Staphylococcus aureus Adenosine Inhibits sPLA2-IIA–Mediated Host Killing in the Airways

Erwan Pernet, Jérémy Brunet, Laurent Guillemot, Michel Chignard, Lhousseine Touqui and Yongzheng Wu
J Immunol June 1, 2015, 194 (11) 5312-5319; DOI: https://doi.org/10.4049/jimmunol.1402665

Abstract

Staphylococcus aureus is a common cause of bacterial infections in respiratory diseases. It secretes molecules to dampen host immunity, and the recently identified adenosine is one of these molecules. The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host protein endowed with antibacterial properties, especially against Gram-positive bacteria such as S. aureus. However, the role of adenosine in sPLA2-IIA–mediated S. aureus killing by host is still unknown. The present studies showed that the S. aureus mutant lacking adenosine production (∆adsA strain) increased sPLA2-IIA expression in guinea pig airways and was cleared more efficiently, compared with the wild-type strain. S. aureus ∆adsA strain induced sPLA2-IIA expression by alveolar macrophages after phagocytic process via NOD2–NF-κB–dependent mechanism. However, S. aureus adenosine (wild-type and adsA-complemented strains) and exogenous adenosine downregulated S. aureus phagocytosis by alveolar macrophages, leading to inhibition of sPLA2-IIA expression. This occurred through inhibition of p38 phosphorylation via adenosine receptors A2a-, A2b-, and protein kinase A–dependent pathways. Taken together, our studies suggest that, in the airway, S. aureus escapes sPLA2-IIA–mediated killing through adenosine-mediated inhibition of phagocytosis and sPLA2-IIA expression.

This article is featured in In This Issue, p.5037

Introduction

Respiratory infections caused by Staphylococcus aureus are a leading cause of community-acquired and health care–associated pneumonia (1–3). In the airways, alveolar macrophages (AMs) are among the first cells that encounter bacteria and are required for the detection and clearance of bacteria. These cells sense the invading pathogen by recognizing specific pathogen-associated molecular patterns (PAMPs) through their pathogen recognition receptors. For instance, TLR2, in association with TLR1 or TLR6, is the major pathogen recognition receptor that recognizes both lipoteichoic acid (LTA) and peptidoglycan (PGN) of S. aureus (4, 5). The intracellular receptor NOD2, which binds to muramyl dipeptide (MDP) motif of PGN, has also been shown to mediate innate immune response and antimicrobial peptide production (6–10).

The type IIA secretory phospholipase A2 (sPLA2-IIA) is one of the most effective antimicrobial peptides produced by the host (11). Its antibacterial property is related to the ability to directly kill bacteria by hydrolyzing their membrane phospholipids (11). Thus, this enzyme represents a major actor in innate host defense against bacterial infections (12), in particular, by Gram-positive (G+) bacteria that are highly susceptible to killing by sPLA2-IIA (13–16). Previous studies showed that constitutively expressed human sPLA2-IIA protected transgenic mice from G+ bacterial infections (16–19). However, the pathophysiological role of the endogenous sPLA2-IIA in the protection against S. aureus infection is still unknown.

Conversely, pathogens can also subvert host immunity via different strategies (20, 21). Adenosine, produced from the degradation of ATP, ADP, and AMP by the highly conserved cell wall–anchored adenosine synthase A (AdsA) (22, 23), has been described recently as an important actor in S. aureus immune escape (24, 25). In mammals, adenosine is sensed by four small G protein–coupled membrane receptors, and, among which, A2a and A2b receptors (A2aR and A2bR) increase cAMP levels (26), leading to protein kinase A (PKA) activation (27). The cAMP/PKA pathway has been shown to regulate sPLA2-IIA expression (28, 29), but the role of adenosine in the regulation of sPLA2-IIA expression is still unknown.

In this work, we show that a S. aureus mutant lacking adenosine production increases sPLA2-IIA expression by AMs of guinea pig, and that S. aureus PGN induces this sPLA2-IIA expression via phagocytosis and NOD2-dependent mechanism. More importantly, we show that S. aureus adenosine downregulates phagocytosis by AMs, leading to inhibition of sPLA2-IIA expression through A2aR/A2bR (in particular A2aR)- and PKA-dependent pathways. In pulmonary infection of guinea pig model, adenosine production decreases S. aureus clearance from airways. To conclude, our work highlights the contribution of NOD2 in sPLA2-IIA expression and a novel mechanism by which S. aureus subverts the host immunity by inhibiting the expression of this antimicrobial molecule.

