EphA2 Is a Lung Epithelial Cell Receptor for Pneumocystis β-Glucans

Abstract

Pneumocystis species interaction with myeloid cells is well known, especially in macrophages; however, how the organism binds to lung epithelial cells is incompletely understood. Ephrin type-A receptor 2 (EphA2) has been previously identified as a lung epithelial pattern recognition receptor that binds to fungal β-glucans. Herein, we also report that EphA2 can also bind Pneumocystis β-glucans, both in isolated forms and also on exposed surfaces of the organism. Furthermore, binding of Pneumocystis β-glucans resulted in phosphorylation of the EphA2 receptor, which has been shown to be important for downstream proinflammatory response. Indeed, we also show that interleukin 6 cytokine is significantly increased when lung epithelial cells are exposed to Pneumocystis β-glucans, and that this response could be blocked by preincubation with a specific antibody to EphA2. Our study presents another Pneumocystis lung epithelial cell receptor with implications for initial colonization and possible therapeutic intervention.

Pneumocystis jiroveciiremains a serious infectious disease threat to the immunocompromised patient. This study suggests that the previously described host β-glucan epithelial cell receptor EphA2 may having importance for initial Pneumocystisbinding and subsequent proliferation in the host lung.

Pneumocystis pneumonia is caused by members of the genus Pneumocystis, which infect the lungs of mammals in both the immunocompetent and immunocompromised state. Pneumocystis jirovecii, the fungal organism that causes P. jirovecii pneumonia (PJP) worldwide in immunocompromised individuals, may contribute to greater than 400000 life-threatening fungal infections per year, with mortality rates as high as 80% [1]. Highly active antiretroviral therapy has significantly reduced PJP in the human immunodeficiency virus (HIV)-infected population, but in resource-limiting settings PJP continues to be a major problem [2]. Data suggest that even in developed countries, the incidence of non-HIV PJP is becoming more prevalent, and groups in this cohort have higher in-hospital mortality and unfavorable outcomes [3]. Therefore, for these reasons, PJP continues to be a major public health threat.

The initial stages of host epithelial colonization by Pneumocystis are incompletely understood. Binding of Pneumocystis to lung epithelial cells is thought to be a central tenet of the life cycle [4]. Trophic forms of the organism, by close interdigitation of their membranes with the epithelial cell, bind tightly without internalization of the fungal organism [5]. These binding events are thought to be vital for subsequent organism proliferation [4]. Unlike in myeloid cells, where the receptor-organism interactions are better characterized [6], little is known about the receptors important for epithelial cell binding of Pneumocystis and, furthermore, how these events might lead to downstream proinflammatory response and initial phases of lung colonization.

Because little is known about Pneumocystis and lung epithelial cells interactions, including the importance of these interactions for organism growth [5, 7], we sought to further characterize the possible role of the previously identified fungal β-glucan receptor EphA2 in Pneumocystis-lung epithelial cell interactions.

METHODS

Cell Culture

Mouse lung epithelial cell line MLE 12 (American Type Culture Collection [ATCC] CRL-2110), a murine cell line expressing some features of normal type II airway epithelial cells [8], were maintained in HITES medium supplemented with 2% fetal bovine serum and antibiotic antimycotic (ThermoFisher Scientific).

Reagents and Strains

All reagents were from Sigma-Aldrich unless specified otherwise. All animal experiments were conducted in accordance with the guidelines of the Mayo Institutional Animal Care and Use Committee. Pneumocystis pneumonia was induced in rats immunosuppressed with dexamethasone as previously described [9]. Pneumocystis carinii organisms were obtained from the ATCC. P. carinii was propagated as previously reported [9]. Whole populations of P. carinii containing both cyst (ascus) and trophic forms were obtained from infected rat lungs by homogenization and filtration through 10-μm filters. To obtain Pneumocystis murina organisms, mice were immune suppressed by CD4 lymphocyte depletion using the anti-mouse CD4 monoclonal antibody GK1.5 as previously reported [10]. A similar technique was utilized for isolation of P. murina from mice. To exclude the presence of other infectious organisms in the fungal isolates, the preparations were stained (Diff-Quik modified Wright-Giemsa stain; Dade Diagnostics) and examined to exclude concurrent infection with bacteria or other fungi. Isolates contaminated with other microorganisms were discarded.

