Allergic rhinitis is among the most common allergic diseases and is increasing in prevalence globally [1
]. Patients with perennial allergic rhinitis continuously suffer from symptoms such as sneezing, itchiness, and rhinorrhea; they, therefore, have to avoid contact with allergens, causing a heavy burden and lifestyle limitations [2
The initiation of allergic rhinitis is dependent on the function of antigen-specific immunoglobulin (Ig) E [3
]. When exposed to an antigen for the first time, antigen-presenting cells take up the antigen and induce the differentiation and proliferation of T cells to type 2 helper T (Th2) cells. Th2 cells induce Ig class-switching to IgE on B cells and stimulate differentiation to antigen-specific IgE-producing plasma cells through the secretion of Th2 cytokines such as interleukin (IL)-4, IL-5, and IL-13 [3
]. Antigen-specific IgE secreted by plasma cells is bound to the cell surface of mast cells via Fc epsilon receptor 1 (FcεRI), and when the individual is re-exposed to the antigen, it initiates FcεRI cross-linking and induces mast cell degranulation [3
]. Degranulated mast cells secrete chemical mediators, including histamine and lipid mediators, that induce the nasal responses of allergic rhinitis, including sneezing, itchiness, and rhinorrhea [5
]. Given these mechanistic processes, promising strategies to control allergic symptoms include the inhibition of Th2 differentiation, IgE production, mast cell degranulation, and the interaction between chemical mediators and their receptors.
Previous studies about the association between the development of allergic diseases and dietary nutrition indicate the involvement of dietary fatty acid (FA) in the acceleration and/or inhibition of allergic responses [6
]. Dietary oils contain various FAs, but their composition is dependent on the materials from which the oils are extracted as well as the extraction process. FA is characterized by the presence or absence of a carbon double bond in its structure; saturated FAs (palmitic acid, stearic acid, etc.) lack a carbon double bond, and unsaturated FAs contain at least one carbon double bond. Among unsaturated FAs, omega-3 FAs (e.g., alpha-linolenic acid [ALA], eicosapentaenoic acid [EPA], and docosahexaenoic acid [DHA]) and omega-6 FAs (e.g., linoleic acid [LA] and arachidonic acid [ARA]) are categorized as essential FAs. Various types of bioactivity of dietary essential FAs have been reported in studies of health and diseases, including immunity, allergy, and inflammation [6
]. Human studies, for example, have shown an association between the quality of dietary FAs and the incidence of allergic diseases [9
]. Animal studies likewise indicate that the quality of dietary FAs is a critical determinant of the development and severity of allergic symptoms [10
]. We previously showed that dietary linseed oil (also called flaxseed oil), which contains high amounts of ALA, ameliorates egg-derived ovalbumin (OVA)-induced food allergy in mice [12
]. Another report suggested that pollen-derived antigen-induced allergic conjunctivitis can be alleviated by dietary linseed oil [13
]. These studies demonstrate the importance of dietary FA quality in the regulation of allergic responses and the contribution of omega-3 FA-rich linseed oil to the inhibition of allergic symptoms.
Dietary FAs are not only an energy source [14
] but also the substrate of bioactive lipid metabolites [15
]. FAs usually exist as components of triglycerides and cell membrane phospholipids in vivo. Under certain circumstances, including cell activation, FAs are degraded from membrane phospholipids by phospholipases. Free FAs can be further converted to various metabolites by oxygenate enzymes (e.g., lipoxygenase [LOX], cyclooxygenase [COX], and cytochrome P450 [CYP]), and the resulting compounds can exacerbate and/or ameliorate allergic diseases [15
]. Recently, the application of a new method of liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) revealed that tremendous numbers of metabolites are generated from dietary FAs, including omega-6 and omega-3 essential FAs [16
]. Further biological studies found novel functions of these FA metabolites in immunity, allergy, and inflammation [16
]. In particular, recent studies have uncovered that omega-3 FA-derived metabolites have anti-allergic functions [9
]. For example, EPA-derived resolvin E1 promotes the resolution of allergic lung inflammation [17
], and DHA-derived resolvin D1 reduces histamine responses in the eye and regulates conjunctival goblet cell secretion [18
]. These studies indicate that omega-3 FA-derived metabolites have potential to regulate inflammation and allergic disease. The accumulation of omega-3 FA-derived metabolites is increased upon the intake of dietary linseed oil. In our previous report, 17,18-epoxyeicosatetraenoic acid (17,18-EpETE), which is converted from EPA by CYPs, was among the main metabolites that accumulated in the mouse large intestine, and it indeed exerted anti-allergic effects in a food allergy model [12
]. However, it remains unclear whether dietary linseed oil has anti-allergic effects at sites other than the intestine, such as the respiratory mucosal compartment, and whether unique anti-allergic FA metabolites accumulate in nasal tissue.