Materials and Methods

Animals and reagents

Male Hartley guinea pigs were purchased from Charles River. Pseudomonas aeruginosa LPS, adenosine, cytochalasin D, and lysostaphin were from Sigma-Aldrich. BAY 11-7082 was purchased from Calbiochem. S. aureus LTA and PGN, Pam3CSK4, MDP, MDP control, CpG, and gefitinib were from Invivogen. MALP-2 and IκBα Ab were from Santa Cruz. ZM241385, PSB603, H-89, and YM26734 were from Tocris. Phospho-CREB, phospho-p38, total-p38, phospho-ERK, and total-ERK Abs were from Cell Signaling Technologies.

Bacterial strains

S. aureus strains RN4220, SA25923, NCTC8325, mu50, and Xen36 were obtained from T. Msadek (Institut Pasteur, Paris, France). S. aureus ∆adsA mutant, AdsA complemented, and Newman wild-type (WT) strains were provided by D. Missiakas (University of Chicago, Chicago, IL) and used as described previously (24).

Infection of guinea pig AMs

AMs from guinea pigs (±450 g) were harvested as previous studies (29, 30). Adhered AMs (1 × 106 cells/ml/well) in culture plate were infected with bacteria (multiplicity of infection 25) for 1 h, followed by wash (3×) with PBS and incubation for additional 24 h in serum-free RPMI 1640 supplemented with antibiotics (penicillin/streptomycin 100 U/ml each, and gentamicin 300 μg/ml). AMs were also stimulated with purified PAMPs for 24 h. In certain experiments, AMs were pretreated with adenosine or inhibitors for 30 min before bacterial infection.

For adhesion assays, AMs were incubated for 30 min with bacteria, washed, and lysed in PBS-Triton 0.1%; and total CFUs were enumerated on agar plates. For phagocytosis studies, 1 h after bacterial infection, AMs were washed and extracellular bacteria were killed by lysostaphin (10 μg/ml) for 10-min incubation (24). Cells were washed again and lysed in PBS-Triton 0.1%, followed by bacteria count on luria broth agar plates. For dynamic killing study, extracellular bacteria were killed by lysostaphin, as described above, and AMs were washed again, followed by incubation for different times. Then AMs were harvested to perform bacterial count.

Quantitative PCR, immunoblot, and IL-8 ELISA

Total RNA was extracted from AMs using RNeasy minikit (Qiagen), and 1 μg total RNA was used for reverse transcription. The cDNA was then used for quantitative RT-PCR to analyze sPLA2-IIA mRNA expression using ABI Prism 7900HT sequencing detection system (Applied Biosystems) and Sybr green master mix (Roche). Primers for β-actin (forward, 5′-CTGTGCTGTCCCTGTATGC-3′; reverse, 5′-CCGTGGTGGTGAAACTGTAG-3′) and sPLA2-IIA (forward, 5′-ACAAGTTATGGCGCCTATGG-3′; reverse, 5′-GCCCAGTGTAGCTGTGAAGC-3′) were synthesized by Sigma-Aldrich. Results are presented as relative quantification of gene expression using the 2−∆∆Ct method.

Total protein extraction from AMs, immunoblot, and IL-8 assay in cultural supernatant were performed, as described previously (29, 31).

Transient plasmid transfection

Chinese hamster ovary cells were cultured in F-12K medium with FBS (10%, v/v) and antibiotics (penicillin/streptomycin 100 U/ml each). Transfection with sPLA2-IIA luciferase plasmids bearing sPLA2-IIA WT promoter (sPLA2-IIA WT) or mutant promoter lacking the binding site of NF-κB (∆NF-κB) (32) was performed for 24 h in FBS-containing medium, using 0.6 μl FuGENE 6 (Promega), 100 ng plasmid DNA, and 30 ng plasmid Renilla. Transfected cells were then stimulated with MDP (10 μg/ml) and negative MDP control (MDPc, 10 μg/ml) in serum-free F-12K for additional 24 h before luciferase assay using Dual-Luciferase Reporter Assay System kit (Promega) and normalized to Renilla.

In vivo infection

Guinea pigs (±150 g) were housed in the Institut Pasteur animal facilities. All animal experiments were performed in compliance with French and European regulations on care and protection of laboratory animals, approved by Ethics Committee 89, and registered under Reference 2014-0014. Animals were infected by introducing intranasally the S. aureus WT or ∆adsA strain (5 × 107 CFUs/strain, suspended in 150 μl PBS). Twenty-four hours postinfection, bronchoalveolar lavages (BALs) were performed with 5 ml PBS (injected/aspirated three times). Bacterial loads were evaluated by CFUs counting on luria broth agar plates. Cells in BALs were counted and analyzed by cytospin. The levels of IL-8 in BAL fluids (BALFs) were measured by ELISA. The sPLA2 activity was assayed using the method described below. The levels of sPLA2-IIA expression in cells (107) isolated from BALs were quantified by quantitative RT-PCR. In certain experiments, the sPLA2 inhibitor YM26734 (2 mg/kg body weight) or vehicle (DMSO) was instilled to animals together with bacteria.