Baseline Expression Analysis of EphA2 and C-Type Lectin Receptor Expression in MLE 12 Cell Line

Total RNA and subsequent cDNA generation from the MLE 12 cell line as well as quantitative polymerase chain reaction (qPCR) analysis were similar to methods previously described [11]. PCR primers used in this analysis are listed in Supplementary Table 1.

Pneumocystis β-Glucan Generation

Purified Pneumocystis β-glucans from P. carinii have been described previously [12], and details are given in the Supplementary Methods.

ELISA Assay for EphA2 Binding

Enzyme-linked immunosorbent assay (ELISA) for EphA2 binding to Pneumocystis β-glucans was similar to that described previously [13]. As a positive control, 2 µg/well EphA2 ligand (EFNA1; BioLegend), was plated. Bovine serum albumin (BSA; negative control) or Pneumocystis β-glucans were plated at 50 µg/well. All ligands were plated overnight at 4°C.

Assays of P. murina Binding to Lung Epithelial Cells

Total P. murina organisms were isolated and purified as described above and labeled with ⁵¹Cr as reported previously [11]. After labeling, the P. murina life forms were enumerated as described previously [14], and approximately 50 P. murina life forms per epithelial cell were applied to MLE 12 cells in 12-well tissue culture plates for 1 hour at 37°C. Competitive inhibition to demonstrate specific EphA2-P. murina surface-exposed β-glucan interactions was conducted by adding soluble EFNA1 ligand or BSA (negative control protein) for 1 hour prior to P. murina addition. After incubations, nonadherent P. murina organisms were removed by gentle washing. For the radiolabeling experiments, the mouse lung epithelial monolayers containing associated P. murina organisms were solubilized in 1N NaOH and the P. murina organisms attached to the lung epithelial cells quantified by scintillation counting. A similar experiment was conducted, and binding measured by qPCR as similar to previously noted [11].

Protein Phosphorylation Analysis of EphA2

MLE 12 cells were plated at 5 × 10⁵ cells per well in 24-well tissue culture plates 24 hours before the assay and placed at 37°C, 5% CO₂. The following day, 50 µg/mL of Pneumocystis β-glucans were applied and plates were spun at 500g for 5 minutes to synchronize infection. After 15, 30, 60, and 90 minutes, cells were lysed in 300 µL radioimmunoprecipitation assay (RIPA) lysis buffer (containing phosphatase and protease inhibitors) and insoluble material pelleted. Total protein, 5 µg per sample, was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the phosphorylated EphA2 protein detected by immunoblotting with the phosphatase-specific antibody, anti-phospho-EphA2 (Cell Signaling; number 6347). Total EphA2 and β₂ microglobulin (B2M) were detected with appropriate antibodies, EphA2 (Cell Signaling; number 6997) and B2M (Cell Signaling; number 59035), respectively. EphA2 phosphorylation was quantified using Image Studio Lite version 5.2.5, and normalized against total EphA2 protein levels.

IL-6 Cytokine Analysis

MLE 12 cells were plated in 96-well plates at 2 × 10⁵ for 24 hours as described above. The next day, 50 µg/mL Pneumocystis β-glucans were applied to the monolayers, and plates incubated for 24 hours. The following day, cell culture media were collected, and the supernatant analyzed for interleukin 6 (IL-6) cytokine production by ELISA (ThermoFisher Scientific).

Statistical Analysis

For multigroup data, initial analysis was first performed with analysis of variance (ANOVA) to determine overall different differences. If ANOVA indicated overall differences, subsequent group analysis was then performed post hoc by Student t test for normally distributed variables. Statistical analysis was performed using Prism version 9.1.2 software (GraphPad, Inc), and differences were considered to be statistically significant when P<.05.

