments.

Cell migration assays

Human peripheral blood monocytes and HEK293/α_Xβ₂ cell migration assays were performed in serum-free RPMI 1640 (monocytes) or DMEM/F-12 (HEK293/α_Xβ₂) medium (Life Technologies) using modified Boyden chambers (Costar Transwell inserts in a 24-well plate format; Corning) with tissue culture-treated polycarbonate filters of 5-μm (for monocytes) or 8-μm pores (for HEK293/α_Xβ₂ cells) as described previously (40, 63–65). The lower chambers contained 600 μl media with 10⁶ C. albicans yeast, which were germinated overnight prior to beginning the analyses. The upper chambers contained final volumes of 200 μl HEK293/α_Xβ₂ cell suspensions. The assays were initiated by addition of 50 μl cell suspension (10⁵ cells/well) to 150 μl media in the upper chambers, and the plates were placed in a humidified incubator at 37°C and 5% CO₂ for 8 h. For inhibition experiments, selected Abs, NIF, and glycans were added simultaneously with the cells to the upper chamber. After migration, nonmigrated cells were removed from upper chamber using cotton swabs. The migrated cells, present on the undersurface of the membrane as well as in the lower chamber, were quantified using the CyQUANT Cell Proliferation kit as described above and previously (63, 64).

Killing (phagocytosis) assay

A total of 10⁵ C. albicans SC5314 strain cells in 0.25 ml high glucose RPMI 1640 medium containing 0.1 M HEPES (pH 7.8) were allowed to germinate in plastic tubes at 37°C for 1 h with slow agitation. The fungal cells were collected by centrifugation, washed twice with D-PBS, suspended in 0.25 ml HBSS/HEPES (pH 7.4), and mixed with 3 × 10⁵ (1:3 ratio), 7 × 10⁵ (1:7 ratio), or 1.1 × 10⁶ (1:11 ratio) HEK293/α_Xβ₂ or mock-transfected HEK293 cells. In control experiments, the HEK293/α_Xβ₂ cells were preincubated with 20 μg/ml anti-α_X mAbs 3.9 for 10 min at room temperature. The cell/fungal mixtures were incubated at 37°C with slow shaking for 2 h. To determine the extent of killing/phagocytosis, aliquots of the cell/fungal suspension were taken every 20 min, diluted with HBSS/HEPES, and subsequently plated in serial dilutions on SDA plates. The CFU were counted manually on day 2 using a Bel-Art Products Colony Counter. Cells were not lysed before plating, and all fungal cells that remained ingested were recorded as “killed.” Results were independently verified by a modification of the method of Lehrer et al. (40, 66). Briefly, at the experiment end point, an equal volume of 1% Tween 80 was added to the fungal/leukocyte mixture to lyse leukocytes, and C. albicans cell pellets obtained by centrifugation were resuspended in 0.25 ml 2.5 mM methylene blue (Sigma-Aldrich) in HBSS/HEPES. The number of viable (nonstained) cells was counted in a hemacytometer. Control samples contained C. albicans incubated without HEK293 cells. Results obtained by both methods of quantitation of fungal viability showed close correlation with variances in the 5–10% range.

Murine model of systemic disseminated candidiasis

The model of systematic candidiasis, described and applied to C57BL/6 by MacCallum and Odds (67), was used. Each experimental group contained 10 mice of 10–12 wk of age, and weights ranged from 19 to 21 g. C57BL/6 (control WT mice) and α_Xβ₂-depleted mice (Δα_X mice) were injected with 10⁵ or 3 × 10⁵ C. albicans SC5314 strain in 0.1 ml sterile saline via the tail vein. Mice were returned to cages and monitored. To determine the degree of distress, we developed a scoring system, similar to one described for the determination of humane end points in a murine model of leukemia (68). The following symptoms were evaluated and scored as follows: coma, 15 points; weight loss of: 20%, 15 points, 15%, 11 points, and 10%, 8 points; abdominal swelling, 6 points; significant decrease in mobility, 4 points; hunched posture, 3 points; porphyrin “red tears,” 3 points; head pressing, 2 points; and spiky coat, 2 points. Each group was monitored daily over the 14-d period at the same time each day. The score was noncumulative and was recalculated for each mouse every 24 ± 1.5 h. The primary end point was the number of mice surviving on day 14 within each experimental group. When mice scored 15 or more points before 14 d, the animals were euthanized. At day 14, all surviving mice were euthanized and subjected to pathological examination. To determine the extent of C. albicans invasion and fungal burden in individual organs, liver, spleen, heart, lungs, and kidney were harvested, weighed, and homogenized in 5 ml PBS, and serial dilutions of the homogenates were plated onto SDA plates for CFU quantitation. In separate experiments, mice from each group of five mice were euthanized after 16 or 40 h of infection, and their organs were removed and examined to determine the degree of the fungal invasion and organ fungal burdens (67).

