UDP-glucose and P2Y14 receptor amplify allergen-induced airway eosinophilia

Abstract

Airway eosinophilia is a hallmark of allergic asthma and is associated with mucus production, airway hyperresponsiveness, and shortness of breath. Although glucocorticoids are widely used to treat asthma, their prolonged use is associated with several side effects. Furthermore, many individuals with eosinophilic asthma are resistant to glucocorticoid treatment, and they have an unmet need for novel therapies. Here, we show that UDP-glucose (UDP-G), a nucleotide sugar, is selectively released into the airways of allergen-sensitized mice upon their subsequent challenge with that same allergen. Mice lacking P2Y14R, the receptor for UDP-G, had decreased airway eosinophilia and airway hyperresponsiveness compared with wild-type mice in a protease-mediated model of asthma. P2Y14R was dispensable for allergic sensitization and for the production of type 2 cytokines in the lung after challenge. However, UDP-G increased chemokinesis in eosinophils and enhanced their response to the eosinophil chemoattractant, CCL24. In turn, eosinophils triggered the release of UDP-G into the airway, thereby amplifying eosinophilic recruitment. This positive feedback loop was sensitive to therapeutic intervention, as a small molecule antagonist of P2Y14R inhibited airway eosinophilia. These findings thus reveal a pathway that can be therapeutically targeted to treat asthma exacerbations and glucocorticoid-resistant forms of this disease.

Keywords: Asthma; Inflammation.

Figure 1. P2ry14 is required for airway eosinophilia and AHR in protease-mediated models of asthma.

(A) Timeline showing allergic sensitization, challenge, and harvest of C57BL/6J wild-type and genetically matched P2ry14^–/– (KO) mice in LPS- and protease-mediated (ASP and PAP) models of asthma. (B) Airway inflammation at 48 hours after allergen challenge in wild-type mice (gray rectangles) and P2ry14^–/– mice (white rectangles) previously sensitized using the indicated adjuvant. Shown are mean values ± SEM (n = 12–17 per group, except for nontreated or OVA-only-treated groups, where n = 3). (C) Airway resistance (R) measurements in OVA-challenged mice previously sensitized using OVA only, ASP/OVA, or LPS/OVA. Mean (± SEM) values are shown (n = 10) for baseline (B) and after administration of the indicated doses of methacholine. Results shown are from a single experiment, representative of 2. ND, not detectable. *P < 0.05; **P < 0.01; ****P < 0.0001; NS, not significant; analyzed using 1-way ANOVA followed by Bonferroni’s post hoc test. (D) Concentrations of IL-5 and IL-13 in BALF after OVA challenge in ASP/OVA asthma model. (E) Concentrations of OVA-specific IgE in serum. Means ± SEM are shown for naive mice, and for OVA with and without ASP-sensitized and OVA-challenged animals. (F) Representative Alcian blue– and periodic acid–Schiff–stained sections showing mucus-producing cells. Sections were prepared from lungs of C57BL/6J wild-type and genetically matched P2ry14^–/– mice (left). Scale bar: 200 μm. Also shown are compiled data for mean numbers ± SEM of mucus-producing cells in individual lung slices from the indicated groups.

Figure 2. P2ry14 is dispensable for allergic sensitization through the airway.

(A) Timeline for injection of OVA-specific (OT-II) T cells into WT or P2ry14-deficient (KO) mice, followed by ASP/OVA sensitization and harvest of mLNs. (B) Concentrations of the indicated cytokines in culture supernatants of mLNs harvested from mice treated with OVA alone or OVA/ASP. (C) Timeline for PPTN treatments, 2 OVA/ASP sensitizations, and a single OVA aerosol challenge. (D) Effect of PPTN treatments during allergic sensitization on cell numbers of the indicated leukocyte subsets following OVA challenge. Data shown represent mean values ± SEM.

Figure 3. P2Y₁₄R blockade during allergen challenge diminishes eosinophilic airway inflammation.

(A) Timeline for ASP/OVA sensitization, PPTN treatment, and OVA challenge. (B) Effect of PPTN administration during challenge on airway inflammation in OVA-challenged mice previously sensitized with ASP/OVA. Data shown represent mean values ± SEM. **P < 0.01; ***P = 0.0003 by 2-tailed Student’s t test.

Figure 4. Expression of P2ry14 in lung eosinophils is essential for eosinophilic airway inflammation.

(A) Cell numbers for the indicated leukocyte types in airways of reciprocal bone marrow–chimeric (BM-chimeric) mice generated using WT and P2ry14^–/– animals and subjected to the ASP/OVA model of asthma. (B and C) Expression of P2ry14 in the indicated populations of leukocytes and stromal cells from lungs of C57BL/6J mice (B), and in eosinophils during their maturation and migration to the periphery (C). (D) Generation of mixed BM chimeras by injecting equal numbers of BM cells from WT (CD45.1⁺) and P2ry14-null (CD45.2⁺) mice into irradiated Cd45.1/Cd45.2 heterozygous recipients. Dot-plot cytograms show representative flow cytometry results for CD45.1 and CD45.2 staining of BM (left) and lung cells (right) of nonchallenged mice, indicating similar reconstitution of leukocyte populations of each donor genotype. (E) Ratio of wild-type (CD45.1⁺) to P2ry14^–/– (CD45.2⁺) eosinophils and neutrophils in the BM and lung parenchyma of naive mice, and in the airspace of OVA-challenged mice previously sensitized with ASP/OVA. (F) Allergic airway inflammation in mice with conditional P2ry14 disruption in LyzM-expressing myeloid cells (left), and disruption in Cd11c-expressing alveolar macrophages and dendritic cells (right). (G) Allergic airway inflammation in mice with P2ry14 disruption in eosinophils. Data shown represent mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Data were analyzed using 2-tailed Student’s t test for pairwise comparisons, or 1-way ANOVA followed by Tukey’s post hoc test for multiple comparisons.

Figure 5. UDP-glucose amplifies eosinophil recruitment to the airway.

(A) Mass spectrometry measurements of UDP-hexoses in BALF 8 hours after OVA challenge of mice previously sensitized using ASP/OVA or LPS/OVA. Data represent sample readouts relative to the internal standard (ITSD), ¹³C₆-UDP-glucose (included in the fluid used for lavage). (B and C) Time course for airway levels of UDP-hexoses (B), and for eosinophil numbers in the indicated locations (C). Analyses were performed on C57BL/6J mice sensitized twice with OVA/ASP, challenged once with OVA, and harvested at the indicated times. Data shown represent means ± SEM (n = 3–5) from a single experiment. (D) Air space, blood, and marginated eosinophils in ASP/OVA-sensitized mice at 16 hours after OVA challenge. (E) Airway levels of CCL11 and CCL24 in ASP/OVA-sensitized mice, 4 hours after OVA challenge. (F) MFI of CCR3 on lung eosinophils. (G) Schematic representation of in vitro cell migration assay. (H) Migration of WT and P2ry14^–/– eosinophils in response to the indicated agents added to the top and/or bottom chambers. U, UDP-glucose. (I) UDP-hexose levels in BALF after adoptive transfer of the indicated cells into the airways. (J) Effect of anti-CCR3 antibody on lung eosinophils. (K) Effect of anti-CCR3 antibody on UDP-hexoses in BALF. IC, isotype control antibody. Data shown represent mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001. Data were analyzed using 2-tailed Student’s t test for pairwise comparisons or 1-way ANOVA followed by Tukey’s post hoc test for multiple comparisons.