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

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.


INTRODUCTION
Asthma is a common disease affecting more than 300 million people worldwide, and the number of patients with asthma is rapidly increasing (1,2). Genetic and environmental factors induce diverse immune responses that are classified into atopic and non-atopic phenotypes, mainly characterized by eosinophilic airway inflammation. Severe asthma is characterized by resistance to standardized treatments, including corticosteroids, and frequent exacerbation, which could worsen the quality of life in these patients. However, the exact mechanism underlying severe asthma has not been fully elucidated. Thus, it is necessary to elucidate its pathophysiological process.
In the present study, we investigated the regulatory roles of 12/15-LOX-derived lipid mediators in IL-33-induced eosinophilic airway inflammation in mice. Lipidomic analysis of inflamed lung tissue and in vitro lipid screening analysis using ILC2s were performed to demonstrate the roles of 12/15-LOX.

Mouse Experiments
Specific pathogen-and virus-antibody-free, 6-8-week-old, male C57BL/6J (C57BL/6) mice, weighing 25-30 g, were purchased from Charles River Laboratories, Japan. 12/15-LOX-deficient mice were obtained from the Jackson Laboratory (002778, Bar Harbor, ME, USA). All animals were housed at the facility in bubble barrier units (bioBubble, Fort Collins, Colo., USA) under positive pressure. The experimental protocol was reviewed and approved by the Laboratory Animal Care and Use Committee of Keio University of Medicine, the Animal Committee of the University of Tokyo, and the Animal Care and Use Committee of the RIKEN.

Administration of Reagents In Vivo
We administered 40 mL of PBS or IL-33 (R&D, 500 ng per mouse) via intranasal administration under anesthesia with intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg). In some experiments, we simultaneously administered 5 mg/day of 14(S)-HDoHE (Cayman Chemical, Ann Arbor, MI, USA) via intraperitoneal injection.

Establishment of IL-33-Induced Airway Inflammation
We administered IL-33 (R&D Systems, Minneapolis, MN, 500 ng per mouse) for 3 consecutive days and analyzed them 1 or 4 days after the last challenge as previously described (40) with technical modification. Bronchoalveolar lavage fluid (BALF) was collected for cell counts and flow cytometric analysis, and lung tissue for the measurement of mRNA expression and histopathologic analysis. Formalin-fixed paraffin-embedded lung slides were stained with hematoxylin and eosin (HE) or periodic acid Schiff (PAS).

Collection of BALF
The mice were sacrificed by an overdose of intravenous pentobarbital at the indicated times after the last challenge. The trachea was cannulated, and the lungs were lavaged by washing twice with 0.7 mL of ice-cold PBS with EDTA (0.6 mM). The total number of cells in BALF was counted using a hemocytometer, and a differential cell count of 200 cells was determined on Diff-Quik-stained cytospin slides (Baxter Scientific Products, McGraw Park, Ill., USA) prepared with Auto Smear CF12D (Sakura Finetek, Tokyo, Japan). Flow cytometric analysis was performed for cell counts of specific types of lymphocytes (ILC2s and Th2 cells).

Targeted Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)-Based Lipidomics
LC-MS/MS-based mediator lipidomics was performed as previously described (41). Lung tissues were homogenized in ice-cold methanol and kept in -20°C overnight. The methanolic extract was then diluted with water, acidified with HCl to a pH of 3.5, and applied to Sep-Pak C18 cartridges (Waters) for solid phase extraction. Deuterated internal standards (1 ng of leukotriene (LT) B 4 -d4, LTD 4 -d5, prostaglandin (PG) E 2 -d4, and 15-HETE-d8 (Cayman Chemical, Ann Arbor, MI, USA) were added to the supernatants prior to extraction. For LC-MS/MS analysis, a triple quadrupole linear ion trap mass spectrometer (QTRAP 5500; AB Sciex, Foster City, CA) equipped with a 1.7-mm, 1.0 × 150 mm Acquity UPLC ™ BEH C18 column (Waters Corp., Milford, MA) was used. MS/MS analyses were conducted in negative ion mode, and the eicosanoids and docosanoids were identified and quantified by multiple reaction monitoring. Calibration curves were obtained over a range of 1-1,000 pg. The LC retention times for each compound were determined using the corresponding synthetic standards. PD1 and PDX, stereoisomers, were not separable under this LC-MS/MS setting.

Functional Assays of ILC2s In Vitro
Mouse ILC2s were isolated from mesentery using a previously reported method (42). Purified ILC2s were cultured in 96-well round-bottom plates in 200 mL RPMI-1640 media containing 10% FCS, HEPES buffer, non-essential amino acids, penicillin, streptomycin, and 2-mercaptoethanol in the presence of IL-2 (10 ng/mL) at 37°C. To comprehensively evaluate the effect of lipid metabolites, cultured ILC2s were seeded at a density of 10,000 cells per well into 96-well round-bottom plates in the absence of IL-2, and IL-33 was added to the culture medium at a final concentration of 10 ng/mL after pretreatment for 30 min with lipid metabolites (10 -11 M -10 -5 M). One hundred microliters of supernatant were collected for the cytokine assay, and ILC2s were counted by flow cytometry on day 4. For apoptosis analysis, ILC2s were stained with Annexin Vand propidium iodide(PI) accordingtothemanufacturer's protocols (Apoptosis Detection Kit, BD Pharmingen), and then analyzed by flow cytometry on day 1 and day 4. All data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Statistical Analysis
Data are presented as the mean ± SEM. Dose-response relationships of lipid metabolites on ILC2 activities were analyzed with repeated measures of analysis of variance, followed by the Bonferroni/Dunn procedure as a post hoc test. Data were analyzed using GraphPad Prism version 4.0c (GraphPad Software, San Diego, CA). Statistical significance was set at P < 0.05.