Statistical analyses

Statistical significance was determined using paired log-rank and Cox regression for mouse survival data or Student t test in all other cases. For all statistical calculations the statistical package in SigmaPlot, version 12.0 (Jandel Scientific Software, Chicago, IL) was used. Differences between groups were considered significant with p < 0.05. Data are expressed as means ± SD unless otherwise noted.

Results

Integrin α_Xβ₂ is required to control C. albicans infection

As a first step to assess the biological significance of α_Xβ₂ in the context of the total host–pathogen relationship, transgenic mice that lack α_X (Δα_X mice) were used in a murine model of disseminated candidiasis. In this assay, mice of both WT (C57BL/6 mice) and Δα_X lines were challenged with 10⁵ or 3 × 10⁵ C. albicans inocula via tail vein injection. All WT mice inoculated with 10⁵ (0.5 × 10⁻⁴/g) C. albicans survived 14 d (336 h). α_Xβ₂ elimination dramatically decreased mice survival; during these same 336 h, 50% of the Δα_X-mice reached the predetermined end point (see Materials and Methods for a complete list of “mortality” criteria) with a median survival time 264 h (Fig. 1A, left panel). A similar level of mortality occurred in WT mice only after introduction of a 3-fold higher inoculum (Fig. 1B). After challenge with the 3 × 10⁵ C. albicans cells (~1.5 × 10⁻⁴/g) inoculum, ~50% of WT mice reached the end point within 12 d with a median survival time at 252 ± 12 h. In contrast, all Δα_X mice reached the end point with this inoculum within the first 9 d with a median survival time of 112 ± 8 h (p < 0.01; log-rank test) (Fig. 1A, right panel).

FIGURE 1.

The effect of α_Xβ₂ elimination on C. albicans virulence in a murine model of disseminated candidiasis. (A) The Kaplan–Meier (cumulative) graphic of murine survival. A total of 10⁵ (left panel) or 3 × 10⁵ (right panel) of (more ...)

Elimination of α_Mβ₂ decreases resistance of brain and liver to C. albicans invasion

