Secreted phospholipase A₂ group X acts as an adjuvant for type-2 inflammation leading to an allergen-specific immune response in the lung

Abstract

Secreted phospholipase A₂ (sPLA₂) enzymes release free fatty acids, including arachidonic acid and generate lysophosphospholipds from phospholipids, including membrane phospholipids from cells and bacteria and surfactant phospholipids. We have shown that an endogenous enzyme sPLA₂ group X (sPLA₂-X) is elevated in the airways of asthmatics and that mice lacking the sPLA₂-X gene (Pla2g10) display attenuated airway hyperresponsiveness (AHR), innate and adaptive immune responses, and type-2 cytokine production in a model of airway sensitization and challenge using a complete allergen that induces endogenous adjuvant activity. This complete allergen also induces the expression of sPLA₂-X/Pla2g10. In the periphery, a sPLA₂ found in bee venom (bee venom PLA₂, bvPLA₂) administered with the incomplete antigen ovalbumin (OVA) leads to an antigen specific immune response. In this study, we demonstrate that both bvPLA₂ and murine sPLA₂-X have adjuvant activity, leading to a type-2 immune response in the lung with features of airway hyperresponsiveness (AHR) and antigen-specific type-2 airway inflammation following peripheral sensitization and subsequent airway challenge with OVA. Further, the adjuvant effects of sPLA₂-X that result in the type-2 biased OVA-specific adaptive immune response in the lung was dependent upon the catalytic activity of the enzyme, as a catalytically inactive mutant form of sPLA₂-X does not elicit the adaptive component of the immune response, although other components of the immune response were induced by the inactive enzyme suggesting receptor mediated effects. Our results demonstrate that exogenous and endogenous sPLA₂s play an important role in peripheral sensitization resulting in airway responses to inhaled antigens.

Introduction

Secreted phospholipase A₂ (sPLA₂) are multi-functional enzymes that act to regulate phospholipid metabolism and bind to specific C-type lectin receptors that are involved in innate immunity (1, 2). Both cytosolic PLA₂s and sPLA₂s mediate the rate-limiting step in the eicosanoid pathway by releasing free fatty acids (FFA)s including arachidonic acid (AA) from the sn2 position of phospholipids, which also leads to the generation of lysophospholipids (LysoPL)s (3–5). The sn2 position of membrane phospholipids is preferentially substituted with poly unsaturated fatty acids (PUFA)s such as AA that initiates the eicosanoid cascade, but FFAs and LysoPLs also play important independent roles as regulators of inflammation. Because sPLA₂s act within the cell prior to secretion and also act in the extracellular environment following secretion (6), the activities attributed to this group of enzymes includes effects mediated on the outer cell membrane of mammalian cells, the cell wall of bacteria and on phospholipids in the airways such as surfactant-associated phospholipids (7).

After the initial discovery that sPLA₂ activity is increased in asthma and allergic rhinitis following allergen challenge (8–10), we found that one specific sPLA₂, sPLA₂ group X (sPLA₂-X), is expressed at high levels in the airways, particularly the airway epithelium and bronchial macrophages (11) and that the levels of this protein are elevated in human asthma in association with severity of asthma and the severity of airway hyperresponsiveness (AHR) (12, 13). Murine models of asthma with mice lacking the sPLA₂-X gene (Pla2g10) have demonstrated an important role for sPLA₂-X in the development of type-2 inflammation and AHR and point to a central role for this enzyme in both the innate and adaptive immune response to inhaled allergen. Deletion of this single enzyme is associated with a marked reduction in several critical pathways including in the formation of eicosanoids, particularly cysteinyl leukotrienes (CysLTs, LTs C₄, D₄, E₄), the type-2 dominated cellular influx into the airways, and IL-33 protein levels in the airways (14, 15). Here we examine the possibility that sPLA₂-X acts during the sensitization phase, leading to features of allergen-induced airways dysfunction upon subsequent antigen exposure in the airways and lung.

Prior work examining bee venom PLA₂ (bvPLA₂), a small PLA₂ that has similar catalytic properties to endogenous mammalian sPLA₂s, demonstrated that bvPLA₂ administered peripherally with an antigen led to an antigen-specific immune response that was type-2 biased and involved type-2 innate lymphoid cells (ILC2)s (16). Thus, we hypothesized that the sPLA₂-X enzyme functions as an endogenous adjuvant to “complete” allergens that can cause sensitization in the absence of an exogenous adjuvant, and that subsequent exposure to the antigen in the lung would lead to a type-2 biased immune response and features of AHR. We base this hypothesis in part on the evidence that the complete allergen house dust mite (HDM) induces sPLA₂-X/Pla2g10 in the lung (15). We conducted initial studies with bvPLA₂ because of the known adjuvant effect, and then examined the function of endogenous sPLA₂-X. We tested our hypothesis using the incomplete antigen ovalbumin (OVA) along with catalytically active and inactive sPLA₂ proteins during the sensitization phase, followed by airway exposure to the antigen alone. This model allowed us to break down the components of the sensitization phase. Our results demonstrate that sPLA₂-X acts as an efficient adjuvant, acting through both the innate and adaptive arms of the immune system, and leading to features of type-2 inflammation and airway dysfunction following exposure to inhaled antigen. Furthermore, our results show that sPLA₂-X adjuvant activity promotes an antigen-specific type-2 inflammatory response upon exposure to antigen in vivo and ex vivo in lung leukocytes. The development of the adaptive immune response, antigen-specific type-2 cytokine response, and bronchoalveolar lavage eosinophilia was dependent upon the active sPLA₂-X enzyme, while an enzymatically inactive mutant sPLA₂-X protein induced AHR and some alterations in lung tissue leukocyte infiltration suggesting that both receptor mediated and enzymatic functions of sPLA₂-X are involved in the development of the type-2 biased immune response to antigen that is triggered upon exposure in the lung following peripheral sensitization.

Materials and Methods

Mice

Wild type (WT) BALB/c mice were used in all experiments and were obtained from Jackson Laboratories. Animals were maintained under specific pathogen-free conditions at the University of Washington and had ad libitum access to food and water. Female mice between 6–8 weeks old were used in experiments in order to reduce heterogeneity. The University of Washington Institutional Animal Care and Use Committee approved all animal studies.