sPLA2 activity assay

The sPLA2 activity in BALFs from S. aureus-infected guinea pigs was measured, as described previously (18). Briefly, aliquots of BALFs (10 μl) were incubated with substrate (3H-OA–labeled Escherichia coli membrane phospholipids) at 37°C in assay buffer (0.1 M Tris [pH 7.5], 1 mM CaCl2, 0.2% sodium azide). After reaction, the released 3H-OA–labeled free fatty acids in supernatant of reaction were monitored, and the sPLA2 activity was presented as percentage of radioactivity in the supernatant compared with that of total count (defined as radioactivity detected in mixed suspension of substrate incubated with PBS). The specificity of this activity assay was verified in the presence of the selective sPLA2 activity inhibitor, YM26734 (10 μM), which was preincubated with the samples for 30 min before adding the substrate.

Statistical analyses

Results are presented as mean ± SEM from at least three independent experiments. The levels of statistical significance were determined by Student t test, one-way or two-way ANOVA followed by Bonferroni’s multiple comparison test, Mann–Whitney U test, and Kruskall–Wallis test, followed by Dunn’s multiple comparisons test.

Results

S. aureus adenosine inhibits sPLA2-IIA expression in AMs

Our previous studies have shown that AMs are the major source of sPLA2-IIA in guinea pig airways and contribute to inflammation by secreting IL-8, a key inflammatory chemokine, in response to various pathogens (29, 30). In this study, we examined the effect of S. aureus adenosine on sPLA2-IIA expression by AMs, in parallel to IL-8 secretion as a control of inflammation. The results showed that various strains of S. aureus failed to induce sPLA2-IIA expression compared with positive control, LPS from P. aeruginosa (Fig. 1A). However, these strains stimulated IL-8 secretion (Supplemental Fig. 1A). We next examined the role of adenosine in sPLA2-IIA expression using the ∆adsA mutant strain, deficient in adenosine production, and adsA-complemented strain (24), as well as exogenous adenosine. Infection with ∆adsA mutant significantly increased sPLA2-IIA expression by AMs compared with noninfected and the WT strain-infected cells (Fig. 1B, 1C). Interestingly, this increased sPLA2-IIA expression was abolished by the complementation of adenosine and exogenous adenosine (Fig. 1B, 1C). However, no difference was observed in IL-8 secretion by AMs infected with these bacterial strains or coincubated with exogenous adenosine (Supplemental Fig. 1B, 1C). This suggests that S. aureus adenosine inhibits sPLA2-IIA expression by AMs, but had no effect on IL-8 expression.

FIGURE 1.
FIGURE 1.

S. aureus adenosine impairs sPLA2-IIA expression in primary AMs. (A) Guinea pig AMs were infected with various strains of S. aureus. sPLA2-IIA expression was quantified 24 h later. Cells were incubated with P. aeruginosa LPS as a positive control. (B) AMs were infected by the WT, ∆adsA mutant, or complemented strain of S. aureus, and sPLA2-IIA expression was quantified 24 h postinfection. (C) AMs were infected by the WT or the ∆adsA strain of S. aureus, or coincubated with exogenous adenosine (100 μM) and ∆adsA strain. Then sPLA2-IIA expression was quantified 24 h postinfection. Results were shown as mean ± SEM from at least three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with control and determined with one-way ANOVA, followed by Bonferroni’s multiple comparisons test.