The substantial increase in susceptibility of the Δα_X mice to the Candida infection indicates that α_Xβ₂ plays a significant role in antifungal protection and innate immunity. This interpretation was further corroborated by pathological examination of infected mice. To determine the impact of α_Xβ₂ deletion on the rate of fungal colonization, WT and the Δα_X mice were challenged i.v. with 10⁵ C. albicans, and selected organs (brain, kidney, lung, heart, spleen, and liver) were recovered 16 and 40 h postinfection and at day 14 from mice that survived. Tissue targeting and invasion, fungal dissemination, and organ fungal burden were assessed in recovered organs. Consistent with previous studies (69), high fungal burden was present in the kidneys at all times postinfection and was similar at 16 and 40 h (p = 0.96; Student t test) in both mouse strains (Fig. 2A, ,2B).2B). There was no significant difference (p > 0.05) in fungal burdens in the spleen, heart, and lungs at both early time points; but at day 14, differences in fungal burden in the surviving WT and Δα_X mice become evident (p < 0.05). At the day 14 survival point, fungal burden in the kidney was significantly elevated (2.8-fold difference; p < 0.01) in Δα_X mice (2 × 10⁴ ± 3.2 × 10³ CFU/g) compared with WT mice (7.7 × 10³ ± 1.2 × 10³) (Fig. 2C). In contrast, fungal burdens in brains and livers recovered from Δα_X mice were substantially elevated compared with the corresponding WT organs: at 16 h, Δα_X brain had 2 × 10⁴ ± 2.4 × 10³ CFU/g tissue, whereas WT brains had 6.1 × 10³ ± 7 × 10² CFU/g (10-fold raise; p < 0.01); Δα_X liver had 7.6 × 10³ ± 1.3 × 10³ CFU/g, 2-fold rise (p < 0.05), whereas WT liver had a fungal burden of 3.5 × 10³ ± 360 CFU/g (Fig. 2A). These differences increased over time: at 40 h, 2.9 × 10⁴ ± 2.3 × 10³ CFU/g Δα_X brain compared with 700 ± 120 CFU/g WT brain (40-fold rise; p < 0.005) and 3 × 10³ ± 220 CFU/g Δα_X liver compared with 480 ± 110 CFU/g WT liver (6-fold rise; p < 0.01) (Fig. 2B). These differences were sustained at day 14 in all surviving mice: 6400 ± 1820 versus 200 ± 160 CFU/g (32-fold; p < 0.01) and 3100 ± 1200 versus 700 ± 400 CFU/g (4-fold; p < 0.01) for brain and liver of Δα_X and WT mice, respectively (Fig. 2C).

FIGURE 2.

The effect of α_Xβ₂ elimination on C. albicans dissemination. Challenged i.v. with SC5314 10⁵ C. albicans, WT ( [filled square]

), or Δα_X (□) mice were euthanized after 16 h (A) or 40 h (B) of infection; their organs (brain, (more ...)

Residential macrophages require α_Xβ₂ to control C. albicans invasion in vivo

The results of fungal burden studies were further confirmed in histological sections of tissues from the infected organs. In the sections of WT brains obtained at 40 h postinfection, staining with periodic acid–Schiff reagent revealed only several scattered fungal hyphae (Fig. 3A, left panel). In contrast, in the brains of Δα_X mice 40 h postinfection, C. albicans formed a visible network of numerous fungal hyphae (Fig. 3A, right panel). After 14 d of infection, in the kidney sections of WT mice, C. albicans formed single scattered colonies (Fig. 3B, left panel), whereas in kidney sections of Δα_X mice, the fungal colonies were numerous and showed evidence of extensive organ colonization (arrows in Fig. 3B, right panel).

FIGURE 3.

The effect of α_Xβ₂ elimination on C. albicans invasion of the brain. Histological sections of murine brains taken after 16 h of infection (A) or of murine kidney obtained after 14 d of infection (B) from WT (left two photos) or from Δα (more ...)

To ensure that α_Xβ₂ deletion affects only leukocytes, the sections of brains from WT (Fig. 3C) and Δα_X (Fig. 3D) mice at 40 h of infection were immunostained with Abs against the common hematopoietic cell surface marker CD45 (anti-Ly5, labeled with Alexa Fluor 488, green fluorescence), anti-CD11c (anti-α_X, labeled with Alexa Fluor 568, red fluorescence), and DAPI (to visualize nuclei, blue fluorescence). The fluorescence overlays demonstrate that only hematopoietic cells (leukocytes) are CD11c⁺ within this organ. The images also show that CD45⁺CD11c⁺ cells in the brain of the WT mice group form filamentous structures most likely along fungal hyphae. In contrast, CD45⁺CD11c⁻ cells in the brains of the Δα_X mice do not organize but instead remained dispersed (Fig. 3C, ,3D).3D). These results indicate that subsets of brain residential leukocytes may use α_Xβ₂ for localization to the fungus.