In this study, we evaluated the effect of dietary linseed oil on allergic rhinitis in the upper respiratory tract by using an OVA-induced nasal allergy model in mice.
2. Materials and Methods
2.1. Mice and Experimental Diet
Female C57BL/6J mice (age, 6–7 weeks) were purchased from Japan SLC. Animals were maintained in the specific pathogen-free facility of the National Institutes of Biomedical Innovation, Health, and Nutrition (NIBIOHN). Mice were maintained for 2 months on diets composed of chemically defined materials and supplemented with 4% of each dietary oil (Table S1
) (Oriental Yeast, Tokyo, Japan) before the induction of allergic rhinitis. The fatty acid composition of dietary oils is shown in Figure S1
. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee and the Committee on the Ethics of Animal Experiments at NIBIOHN (DS27-47 and DSR01-2).
2.2. Induction of Allergic Rhinitis
Allergic rhinitis was induced as described previously, with modifications [19
]. In brief, mice were sensitized by intraperitoneal injection of 5 μg of OVA (Sigma-Aldrich, St. Louis, MO, USA), with 1 mg of alum hydroxide (Thermo Fisher Scientific, Waltham, MA, USA) in 200 μL of phosphate-buffered saline (PBS) (Nacalai Tesque, Kyoto, Japan). After 7 days, awake mice were intranasally challenged with either 250 μg of OVA in 20 μL of PBS or PBS alone as a vehicle control for 4 consecutive days. Allergic symptoms were assessed by counting the number of sneezing behaviors in a 5 min period after the nasal challenge. Soon after nasal administration, mice showed sneezing, which could be recognized by sound and neck movement. The enumeration of sneezing numbers was performed in a quiet room in order to reduce noises and to detect sneezing sound clearly. The measurement was conducted one by one and the mouse was put in a cage for a 5 min period after the nasal challenge.
2.3. Administration of Reagents to Mice
The 15-hydroxyeicosapentaenoic acid (15-HEPE) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Mice were intranasally treated with either 10 ng of 15-HEPE in 20 μL of PBS or 0.5% (vol/vol) ethanol in PBS as a vehicle control, 5 min before the nasal challenge. To evaluate the ability of 15-HEPE to prevent the development of allergic rhinitis, lipid administration began on the first day of the nasal challenge. To evaluate the therapeutic effect of 15-HEPE, lipid administration was started from the fifth day of the nasal challenge.
GW9662 (Abcam, Cambridge, United Kingdom), a peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, was dissolved in PBS containing 0.5% (vol/vol) ethanol. PBS with 0.5% ethanol was used as a mock-treatment control. GW9662 solution or the mock-treatment was intraperitoneally injected daily at 1 mg/kg of body weight in a 200 μL volume, at 30 min before nasal administration of the lipid solution.
PD146176 (Cayman Chemical) was dissolved in PBS containing 1% (vol/vol) dimethyl sulfoxide (DMSO). PBS with 1% (vol/vol) DMSO was used as a mock-treatment control. This solution was intraperitoneally injected daily at 1 mg/kg of body weight in a 200 μL volume, at 30 min before the nasal challenge.
The mixture of anti-C-C motif chemokine ligand (CCL) 11 antibody (24 μg: Biolegend, San Diego, CA, USA) and anti-IL-5 antibody (3 μg: Biolegend) in a 100 μL volume was intravenously administrated to the allergic rhinitis mouse model on the first and third days of the nasal challenge. Rat immunoglobulin G1
) (27 μg: Biolegend) was used as an isotype control [23
2.4. Tissue Collection and Preparation of a Single Cell Suspension
Nasal passage (NP) cells were collected from mice as previously reported, with modifications [19
]. The cell suspension was filtered through 100 μm cell strainers (BD Biosciences, Franklin Lakes, NJ, USA).