OVA with PLA₂ Adjuvant Model of Experimental Asthma

During the peripheral sensitization phase, WT BALB/c mice received intraperitoneal sensitization with 100 μg low-endotoxin OVA (EndoFit™ OVA, Invivogen, San Diego, CA) with either 50 μg bvPLA₂ (Sigma, St.Louis, MO), recombinant murine sPLA₂-X or the enzymatically inactive H46Q mutant form of sPLA₂-X on days 0 and 14 (17)(Supplemental Figures S1 and S2). The levels of endotoxin were measured in key reagents using a Limulus Assay as reported in Supplemental Table 1 (Genscript, Piscataway, NJ). On days 26–28, mice received oropharyngeal doses of 50 μg OVA in 50 μl saline. An additional control group received an oropharyngeal dose of saline following the administration of bvPLA₂ in the peritoneum. On day 29, mice were intubated and ventilated using a flexiVent small animal ventilator (SCIREQ Inc., Montreal, Quebec, CA). Lung function was measured following increasing doses of methacholine (0, 6.25, 12.5, 25, and 50 mg) and dynamic resistance (R) and elastance (E) were calculated using the single frequency forced oscillation technique.

Assessment of Airway Inflammation

The lungs were lavaged post-mortem via tracheal cannula with ice-cold PBS and total bronchoalveolar lavage (BAL) cell counts were determined using a Cellometer Auto 2000 (Nexcelom Bioscience, Lawrence, MA). The BAL fluid was spun down and cells were aliquoted for cell differential analysis via multi-color flow cytometry and the supernatant stored at −80°C with 1× protease inhibitor cocktail.

Lung Leukocyte Isolation

The left lung was minced in warmed digestion solution (RPMI 1640 with 5% FCS, 0.5 mg/ml Liberase TL and 40 U/ml DNase I). Digestion was stopped with cold HBSS containing 10 mM EDTA and the digested tissue was pushed through a 40 μm cell strainer and washed several times with wash buffer (RPMI 1640 with 5% FCS). Red blood cells were removed using ACK red blood cell lysis buffer (eBioscience, San Diego, CA). Lung digest was resuspended in wash buffer, and an aliquot was removed for cell count and viability using the Cellometer Auto 2000. Cells were centrifuged and resuspended in MACS/FACS buffer (HBSS w/o Ca and Mg, 2 mM EDTA and 0.5% BSA) containing CD45 MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD45+ lung leukocytes were isolated using a Miltenyi LS column and magnetic separator.

Multi-Color Flow Cytometry for Cell Differentials

Single cell suspensions from lung leukocytes and BAL cells were stained for cell differentials using a cocktail of the following antibodies: CD11c (clone N418, eBioscience), Siglec-F (clone E50–2440, BD Bioscience), Ly-6G (clone RB6–8C5, eBioscience), CD45 (clone 30-F11, Biolegend, San Diego, CA) CD3e (clone 145–2C11, eBioscience) and MHCII (M5/114.15.2, BD Biosciences, San Jose, CA). Cells were blocked using Fc block (BD Biosciences) and incubated with the primary antibody cocktail for 30 min at 4°C in the dark. Cells were washed and resuspended in PBS containing eFluor 450 fixable viability dye (eBioscience) and incubated for 15 min at 4°C in the dark. Cells were then fixed using 4% paraformaldehyde and stored at 4°C in FACS buffer until analyzed via flow cytometry. Cells were analyzed using a FACS LSR II flow cytometer (Becton Dickinson Instrument Systems, Franklin Lakes, NJ) and gating was performed using FlowJo software (TreeStar, Ashland, OR). Single stained controls of lung leukocytes were used to set voltages and for color compensation, and leukocyte populations were determined as previously reported (18, 19). Briefly, our gating strategy consisted of first selecting singlets using forward scatter width and area, followed by selection of viable cells using the fixable viability dye. Leukocytes were then identified using side scatter and CD45. CD45⁺ cells were gated for CD3⁺ T cells and the remaining CD3⁻CD45⁺ cells were further subdivided into MHCII^high and MHCII^low populations. In the MHCII^low population, eosinophils were identified as CD11c⁻SiglecF⁺ leukocytes, resident macrophages as CD11c^highSiglecF^high leukocytes and neutrophils were identified as CD11c⁻SiglecF⁻ leukocytes that were also Ly-6G⁺. In the MHCII^high population, CD11c⁺SiglecF⁻ dendritic cells (DC) and CD11c⁻SiglecF⁻ monocyte-derived macrophages (recruited macrophages) were identified. A similar gating strategy was used to identify leukocyte subsets in the BAL fluid.

Multi-Color Flow Cytometry for ILC2s

Single cell suspensions from peritoneal cells were stained for detection of ILC2s using the following antibodies: Mouse Hematopoietic Lineage eFluor 450 Cocktail (eBioscience), containing CD3 (17A2), CD45R/B220 (RA3–6B2), CD11b (M1/70), Ter119 (TER-119) and Gr-1/Ly6G (RB6–8C5) with the addition of eFluor 450-conjugated antibodies for CD4 (RM4–5, eBioscience), CD8a (53–6.7, eBioscience), CD11c (N418, eBioscience), CD19 (1D3, eBioscience), NK1.1 (PK136, eBioscience) and Fc epsilon Receptor I alpha (FcεR1) (MAR-1, eBioscience). Further selection was performed on the lineage negative (Lin⁻) population using the following antibodies: CD45 (30-F11, eBioscience), CD90.2/Thy-1.2 (53–2.1, eBioscience), CD25 (PC61.5, eBioscience), T1/ST2 (DJ8, MD Biosciences, Oakdale, MN).

Cells were washed and resuspended in PBS, blocked using Fc block (BD Biosciences), washed and subsequently incubated with the primary antibodies for 30 min at 4°C in the dark. The cells were then washed and resuspended in PBS containing fixable UV blue dead cell viability stain (eBioscience) and incubated for 15 min at 4°C in the dark. The cells were fixed in 4% paraformaldehyde for 5 min at room temperature and stored at 4°C in FACS buffer until analyzed via flow cytometry. Cells were analyzed using a FACS LSR II flow cytometer and gating was performed using FlowJo software. Our gating strategy for ILC2s consisted of selecting singlets using forward scatter width and area, followed by selection of viable leucocytes using the fixable viability dye and CD45. We then identified Lin- cells expressing CD90.2 as ILC2s and further enumerated CD25+ and ST2+ cells among this population. Single stained controls and color compensation was conducted using anti-CD3 antibodies containing the different fluorophores. Fluorescence minus one (FMO) controls were prepared by omitting CD90.2, CD25, and T1/ST2 antibodies to set the appropriate thresholds for these gates.