Activated monocytes use both α_Mβ₂ and α_Xβ₂ for fungal recognition

Previous studies demonstrated that NK lymphocytes (35, 61) and neutrophils (40) use integrin α_Mβ₂ but not α_Xβ₂ for C. albicans recognition. With our data on organ fungal burden suggesting that α_Xβ₂ deficiency affected residential tissue macrophages, the microglia in a brain, and Kupffer cells a in liver, we chose monocytes as representative primary cell to assess α_Xβ₂ involvement in leukocyte adhesion to C. albicans. Human peripheral blood monocytes were isolated, stimulated with PMA to activate their integrins, and added to germinated SC5314 fungus. In the absence of inhibitors, 28 ± 6% of the activated monocytes adhered to the fungal hyphae as compared with only 2% monocyte adhesion to the fungal yeast form (defined as nonspecific adhesion). Both anti-α_M I-domain mAb 44a and the α_Mβ₂-specific ligand NIF decreased monocyte adhesion to the fungal hyphae by ~3-fold, to 8 ± 4%. Anti-α_X mAb inhibited cell adhesion only ~50%, to 15 ± 6% of the total cells. The anti-α_M lectin domain mAb OKM1 and anti-β₂ mAb IB4 completely inhibited monocyte adhesion, whereas the irrelevant control, mAb W6/32, had no effect (Fig. 4A). A similar specificity was demonstrated in migration assays (Fig. 4B). When monocytes were allowed to migrate (through 5-μm pores) overnight to germinated C. albicans, anti-β₂ mAb completely inhibited the migration, anti-α_M I-domain mAb, and anti-α_X mAb inhibited 40–50%, and the control irrelevant anti-MHC class II (MHC-II) mAbs had no effect. However, a difference between migration and adhesion assays was noted. Anti-α_M lectin-domain mAb OKM1 completely inhibited adhesion of monocytes to the fungus, whereas this mAb had no effect on the monocyte migration to the fungus. This difference may reflect different roles of the fungal carbohydrates (e.g., β-glucans and mannans) recognized by the lectin domain of α_Mβ₂ on the migratory and adhesive responses of monocytes. These results demonstrate that monocytes can use α_Xβ₂ as well as α_Mβ₂ to engage C. albicans, and taken together, the two integrins are the major mediators of monocyte adhesion and migration to the fungus.

FIGURE 4.

Effect of selected Abs and inhibitors on adhesive (A) and migratory (B) responses to C. albicans of PMA-activated human monocytes. Isolated human peripheral blood monocytes were preincubated for 10 min on ice in the presence of 5 mM PMA with or without (more ...)

Purified recombinant α_Xβ₂ recognizes the hyphae of WT C. albicans but not Pra1-deficient fungi

To access the specificity of α_Xβ₂–C. albicans interaction, a HEK293 cell line stably expressing the integrin was developed. Full-length cDNAs of human α_X and β₂ were cotransfected into HEK293, and positive cells were sorted and subcloned. The established cell line (HEK293/α_Xβ₂ cells) then was examined by FACS using a panel of Abs: anti-human α_X (mAb 3.9), anti-human β₂ (IB4), anti-human α_M I-domain (44a), anti-human α_M lectin domain (OKM1), and control irrelevant mAb W6/32 (anti-human MHC-II). As expected, α_X and β₂ were highly expressed on the HEK293/α_Xβ₂ cell surface (Fig. 5A). Although α_X is highly homologous to α_M, the anti-α_M I-domain mAb 44a, which block binding of most ligands to α_Mβ₂, did not recognize the HEK293/α_Xβ₂ cells. Surprisingly, the anti-α_M lectin domain mAb OKM1, which inhibits the binding of polysaccharides such as β-glucans and mannans to α_Mβ₂ (70), also showed weak reactivity with α_Xβ₂ (Fig. 5A) but not with mock-transfected HEK293 cells (data not shown). Thus, the α_X subunit may contain structures similar to the α_M lectin domain and therefore may recognize fungal polysaccharides and serve as a pattern recognition receptor.

FIGURE 5.

(A) Characterization of HEK293/α_Xβ₂ cells by reactivity mAbs; (B, C) binding of purified α_Xβ₂ to germinated C. albicans. (A) Characterization of HEK293/α_Xβ₂ cells by FACS. A panel of Abs was used to examine (more ...)

