Eosinophil Phenotypes Are Functionally Regulated by Resolvin D2 during Allergic Lung Inflammation

Abstract

Eosinophils (Eos) reside in multiple organs during homeostasis and respond rapidly to an inflammatory challenge. Although Eos share chemical staining properties, they also demonstrate phenotypic and functional plasticity that is not fully understood. Here, we used a murine model of allergic lung inflammation to characterize Eos subsets and determine their spatiotemporal and functional regulation during inflammation and its resolution in response to resolvin D2 (RvD2), a potent specialized proresolving mediator. Two Eos subsets were identified by CD101 expression with distinct anatomic localization and transcriptional signatures at baseline and during inflammation. CD101^low Eos were predominantly located in a lung vascular niche and responded to allergen challenge by moving into the lung interstitium. CD101^high Eos were predominantly located in bronchoalveolar lavage (BAL) and extravascular lung, only present during inflammation, and had transcriptional evidence for cell activation. RvD2 reduced total Eos numbers and changed their phenotype and activation by at least two distinct mechanisms: decreasing interleukin 5-dependent recruitment of CD101^low Eos and decreasing conversion of CD101^low Eos to CD101^high Eos. Collectively, these findings indicate that Eos are a heterogeneous pool of cells with distinct activation states and spatiotemporal regulation during resolution of inflammation and that RvD2 is a potent proresolving mediator for Eos recruitment and activation.

Keywords: allergic inflammation; eosinophils; resolvin D2; specialized proresolving mediators.

Figure 1.

Eosinophils (Eos) are spatiotemporally regulated in a self-limited model of allergic lung inflammation. (A) Allergic lung inflammation model (see Methods). (B) Time course of Eos numbers in whole lung. (C) Time course of Eos numbers in BAL (red) and lung tissue after BAL (black). (D) Representative flow cytometry plots of CD101^low and CD101^high Eos from lung at Day 17. Full gating strategy in Figure E2A. (E) Representative flow cytometry plots of CD101^low and CD101^high Eos from BAL and lung at Days 0 and 17. Full gating strategy in Figures E2A and E2B. (F) Representative flow cytometry plot of whole-lung CD101^low Eos at Day 0 after CD45-BV421 staining (see Methods), with quantification of CD45-BV421 positive or negative Eos on the right. (G) Representative flow cytometry plot of whole-lung CD101^low and CD101^high Eos at Day 17 after CD45-BV421, with quantification of CD45-BV421 positive or negative CD101^low and CD101^high Eos on the right. ^#P < 0.05 compared with Day 0. ^δP < 0.05 compared with Day 15. ^ΦP < 0.05 compared with Day 21. *P < 0.05 compared with BV421⁺ within same cell type (CD101^low and CD101^high). Comparisons between two groups were performed by unpaired t test. Bars represent mean ± SEM. HDM = house dust mite.

Figure 2.

CD101^low and CD101^high Eos have distinct transcriptional signatures. (A) Representative images of cytospin performed on flow-sorted CD101^low Eos from Days 0 and 17 and CD101^high Eos from Day 17 of house dust mite (HDM) protocol. Scale bars, 50 μm. (B and C) mRNA analysis of flow-sorted CD101^low and CD101^high Eos using NanoString (see Methods). Data are represented as a heatmap of differentially expressed genes (P < 0.05 by unpaired t test) comparing CD101^low Eos from Day 0 with Day 17 of HDM protocol (B) and comparing CD101^low Eos with CD101^high Eos at Day 17 of HDM protocol (C). (D–F) Concentrations of 14-hydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14-HDHA), 17-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17-HDHA), and resolvin D2 (RvD2) from flow-sorted CD101^low and CD101^high Eos stimulated with docosahexaenoic acid (DHA) by liquid chromatography–tandem mass spectrometry. (G) Level of DRV2 expression on CD101^low and CD101^high Eos at Day 17 of HDM protocol, as mean fluorescence intensity measured by flow cytometry. *P < 0.05 and **P < 0.01 by unpaired t test. Bars represent mean ± SEM.

Figure 3.