Antigen Re-stimulation of Cultured Lung Leukocytes

For OVA re-stimulation of isolated lung leukocytes, 2 × 10⁵ cells were plated in each well of a 96-well tissue culture plate. Cells were stimulated with 100 μg/ml OVA or with saline as a control. Following stimulation for 24h, the cell culture supernatant was isolated and stored at -80°C with 1× protease inhibitor cocktail (Pierce). Cell culture supernatant was later analyzed for cytokine levels via ELISA.

Cytokine and Immunoglobin Analysis by ELISA

Murine IL-33 protein content from BAL fluid, lung tissue lysates and peritoneal fluid were determined by ELISA per manufacturer’s instructions (R&D, Minneapolis, MN) and analyzed on a Biotek EL800 platereader (Winooski, VT). Detection of other murine cytokines (IL-4, IL-5, IL-13, IL-10, IL-6, IL-2, TNF, KC) from antigen restimulated leukocytes was performed using the Mouse V-PLEX Pro-inflammatory Panel 1 assay and the Mouse IL-13 Ultrasensitive assay per manufacturer’s instructions (Meso Scale Diagnostics, Rockville, MD) and plates were analyzed using a Sector S 600 plate reader. The number of samples assayed was based on the number of wells available on the plate and all data were reported. OVA-specific IgE and IgG1 from mouse plasma samples were assayed by ELISA (Cayman, Ann Arbor, MI).

Statistical Analysis

Statistical analysis was conducted using GraphPad Prism software (version 8.3.1; GraphPad, La Jolla, CA). Differences in AHR to MCh were evaluated with a 2-way ANOVA test with contrasts between MCh dose and sensitization condition. Post hoc analysis was performed at each dose after correction for multiple comparisons. Differences in features of inflammation including cell differentials in BAL fluid and lung tissue, and immunoglobulin levels were assessed with a 1-way ANOVA with contrasts based on the type of sensitization and challenge. The production of cytokines from restimulated leukocytes for the bvPLA₂ studies were assessed by an unpaired 2-tailed Welch’s t test to account for the lower variance of the unstimulated leukocytes. Cytokine production from restimulated leukocytes for the sPLA₂-X studies were assessed with a 2-way ANOVA test with contrasts between sensitization condition and OVA restimulation. Correction for multiple comparisons was made with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli (20). For the analysis of ILC2s, the data were assessed with an unpaired t test or 1-way ANOVA with contrasts based on the type of sensitization and challenge without further correction for multiple comparisons. For all tests, a P value less than 0.05 was considered significant and in the case of correction for multiple comparisons, a q value of less than 0.05 was considered significant.

Results

Bee venom PLA₂ acts as an adjuvant that facilitates an antigen-specific type-2 immune response in the lung following peripheral sensitization.

Prior work demonstrated that in the periphery bvPLA₂ delivered along with an antigen leads to the development of a type-2 biased antigen-specific immune response (16). Thus, we hypothesized that both exogenous and endogenous secreted PLA₂s could serve as effective adjuvants leading to features of type-2 allergic inflammation upon re-exposure of the airways to antigen. There is strong evidence that such peripheral sensitization is important for the development of allergic asthma in humans (21). To examine this question, we established a model using bvPLA₂ as an adjuvant in combination with the antigen OVA, as OVA alone does not elicit a strong type-2 immune response. In our model, we administered sensitization doses 2 weeks apart in the peritoneum, followed by airway challenge with antigen on days 26–28 (Supplemental Fig. 1A). One day after the last airway challenge, AHR was assessed with increasing concentrations of inhaled methacholine. Mice sensitized with bvPLA₂ plus OVA (bvPLA₂/OVA) and subsequently challenged with OVA had increased AHR compared to mice given bvPLA₂ alone (bvPLA₂/Sal) and challenged with saline (Fig. 1A). Mice sensitized with saline plus OVA (Sal/OVA) and challenged with OVA also displayed elevated AHR, similar to previous observations (22, 23); however, these mice did not develop AHR to the same extent as mice in the bvPLA₂/OVA group (Fig. 1A). Sensitization in the presence of bvPLA₂ led to a significant increase in the bronchoalveolar lavage (BAL) leukocytes relative to OVA alone or bvPLA₂ without antigen (Fig 1B), but the number of lung leukocytes was not altered in this model (Fig. 1C).

FIGURE 1.

Bee venom PLA₂ (bvPLA₂) serves as an effective adjuvant for a type-2 inflammation and airway hyperresponsiveness. (A) Sensitization to OVA in the presence of bvPLA₂ (bvPLA₂/OVA) followed by OVA challenge caused increased airway hyperresponsiveness (AHR) in response to progressive concentrations of methacholine compared to the administration of OVA in the absence of an adjuvant during the sensitization phase (Sal/OVA) and relative to bvPLA₂ alone with no antigen (bvPLA₂/Sal). (B) The concentration of BAL cells was increased when bvPLA₂ was administered during the sensitization phase followed by re-exposure to OVA in the airways. (C) In contrast, the concentration of CD45+ leukocytes in the lung did not differ between the groups. (D-G) The concentrations of eosinophils (D), T cells (E), recruited macrophages (F, Rec Macs), and dendritic cells (G, DCs) assessed by multicolor flow cytometry were all increased in the BAL fluid when bvPLA₂ was administered during the sensitization phase along with the antigen OVA. The data are represented as the mean +/− SEM. There were 2 experimental replicates with n=4 mice per group for a total of 8 mice in each group. The reported P values were corrected for multiple comparisons as described in the statistical methods.

Evaluation of the cellular influx into the BAL fluid by multicolor flow cytometry revealed marked increases in the concentration of eosinophils, T cells, recruited (CD3-CD45+MHCII^highCD11c⁻SiglecF⁻) macrophages and dendritic cells (DC)s facilitated by sensitization to OVA in the presence of bvPLA₂ (Figs. 1D-G), while OVA without an adjuvant also increased the concentration of eosinophils (P=0.04), T cells (P=0.007), and DCs (P=0.0007), but not recruited macrophages (P=0.14) relative to the bvPLA₂/Sal control. In the BAL fluid there was also an increase in the concentration of neutrophils (PMN) with either OVA alone (P<0.0001) or with bvPLA₂/OVA (P<0.0001) relative to the bvPLA₂/Sal control, but there is no difference between these conditions (P=0.09) and the magnitude of the PMN influx was small relative to the influx of eosinophils. We also found that there was an increase in the number of resident (CD3-CD45+MHCII^lowCD11c⁺SiglecF⁺) macrophages when bvPLA₂/OVA was administered relative to bvPLA₂/Sal (P=0.0003) or relative to OVA without an adjuvant (P=0.005); while ova without an adjuvant was modestly different than the bvPLA₂/Sal control (P=0.06).