Both α_Mβ₂ and α_Xβ₂ integrins have many ligands in common. Because the C. albicans hyphae surface protein Pra1 was identified as a major C. albicans ligand for α_Mβ₂ (40), we considered whether this protein might also serve as a ligand for α_Xβ₂. We tested HEK293/α_Xβ₂ cells as a source of α_Xβ₂ protein and the ability of purified α_Xβ₂ to recognize two C. albicans strains, SC5314 (WT) and Pra1-nul mutant (CAMB5-18). The yeast of both fungal strains were allowed to germinate overnight for maximal Pra1 expression in the WT strain and the fungal cell wall chitin and glucans were stained with blue fluorescent dye Blankophor (62). Labeled fungi then were incubated with FITC-labeled α_Xβ₂ and examined by fluorescence microscopy. The microphotograph shown in Fig. 5B clearly demonstrates that soluble α_Xβ₂ (green fluorescence) bound the hyphal form of WT fungus but not the rounded yeast form of C. albicans (Fig. 5B). No binding to the Pra1-deficient strain CAMB5-18 (Fig. 5C) was detected, indicating that α_Xβ₂ binds to Pra1 or to a C. albicans hyphal constituent regulated by Pra1.

Upon expression of α_Xβ₂ HEK293 cells acquire ability to recognize, bind, and phagocytose C. albicans

In the next set of experiments, the ability of α_Xβ₂ to support HEK293 cell migration to C. albicans and adhesion to the fungi with subsequent phagocytosis was explored. HEK293 cells, expressing either α_Xβ₂ or mock transfected, were added to plates with germinated C. albicans of SC5314 or CAMB5-18 strains. In some cases, plates with nongerminated fungi were also tested. After 30 min, unbound cells were washed away, and adherent cells were quantified using the CyQUANT fluorescent dye. In the absence of inhibitors, 58 ± 5% of α_Xβ₂ cells adhered to the hyphal form of SC5314 strain, whereas they failed to adhere to the yeast form of this fungal strain. The control mock-transfected HEK293 cells did not adhere to any form of C. albicans SC5314 strain (Fig. 6A), suggesting that the adhesion is α_Xβ₂ dependent. This conclusion was confirmed using a panel of α_Xβ₂ and α_Mβ₂ inhibitors. The adhesion of HEK293/α_Xβ₂ was completely inhibited with anti-α_X (3.9) and anti-β₂ (IB4) mAbs, whereas the anti-α_M lectin domain mAb OKM1 inhibited 80 ± 10% of the cell adhesion. In contrast, the anti-α_M I-domain blocking mAbs 44a and NIF, a specific inhibitor of ligand binding to α_Mβ₂ (58, 71) (NIF) as well as control W6/32 anti–MHC-II mAb, were ineffective. Soluble β-glucans at a concentration of 1 μg/ml inhibited adhesion of the HEK293/α_Xβ₂ cells by 50 ± 5%. Surprisingly, baker yeast mannans, which blocks HEK293/α_Mβ₂ adhesion to C. albicans (36), did not block adhesion of HEK293/α_Xβ₂ cells (Fig. 6A). In control experiments, neither β-glucan nor mannan was able to inhibit α_Xβ₂-supported adhesion to another α_Mβ₂ ligand—the fibrinogen peptide P2C, a nonglycosylated ligand that is also recognized by α_Xβ₂ (59, 72) (results not shown). Both cell lines were not able to adhere to germinated Pra1-deficient C. albicans strain CAMB5-18 (40) (Fig. 6A), again indicating that C. albicans recognition by α_Xβ₂ is Pra1 dependent.

FIGURE 6.

Upon expression on surface of HEK293 cells α_Xβ₂ supports adhesive, migratory and phagocytic activities of the cells toward C. albicans. (A) HEK293/α_Xβ₂ cell adhesion to C. albicans. A total of 5 × 10⁵ HEK293/α (more ...)