RESULTS

EphA2 Is a Pneumocystis β-Glucan Receptor

For these studies we used the mouse epithelial cell line MLE 12. Analysis of baseline expression by qPCR demonstrated EphA2 receptor expression, and little to no other C-type lectin receptor expression (Supplementary Figure 1). These data verify the presence of the EphA2 receptor in this cell line. Next, we investigated the tyrosine kinase receptor EphA2 and its ability to bind Pneumocystis β-glucans. Previously, it has been reported that this receptor is involved in the host epithelial cell binding of fungal β-glucans, and subsequent endocytosis/internalization and cytokine production in fungal pathogens such as Candida albicans and Aspergillus fumigatus, respectively [13, 15]. First, to determine whether EphA2 directly binds Pneumocystis β-glucans, an ELISA was utilized. As a positive control, the known ligand for EphA2, EFNA1, was included. As expected, EFNA1 bound to its native receptor with high significance. Similar to what has been observed with β-glucans from other fungal pathogens, as noted above, EphA2 receptor could also bind Pneumocystis β-glucans to a significant degree (Figure 1A). Next, others have suggested that the major surface glycoprotein (MSG/gpA) of Pneumocystis may cloak β-glucans that are present near the organism’s plasma membrane [16]. To determine if EphA2 receptor could bind P. murina organisms that were heat-inactivated at 56°C for 1 hour to remove organism gpA and possibly expose more plasma-associated β-glucans, the ELISA assay was repeated with these conditions. Indeed, as Supplementary Figure 2 demonstrates, native P. murina organisms bound EphA2 receptor to a significant degree as compared to the BSA control alone. Furthermore, heat-treated P. murina bound EphA2 receptor to an even higher degree than the BSA control alone as well as significantly binding EphA2 receptor greater than untreated organisms alone. These data suggest that the EphA2 receptor can bind exposed β-glucans on the Pneumocystis organism cell wall, and that this occurs to an even greater degree than plasma membrane-associated Pneumocystis β-glucans, giving further credence to the EphA2 receptor recognizing this Pneumocystis carbohydrate.

Figure 1.

A, Recombinant EphA2 binding to EFNA1 and Pneumocystis β-glucans as determined by ELISA. B, Binding of Pneumocystis murina organisms to MLE 12 lung epithelial cells. Statistical analysis of binding is shown relative to fungal organisms binding to MLE 12 cells (100%). All experiments were conducted at least 3 times. Initial analysis was first performed with ANOVA. If ANOVA indicated overall differences, subsequent group analysis was then performed by 2-sample unpaired Student t test for normally distributed variables. Error bars show SD from the mean. ∗P<.05, ∗∗P<.01. Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; EphA2, ephrin type-A receptor 2; MLE, mouse lung epithelial cell; OD, optical density.

EphA2 on Lung Epithelial Cells Can Bind Live Pneumocystis Organisms

We have previously shown that intact Pneumocystis organisms (asci) exhibit exposed β-glucans on their surface as measured by dectin-1 binding [17]. To assess organism binding, ⁵¹Cr-labeled P. murina life forms were incubated with the MLE 12 cells and washed to remove nonadherent P. murina life forms. Bound P. murina life forms were then enumerated by scintillation counting or qPCR. Indeed, live P. murina organisms bound to the mouse lung epithelial line with high significance as compared to plastic alone. Competitive inhibition with the natural ligand for EphA2, EFNA1, significantly reduced interactions between P. murina and MLE 12, as measured by scintillation counts (Figure 1B) and qPCR (Supplementary Figure 3). These results indicate that EphA2 receptors expressed on the surface of epithelial cells can bind Pneumocystis organisms.

Pneumocystis β-Glucans Activate EphA2

We sought to determine if EphA2 not only binds Pneumocystis β-glucans and the fungal organisms themselves, but also when bound to the carbohydrate can activate the tyrosine kinase receptor. Similar to what has been reported previously with other β-glucans, particularly laminarin and zymosan [13], Pneumocystis β-glucans significantly stimulated EphA2 phosphorylation in a time course-dependent fashion, with greatest phosphorylation noted at 15 minutes, and levels steadily declining to near baseline levels by 90 minutes (Figure 2A and 2B). These data indicate the Pneumocystis β-glucans can activate EphA2 signaling in lung epithelial cells.

Figure 2.

A, Pneumocystis β-glucans can activate lung epithelial cell EphA2 receptors and release IL-6 proinflammatory cytokine. Western blot analysis showing the time course of EphA2 phosphorylation (pEphA2) in MLE 12 cells in the presence of Pneumocystis β-glucans for the indicated times (minutes). B2M was used as a loading control. B, pEphA2 signals were quantified with Image Studio Lite software and normalized to total EphA2 levels. C, IL-6 cytokine analysis of MLE 12 epithelial cells after stimulation with Pneumocystis β-glucans and blockage of cytokine response by preincubation with an EphA2-specific antibody. All experiments were conducted at least 3 times. Initial analysis was first performed with ANOVA. If ANOVA indicated overall differences, subsequent group analysis was then performed by 2-sample unpaired Student t test for normally distributed variables. Error bars show SD from the mean. First lane is time zero. ∗P < .05, ∗∗P < .01. Abbreviations: ANOVA, analysis of variance; B2M, β₂-microglobulin; EphA2, ephrin type-A receptor 2; IL-6, interleukin 6; MLE, mouse lung epithelial cell.