Lung leukocyte populations were altered to less of a degree by the adjuvant properties of bvPLA₂ in this model, suggesting that antigen-challenge following peripheral sensitization mediated by bvPLA₂ led predominantly to an immune response that was largely confined to the airways following antigen administration. Although the number of eosinophils in the lung was higher in both groups that were challenged with OVA (P=0.04 OVA alone relative to bvPLA₂/Sal), the concentration of eosinophils was highest when bvPLA₂ was administered during sensitization relative to either group (Supplemental Fig. 1B). In contrast, lung T cells, recruited macrophages and resident macrophages were not significantly altered by the administration of bvPLA₂ during the sensitization phase (Supplemental Figs. 1C-E); however, it is also notable that when OVA was administered alone, there was a significant decrease in resident macrophages (P=0.02). We also noted that the number of DCs was increased either by the administration of OVA alone (P<0.0001) or with bvPLA₂ as an adjuvant, while there were no notable differences in the concentration of PMNs (Supplemental Figs. 1F-G). These findings demonstrate that bvPLA₂ acts as an adjuvant, and subsequent airway challenge with antigen drives a type-2 skewed immune response in the lung characterized by AHR and leukocyte recruitment predominantly to the airways.

Peripheral sensitization to OVA with bvPLA₂ facilitates antigen-specific cytokine production by lung leukocytes following antigen challenge.

To gain a better understanding of the events that are established following peripheral sensitization in the presence of bvPLA₂ followed by antigen challenge in the lung, we isolated leukocytes from the lungs of sensitized and challenged mice and stimulated these cells ex vivo with OVA for 24 hours. Cells from bvPLA₂/OVA sensitized mice produced more of the type-2 cytokines IL-4 and IL-5 than cells from saline/OVA sensitized mice; however, we did not observe a difference in IL-13, although the levels of IL-13 were surprisingly low in this model, while the levels of IL-5 were markedly elevated (Figs. 2A-C). We also detected significantly higher levels of IL-10, IL-6, IL-2 (Figs. 2D-H), as well as KC (P=0.001) and TNF (P=0.003) in lung leukocytes isolated from bvPLA₂/OVA mice. Leukocytes that were sensitized with bvPLA₂ alone in the absence of the antigen challenge with OVA did not produce measurable cytokines following in vitro OVA stimulation (data not shown). These data suggest that peripheral sensitization mediated through bvPLA₂ enhances activation of leukocytes in the lung following antigen exposure in the lung and specifically leads to the antigen-specific generation of both type-1 and type-2 cytokines.

FIGURE 2.

Cytokine production by lung leukocytes in response to OVA restimulation is increased when bvPLA₂ was administered during the sensitization phase. (A-C) Cytokine production following in vitro OVA restimulation of CD45+ lung leukocytes following sensitization and challenge had increased production of the type-2 cytokines IL-4 (A), IL-5 (B), but not IL-13 (C). (D-F) The production of cytokines that are not classically type-2 including IL-10 (D), IL-6 (E) and IL-2 (F) were also increased in mice that received bvPLA₂ during sensitization. The data are represented as the mean +/− SEM. There were 8 lung samples in the control group that received OVA and 5 in the group that received bvPLA₂/OVA; P values represent the Welch’s t test.

Sensitization with bvPLA₂ promotes an adaptive immune response to antigen in the lung.

To evaluate the effects of bvPLA₂ on the adaptive immune response to OVA, we measured antigen-specific IgE and IgG. Although the levels of OVA-IgE were increased relative to the bvPLA₂/Sal control, the levels of OVA-IgE were significantly higher in the bvPLA₂/OVA group compared to both controls (Fig. 3A) In contrast, OVA-IgG was modestly elevated in the bvPLA₂/OVA group, but this increase was not seen in the group that received OVA in the absence of an adjuvant and there was also a modest trend towards an increase in OVA-IgG compared to the Sal/OVA group (Fig. 3B). These results indicate that bvPLA₂ adjuvant activity enhances the adaptive immune response in an antigen-specific manner and that this response is more prominent for antigen-specific IgE, although the levels of OVA-specific IgE are lower than OVA-specific IgG.

FIGURE 3.

The antigen-specific immune response is enhanced by bvPLA₂ during the sensitization phase. (A) OVA-specific IgE in serum following sensitization and challenge was significantly increased when bvPLA₂ was administered during the sensitization phase. (B) A similar trend was identified for the levels of OVA-specific IgG in this model. The data are represented as the mean +/− SEM. There were 8 samples in each group and the reported P values were corrected for multiple comparisons.

Adjuvant activity of sPLA₂-X facilitates the development of allergic airway disease following sensitization in the periphery.

Next we sought to expand upon these findings by investigating the potential adjuvant properties of a recombinant version of endogenous sPLA₂-X that we have implicated in human asthma (12, 14, 15, 24). Both sPLA₂-X and bvPLA₂ are small PLA₂s that act on the outer cell membrane of mammalian cells with similar enzymatic activity, but these sPLA₂s are structurally dissimilar, a critical distinction since they both act through receptors but are divergent in this regard (25, 26). In prior studies, we demonstrated that mice lacking sPLA₂-X/Pla2g10 are protected from developing allergic airway disease and have impaired innate and adaptive immune responses to the complete allergen HDM (15) and in a model with the exogenous adjuvant alum (14). Here we sought to determine the ability of sPLA₂-X to act as an adjuvant and to investigate whether peripheral sensitization in the presence of this endogenous enzyme facilitated a type-2 response in the lung. Further, we examined the dependence on the enzymatic activity of sPLA₂-X through the use of a mutant version of this enzyme in which a single amino acid substitution from histidine to glutamine in the active site (H46Q mutant sPLA₂-X) renders it enzymatically inactive but maintains the secondary protein structure preserving receptor binding (17). We exposed mice to OVA plus recombinant WT sPLA₂-X or to H46Q mutant sPLA₂-X according to our model (Supplemental Fig. 2A). Following sensitization in the periphery with each protein followed by airway challenge with antigen, both the WT version of sPLA₂-X and the H46Q mutant sPLA₂-X tended to induce AHR above the saline controls (P=0.07 H46Q vs. Sal), but the WT sPLA₂-X increased AHR over both the saline control and the H46Q mutant administered during the sensitization phase resulting in significant difference overall based on the type of protein administered during sensitization and methacholine dose (P=0.03, Fig. 4A). In this model, we excluded the 50 mg/ml concentration of methacholine because several of the WT mice died at this concentration due to enhanced AHR. The cellular influx into the airways was notable for a significant increase of leukocytes in the BAL fluid of mice sensitized with WT sPLA₂-X plus OVA that was not seen in mice sensitized with the H46Q mutant form of sPLA₂-X (Fig. 4B). The concentration of lung tissue leukocytes tended to increase in the mice that received WT sPLA₂-X plus OVA, but this was not significantly different from the group that received the inactive form of sPLA₂-X (Fig. 4C).