Next, we tested migration of α_Xβ₂ cells to C. albicans conditioned medium, a source of soluble Pra1 (40), in the presence or absence of integrin inhibitors. After 8 h, in the absence of inhibitors, 17 ± 4% α_Xβ₂ cells migrated to fungal supernatant. The anti-β₂ mAb IB4, at 20 μg/ml, reduced cell migration to fungal supernatant to 4 ± 1%. The same effect was observed in the presence of anti-α_X mAb 3.9, which also inhibited migration to C. albicans supernatant. As a control, we also measured migration of the cells to vitronectin, which is mediated primarily by endogenous α_V integrins on these cells. Anti-α_V mAb 272-17E6 inhibited migration of the cells to vitronectin to 4.4 ± 3.5% but had no affect on migration of the cells to the fungal supernatant. Echistatin, a snake venom disintegrin that inhibits ligand recognition by most integrins, including the β₂ and α_V integrins (61), completely reduced cell migration to both Candida supernatant and vitronectin (Fig. 6B). Taken together, these data indicate that migration of α_Xβ₂ cells to C. albicans proteins is integrin dependent, and α_Xβ₂ is implicated in this response of the HEK293/α_Xβ₂ cells. Notably, β-glucan and anti-α_M lectin domain mAb OKM1, which inhibited adhesion of α_Xβ₂ cells to C. albicans, did not affect migration of these cells to the fungal extracellular proteins (Fig. 6B), suggesting that the lectin specificity of α_Xβ₂ is involved in adhesion but not migration to C. albicans.

The ability of α_Xβ₂ to promote antifungal activity of HEK293 cells was also tested. Because fibroblasts such as HEK293 possess weak endogenous antifungal activity and are able to internalize pathogens by passive endocytosis (73, 74), we wished to distinguish passive nonspecific endocytosis from specific phagocytosis, and various ratios of fungi/cells were tested in killing assays. Germinated C. albicans cells of the SC5314 strain were coincubated with α_Xβ₂ or mock-transfected HEK293 at fungi/HEK293 cell ratios of 1:3, 1:7, or 1:11 for 2 h. Aliquots were taken every 20 min, and the amounts of viable fungi remaining were quantified as CFU by plating aliquots in a series of dilutions onto agar plates. Only 58 ± 6% of the initial fungi remained viable after a 2-h incubation with the α_Xβ₂ cells at the 1:3 ratio. At a 1:7 ratio, the amount of surviving fungi decreased to 42 ± 5% and fell to 28 ± 6% at a 1:11 ratio. In contrast, with mock-transfected HEK293 cells, fungal survival was 76 ± 6 and 82 ± 5% at the 1:11 and 1:7 ratios, respectively, and all fungi survived at the ratio 1:3 (Fig. 6C). Preincubation of α_Xβ₂ cells with anti-α_X blocking mAb 3.9 completely inhibited the antifungal activity of the α_Xβ₂ cells to levels observed with mock-transfected HEK293 cells (p > 0.5). These results indicate that α_Xβ₂ is able to promote phagocytosis of C. albicans.

Discussion

In the present work, we demonstrate the importance of integrin α_Xβ₂ for protection against C. albicans systemic infection by the innate immune system. Although the ability of α_Xβ₂ to recognize C3bi and thereby to assist in the elimination of opsonized particles has been described previously (48), to our knowledge, our study is the first demonstration of involvement of this integrin in direct pathogen recognition and elimination by leukocytes as well as its critical importance in the control of fungal invasion to brain and liver by certain subsets of tissue residential macrophages (see below).

Our data on the murine organ fungal burdens indicate that the α_Xβ₂ elimination affects mainly the liver and brain, dramatically increasing invasion and propagation of the fungus in these organs. This effect of α_Xβ₂ became evident at the earliest stages of infection: as early as16 h after the challenge, a 2-fold difference (p < 0.05) emerged in fungal burdens in the livers of Δα_X and WT mice and a 10-fold difference (p < 0.01) in the brains. At 40 h postinfection, this difference in the susceptibility of the α_Xβ₂-deficient mice and WT animals reached 6-fold (p < 0.01) in the liver and >40-fold (p < 0.005) in the brain.