EphA2 Signaling via Pneumocystis β-Glucan Interactions Contribute to the Proinflammatory Response in Lung Epithelial Cells

Previously, in the context of lung epithelial cell and Pneumocystis organism interactions, we and others have shown that these events can lead to proinflammatory release that may be important for host immune response and limiting early fungal proliferation [18, 19]. As noted in Figure 2C, stimulation with Pneumocystis β-glucans resulted in a significant release of IL-6 cytokine, and this response was inhibited by preincubation with a specific antibody for EphA2. These data demonstrate the specific role of the EphA2 receptor as an epithelial cell receptor for Pneumocystis β-glucans.

DISCUSSION

We and others have shown the importance of pattern recognition receptors, especially C-type lectin receptors, in host response to Pneumocystis. These receptors have been shown to be critical in the myeloid cell response both in vitro and in vivo. A thorough review of these receptors has recently been published [6]. Known Pneumocystis ligands that can bind this class of receptors include β-glucans and major surface glycoprotein (MSG/gpA) [6]. Although the list of myeloid receptors that bind Pneumocystis organisms is extensive and growing, little information is known about receptors that might be present on lung epithelial cells that interact with the fungus and are important for early host response. For instance, cytokines such as IL-6, IL-8, macrophage-inflammatory protein 2 (MIP-2), and tumor necrosis factor-α (TNF-α) are known to be released by these epithelial cell-fungal interactions, but receptors leading to these events are largely unknown [20]. Some studies have suggested that interactions between Pneumocystis and lung epithelial cells involving lactosylceramide, a host cell glycolipid, lead to subsequent cytokine release [9]. More recently, we have shown a specific epithelial receptor, HSAP5, is involved in Pneumocystis attachment [20]. Here, we present new evidence that EphA2 receptor present in lung epithelial cells contributes to the binding of Pneumocystis organisms to mouse lung epithelial cells in vitro.

The Eph (erythropoietin-producing hepatocellular carcinoma) receptor family consists of the largest known tyrosine kinase receptor family known. Their functions have been shown to be important in normal embryonic development and homeostatic tissue maintenance in adults [21]. Currently, there are 9 EphA members and 5 EphB members [22]. Both EphA1 and EphA2 receptors are linked to tumor malignancy and cancer progression [23]. However, more recent data have also implicated the cell surface Epha2 receptor as important for fungal binding in both epithelial cells and neutrophils [13]. For instance, recent reports show that EphA2 is utilized by A. fumigatus as a portal of entry for conidia [15]. Similarly, elegant work by Swidergall et al demonstrated for the first time that EphA2 can serve as a receptor for β-glucans present in C. albicans, and that this receptor is paramount for not only initial colonization of oral epithelial cells, but also in subsequent fungal growth during initial oropharyngeal infection [13]. These researchers also demonstrated that in this infection EphA2 is needed for mitogen-activated protein kinase (MAPK) and activation of signal transducer and activator of transcription 3 (Stat3), whereas dectin-1 activates nuclear factor-κB (NF-κB), via a Ca²⁺ requirement [13]. Future studies in our laboratory will include determining a possible role of dectin-1/EphA2 binding events on lung airway epithelium with Pneumocystis.

In conclusion, data presented here suggest that the host epithelial cells EphA2 receptor can serve as a receptor for Pneumocystis. Previous studies have suggested that early colonization of the lung by Pneumocystis is an important first step in PJP and organism proliferation. For instance, in early P. jirovecii colonization of preterm infants, data suggest that this event could be important for respiratory distress syndrome [24]. Therefore, possibly targeting inhibition of the EphA2 receptor-Pneumocystis organism interactions, such as through specific EphA2-directed antibody therapy, might be a viable therapeutic approach to PJP.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions. T. J. K. designed the studies. T. J. K. and K. S. performed the studies and analyzed the data. T. J. K., E. M. C., and A. H. L. wrote the paper.

Financial support. This work was supported by the Mayo Foundation; the Walter and Leonore Annenberg Foundation; and the National Institutes of Health (grant number R01-HL62150 to A. H. L.).

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.