RvD2 regulates CD101^low and CD101^high Eos numbers during inflammation. (A) Animal model (see Methods). (B and C) Authentication of RvD2 (see Methods). (D–F) Eos numbers in whole lung (D), BAL (E), and lung after BAL (F). (G) Flow cytometry plots of CD101^low and CD101^high Eos from lung (top) and BAL (bottom) at Days 0 and 17. (H and I) Number of CD101^low (H) and CD101^high (I) Eos in lung and BAL at Days 0 and 17. (J) CD101^high/CD101^low Eos ratio in whole lungs at Day 17. (K and L) Number of CD101^low (K) and CD101^high (L) Eos in lung and BAL at Day 17. ^#P < 0.05, ^###P < 0.001, ^####P < 0.0001 compared with Day 0. ^δP < 0.05 compared with Day 15 in D–F. ^ΦP < 0.05 compared with Day 21 in D and E. ^ɵP < 0.05 comparing vehicle (Veh) with RvD2 within the same day in D and E. *P < 0.05, ***P < 0.001, ****P < 0.0001 compared with Day 17 within the same compartment lung or BAL in H–L. ^ΦΦΦP < 0.001 compared with Day 17 RvD2 in K and L. Comparisons between more than two groups were performed by one-way ANOVA and Tukey test for multiple comparisons (D–F and H, I, K, and L). Comparison between two groups was performed by unpaired t test (J). Bars represent mean ± SEM.

Figure 4.

RvD2 regulates CD101^low and CD101^high Eos gene signature and function during inflammation. (A and B) Volcano plot of RNA analysis using NanoString in CD101^low (A) and CD101^high (B) Eos. Fold change of differentially expressed genes (P < 0.05 by unpaired t test; red dots) comparing CD101^low (A) and CD101^high (B) Eos at Day 21 of RvD2 versus Veh. (C and D) Gene expression of Alox5 (C) and Alox15 (D) by RT-PCR. (E) Lipoxin A₄ (LXA₄) concentrations in BAL fluid at Day 17. (F) Prostaglandin D2 (PGD₂) concentrations in BAL fluid at Day 17. (G) CD11b expression on CD101^low and CD101^high Eos. (H) CRTH2 expression on CD101^low and CD101^high Eos. (I) Eos peroxidase (EPO) release from flow-sorted CD101^low and CD101^high Eos (see Methods). ^#P < 0.05 compared with Day 0 in D and F and CD101^low Day 17 in G and H. ^##P < 0.01 compared with CD101^low Day 17 in G. *P < 0.05 compared with Day 21 within CD101^high Eos in C and D and with CD101^high Day 17 in G. ^ϕP < 0.05 compared with Veh within CD101^high and ^δP < 0.05 compared with RvD2 within CD101^high in I. Comparisons were performed by one-way ANOVA and Tukey test for multiple comparisons. Bars represent mean ± SEM.

Figure 5.

RvD2 regulates IL-5–induced Eos responses. (A) IL-5 concentrations in BAL fluid by cytometric bead array. (B) Blood Eos numbers at Days 0 and 17. (C) CD125 expression. (D) Timeline of recombinant mouse IL-5 (rmIL-5) exposure; quantification of Eos in E–G. (E) Blood Eos numbers at Day 14 in D. (F and G) Lung and BAL CD101^low and CD101^high Eos, respectively. (H) Number of Eos in BAL at Day 15 of the HDM protocol (Figure 1A). (I) Timeline of lung cells ex vivo stimulation (see Methods). (J) Flow cytometry plots of CD101^low Eos after ex vivo stimulation. (K) Percentage of CD101^low and CD101^high Eos after ex vivo stimulation. ^ϕP < 0.05 and ^ϕϕP < 0.01 compared with Day 0 in A, B, and H. *P < 0.05 compared with Day 17 in A–C (within CD101^high). ^#P < 0.05 compared with CD101^low d17 in C. ^δP < 0.05 and ^δδδP < 0.001 compared with saline within the same cell type. ^θP < 0.05 compared with saline and ^εP < 0.05 and ^εεP < 0.01 compared with HDM and rmIL-5 in K. Comparisons between two groups were performed by unpaired t test (C and E–H). Comparisons between more than two groups were performed by one-way ANOVA and Tukey test for multiple comparisons (A, B, and K). Bars represent mean ± SEM.