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Secreted PLA2 group X orchestrates innate and adaptive immune responses to inhaled allergen - PubMed

  • ️Sun Jan 01 2017

Secreted PLA2 group X orchestrates innate and adaptive immune responses to inhaled allergen

James D Nolin et al. JCI Insight. 2017.

Abstract

Phospholipase A2 (PLA2) enzymes regulate the formation of eicosanoids and lysophospholipids that contribute to allergic airway inflammation. Secreted PLA2 group X (sPLA2-X) was recently found to be increased in the airways of asthmatics and is highly expressed in airway epithelial cells and macrophages. In the current study, we show that allergen exposure increases sPLA2-X in humans and in mice, and that global deletion of Pla2g10 results in a marked reduction in airway hyperresponsiveness (AHR), eosinophil and T cell trafficking to the airways, airway occlusion, generation of type-2 cytokines by antigen-stimulated leukocytes, and antigen-specific immunoglobulins. Further, we found that Pla2g10-/- mice had reduced IL-33 levels in BALF, fewer type-2 innate lymphoid cells (ILC2s) in the lung, less IL-33-induced IL-13 expression in mast cells, and a marked reduction in both the number of newly recruited macrophages and the M2 polarization of these macrophages in the lung. These results indicate that sPLA2-X serves as a central regulator of both innate and adaptive immune response to proteolytic allergen.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Proteolytic allergens increase sPLA2-X in human and mouse airways.

(A) Four individuals with asthma were challenged via the airways with aerosolized allergen, and levels of sPLA2-X protein were measured using time-resolved fluorescence immunoassay (TRFIA) in induced sputum at 7 hours and 24 hours after challenge. Mean ± SEM (n = 4/time point) with 1-way ANOVA. BL, baseline. (B) Whole lung Pla2g10 gene expression from WT and Pla2g10–/– (–/–) mice exposed to saline or HDM (n = 3/group). Mean ± SEM, Mann-Whitney test. (C) BALF sPLA2-X protein levels from WT and Pla2g10–/– mice exposed to HDM were analyzed via Western blot (20 μl/lane) and quantified using densitometry (n = 4 for saline [Sal] and 7 for HDM). Mean ± SEM, Mann-Whitney test. A.U., arbitrary units.

Figure 2
Figure 2. Pla2g10–/– mice are protected from developing airway hyperresponsiveness (AHR) and aberrant lung inflammation.

(A) HDM sensitization and challenge model used to induce allergic airway disease. Animals were instilled with 100 μg HDM (protein) on day 0, and then 25 μg HDM (protein) on days 7–11 and 14–18. On day 21, airway mechanics were assessed and mice were sacrificed. o.p., oropharyngeal administration. (B) Cells in the BALF following HDM sensitization and challenge were isolated and counted using an automated cell counter (n = 3/group for Sal, 7 for WT HDM, and 8 for Pla2g10–/– HDM). Mean ± SEM. (C) Single cell suspensions of lung leukocytes following HDM sensitization and challenge were isolated from digested lung tissue and counted using an automated cell counter (n = 3/group for Sal, 7 for WT HDM, and 8 for Pla2g10–/– HDM). Mean ± SEM. (D and E) Measurement of AHR to increasing doses of methacholine following HDM sensitization and challenge using the forced oscillation technique to measure lung mechanics (n = 3/group for Sal, 10 for WT HDM, and 12 for Pla2g10–/– HDM). Mean ± SEM. Statistical significance was determined by 2-way ANOVA with uncorrected Fisher’s LSD.

Figure 3
Figure 3. Leukocyte influx into the airways is decreased in Pla2g10–/– mice.

(A–D) Representative images of lung tissue H&E staining in WT and Pla2g10–/– mice exposed to either saline or HDM as outlined in scheme in Figure 2A. Original magnification, 20×; scale bar: 50 μm. (E and F) Concentration of eosinophils and T cells in (E) BALF and (F) single cell lung suspension following sensitization and challenge with saline or HDM (n = 3/group for Sal, 7 for WT HDM, and 8 for Pla2g10–/– HDM). Cell differentials were determined by multicolor flow cytometry and were defined by the following surface markers: Eosinophils (CD45+CD3–MHCIIloCD11c–SiglecF+), T cells (CD45+CD3+). Statistical significance was determined by 2-way ANOVA with uncorrected Fisher’s LSD.

Figure 4
Figure 4. Peribronchiolar eosinophilic inflammation is decreased in mice lacking Pla2g10.

(A–D) Representative images of eosinophil major basic protein (MBP) immunostaining in lungs from saline- and HDM-exposed WT and Pla2g10–/– mice. Original magnification, 20×; scale bar: 50 μm. (E) Total number of peribronchial eosinophils and (F) percentage of peribronchial MBP immunostaining per total lung tissue quantified using Visiopharm analysis software (n = 3 mice/group). (G) Western blotting and densitometric analysis of MBP in BALF (20 μl each sample) from saline- and HDM-exposed WT and Pla2g10–/– mice (n = 4/group for Sal and 7/group for HDM). Statistical significance was determined by 2-way ANOVA with uncorrected Fisher’s LSD.