The integrins of the β₂ subfamily, known collectively as “leukocyte integrins,” are expressed predominantly on the surface of leukocytes (23, 75). In our experiments, immunostaining of infected brain and liver sections revealed that only the CD45⁺ hematopoietic cells in these tissues express α_X [also see (76)], and thus, α_Xβ₂ elimination is likely to affect leukocyte function only.

The kidney and the brain are the primary targets for C. albicans during systemic infection. The fungi invade these organs directly from the bloodstream, and invasion can start during the first minutes postinfection (67). The blood immune mechanisms (e.g., monocytes, neutrophils, NK lymphocytes, and cells of the blood–brain barrier) provide little protection from neuroinvasion during the initial stages of systemic infection. Blood cells can clarify the bloodstream of sublethal doses of C. albicans only after 20 h of infection, and, in the case of near-lethal doses, fungal CFUs can be detected in the blood even after 24–30 h postinfection (67). The i.v. route for the fungal injection bypasses possible contact of the fungi with tissue macrophages. To circumvent the blood–brain barrier, C. albicans uses a unique mechanism of invasion: upon binding to gp96 heat shock protein and/or to N-cadherin on the surface of normally nonphagocytic brain microvascular epithelial cells, fungi stimulate their own uptake (77, 78). Therefore, in our model, the difference in organ fungal burdens of WT and Δα_X in mice appears to be due to differences in activity of the organ-resident macrophage subsets, microglial cells in brain, and Kupffer cells in liver.

Existing literature present extensive evidence that microglia play the principal role in the protection against C. albicans intracerebral infections. Direct proof of their crucial role was provided by the demonstration that intracerebral transfer of microglial cells provides complete protection (100% survival) against subsequent intracerebral challenge with a lethal inoculum of the fungus. After i.v. challenge with near-lethal C. albicans inoculums, the concentration of fungal CFUs in the brain rapidly increases and reaches maximal level at ~24 h infection. Then, the fungal burden in brain stabilizes and remains at this level until days 7–8 with a subsequent slow decline (67). This time course implies that 24 h is sufficient for microglial activation and conversion to “brain macrophages,” and the migration to the fungus to contain infection and corresponds well with our data, demonstrating that after 40 h most brain CD45⁺CD11c⁺ cells in WT have migrated and assembled around the hyphal-like structures of C. albicans. In α_Xβ₂-deficient mice, the CD45⁺CD11c⁻ cells in the brain remained diffusely distributed, suggesting that α_Xβ₂ is required for these cells to migrate to and recognize C. albicans.

Kupffer cells are the specialized phagocytic cells found on the luminal surface of hepatic sinusoids (79). These cells are of monocytoid lineage (79, 80) and express both α_Xβ₂ and α_Mβ₂ integrins (81–83), and their importance for protection against C. albicans invasion has been demonstrated previously (84–87). The possible involvement of integrin α_Xβ₂ in phagocytosis of C. albicans by microglial and Kupffer cells has been proposed (84, 88, 89). Taken together, these data suggest that α_Xβ₂ but not α_Mβ₂ is critical in antifungal activity of tissue-resident macrophage subsets. This conclusion is consistent with the previous report demonstrating that increased expression of α_Xβ₂ results in enhanced phagocytosis of M. tuberculosis by human macrophages (49).

Integrins on the surface of nonstimulated leukocytes are expressed in inactive “closed” conformation and require activation to recognize their ligands with high affinity. During inflammation, various physiological agonists induce activation of specific integrins. Thus, α_Xβ₂ may become activated, whereas α_Mβ₂ remains in an inactive conformation or vice versa, and therefore, these two β₂ integrins may differentially participate in leukocyte function despite both being expressed on the leukocyte surface (19). Activation of peripheral blood monocytes in vitro with PMA results in activation of all leukocyte integrins. For this reason, anti-α_Xβ₂ mAbs block adhesion of PMA-activated monocytes only partially. The only anti-α_M mAb that blocks adhesion to the fungus is directed to the α_M lectin domain, and as we have shown, this mAb also cross-reacts with a previously unrecognized lectin domain within the α_X subunit.