2.5. Flow Cytometry
Flow cytometric analysis was performed as described previously, with modifications [24
]. In brief, the cell suspension in 2% newborn calf serum (NCS) PBS was stained with an anti-CD16/32 antibody (TruStain fcX) (Biolegend) to avoid non-specific staining. After being washed with 2% NCS PBS, the cells were further stained with the following antibodies: FITC-anti-Ly6G (Biolegend), FITC-anti-CD63 (gift from Dr. Kurashima, The University of Tokyo) [25
], APC-Cy7-anti-CD11b (Biolegend), APC-anti-FcεRI (eBioscience), PE-anti-c-kit (BD Biosciences), PE-Cy7-anti-CD45 (Biolegend), BV421-anti-CD45 (Biolegend), and BV421-anti-Siglec-F (Biolegend). Dead cells were detected by using 7-AAD (Biolegend) and were excluded from the analysis. Flow cytometry analysis was performed by using MACSQuant (Miltenyi Biotec, Bergisch Gladbach, Germany) or FACSAria (BD Biosciences). Data were analyzed by using FlowJo 9.9 (Tree Star, Ashland, OR, USA).
2.6. OVA-Specific IgE ELISA
Plasma was collected 5 min after the last nasal challenge and heparinized. OVA-specific IgE levels in the plasma were determined by using an ELISA kit (DS Pharma, Osaka, Japan) according to the manufacturer’s protocol.
2.7. Mast Cell Degranulation Assay
Mouse bone marrow-derived mast cells (BMMCs) were prepared and degranulation was induced as previously reported [25
] In brief, bone marrow cells were prepared from the femur and tibia of 8-week-old C57BL/6J wild-type mice and were cultured in RPMI1640 (Sigma-Aldrich) supplemented with 10% FBS, 100 IU/mL penicillin plus 100 μg/mL streptomycin (Nacalai Tesque), 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque), and 5 ng/mL mouse recombinant IL-3 (PeproTech, Rocky Hill, CT, USA) for 4 weeks. After that, 50 ng/mL stem cell factor (SCF) (PeproTech) was added to the culture medium, and the cells were incubated for 2 to 4 weeks. The efficacy and percentage of differentiation to BMMCs were confirmed by flow cytometry as the FcεRI+, c-Kit+, CD45+ population; a > 90% pure population was used for the degranulation assay.
For the degranulation assay, BMMCs were sensitized with 0.2 mg/mL anti-dinitrophenyl (DNP)-IgE (Sigma-Aldrich) for 24 h. Cells were then washed and stimulated with 100 ng/mL DNP-BSA (LSL, Tokyo, Japan) at 37 °C for 30 min. To assess the effect of lipid mediators on degranulation, 15-HEPE (100 nM) or 0.5% (vol/vol) EtOH in PBS as a vehicle control was added 30 min before stimulation with DNP-BSA. Degranulation of BMMCs was evaluated by using flow cytometry and staining the BMMCs with CD63 as a marker for degranulation.
2.8. Culture of Bone Marrow-Derived Neutrophils and Eosinophils
Neutrophils were collected from bone marrow cells of female C57BL/6J mice as previously reported [26
]. Bone marrow cells were suspended in RPMI 1640 medium containing 2% NCS and carefully added onto 62% Percoll (GE Healthcare, Chicago, IL, USA) in RPMI 1640 medium. After centrifugation at 1000× g
for 20 min at 25 °C, neutrophils were precipitated.
Eosinophils were prepared by differentiation from mouse bone marrow cells as previously reported [27
]. Briefly, mouse bone marrow cells were cultured at 1.0 × 106 cells/mL in medium containing RPMI 1640 with 20% FBS, 100 IU/mL penicillin plus 10 μg/mL streptomycin (Nacalai Tesque), 25 mM HEPES (Nacalai Tesque), 1× nonessential amino acids (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque) and 50 μM 2-ME (Thermo Fisher Scientific). From day 0 through 4, 100 ng/mL stem cell factor (SCF; [PeproTech]) and 100 ng/mL FLT3 ligand (PeproTech) were supplemented. On day 4, the medium with SCF and FLT3 ligand was replaced to the medium containing 10 ng/mL mouse recombinant IL-5 (Peprotech). From day 8 through 10, the cells were transferred to a new flask and the medium was changed. The cell concentration was adjusted to 1.0 × 106 cells/mL every day. On day 12, eosinophil differentiation was assessed by FACS staining with BV421-anti-Siglec-F and the cells were used for experiments between day 12 and day 19.
2.9. Lipid Metabolism of Cultured Neutrophils and Eosinophils
A lipid metabolism assay was performed as previously reported with modification [28
]. Neutrophils or eosinophils were suspended in RPMI 1640 at 1.0 × 106
cells/mL. A lipid production assay was performed with the addition of 1 μM EPA or ARA, together with 2 μM calcium ionophore in the culture medium. After 30 min, the reaction was stopped by adding 2 times the amount of ice-cold methanol to the medium.