FIGURE 4.

The endogenous protein sPLA₂-X acts as an effective adjuvant for type-2 inflammation in the lung and the adaptive component of this responses is dependent upon the PLA₂ activity of this enzyme. (A) Sensitization to OVA in the presence of sPLA₂-X (sPLA₂-X/OVA) followed by OVA challenge caused increased airway hyperresponsiveness (AHR) in response to progressive concentrations of methacholine compared to the administration of OVA in the absence of an adjuvant during the sensitization phase (Sal/OVA). The administration of a catalytically inactive mutant sPLA₂-X (H46Q sPLA₂-X) in place of the wild type (WT) enzyme followed by OVA challenge (H46Q/OVA) also caused an increase in AHR relative to Sal/OVA. (B) In contrast, the concentration of BAL cells was increased only when catalytically active sPLA₂-X was administered during the sensitization phase followed by re-exposure to OVA in the airways. (C) The concentration of CD45+ leukocytes in the lung tended to increase in the lung when catalytically active sPLA₂-X was administered during the sensitization phase. (D-G) The concentrations of eosinophils (D), T cells (E), recruited macrophages (F, Rec Macs), and dendritic cells (G, DCs) assessed by multicolor flow cytometry were all increased in the BAL fluid when catalytically active sPLA₂-X was administered during the sensitization phase along with the antigen OVA. The data are represented as the mean +/− SEM. There were 2 experimental replicates with n=4 and n=3 mice per group for a total of 7 mice in each group and the reported P values were corrected for multiple comparisons as described in the statistical methods.

Multi-color flow cytometry to decipher leukocyte populations in the BAL fluid revealed marked increases in eosinophils, T cells, recruited macrophages and DCs in mice sensitized with WT sPLA₂-X compared to saline controls (Figs. 4D-G). Sensitization with the inactive H46Q mutant did not result in any notable change in leukocyte populations (Figs. 4D-G). We also noted an increase in the concentration of PMNs in the BAL fluid mediated by WT sPLA₂-X compared to either control group (P=0.0004), but the concentration of PMNs was very low in this model. We also did not observe any differences in the concentrations of resident macrophages.

Further characterization of leukocyte subsets in the lung tissue revealed that there were significant increases in lung tissue eosinophils with WT sPLA₂-X that did not occur with the mutant H46Q protein, while there were no significant differences in T cells or recruited macrophages (Supplemental Figs. 2B-D). We also noted that the concentration of resident macrophages was reduced when WT sPLA₂-X was administered during sensitization, while the concentrations of DCs and PMNs in lung tissue were both increased following sensitization in the presence of WT sPLA₂-X followed by subsequent challenge of the lung with OVA (Supplemental Figs. 2E-G). Together, these data indicate that sPLA₂-X activity is required for the activation and recruitment of leukocyte populations to the airways in response to inhaled antigen, and that these responses are accompanied by similar changes of lesser magnitude in lung tissue leukocytes following OVA rechallenge of the airways.

Enzymatic activity of sPLA₂-X is required for the adaptive type-2 immune response and airway inflammation.

Lung leukocytes isolated from control mice sensitized with saline plus OVA (Sal) displayed modest changes in type-2 cytokines following ex vivo stimulation with OVA (Figs. 5A-C). In contrast, leukocytes from mice sensitized with WT sPLA₂-X protein plus OVA (WT) produced robust levels of IL-4, IL-5 and IL-13, whereas leukocytes from mice sensitized with the inactive sPLA₂-X mutant (H46Q) plus OVA produced levels of IL-4, IL-5 and IL-13 following OVA rechallenge that were similar to leukocytes from saline control mice (Figs. 5A-C). In addition to type 2 cytokines, leukocytes from mice sensitized with WT sPLA₂-X produced significantly higher levels of IL-10, IL-6 and IL-2 compared to leukocytes from mice sensitized with inactive H46Q sPLA₂-X or leukocytes isolated from saline control mice in response to OVA restimulation (Figs. 5D-F). An exception was that the levels of IL-6 increased significantly following OVA restimulation when inactive H46Q sPLA₂-X was administered during the sensitization phase (Fig 5E, P=0.04). In contrast to the results with bvPLA2/OVA, we did not observe any differences in the levels of TNF or KC with either WT sPLA₂-X or H46Q sPLA₂-X at baseline or after antigen restimulation.

FIGURE 5.

The antigen-specific cytokine production by lung leukocytes following restimulation with OVA restimulation is increased primarily when catalytically active sPLA₂-X was administered during the sensitization phase. (A-C) Cytokine production following in vitro OVA restimulation of CD45+ lung leukocytes following sensitization and challenge revealed marked increases in the production of multiple different cytokines. Specifically, the administration of catalytically active sPLA₂-X during the sensitization phase caused the marked increase in the production of the type-2 cytokines IL-4 (A), IL-5 (B), and IL-13 (C) in the lung. (D-F) The production of cytokines that are not classically type-2 including IL-10 (D), IL-6 (E) and IL-2 (F) were also increased in mice that received catalytically active sPLA₂-X during the sensitization phase. The catalytically inactive mutant sPLA₂-X (H46Q sPLA₂-X) administered during the sensitization phase, also led to the subsequent increase in IL-6 following OVA restimulation (E, P =0.04). The data are represented as the mean +/− SEM. There were 4 lung samples that were incubated with saline from each of the groups and 5–6 lung samples from each of the groups that were incubated with OVA; P values represent a 2-way ANOVA with correction for multiple comparisons.

To further evaluate ex vivo cytokine production in immune cell populations, we stimulated splenocytes from these mice with OVA and analyzed cytokine production as with lung leukocytes above. Compared to lung leukocytes, production of cytokines by splenocytes in response to restimulation with OVA was attenuated, with no differences observed in IL-13, IL-10, IL-6 or IL-2 based on the type of sensitization received (Supplemental Fig. 3A-F); However, OVA restimulated splenocytes from mice sensitized with OVA plus sPLA₂-X produced significantly more IL-5 than splenocytes from mice sensitized with OVA plus the inactive H46Q sPLA₂-X or with saline control and there was a trend towards more IL-4 from splenocytes from mice sensitized with WT sPLA₂-X relative to inactive H46Q sPLA₂-X (Supplemental Figs. 3A&B). There were no differences in the levels of TNF or KC with either WT sPLA₂-X or H46Q sPLA₂-X. These findings suggest that sPLA₂-X activity is essential for the type-2 skewed adaptive immune response to antigen and that these responses are markedly accentuated in the lung following 3 days of antigen rechallenge in the airways.