S. aureus induces sPLA2-IIA expression via NOD2 receptor

Based on the above results, we then investigated how the ∆adsA strain of S. aureus induced sPLA2-IIA expression. We first searched for the S. aureus PAMP(s) and associated receptor(s) involved in sPLA2-IIA expression by AMs using purified PGN (ligand of TLR2 and NOD2) and LTA (ligand of TLR2), two major membrane components of S. aureus. Our results showed that PGN significantly induced sPLA2-IIA expression by AMs, whereas LTA had limited effect (Fig. 2A), suggesting the potential implication of NOD2 receptor in this PGN-induced sPLA2-IIA expression. However, both PGN and LTA induced IL-8 secretion (Supplemental Fig. 1D). To further verify the role of NOD2, AMs were then incubated with the minimal bioactive PGN motif MDP (ligand of NOD2), MDPc (inactivated MDP), MALP-2 (ligand of TLR2/6), Pam3CSK4 (ligand of TLR2/1), and CpG (agonist of TLR9). Remarkably, only MDP strongly induced sPLA2-IIA expression by AMs (Fig. 2B). However, both TLR2 and NOD2 ligands induced IL-8 secretion (Supplemental Fig. 1E). These results indicated that NOD2 might be the unique receptor mediating PGN-induced sPLA2-IIA expression in AMs. Due to the fact that NOD2 is an intracellular PAMP sensor, we next examined the impact of adenosine on S. aureus phagocytosis in our cell model. We showed that the ∆adsA strain was more phagocytized by AMs compared with the WT, complemented, and adenosine-coincubated ∆adsA strains (Fig. 2C, 2D), although these strains exhibited similar adhesion to AMs (data not shown). Interestingly, we also found that the phagocytic killing of ∆adsA strain was faster than that of WT strain after a 2-h uptake by AMs. Nonetheless, 4 h postinfection, a similar level of bacteria was observed within AMs (Fig. 2E). In addition, cytochalasin D also significantly decreased sPLA2-IIA expression induced by the ∆adsA strain (Fig. 2F) without significant effect on IL-8 secretion (Supplemental Fig. 1F). Taken together, these results indicated that phagocytized ∆adsA S. aureus induces sPLA2-IIA expression through PGN-NOD2 signals. Conversely, S. aureus produces adenosine that inhibits its phagocytosis, leading to decreased sPLA2-IIA expression.

FIGURE 2.
FIGURE 2.

S. aureus-induced sPLA2-IIA expression is mediated by intracellular NOD2 receptor. (A) AMs were stimulated for 24 h with purified PGN (10 μg/ml) or LTA (1 μg/ml), before quantification of sPLA2-IIA expression. (B) AMs were incubated with MDP (10 μg/ml), MDPc (10 μg/ml), Pam3CSK4 (300 ng/ml), MALP-2 (250 nM), and CpG (1 μg/ml) for 24 h, followed by examination of sPLA2-IIA expression. (C) Phagocytosis of the WT, the ∆adsA mutant, and the complemented strain of S. aureus by AMs. (D) Phagocytosis of the WT and the ∆adsA strain (in the absence or presence of exogenous adenosine [100 μM]) of S. aureus by AMs. (E) Dynamic phagocytic killing of the WT and the ∆adsA strain of S. aureus by AMs. (F) AMs pretreated with cytochalasin D (5 μM) or DMSO were infected with the S. aureus WT or ∆adsA strain, and sPLA2-IIA expression was analyzed. Data were presented as mean ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control or as indicated. §§p < 0.01 compared with phagocytosis at 1 h postinfection. The statistical significances were determined with Student t test (C and D) or one-way ANOVA, followed by Bonferroni’s multiple comparisons test (A, B, and F).

S. aureus-induced sPLA2-IIA expression is dependent on receptor interacting protein 2 kinase and NF-κB transcription factor

We next investigated the downstream signals by which NOD2-mediated ∆adsA S. aureus-induced sPLA2-IIA expression in AMs, starting with the NOD2-activated receptor interacting protein 2 (RIP2) kinase (33). Incubation of AMs with the RIP2 pharmacological inhibitor Gefitinib abolished the effect of the S. aureus ∆adsA mutant on sPLA2-IIA expression (Fig. 3A), but had no effect on IL-8 secretion (Supplemental Fig. 2A). In addition, we also investigated the role of NF-κB, located downstream of RIP2 and major regulator of sPLA2-IIA expression (31, 34). In agreement, our results showed that BAY 11-7082 (an inhibitor of IκBα phosphorylation) significantly reduced ∆adsA S. aureus-induced sPLA2-IIA expression (Fig. 3B) as well as IL-8 secretion (Supplemental Fig. 2B). Furthermore, IκBα degradation, which reflects NF-κB activation, was increased in AMs infected by the ∆adsA mutant compared with those infected by the WT strain (Fig. 3C). We further confirmed the role of NF-κB using the Chinese hamster ovary cell line transfected with a luciferase plasmid bearing the sPLA2-IIA WT promoter or the mutant promoter in which the NF-κB binding site was deleted (∆NF-κB) (32). MDP, but not MDPc, significantly increased sPLA2-IIA promoter activity (Fig. 3D). However, deletion of the NF-κB binding site abolished this induction (Fig. 3D). Together, these data indicated that ∆adsA mutant of S. aureus induced sPLA2-IIA expression through a NOD2–RIP2–NF-κB–dependent pathway.

FIGURE 3.
FIGURE 3.