2.10. Lipid Extraction from Cells, Culture Supernatant, and Plasma
Lipid extraction was performed as previously reported [29
]. Cells were suspended in PBS and transferred to a polypropylene tube. After centrifugation to remove the PBS, methanol was added to extract the lipids. For culture supernatant and plasma, 9 volumes of methanol were used for the extraction. After centrifugation at 1600× g
for 10 min at 4 °C, the supernatant was collected and diluted to 50% methanol. Solid-phase extraction was then performed by using a Mono Spin C18-AX cartridge (GL Science, Tokyo, Japan) with internal standards (arachidonic acid-d8 [Cayman Chemical], 15-hydroxyeicosatetraenoic acid-d4 [Cayman Chemical], and leukotriene B4-d5, [Cayman Chemical]). Briefly, the cartridge was washed with methanol and then water. The extracted sample in 50% methanol was then applied to the cartridge and washed with water and 50% methanol. The lipid sample was subsequently eluted by using 90% methanol containing 2% acetic acid.
2.11. LC–MS/MS Analysis of Free FAs and Their Metabolites
Lipid metabolites were analyzed by using a UPLC system (ACQUITY) (Waters, Milford, MA, USA) coupled with mass spectrometry (Orbitrap ELITE) (Thermo Fisher Scientific), with modifications of the previously reported protocol [29
]. UPLC application was performed with a 1.7 mm, 1.0 × 150 mm ACQUITY UPLC BEH C18 column (Waters). Mass spectrometric analysis for quantification was based on the ion trap MS2 detection method. Data analysis was performed by using the software Xcalibur 2.2 (Thermo Fisher Scientific).
For quantification, calibration curves were drawn by using the following lipid standards: LA (Cayman Chemical), ALA (Cayman Chemical), ARA (Cayman Chemical), EPA (Cayman Chemical), DHA (Cayman Chemical), 18-hydroxyeicosapentaenoic acid (18-HEPE; Cayman Chemical), 15-HEPE (Cayman Chemical), 12-hydroxyeicosapentaenoic acid (12-HEPE; Cayman Chemical), 5-hydroxyeicosapentaenoic acid (5-HEPE; Cayman Chemical), and 17,18-epoxyeicosatetraenoic acid (17,18-EpETE; Cayman Chemical).
2.12. Reverse Transcription and Quantitative PCR
Reverse transcription and quantitative PCR analysis were performed as described previously [30
]. In brief, RNA from cell suspensions was isolated by using Sepazol (Nacalai Tesque) and chloroform (Nacalai Tesque). After precipitation with 2-propanol (Nacalai Tesque) and washing with 75% (vol
) ethanol (Nacalai Tesque), the residue was incubated with DNaseI (Thermo Fisher Scientific) and reverse-transcribed to cDNA (Superscript 3 reverse transcriptase, VIRO cDNA Synthesis Kit; Invitrogen).
Real-time PCR was performed by using the LightCycler 480 System II (Roche, Basel, Switzerland) and SYBR Green I Master reagents (Roche). Primer sequences were: 5′-ggggatggagaagctacagg-3′ (sense) and 5′-tccgcttcaaacagagtgc-3′ (anti-sense) for Alox15, 5′-gaaagacaacggacaaatcacc-3′ (sense) and 5′-gggggtgatatgtttgaacttg-3′ (anti-sense) for Pparg and 5′-aaggccaaccgtgaaaagat-3′ (sense) and 5′-gtggtacgaccagaggcatac-3′ (anti-sense) for Actb.
2.13. Statistical Analysis
Statistical significance was evaluated by using one-way ANOVA followed by Dunn’s Kruskal–Wallis Multiple Comparisons or Tukey’s multiple comparison test for multiple groups and the Mann–Whitney test for 2 groups. In some analyses, outliers were excluded by the Smirnov–Grubbs test. All analyses were performed by using Prism 6 software (GraphPad Software, San Diego, CA, USA). A P value of less than 0.05 was considered significant.
Numerous studies suggest that dietary FA affects health and diseases. In particular, omega-3 FA and its metabolites have several beneficial functions including anti-hypertension [35
], anti-metabolic syndrome [36
] and anti-allergy functions [12
]. In the current study, we investigated the effect of dietary omega-3 FA on allergic rhinitis in mice. We found that dietary linseed oil dampened allergic rhinitis response in mice. Lipidomics revealed that ALA and EPA were increased in linseed oil-fed mice and 15-HEPE was accumulated in NP of linseed oil-fed mice after the induction of nasal allergy. The 15-HEPE was produced by the 15-LOX activity of eosinophil which infiltrated into NP and deletion of eosinophil exacerbated allergic responses in linseed oil-fed mice. The 15-HEPE reduces mast cell degranulation in vitro and dampened allergic responses in mice in a PPARγ-dependent manner.