To assess the potential for sPLA₂-X adjuvant activity to elicit an adaptive immune response, we measured OVA-specific IgE and IgG using the same method as for the bvPLA₂ experiments. Both OVA-IgE and OVA-IgG were significantly elevated in the serum of mice sensitized with WT sPLA₂-X, a response that was completely abrogated in mice sensitized with the H46Q mutant form of sPLA₂-X (Figs. 6A&B). The OVA-specific IgE and IgG did not differ between the saline controls and those sensitized in the presence of H46Q mutant sPLA₂-X. It should also be noted that the absolute levels of the antigen-specific immunoglobins were substantially higher than in the studies with bvPLA₂. These data show that the adjuvant properties of sPLA₂-X that drive activation of both the innate and adaptive immune responses are dependent on the enzymatic activity of sPLA₂-X.

FIGURE 6.

The production of antigen-specific antibodies is increased when catalytically active sPLA₂-X was administered during the sensitization phase and is dependent upon the catalytic activity of the enzyme. OVA-specific IgE in serum following sensitization and challenge was significantly increased when catalytically active sPLA₂-X was administered during the sensitization phase, but not when catalytically inactive H46Q mutant sPLA₂-X was administered during the sensitization phase. (B) Catalytically active sPLA₂-X during the sensitization phase increased the levels of OVA-specific IgG in this model. The data are represented as the mean +/− SEM. There were 7 samples in each group for the OVA-specific IgE and 5–7 samples in each group for the OVA-specific IgG based on the availability of serum; the reported P values were corrected for multiple comparisons.

Enzymatic activity of sPLA₂-X is not necessary for the leukocyte influx into the peritoneum following intraperitoneal administration.

Prior work using bvPLA₂ demonstrated dependence upon ILC2s during the sensitization process and also identified release of IL-33 in response to this enzyme both in vivo and ex vivo (16). Previously, we found that mice lacking sPLA₂-X/Pla2g10 have lower levels of IL-33 in the airways following sensitization and challenge with HDM (15). To further characterize this response and examine the dependence upon enzymatic activity, we performed intraperitoneal (i.p.) injections of WT sPLA₂-X or saline on three consecutive days and evaluated the number and activation state of ILC2s in the peritoneum (Model, Fig. 7A). From the peritoneal lavage, we found a significant leukocyte influx induced by sPLA₂-X (Fig. 7B) that included a modest increase in ILC2s (Fig. 7C), but a more notable increase in the number of ILC2s that expressed both CD25 and the IL-33 transmembrane receptor ST2 (Fig. 7D) as well as the total number of ILC2s that expressed ST2 (Fig. 7E). We repeated this experiment with both the WT sPLA₂-X and the inactive H46Q sPLA₂-X and confirmed that the active enzyme induced a cellular influx into the peritoneum (P=0.03), but this cellular influx did not differ from that of the inactive enzyme (P = 0.74). Similarly, the number of viable CD45+ leukocytes did not differ between the active and inactive enzymes by flow cytometry (Fig. 7F) and there were similar increases in ILC2s expressing CD25 and ST2 (Fig. 7G) as well as the total number of ILC2s that expressed ST2 (Fig. 7H) as we had seen with the experiments using saline or the active enzyme and the enzymatic activity did not appear essential for this response. We also measured the level of IL-33 in the peritoneum following sequential injections of both the WT and H46Q sPLA₂-X and found that the levels of IL-33 were below the level of detection of the assay. We also assess the numbers of cells in the BAL fluid and the lung following multiple i.p. injections of the WT sPLA₂-X enzyme without challenging the airways with antigen and found no evidence of a cellular influx in either the lung or BAL fluid, including no notable differences in the cellular differential by flow cytometry (data not shown). These results suggest that enzymatic activity of sPLA₂-X is not necessary for the generation of a cellular influx in the peritoneum or for activation of ILC2s, suggesting that receptor mediated effects may be involved.

FIGURE 7.

Catalytically active sPLA₂-X and catalytically inactive H46Q mutant sPLA₂-X caused a cellular influx including activated ILC2s in the peritoneum. (A) Saline, active sPLA₂-X or catalytically inactive H46Q mutant sPLA₂-X were administered in the peritoneum (i.p.) on 3 subsequent days follow by peritoneal lavage (PL). (B-E) Flow cytometry comparing saline to wild type (WT) sPLA₂-X revealed an increase in the total number of CD45+ leukocytes (B), including an increase in the number of Lin-CD90.2+ type-2 innate lymphoid cells (ILC2)s (C), particularly ILC2s expressing both CD25 and ST2 (D), and total ILC2s expressing ST2 (E). (F-H) Further experimentation including the inactive H46Q mutant sPLA₂-X identified an increase in total CD45+ leukocytes that occurred in the absence of enzymatic activity (F), and similar trends for the number of ILC2s expressing both CD25 and ST2 (G), and total ILC2s expressing ST2 (H). There were 2 experimental replicates with n=2 saline and n=3 WT sPLA₂-X treated mice per group in the first experiment (B-E) and one experimental group with n=3 treated mice per group comparing saline, WT sPLA₂-X and H46Q mutant sPLA₂-X (F-H). The data are represented as the mean +/− SEM; P values represent either an unpaired t test (B-E) or a 2-way ANOVA(F-H).

Discussion

In the present study we used two different small extracellular PLA₂s to examine the potential adjuvant effects of PLA₂ enzymes focusing on the possibility that these enzymes serve to facilitate the development of airway inflammation and AHR following the subsequent inhalation of an allergen that itself does not have significant intrinsic adjuvant activity. This work is particularly relevant to human asthma as our lab has identified a specific endogenous sPLA₂ (sPLA₂-X) that is increased in human asthma (11–13, 15) and plays a central role in both the innate and adaptive immune response in mouse models of asthma (14, 15, 24). We used bvPLA₂ initially because a recent study demonstrated that in the periphery, bvPLA₂ serves as an effective adjuvant leading to an antigen specific immune response in the hind leg (16). We extend these findings here to examine whether such peripheral sensitization leads to an immune response relevant to asthma in the lung when the antigen is subsequently inhaled. We found that both bvPLA₂ as well as a recombinant version of endogenous sPLA₂-X serve as effective adjuvants in the periphery leading to a type-2 skewed immune response in the lung after re-exposure to antigen in the lung, although the severity of the immune response and AHR appeared to be greater when sPLA₂-X was used as the adjuvant. Through the use of a recombinant mutant sPLA₂-X enzyme with a targeted substitution in the active site that renders it catalytically inactive (17), we demonstrated that the adaptive immune response to the antigen in the lung, including the antigen-specific generation of cytokines by lung leukocytes and allergen-specific IgE were dependent upon the enzymatic activity of the sPLA₂, while features of AHR as well as some aspects of leukocyte trafficking to the lung were not dependent upon the enzymatic activity and are more likely a consequence of receptor-mediated actions. Further, the generation of ILC2s expressing markers of activation in the peritoneum could be induced by either the active or inactive enzyme. Another possibility is that residual endotoxin in the reagents led to the non-enzymatic features of inflammation, but this is unlikely given the low levels of endotoxin in the reagents.