RIP2 and NF-κB are involved in S. aureus-induced sPLA2-IIA expression. (A and B) AMs were pretreated with (A) Gefitinib (500 nM) or (B) BAY-117082 (3 μM) for 30 min before infection with the S. aureus WT or ∆adsA strain. Twenty-four hours later, sPLA2-IIA expression was evaluated. (C) Quantification of IκBα degradation in AMs postinfection by the S. aureus WT or ∆adsA strain at different time points. (D) MDP-induced sPLA2-IIA expression by CHO cells was dependent on the activation of NF-κB. sPLA2-IIA–luciferase plasmid (bearing sPLA2-IIA WT promoter [WT] or mutant promoter in which the binding site of NF-κB was deleted [∆NF-κB])-transfected CHO cells were stimulated with MDP or MDPc (10 μg/ml/each) for 24 h before luciferase assay. (A, B, and D) Data were presented as mean ± SEM from at least three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with uninfection group or as indicated and determined with one-way ANOVA, followed by Bonferroni’s multiple comparisons test. Blot presented in (C) was representative of at least three experiments.

Adenosine impairs sPLA2-IIA expression by inhibiting S. aureus phagocytosis via A2a/A2b receptors and PKA pathway

Adenosine regulates intracellular cAMP levels via its receptors, leading to PKA activation, and, among adenosine receptors, only A2a and A2b receptors (A2aR and A2bR) increase cAMP levels (26). This led us to investigate the role of adenosine and its receptors in the modulation of sPLA2-IIA expression by AMs. Our results showed that the pharmacological inhibitors of both the A2aR (ZM241385) and A2bR (PSB603) enhanced sPLA2-IIA expression induced by the S. aureus WT strain (Fig. 4A), which exhibited similar levels as that by the ∆adsA mutant of S. aureus. However, these inhibitors had no effect on IL-8 secretion (Supplemental Fig. 2C), supporting our hypothesis that different signaling pathways modulate sPLA2-IIA and IL-8 expression.

FIGURE 4.
FIGURE 4.

Adenosine impairs sPLA2-IIA expression by inhibiting phagocytosis through A2aR/A2bR–PKA pathway. (A) AMs were pretreated with ZM241385 (1 μM), PSB603 (1 μM), or DMSO for 30 min. Cells were then infected with the S. aureus WT or ∆adsA strain, followed by analysis of sPLA2-IIA expression 24 h later. (B) sPLA2-IIA expression by AMs pretreated with H-89 (500 nM) before infection with the WT or the ∆adsA strain of S. aureus. (C and D) Phagocytosis assay of the S. aureus WT strain by AMs pretreated with ZM241385 (1 μM) and PSB603 (1 μM) (C), or H-89 (500 nM) (D). The ∆adsA strain was also included. (E) Expression of adenosine receptors (A2aR and A2bR) 24 h after S. aureus infections. Data were presented as mean ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control or as indicated. §p < 0.05, §§p < 0.01 compared with control of A2aR. The significances were determined with Student t test or one-way ANOVA, followed by Bonferroni’s multiple comparisons test.

These results prompted us to investigate the role of PKA in the modulation of sPLA2-IIA expression. Our results showed that the PKA pharmacological inhibitor H-89 increased sPLA2-IIA expression by AMs infected with the S. aureus WT strain (Fig. 4B). Here again, this inhibitor had no effect on IL-8 production by AMs (Supplemental Fig. 2D). We also examined the role of A2aR/A2bR and PKA on S. aureus phagocytosis by AMs. Inhibition of either A2aR, A2bR, or PKA strongly increased phagocytosis of S. aureus WT strain (Fig. 4C, 4D). These results demonstrated that the A2aR/A2bR–PKA pathway modulates sPLA2-IIA expression via inhibition of S. aureus phagocytosis. In addition, we examined the effect of S. aureus infection on the expression of A2aR and A2bR in AMs. Our result showed that A2bR exhibited a higher expression level compared with that of A2aR at basal conditions. However, after S. aureus infection, a dramatic increase of A2aR expression was observed (Fig. 4E), whereas the A2bR expression remained unchanged.

Adenosine inhibits phagocytosis of S. aureus through p38 inactivation

Next, we attempted to investigate the mechanism by which adenosine inhibited S. aureus phagocytosis. It has been reported that MAPK modulate S. aureus phagocytosis (35). In this work, we showed that, compared with the WT strain, the S. aureus ∆adsA mutant increased p38 phosphorylation, but had no effect on ERK phosphorylation (Fig. 5A). Incubation of AMs with the p38 inhibitor, SB203580, decreased phagocytosis of both the S. aureus WT and ∆adsA strains to similar levels (Fig. 5B). These results suggested that increased p38 activation was responsible for the enhanced phagocytosis of the ∆adsA strain. As expected, p38 inhibition also abolished S. aureus-induced sPLA2-IIA expression (Fig. 5C) and decreased IL-8 secretion by AMs (Supplemental Fig. 3A). Importantly, we also showed that inhibition of A2aR/A2bR and PKA increased the p38 phosphorylation (Fig. 5D). Taken together, these data suggested that the A2a/A2b–PKA pathway inhibits S. aureus phagocytosis by AMs through modulation of p38 phosphorylation.