Type I allergy is initiated by the production of allergen-specific IgE and subsequently mediated via cross-linking with FcεRI on mast cells accompanied by degranulation and release of chemical mediators [3
]. In this study, we found that 15-HEPE levels were increased by dietary intake of linseed oil and exerted an anti-allergic effect by inhibiting mast cell degranulation without affecting allergen-specific IgE production. The inhibition of mast cell degranulation is among the most common strategies to ameliorate type I allergic symptoms, such as those associated with atopic dermatitis [34
], food allergy [12
], and rhinitis [38
]. Previous reports indicated that a PPARγ agonist inhibited atopic dermatitis in mice through the suppression of mast cell maturation and mediator release [34
]. In the present study, we found that mast cells in the NP of allergic mice expressed PPARγ, which acts as a receptor for 15-HEPE to inhibit degranulation in vitro.
We also found that the induction of allergic rhinitis changed FA metabolism in the NP. A previous study reported that some anti-inflammatory lipid mediators were endogenously generated during the resolution phase of zymosan-initiated peritonitis and acted as resolving molecules [31
]. In the present study, EPA-derived 15-HEPE levels increased in linseed oil-fed mice with allergic rhinitis.
Increased 15-HEPE levels were found in the NP but not in the plasma of LinOVA. Several reports have indicated that FA metabolism differs between tissues. For example, EPA-derived 17,18-EpETE uniquely accumulates in the large intestine in linseed oil-fed mice [12
]. Another report suggested that 12-hydroxyeicosatetraenoic acid and 13-hydroxyoctadecadienoic acid levels were preferentially increased in the lungs of asthmatic mice [40
]. These differences can be explained, at least in part, by the cells that express the responsible enzymes. Indeed, macrophages in the heart of fat-1
transgenic mice produce 18-HEPE, which inhibits macrophage-mediated proinflammatory activation of cardiac fibroblasts [41
]. The 18-HEPE is further transformed to resolvin E1 by 5-LOX expressed on neutrophils [42
]. We also recently showed that neutrophil 5-LOX contributes to the production of leukotriene B4
from ARA and influences the development of inflammation [26
In the present study, we found that eosinophils highly express 15-LOX. Generally, eosinophil numbers are considered to reflect the severity of the allergic symptoms of asthma and rhinitis. Indeed, eosinophils release cytotoxic proteins, including major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPx), which exacerbate inflammation [44
]. In addition, eosinophils are recognized as activators of mast cells and basophils [45
]. In contrast, some recent studies have investigated the anti-inflammatory activities of eosinophils. These studies have revealed that eosinophils also regulate infectious bacteria-induced intestinal inflammation by suppressing Th1 responses [47
]. In the lung, pulmonary resident eosinophils have regulatory profiles against Th2 responses [48
]. Moreover, recent studies have revealed that eosinophils participate in the lipid network for the production of anti-inflammatory and/or pro-resolution lipid mediators [49
]. It is also indicated that eosinophils may be involved in the control of nasal allergy, but details on the mechanisms have been unclear [52
]. In the present study, we demonstrated the novel function of eosinophils in the control of allergic responses. We found that eosinophils infiltrated the NP upon the induction of allergic rhinitis and produced 15-HEPE for the inhibition of mast cell degranulation. Thus, eosinophils produce several types of lipid mediators for the regulation of inflammation and allergy by targeting different types of immune cells such as neutrophils, phagocytes and mast cells. Eosinophil infiltration occurs regardless of dietary oils; however, the intake of linseed oil resulted in an EPA-enriched environment, where eosinophils metabolized EPA to produce 15-HEPE for the inhibition of allergic rhinitis responses.
In conclusion, our findings suggest that ALA, which is abundant in linseed oil, reduces allergic responses through the local production of 15-HEPE by eosinophils that infiltrate the NP. The 15-HEPE dampens allergic responses in a PPARγ-dependent manner and inhibits mast cell degranulation. These findings identify unique eosinophil functions in the development of allergic rhinitis which are determined by the surrounding lipid environment. Our study also provides valuable information for both dietary and pharmaceutical strategies to ameliorate allergic symptoms.