Figure 5
Figure 5. Epithelial mucus production in HDM-exposed Pla2g10–/– mice is dampened compared with WT mice.

(A) Representative images of PAS staining from a WT mouse exposed to saline or HDM. Original magnification, 20x; scale bar: 50 μm. (B) Percentage of occluded airways in saline- and HDM-exposed mice, quantified based on the extent of PAS-positive staining in the airway lumen (n = 3/group for Sal and 4/group for HDM). Mean ± SEM, 2-way ANOVA with uncorrected Fisher’s LSD. (C) Individual airways were assessed to determine the ratio of epithelial area that stained positive for PAS to the total epithelial area as determined by Visiopharm analysis in HDM-exposed WT and Pla2g10–/– mice (–/–, Pla2g10–/–; n = 122 airways for WT and 149 airways for –/–). Mean ± SEM, unpaired t test.

Figure 6
Figure 6. Lack of Pla2g10 alters cytokine and CysLT production.

Concentration of (A) IL-4, (B) IL-5, (C) IL-13, and (D) IL-6 from supernatant of cultured WT or Pla2g10–/– (–/–) lung leukocytes isolated from saline or HDM sensitized mice (Sens.) and stimulated with either saline or HDM. Only IL-13 was significantly lower in Pla2g10–/– mice (n = 6–8 mice/group). Mean ± SEM, 2-way ANOVA with uncorrected Fisher’s LSD. (E) CysLT levels measured in the BALF from HDM sensitized and challenged mice (n = 3 for Sal, 9 for WT HDM, and 11 for Pla2g10–/– HDM). Mean ± SEM, 2-way ANOVA with uncorrected Fisher’s LSD. (F) IL-33 release into the BALF following sensitization and challenge with HDM (n = 5 mice/group). Mean ± SEM, unpaired t test.

Figure 7
Figure 7. ILC2 polarization is impaired in mice lacking Pla2g10.

Gating was performed to isolate lineage negative (Lin–), CD45+ leukocytes (Lin–CD45+, see Supplemental Figure 3). Further gating on the Lin–CD45+ leukocyte population was performed to isolate (A) ST2+ cells and (B) ST2+CD127+ cells from WT and Pla2g10–/– mice exposed to saline or HDM. (C) Additional analysis revealed no significant differences in ST2+CD127+Sca1+ cells between WT and Pla2g10–/– mice (n = 3 mice/group). Mean ± SEM, 2-way ANOVA with uncorrected Fisher’s LSD.

Figure 8
Figure 8. Lack of Pla2g10 alters mast cell activation.

(A) Lung tissue mast cells were visualized and enumerated using toluidine blue staining in mice exposed to saline or HDM (n = 6 mice for Sal and 8 mice for HDM). Mean ± SEM, Mann-Whitney test. (B) The expression of Il13 in WT BMMCs was evaluated in response to 4-hour stimulation with IL-33 (10 ng/ml), IL-13 (10 ng/ml), or HDM (250 μg/ml) (n = 4/group for Sal, IL-33, and IL-13 and 2 for HDM). (C) WT and Pla2g10–/– BMMC lysates were analyzed for sPLA2-X protein content via Western blot. WT and Pla2g10–/– BMMCs were stimulated with IL-33, IL-25, and TSLP (all at 10 ng/ml), and expression of Il13 was evaluated (n = 6–8/group for Sal and IL-33, 2–4/group for IL-25 and TSLP). Mean ± SEM, 1-way ANOVA with uncorrected Fisher’s LSD.

Figure 9
Figure 9. Macrophage polarization and activation are influenced by Pla2g10 status.

(A) Concentrations of resident macrophages in lung tissue from WT and Pla2g10–/– mice were similar after saline and HDM challenge. The surface expression of (B) CD206/MR and (C) CD71/TfR on these resident macrophages was assessed to measure the extent of M2 polarization. (D) HDM-induced recruited macrophages in lung tissue were also evaluated by multicolor flow. In this recruited macrophage population, the surface expression of (E) CD206/MR and (F) CD71/TfR was also evaluated to determine M2 polarization. n = 4–5 mice/group. Mean ± SEM, 2-way ANOVA with uncorrected Fisher’s LSD.

Figure 10
Figure 10. Pla2g10–/– mice display an impaired adaptive immune response to HDM.

The serum levels of (A) total IgE and (B) DerP1-specific IgG were assessed by ELISA (n = 3–5 mice/group for Sal and 7–10 mice/group for HDM). A.U., arbitrary units. Mean ± SEM, 2-way ANOVA with uncorrected Fisher’s LSD.

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