FIGURE 5.
FIGURE 5.

S. aureus adenosine-impaired phagocytosis by AMs is mediated through inhibition of p38 phosphorylation. (A) Phosphorylation of p38 and ERK following infection of AMs by the S. aureus WT or ∆adsA strain (multiplicity of infection 25). (B) Phagocytosis assay of the S. aureus WT or ∆adsA strain by AMs pretreated with p38 inhibitor SB203580 (10 μM) for 30 min. (C) sPLA2-IIA expression by AMs pretreated with DMSO or SB203580 and infected with the S. aureus WT or ∆adsA strain. (D) Western blot analysis of p38 phosphorylation 30 min postinfection of AMs by the S. aureus WT strain. The cells were treated with inhibitors of PKA (H-89, 500 nM), A2aR (ZM241385, 1 μM), and A2bR (PSB603, 1 μM), respectively. (B and C) Data were presented as mean ± SEM from at least three independent experiments. (A and D) Blots were representative of at least three experiments. **p < 0.01, ***p < 0.001 compared with control or as indicated and determined with Student t test or one-way ANOVA, followed by Bonferroni’s multiple comparison test.

Adenosine protects S. aureus from sPLA2-IIA–mediated killing in guinea pig airways

All above findings prompted us to examine the pathophysiological relevance of adenosine-mediated sPLA2-IIA inhibition in airways of guinea pigs. Animals were infected by intranasal instillation of the S. aureus WT or ∆adsA strain. Compared with PBS instillation, S. aureus infection increased (although not significant) the total cell counts and neutrophil recruitment in the airways (Fig. 6A). No significant difference in cell recruitment (Fig. 6A) and IL-8 secretion (Supplemental Fig. 3B) was observed between the S. aureus WT and ∆adsA strains. Interestingly, cells isolated from BALs of ∆adsA-infected animals had higher levels of sPLA2-IIA expression compared with those from WT strain-infected animals (Fig. 6B), which was correlated with a higher clearance of the ∆adsA strain of S. aureus from airways compared with that of the WT strain (Fig. 6C). Moreover, a higher level of sPLA2 activity was detected in the BALFs from ∆adsA-infected compared with WT strain-infected guinea pigs (Fig. 6D), which was inhibited by the sPLA2 pharmacological inhibitor YM26734 (Fig. 6D). Remarkably, instillation of the YM26734 inhibitor reversed the clearance of the ∆adsA strain, but had no effect on that of the WT strain (Fig. 6E). As a whole, these findings demonstrated that inhibition of sPLA2-IIA expression by adenosine improved S. aureus survival in the airways of guinea pigs.

FIGURE 6.
FIGURE 6.

Adenosine secretion promotes S. aureus persistence in airways of guinea pigs through inhibition of sPLA2-IIA expression. Guinea pigs were infected intranasally with 5 × 107 CFUs of the S. aureus WT or ∆adsA strain. (A) Total cells in BALs were counted, and cellular differentiation (macrophages [Mϕ]/monocytes and neutrophils) was analyzed by cytospin in PBS-treated (n = 6) and bacterial-infected animals (n = 8 per group), respectively. (B) sPLA2-IIA expression was quantified by quantitative PCR in cells isolated from BALs of guinea pigs after 24-h infection with the WT or the ∆adsA strain of S. aureus. (C) Bacterial loads in BALs of the WT or the ∆adsA-infected animals 24 h postinfection (n ≥ 8 animals per group). (D) sPLA2 activity assay (in the absence or presence of YM26734) in BALFs from guinea pigs infected with S. aureus WT or ∆adsA strain for 24 h. (E) Animals were infected with S. aureus WT or ∆adsA strain in the presence or absence of sPLA2 inhibitor (YM26734, 2 mg/kg body weight). Bacterial loads in BALs of infected animals were then analyzed (n ≥ 5 per group). Data were presented as mean ± SEM. *p < 0.05, **p < 0.01 determined using (B and C) Mann–Whitney test and (D) Kruskal–Wallis test, followed by Dunn's multiple comparisons test.