Both bvPLA₂ and sPLA₂-X cleave the sn2 position of phospholipids derived from cellular membranes and other sources such as surfactant phospholipids leading to the release of FFAs and LysoPLs. The sn2 position is particularly important because it is preferentially substituted with PUFAs including AA that lead to the generation of eicosanoids. Through the use of an inactive mutant sPLA₂-X that maintains the secondary enzymatic structure of the enzyme, but is devoid of PLA₂ activity, we demonstrated with certainty that the adaptive immune responses facilitated by the sPLA₂-X enzyme was dependent upon the catalytic activity of the enzyme. We found that although AHR was not entirely dependent upon the enzymatic activity, the generation of an adaptive immune response to inhaled antigen was dependent upon enzymatic activity during the sensitization phase, leading to the recruitment of leukocytes to the lung that responded to antigen in an allergen-specific manner with the generation of key cytokines by lung leukocytes. Additionally, sensitization in the presence of either bvPLA₂ or WT sPLA₂-X caused the same features of the adaptive immune response including the allergen-specific generation of cytokines by lung leukocytes and allergen-specific IgE, although it is notable that the intensity of the immune response was greater for sPLA₂-X, particularly the higher levels of antigen-induced IL-13 in lung leukocytes and allergen-specific immunoglobins. It is also notable that while there was a clear signal that the immune response was skewed towards a type-2 response, there were also allergen-induced generation of cytokines that are not classically type-2 including IL-2, IL-10, and IL-6 for both sPLA₂s, as well as an increase in both TNF and KC following the administration of bvPLA₂ during the sensitization phase. It should also be noted that the antigen-induced levels of IL-5 were particularly elevated in both models and antigen-induced increases in splenocytes could be seen following sensitization in the presence of sPLA₂-X. Lung leukocytes were thus primed in this context to generate many different cytokines in an allergen-specific manner since OVA elicits very little immune response in the absence of adjuvant-driven sensitization. Our data also demonstrates that while sensitization leads to antigen-induced production of IL-5 in the lung and spleen and was dependent upon active sPLA₂-X, the antigen-specific generation of IL-13 only occurred in the lung and was dependent upon the enzymatic activity of sPLA₂-X.

Our results demonstrate a significant dissociation between tissue leukocyte infiltration and leukocytes in the BAL fluid that tend to track closely with peri-airway inflammation in our prior studies (15, 19). The changes in BAL leukocyte trafficking were also closely associated with the adaptive immune response that is dependent upon enzymatic activity of the sPLA₂. These results indicate that the response to a simple antigen such as OVA in the lung requires the generation of allergen-specific IgE that was facilitated by the enzymatic activity of either bvPLA₂ or sPLA₂-X leading to an airway centered immune response that is very similar to the findings in human asthma, with less of a difference in tissue infiltration with leukocytes. Because enzymatic activity was necessary for OVA-specific IgE, these findings are consistent with recent work revealing that IgE binding to the low affinity IgE receptor CD23 on the epithelium is necessary for efficient epithelial transcytosis of antigen (27). Another possibility is that efficient generation of high-affinity IgE mediated through specialized T cell subsets (28) is facilitated by a product of sPLA₂ activity in the periphery, altering the subsequent response in the airways through cells bearing the high-affinity IgE receptor (FcεRI).

These results are in contrast to the inhalation of complex antigens such as HDM that can induce features of airways disease in the absence of IgE (29), and specifically that C-type lectin binding of HDM proteins to dectin-2 during the effector phase is independent of IgE (30). We found here that the generation of antigen-specific IgG is dependent on the enzymatic activity of sPLA₂-X. Since we demonstrated previously that in the lung sPLA₂-X/Pla2g10 expression and protein levels are elevated in response to inhaled HDM allergen, and that the generation of antigen-specific IgG is dependent on sPLA₂-X/Pla2g10, these results suggest that the induction of sPLA₂-X provides a mechanism for sensitization endogenous antigens such as HDM that may be dependent upon CysLTs (31, 32) and also the ability of complete antigens to assist in the sensitization to “incomplete” allergens such as OVA that require the presence of an adjuvant for effective allergenicity.

Although prior work with bvPLA₂ indicates that sensitization in the periphery is dependent upon IL-33 signaling through the ST2 receptor, our results indicate that the allergen-specific generation of IL-13 that occurs in the lung, but not in the spleen is dependent upon the enzymatic activity of sPLA₂-X, although the induction of activated ILC2s expressing the ST2 receptor does not require this enzymatic activity. These results suggest that there are several PLA₂-mediated processes that lead to the subsequent response to inhaled allergen and that multiple different epithelial/structural cell-derived cytokines fully regulate type-2 immunity (33). Prior studies have identified PLA₂ products as important elements of the sensitization process relevant to the lung, particularly the sensitization to HDM that occurs in the absence of adjuvant, but is dependent upon the endogenous generation of CysLTs by C-type lectin binding of HDM proteins to dectin-2 in dendritic cells (32) and that sensitization is dependent upon the CysLT1 receptor (CysLT1R) in this context (31). For human sPLA₂-X, we have demonstrated that this enzyme serves as regulator of the generation of CysLTs by human eosinophils (34, 35), and in mice the deletion of sPLA₂-X/Pla2g10 causes a marked reduction in the elevated levels of CysLTs found in murine models of asthma (14, 15). In addition to the generation of eicosanoids, PLA₂s also release other FFAs and LysoPLs. As inhaled proteolytic allergens leads to the generation of IL-33 (15, 36), and the deletion of Pla2g10 in this context leads to a marked reduction in IL-33 (15), these results suggest that signals in addition to CysLTs are involved. In fact, recent work demonstrates that FFAs act along with IL-33 to regulate the activation of ILC2s through FFA-receptor-1 (37). Thus, while sensitization in the periphery was dependent on signaling through ST2, leading to a subsequent and allergen specific T-cell response (16), the subsequent response in the lung appears more complex and requires additional PLA₂ related signals, particularly catalytic activity of the enzyme for the adaptive immune response and other factors that are not dependent upon the catalytic activity that serve to regulate the development of AHR.