β-Glucans and mannans are important immunomodulators, and their binding by leukocytes is implemented by integrin α_Mβ₂ (90, 91). Upon ligation with the integrin, β-glucans activate α_Mβ₂ and stabilize it in an intermediate active conformation (92).Unlike α_Mβ₂, where the carbohydrate binding and sugar selectivity of its α_M-lectin domain are well characterized (e.g., (93–95)), there is no evidence in the literature for recognition of fungal glycans or bacterial LPS by α_Xβ₂. Therefore, our observation that activity of α_Xβ₂ is modulated by fungal β-glucans is a novel finding of our present work. On the basis of ~70% homology between α_M and α_X and that the OKM1 mAb, which blocks glycan binding to α_M (70), weakly cross-reacts with α_X, we anticipate certain similarities in the sugar specificity of these integrin subunits. However, α_Mβ₂ and α_Xβ₂ demonstrated clear distinction in carbohydrate selectivity: although α_Mβ₂ recognizes both β-glucans and mannans, the activity of α_Xβ₂ appears to be modulated by β-glucans but not by mannans. In our experiments, mannans, unlike β-glucans, were not able to inhibit adhesion of HEK293/α_Xβ₂ to C. albicans hyphae. The observed differences in sugar selectivity of the integrins may play an important role in the regulation of leukocyte activation and differentiation (95, 96).

In the present work, direct interaction between purified α_Xβ₂ and Pra1 was not tested directly. Therefore, we cannot exclude the possibility that another C. albicans hyphal protein that is regulated by Pra1 may serve as a ligand for α_Xβ₂. However, the existing literature provides no evidence for such a molecule. Thus, our findings that purified α_Xβ₂ interacts with C. albicans hyphae but not with the yeast form and that the HEK293/α_Xβ₂ cells recognize and adhere to hyphae of WT C. albicans strain SC5314, but not of Pra1-deficient strain CAMB5-18, provide strong evidence that Pra1 serves as C. albicans ligand for α_Xβ₂.

The integrin α_Xβ₂ is usually present on the surface of leukocyte subsets together with another member of the β₂ integrin family, α_Mβ₂, to which such primary antipathogen leukocyte activities, such as recognition of bacterial LPS and fungal mannoproteins, are traditionally ascribed (12, 17, 24). We speculate that α_Mβ₂ and α_Xβ₂ integrins may play complementary roles in executing cellular immunity or that different cellular agonists may favor activation and utilization of one particular integrin. Our data showing significantly reduced resistance of α_Xβ₂-deficient mice to Candida invasion and the α_Xβ₂ requirement for fungal recognition and killing by macrophages clearly demonstrate that α_Xβ₂ plays an independent role in the defense against fungal infections and does not simply serve as an auxiliary receptor for pathogens, secondary to α_Mβ₂.

Taken together, these data clearly demonstrate the importance of α_Xβ₂ in protection against C. albicans systemic infection, and this protective effect is mediated by subsets of tissue residential macrophages.

Acknowledgments

We thank Dr. Christy A. Ballantyne of Baylor College of Medicine for the Δα_X mice, Dr. William A. Fonzi of Georgetown University for C. albicans ΔPra1 strain, Earl Poptic from Cleveland Clinic Hybridoma Core for help with large-scale Ab preparation, Carla Drumm (Cleveland Clinic) for help with mouse husbandry, and Rajani Tendulkar (Cleveland Clinic) for excellent administrative support of the project.

This work was supported by National Institute for Allergy and Infectious Diseases Grant AIO 80596.

Abbreviations used in this article:


D-PBS	Dulbecco's PBS

HEK293	human epithelial kidney 293

HEK293/α_Xβ₂ cell	the HEK293 cell expressing α_Xβ₂ on its surface

KO	knockout

LAD-1	leukocyte adhesion deficit, type 1

MHC-II	MHC class II

NIF	canine hookworm neutrophil inhibitory factor

OG	octyl-β-d-glucopyranoside

Pra1	pH-regulated Ag 1

SDA	Sabouraud Dextrose Agar

WT	wild-type

Δα_X mice	mice depleted in α_Xβ₂.

Disclosures

The authors have no financial conflicts of interest.

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