Discussion

S. aureus is a major opportunistic human pathogen known to cause a wide range of infections such as pneumonia (1, 2). Using a guinea pig model of pulmonary infection, we demonstrated the role of the endogenous sPLA2-IIA in the host response against S. aureus. Our results showed that S. aureus airways infection initiated the phagocytosis process by AMs. The phagocytized S. aureus was sensed by the intracellular NOD2 receptor via PGN motifs of this bacterium. Engagement of NOD2 led to sPLA2-IIA induction and subsequent bacterial killing by this enzyme. However, the present studies also showed that S. aureus enabled evasion of host defense through a process involving adenosine production. We showed that adenosine inhibited S. aureus phagocytosis through the host A2aR/A2bR–PKA pathway, resulting in the inhibition of sPLA2-IIA expression. Interestingly, this inhibition was specific to sPLA2-IIA, as IL-8 expression was not affected by adenosine both in vivo and in vitro. All of these findings might have important implications for understandings of the mechanisms of S. aureus pathogenesis in the airways.

The sPLA2-IIA enzyme is one of the most efficient antimicrobial peptides of the host against bacterial infections, in particular by G+ bacteria (11, 14). It hydrolyzes membrane phospholipids, major structural components of bacterial cell wall, leading to bacteria death. In a transgenic mouse model, sPLA2-IIA has been shown to protect animals from i.p. or pulmonary infections by S. aureus or Bacillus anthracis, respectively (17–19). However, these transgenic mice are artificial models with constitutive overexpression of sPLA2-IIA (36). Moreover, mice are known to exhibit no or very low sPLA2-IIA expression in the lung (37). Therefore, we used guinea pigs as animal model known to produce high pulmonary levels of sPLA2-IIA (18, 30). Our results showed that pulmonary infection with the S. aureus ΔadsA strain, deficient in adenosine production, increased sPLA2-IIA expression in guinea pigs lungs, leading to an enhanced clearance of S. aureus ΔadsA strain from the airways. In line with our previous study (18), pretreatment of animals with the pharmacological inhibitor of sPLA2, YM26734, abolished this clearance. Indeed, sPLA2 activity present in the BALFs from S. aureus-infected guinea pigs was inhibited by YM26734 (Fig. 6D). Although YM26734 is not a specific inhibitor of sPLA2-IIA, this enzyme has been demonstrated as the major sPLA2 in guinea pig airways, and AMs are the major source of this enzyme (30). Different virulence factors from both Gram-negative (G−) and G+ bacteria have been shown to induce sPLA2-IIA expression (29, 30, 38). For instance, we showed that B. anthracis PGN induces sPLA2-IIA expression in AMs (29). We found in the present work a similar effect of S. aureus PGN on this expression. We demonstrated in this work that NOD2–RIP2 signaling cascade, but not the surface receptors such as TLRs, mediated PGN-induced sPLA2-IIA expression. In addition, we also showed that phagocytosis of S. aureus by AMs was a key step in the induction of sPLA2-IIA expression. We observed a faster phagocytic killing of ΔadsA (accompanied with an increased sPLA2-IIA expression) compared with that of WT strain (Fig. 2E). Indeed, engulfed bacteria are known to be degraded into phagolysosomes and to release PGN (39). The latter is sensed by the cytoplasmic receptor NOD2 (6). NOD2 is a powerful inducer of host immune response and is essential to control bacterial infections (7, 8). Consistent with our results on sPLA2-IIA expression, NOD2 has been shown to regulate expression of mouse antimicrobial peptides, such as α- and β-defensins (8, 10). Thus, it supports that NOD2 signaling pathway might be a general inducer of antimicrobial peptides production.

It is likely that pathogens establish different mechanisms to escape the phagocytic clearance. One of these mechanisms involves the cAMP/PKA pathway, used by Bordetella pertussis and B. anthracis to circumvent phagocytosis (40, 41). Consistent with these results, we observed in this work that adenosine inhibited S. aureus phagocytosis and the subsequent sPLA2-IIA expression through cAMP/PKA-dependent pathway. We also identified the role of the A2aR and A2bR in S. aureus phagocytosis and sPLA2-IIA expression. Our results indicated that A2aR played a major role in S. aureus phagocytosis by AMs (Fig. 4C). However, it was not possible from our data to conclude which receptor was predominantly involved in the modulation of sPLA2-IIA expression by adenosine (Fig. 4A), although S. aureus infection increased the expression of A2aR at higher levels compared with those of A2bR (Fig. 4E). In line with our results, higher A2aR expression has been reported in LPS-stimulated cells (42). Given that S. aureus phagocytosis modulates sPLA2-IIA expression in AMs (Fig. 2F), we can speculate that A2aR may indirectly play a predominant role in the inhibition of sPLA2-IIA expression. In addition, compared with A2bR, we showed A2aR also exhibited a main role in S. aureus-induced p38 phosphorylation (Fig. 5D). The latter was proven to be involved in both S. aureus phagocytosis and sPLA2-IIA expression by AMs, respectively (Fig. 5B, 5C). Taken together, these evidences demonstrated a predominant role of A2aR in S. aureus phagocytosis and modulation of sPLA2-IIA expression via a p38-dependent mechanism.