12/15-LOX Deficiency Augmented IL-33-Induced Airway Eosinophilic Inflammation
To determine whether 12/15-LOX affects the disease onset and/ or progression of airway eosinophilic inflammation, we used mice deficient in the gene encoding 12/15-LOX (alox15) in a murine model of IL-33-induced innate airway eosinophilic inflammation. 12/15-LOX-deficient (12/15-LOX -/-) mice developed more severe airway inflammation associated with an increased number of eosinophils and lymphocytes in BALF compared to controls ( Figure 1A). Lymphocyte subset analysis revealed a significant increase in the number of ILC2s and Th2 cells (CD4 + ST2 + cells) in BALF from IL-33-challenged 12/15-LOX -/mice compared to wild-type mice ( Figure 1B). Importantly, the number of ILC2s was greater than that of Th2 cells ( Figure 1B). Expression of mRNA levels of type 2 cytokines (il5, il13) and chemokines for eosinophils (ccl11, ccl24, and ccl26), which are critical inducers of eosinophilic inflammation, in the lungs was significantly higher in IL-33-treated 12/15-LOX -/mice than in wild-type mice ( Figure 1C). Histological analysis demonstrated prominent accumulation of inflammatory cells around the bronchus and increased mucus production in the lung tissue stained with HE and PAS, respectively ( Figures 1D, E). In addition, the absence of 12/15-LOX had no impact on the number of cells and lymphocyte subset including Th2 cells and ILC2s in BALF at the steady state ( Supplementary Figures 1A, B).

14(S)-HDoHE Suppressed IL-33-Induced Airway Eosinophilic Inflammation In Vivo
Next, we determined the potential preventive effect of 14(S)-HDoHE, a major product of 12/15-LOX in the lung during

DISCUSSION
In the present study, we demonstrated that 12/15-LOX, a key enzyme for the biosynthesis of specialized pro-resolving lipid mediators (SPMs), conferred a protective effect on innate pulmonary eosinophilic inflammation in vivo. Lipidomic analysis revealed a series of 12/15-LOX-derived mediators present at substantial levels in the inflamed lung. Using bioactive lipid screening, the potent effect of 14(S)-HDoHE and 10(S),17(S)-diHDoHE in suppressing ILC2 function was observed in vitro. These findings demonstrate the direct effect of DHA-derived pro-resolving mediators in suppressing ILC2 activation. 14(S)-HDoHE also displayed potent antiinflammatory effects on IL-33-induced eosinophilic airway inflammation when administered in vivo. These results provide a new therapeutic option for 12/15-LOX-derived pro-resolving mediators in ILC2-mediated allergic diseases.
Previous studies have shown the regulatory effects of 14(S)-HDoHE on murine platelets and human alveolar macrophages in vitro (43,44). PD1/PDX also possesses potent anti-inflammatory functions in regulating neutrophil activation in vitro (45,46) and ameliorates pulmonary inflammation and fibrosis in vivo (47,48). Interestingly, we previously reported that peripheral blood eosinophils isolated from patients with severe asthma had a defective biosynthetic capacity of 14(S)-HDoHE, 17(S)-HDoHE, and PD1/PDX (7). Similarly, their biosynthetic capacities have been reported to be impaired in obese mice, and systemic administration of these metabolites ameliorated obesityinduced inflammatory states (49). These findings highlight the therapeutic potential of 14(S)-HDoHE and related SPMs in the regulation of chronic inflammation through DHA metabolism. However, the precise mechanism underlying the 14(S)-HDoHEmediated effects thorough specific receptors and/or downstream metabolites remains undetermined.
The cellular sources of 12/15-LOX-derived mediators during allergic inflammation are of particular interest. 12/15-LOXexpressing eosinophils play pro-resolving functions by enhancing the resolution of neutrophilic inflammation in acute peritonitis (8,9) and by promoting corneal wound healing in the eye (59). In addition, 12/15-LOX-expressing resident macrophages play an important role in the efferocytosis of apoptotic neutrophils in acute peritonitis (10). Further studies are required to identify the cell types that locally produce 12/15-LOX-derived SPMs to suppress ILC-2-mediated eosinophilic inflammation.
In conclusion, our results demonstrate that 12/15-LOX-derived mediators regulate IL-33-induced eosinophilic airway inflammation. Bioactive lipid screening identified 14(S)-HDoHE and 10(S),17(S)-diHDoHE as potent endogenous suppressors of ILC2 activation. These findings contribute to a better understanding of the cellular and molecular mechanisms underlying the resolution of eosinophilic airway inflammation. Thus, 12/15-LOX-derived lipid mediators and/or 12/15-LOX-mediated lipid metabolism may represent a potential therapeutic strategy for ameliorating airway inflammation-associated conditions.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Care and Use Committee of the RIKEN, the Animal Committee of the University of Tokyo, the Laboratory Animal Care and Use Committee of Keio University of Medicine.