Although both enzymes used in this study have PLA₂ enzymatic activity, bvPLA₂ enzymatically is most like the mammalian group III sPLA₂ (sPLA₂-III) enzyme; however, the secondary structures of bvPLA₂ and sPLA₂-III are divergent (26). In our study, we found that the adaptive immune response to inhaled allergen could be facilitated by either enzyme, but there were differences as either active or inactive sPLA₂-X caused a degree of lung leukocyte infiltration, while bvPLA₂ caused much less of an alteration in lung leukocytes. With regard to sPLA₂-X, the murine enzyme binds with high affinity to the “M-type” PLA₂ receptor Pla2r1, which is a paralog of the mannose receptor (CD206) and has reported signaling functions as well as serving as a soluble receptor that serves to decrease the levels of several different sPLA₂ (19, 25, 38). Functions attributed to Pla2r1 include cell proliferation (39), vascular smooth muscle contraction (40) and activation of the mitogen activated kinase (MAPK) cascade (41). We found that deletion of the Pla2r1 gene leads to higher basal levels of sPLA₂-X, heightened AHR and increased levels of cytokines produced by lung leukocytes both with and without antigen stimulation, suggesting that Pla2r1 is functioning in the context of the OVA/alum model primarily to reduce the levels of specific sPLA₂s (19). In contrast, bvPLA₂ does not bind to Pla2r1 but binds to the “N-type” PLA₂ receptor which may be responsible for neurotoxicity (25, 42, 43) and also binds to the mannose receptor CD206 (44). The possibility that bvPLA₂ binds directly to the IL-33 receptor ST2 has been raised (26) based on the effects of bvPLA₂ in the periphery that are mediated through ST2 (16), but there has been no direct evidence of binding to this receptor.

In regard to the potential non-enzymatic functions of sPLA₂-X, we demonstrated that either active sPLA₂-X or the inactive H46Q mutant sPLA₂-X lead to both a cellular influx as well as the activation of ILC2s in the peritoneum. The generation of ILC2s and release of IL-33 in the peritoneum was previously demonstrated for bvPLA₂, but the dependence on enzymatic activity was not ascertained (16). As the global deletion of sPLA₂-X/Pla2g10 causes a reduction in both IL-33 and ILC2s in the lung (15), we examined the administration of sPLA₂-X on 3 subsequent days in the peritoneum. In contrast to the direct administration of IL-33 in the peritoneum (45), we did not observe any increase in lung leukocytes early after the administration of sPLA₂-X in the peritoneum. Thus, the majority of our results indicate that an enzymatic product is necessary for both the increase in antigen specific generation of IL-5 in the spleen and antigen specific generation of IL-4, IL-5, and IL-13 by lung leukocytes; however, sensitization with the inactive enzyme leads to the local generation of ILC2s in the peritoneum and subsequently a modest increase in lung eosinophil infiltration and features of AHR through a mechanism that is not dependent upon the catalytic activity of the enzyme.

Although bvPLA₂ was sufficient to act as an effective adjuvant leading to sensitization and an allergen specific immune response in the lung, it is known that bvPLA₂ also has suppressive effects when administered on a regular basis around the time of sensitization in the OVA/alum model, and that this effect is mediated through the mannose receptor CD206, leading to regulatory T-cell dependent suppression of OVA-induced airway dysfunction (46, 47). Remarkably, features of airway inflammation and AHR in the OVA/alum model were suppressed by the intratracheal administration of bvPLA₂ given 3 days subsequent to the sensitization doses of OVA/alum (48). Thus, bvPLA₂ is sufficient to induce an allergen specific immune response in the periphery (16), which leads to a subsequent airway-centered adaptive immune response in the lung, as we have shown here, while at the same time bvPLA₂ has a suppressive effect on inflammation acting directly through receptor binding to the mannose receptor CD206 (47). These considerations could explain the relatively attenuated immune response with bvPLA₂ relative to sPLA₂-X, or simply other differences in the adjuvant properties of the enzymes. The suppressive effects of bvPLA₂ are most well-established for the anaphylactic response to bvPLA₂, where the generation of IgE antibodies leads to suppression of the anaphylactic response to bee venom (16, 49). Our results would suggest that this direct antibody-mediated protection is most likely due to the enzymatic function of bvPLA₂, while more general suppressive functions may be receptor-mediated. Transgenic mice expressing the murine sPLA₂-III/Pla2g3 which closely mirrors the enzymatic but not receptor binding function of bvPLA₂ spontaneously develop multiple features of inflammation including skin inflammation and lymphoid aggregates in the airway as well as mucus hypersecretion in the airway (50), suggesting as we have found here that the catalytic activity of bvPLA₂ facilitates an antigen-specific response in the lung. Earlier work demonstrated that bvPLA₂ causes the generation of leukotrienes from peripheral blood basophils (51) as we have demonstrated for human sPLA₂-X acting on eosinophils (34, 35). To our knowledge, no suppressive functions have been directly attributed to sPLA₂-X through receptor-mediated functions; however, we have provided evidence that the receptor that it binds (Pla2r1) has suppressive functions primarily through binding and removing sPLA₂-X (19).

Our results indicate that the endogenous murine sPLA₂-X enzyme plays a role in the sensitization phase of allergen-induced airway immunopathology. Sensitization with either this enzyme or bvPLA₂ was sufficient to induce an adaptive immune response in the lung upon the subsequent inhalation of allergen. These results indicate that both sPLA₂s have the capacity to act as an adjuvant relevant to the development of immunopathology in the lung. The adaptive immune response and the antigen-induced generation of IL-13 in the lung was dependent on the catalytic activity of the enzyme, but the results also suggest receptor-mediated effects can alter some aspects of the innate immune response and AHR. As endogenous sPLA₂-X is increased in human asthma, can be upregulated at mucosal surfaces and is upregulated in the skin during inflammatory responses (52), these results have important implications for the underlying etiology of allergen-induced asthma.

Key Points.

1. sPLA₂-X serves as an effective adjuvant for a T2 immune response in the lungs.

2. The adaptive immune response requires the enzymatic activity of sPLA₂-X.

3. The innate immune response is not fully dependent upon sPLA₂-X enzymatic activity.