Adenosine is produced by many bacterial species via 5′-nucleotidases (24, 43). Recently, Liu et al. (43) reported that adenosine secreted by Streptococcus suis inhibits neutrophil degranulation and oxidative activity through the A2aR, favoring bacterial multiplication. Thus, adenosine secretion may represent a general mechanism used by bacteria to escape both macrophages and neutrophil-mediated innate immune response.

In addition to its effects on phagocytosis, adenosine also functions as an anti-inflammatory molecule in macrophages through the cAMP/PKA pathway (44, 45). This effect occurs through the downregulation of NF-κB activity by PKA (46). Surprisingly, in the current study, we did not observe any effect of adenosine produced by S. aureus on IL-8 secretion, regulated by NF-κB (Supplemental Fig. 2B). Moreover, inhibition of A2aR/A2bR and PKA also had no effect on IL-8 secretion (Supplemental Fig. 2C, 2D). These results suggest that the cAMP/PKA specifically regulates sPLA2-IIA expression. These data were consistent with our previous observations showing that adenylate cyclases from B. anthracis and B. pertussis, which raised cAMP levels, had no effect on IL-8 production by AMs, but inhibited sPLA2-IIA expression (11, 29). Nonetheless, S. aureus adenosine may regulate other inflammatory mediators. Indeed, recent works highlighted the essential role of phagocytosis and PGN/NOD2 in IL-1β secretion (39, 47), a key inflammatory cytokine in the host response to S. aureus (48). In this work, we showed that adenosine decreased S. aureus phagocytosis through A2aR (Fig. 4C), affecting subsequent PGN/NOD2 interaction, which may impair IL-1β secretion. However, in contrast, others showed that adenosine signaling was required to induce IL-1β secretion through the A2aR (45, 49). Thus, it would be interesting to investigate the role of adenosine produced by S. aureus on IL-1β secretion.

In conclusion, as shown in Fig. 7, our work highlights that upon its phagocytosis, S. aureus is sensed by AMs to induce sPLA2-IIA expression via NOD2-dependent pathway. This represents a new pathway used by the host to eliminate S. aureus via sPLA2-IIA. However, we also show that S. aureus is enabled to escape phagocytosis and subsequent inhibition of sPLA2-IIA expression through adenosine-dependent process. This allows S. aureus to subvert sPLA2-IIA killing, thus promoting its survival in the pulmonary microenvironment.

FIGURE 7.
FIGURE 7.

Schematic representation of the sPLA2-IIA modulation by S. aureus. S. aureus infection initiates p38-mediated phagocytosis of host cells by recognizing PGN and LTA through its TLR2 receptor (35), leading to bacterial elimination. In addition, the phagocytosis results in bacterial engulfment, degradation in phagolysosomes, and release of PGN. The latter is further sensed by NOD2, leading to sPLA2-IIA induction via RIP2 and NF-κB signals. The induced sPLA2-IIA exhibits the bactericidal function to further clear invading S. aureus. However, S. aureus-producing adenosine triggers host PKA activation via A2a and A2b adenosine receptors, which inhibits p38-mediated phagocytosis and subsequent sPLA2-IIA expression, and thus escapes from host clearance.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. D. Missiakas (University of Chicago, Chicago, IL) for providing the S. aureus Newman WT, ∆adsA mutant, and AdsA complemented strains, and Dr. Tarek Msadek (Institut Pasteur, Paris, France) for the different WT strains of S. aureus.

Footnotes

  • ↵1 L.T. and Y.W. conjointly supervised this work.

  • This work was supported by Domaine d’Intérêts Majeur-Maladies Infectieuses from the Région Ile de France (a Ph.D. fellowship to E.P.) and Vaincre la Mucoviscidose Grants RF20120600672 and RF20130500830 (to Y.W.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AdsA
    adenosine synthase enzyme
    AM
    alveolar macrophage
    BAL
    bronchoalveolar lavage
    BALF
    BAL fluid
    LTA
    lipoteichoic acid
    MDP
    muramyl dipeptide
    MDPc
    negative MDP control
    PAMP
    pathogen-associated molecular pattern
    PGN
    peptidoglycan
    PKA
    protein kinase A
    RIP2
    receptor interacting protein 2
    sPLA2-IIA
    type IIA secretory phospholipase A2
    WT
    wild-type.

  • Received October 21, 2014.
  • Accepted March 17, 2015.

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