12/15-Lipoxygenase Regulates IL-33-Induced Eosinophilic Airway Inflammation in Mice

Abstract

Dysregulated fatty acid metabolism is clinically associated with eosinophilic allergic diseases, including severe asthma and chronic rhinosinusitis. This study aimed to demonstrate the role of 12/15-lipoxygenase (12/15-LOX) in interleukin (IL)-33-induced eosinophilic airway inflammation; to this end, we used 12/15-LOX-deficient mice, which displayed augmented IL-33-induced lung inflammation, characterized by an increased number of infiltrated eosinophils and group 2 innate lymphoid cells (ILC2s) in the airway. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics revealed that the levels of a series of 12/15-LOX-derived metabolites were significantly decreased, and application of 14(S)-hydroxy docosahexaenoic acid (HDoHE), a major 12/15-LOX-derived product, suppressed IL-33-mediated eosinophilic inflammation in 12/15-LOX-deficient mice. Using bioactive lipid screening, we found that 14(S)-HDoHE and 10(S),17(S)-diHDoHE markedly attenuated ILC2 proliferation and cytokine production at micromolar concentration in vitro. In addition, maresin 1 (MaR1) and resolvin D1 (RvD1), 12/15-LOX-derived specialized proresolving mediators (SPMs), inhibited cytokine production of ILC2s at nanomolar concentration. These findings demonstrate the protective role of endogenous 12/15-LOX-derived lipid mediators in controlling ILC2-mediated eosinophilic airway inflammation and related diseases. Thus, 12/15-LOX-derived lipid mediators may represent a potential therapeutic strategy for ameliorating airway inflammation-associated conditions.

Keywords: 12/15-lipoxygenase; 14(S)-HDoHE; IL-33; docosahexaenoic acid; group 2 innate lymphoid cell; lipidomics; maresin; specialized proresolving mediator.

Figure 1

12/15-lipoxygenase deficiency augments IL-33-induced airway eosinophilic inflammation. Airway inflammation was induced by administration of IL-33 (500 ng per mouse, intranasal), for 3 consecutive days, in C57BL/6 and 12/15-lipoxygenase deficient mice. Analysis was performed 4 days after the final administration of IL-33. (A) Number of total cells, eosinophils, lymphocytes, and macrophages in BALF. (B) Number of ILC2 and Th2 cells in BALF by flow cytometric analysis. (C) Relative mRNA expression of cytokines (il5, il13, ccl11, ccl24, and ccl26) was determined by RT-PCR and quantitative real-time PCR analysis. Airway histology was assessed by (D) hematoxylin and eosin and (E) periodic acid–Schiff (PAS)-Alcan blue staining. The data are representative of three independent experiments. Mean ± SEM, n=4 for each group.

Figure 2

Lipidomic profiles of lungs during IL-33-induced airway eosinophilic inflammation. Airway inflammation was induced by administration of IL-33 (500 ng per mouse, intranasal), for 3 consecutive days, in C57BL/6 and 12/15-lipoxygenase (LOX)-deficient mice. Analysis was performed 1 day after the final administration of IL-33. (A) Lipidomic analysis showed quantitative alterations of arachidonic acid (AA) and docosahexaenoic acid (DHA)-derived metabolites via cyclooxygenase (COX), 5-LOX, and 12/15-LOX, including prostaglandins (PG), thromboxanes (Tx), 12- hydroxyheptadecatrienoic acid (HHT), hydroxyeicosatetraenoic acid (HETE), leukotriene B₄ (LTB₄), and hydroxy docosahexaenoic acid (HDoHE). (B) Comparative analysis of DHA-derived monohydroxy metabolites in wild-type or 12/15-LOX deficient mice. (C) Comparative analysis of the amounts of polyunsaturated fatty acids [AA, DHA, and eicosapentaenoic acid (EPA)] in wild-type or 12/15-LOX deficient mice. (D) Comparative analysis of 12/15-LOX-derived dihydroxy- or trihydroxy-lipid metabolites, including lipoxin (LX), Rv, MR, and protectin (PD). Mean ± SEM, n=3 for each group. **P < 0.01 (Student’s t-test). NS, not significant.

Figure 3

14(S)-HDoHE inhibit proliferation and cytokine production of ILC2s, with pro-apoptotic effects. ILC2 cells (10,000 cells per well) were cultured with IL-33 (10 ng/mL) for 1 or 4 days in the presence or absence of (A) DHA-derived monohydroxy-lipid metabolites (HDoHE, hydroxy docosahexaenoic acid: 10^-5 M) or (B–G) in the presence or absence of 14(S)-HDoHE (3 × 10^-7 – 10^-5 M: (B, C); or 10^-5 M: (D–G), (D) 12-hydroxyeicosatetraenoic acid (HETE) and 12-hydroxyeicosapentaenoic acid (HEPE). The total cell count (A, B) and concentrations of IL-5 (C, D) in the culture supernatant were measured. (E–G) Flow cytometric analysis of Annexin V- and PI-stained ILC2 cells cultured with or without IL-33 (10 ng/mL) in the presence or absence of 14(S)-HDoHE (10^-5 M) for 1 or 4 days. The data are representative of three independent experiments. Mean ± SEM, n=3-8 for each group. *P < 0.05, **P < 0.01 (Student’s t-test).

Figure 4

Effects of SPMs on proliferation and cytokine production of ILC2s. (A) ILC2 cells (10,000 cells per well) were cultured with IL-33 (10 ng/mL) for 4 days in the presence or absence of (A) dihydroxy- or trihydroxy-lipid metabolites (LX, lipoxin; Rv, resolvin; MR, maresin; PD, protectin D: 10^-5 M) or in the presence or absence of (B, C) 10(S),17(S)-diHDoHE (PDX, 3 × 10^-7 – 10^-5 M), (D, E) 17(S)-HDoHE-derived SPMs (RvD1, resolvin D1; RvD2, resolvin D2; PD1, protectin D1: 3 x 10^-7 nM), (F, G) RvD1 (1 × 10^-11 – 3 x 10^-7 M: (D, E), or (H, I) MaR1 (MaR1; maresin 1: 10^-11 – 10^-5 M). The total cell count (A, B, D, F, H) and concentrations of IL-5 (C, E, G, I) in the culture supernatant were measured. Data are shown as Mean ± SEM, n = 3 for each group. *P < 0.05, **P < 0.01 (Student’s t-test).

Figure 5

14(S)-HDoHE suppresses IL-33-induced airway eosinophilic inflammation. Airway inflammation was induced by administration of IL-33 (500 ng per mouse, intranasal), for 3 consecutive days, in C57BL/6 and 12/15-lipoxygenase deficient mice. 14(S)-hydroxy docosahexaenoic acid (HDoHE) was administered via intraperitoneal injection prior to IL-33 administration. Flow cytometric analysis was performed 4 days after the final administration of IL-33. (A) Number of eosinophils and macrophages in the BALF. (B) Number of lymphocyte subsets, including ILC2 and Th2 cells, in the BALF. Mean ± SEM, n=4 for each group. NS, not significant.