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. 2019 Mar;33(3):3392-3403.
doi: 10.1096/fj.201801805R. Epub 2018 Nov 1.

Dietary ω-3 fatty acids alter the lipid mediator profile and alleviate allergic conjunctivitis without modulating Th2 immune responses

Toshiaki Hirakata  1   2 , Hyeon-Cheol Lee  2 , Mai Ohba  2 , Kazuko Saeki  2 , Toshiaki Okuno  2 , Akira Murakami  1 , Akira Matsuda  1 , Takehiko Yokomizo  2
Affiliations

Dietary ω-3 fatty acids alter the lipid mediator profile and alleviate allergic conjunctivitis without modulating Th2 immune responses

Toshiaki Hirakata et al. FASEB J. 2019 Mar.

Abstract

Allergic conjunctivitis (AC) is one of the most common ocular surface diseases in the world. In AC, T helper type 2 (Th2) immune responses play central roles in orchestrating inflammatory responses. However, the roles of lipid mediators in the onset and progression of AC remain to be fully explored. Although previous reports have shown the beneficial effects of supplementation of ω-3 fatty acids in asthma or atopic dermatitis, the underlying molecular mechanisms are poorly understood. In this study, a diet rich in ω-3 fatty acids alleviated AC symptoms in both early and late phases without affecting Th2 immune responses, but rather by altering the lipid mediator profiles. The ω-3 fatty acids completely suppressed scratching behavior toward the eyes, an allergic reaction provoked by itch. Although total serum IgE levels and the expression levels of Th2 cytokines and chemokines in the conjunctiva were not altered by ω-3 fatty acids, eosinophil infiltration into the conjunctiva was dramatically suppressed. The levels of ω-6-derived proinflammatory lipid mediators, including those with chemoattractant properties for eosinophils, were markedly reduced in the conjunctivae of ω-3 diet-fed mice. Dietary ω-3 fatty acids can alleviate a variety of symptoms of AC by altering the lipid mediator profile.-Hirakata, T., Lee, H.-C., Ohba, M., Saeki, K., Okuno, T., Murakami, A., Matsuda, A., Yokomizo, T. Dietary ω-3 fatty acids alter the lipid mediator profile and alleviate allergic conjunctivitis without modulating Th2 immune responses.

Keywords: eicosanoid; fish oil; hay fever; inflammation; lipidomics.

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Conflict of interest statement

The authors thank members of the Laboratory of Morphology and Image Analysis Research Support Center at the Juntendo University Graduate School of Medicine for technical assistance with microscopy. This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT)/Japan Society for the Promotion of Sport Grants-in-Aid for Scientific Research (KAKENHI) (18K16246 to H.-C.L.; 15K08316 and 18K06923 to K.S.; 15KK0320 and 16K08596 to T.O.; 16K11303 to A.Matsuda; and 15H05904, 15H04708, and 18H02627 to T.Y.), and grants from the Naito Foundation, the Ono Medical Research Foundation, the Uehara Memorial Foundation, the Mitsubishi Foundation, the Takeda Science Foundation, and, in part, by Grant-in-Aid S1311011 from the Foundation of Strategic Research Projects in Private Universities from MEXT, and institutional grants for Environmental and Gender-Specific Medicine and the Atopy Research Center at the Juntendo University Graduate School of Medicine. The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
An ω-3 diet attenuates experimental AC. A) The protocol used in the mouse model of RW pollen–induced EAC is shown. Mice were fed with chow containing 4% linseed (ω-3 diet; n-3) or soy (ω-6 diet; n-6) oil for 30 d before sensitization with RW pollen. PBS was used as a control. B) Representative photos of mouse eyes. C) Clinical score was calculated at d 0, 29, and 30 (n = 6–7 at each time point). The total score consisted of the sum of scores for 4 factors—chemosis, conjunctival redness, lid edema, and discharge—each scored from 0 to 3 points, depending on the severity. Data are representative of 4 independent experiments, in which comparable results were obtained. Data were analyzed by Mann-Whitney U test. Data are expressed as means ± se. **P < 0.01, ω-6 vs. ω-3; ††P < 0.01, PBS vs. RW; §§P < 0.01, d 29 vs. d 30.
Figure 2
Figure 2
An ω-3 diet attenuates AC-induced itch. A) Scratching behavior was monitored immediately after the final challenge and counted for 40 min. B) The total number of scratching actions during 40 min (n = 6). Data were analyzed by 1-way ANOVA and Tukey’s multiple-comparison test. Data are expressed as means ± se. ****P < 0.0001, ω-6 vs. ω-3; ††††P < 0.0001, PBS vs. RW.
Figure 3
Figure 3
Reduced eosinophil migration in the conjunctiva of ω-3–fed mice. A–D) Sections containing conjunctiva were stained with Giemsa stain, and eosinophils were counted. Arrows: eosinophils. Scale bars, 50 µm. The conjunctiva of a mouse fed an ω-6 diet (n-6) treated with PBS (A) or with RW pollen (C). The conjunctiva of a mouse fed an ω-3 diet (n-3) treated with PBS (B) or created with RW pollen (D). E) The number of eosinophils infiltrating conjunctiva. F) mRNA level of eosinophil-associated RNase subfamily B mRNA measured by real-time PCR (n = 3). E, F) Data are expressed as means ± se. Data were analyzed by 1-way ANOVA and Tukey’s multiple-comparison test. **P < 0.01, ***P < 0.001, ω-6 vs. ω-3; ††P < 0.01, ††††P < 0.0001, PBS vs. RW.
Figure 4
Figure 4
Dietary ω-3 fatty acids do not affect eosinophils in the peripheral blood. A, B) The percentage of eosinophils in the peripheral blood was analyzed by flow cytometry (n = 6). Data are expressed as means ± se and the individual values are plotted on the bar graph. Data were analyzed by 1-way ANOVA and Tukey’s multiple comparison test.
Figure 5
Figure 5
Th2 immune responses are not modulated by dietary ω-3 fatty acids. A) mRNA expression levels of Th2 cytokines and chemokines in the conjunctiva were measured by real-time PCR (n = 6–7). B) Serum IgE level was measured by ELISA (n = 6–7). A, B) Data are expressed as means ± se, analyzed by 1-way ANOVA and Tukey’s multiple-comparison test. ***P < 0.001, ω-6 vs. ω-3; ††P < 0.01, †††P < 0.001, ††††P < 0.0001, PBS vs. RW.
Figure 6
Figure 6
Lipidomics analysis of the conjunctiva. Various lipid mediators derived from AA (A), DHA (B), and EPA (C) in conjunctivae were measured by LC/MS/MS (n = 4–5). Data are expressed as means ± se, analyzed by 1-way ANOVA and Tukey’s multiple-comparison test. **P < 0.01, ***P < 0.001, ****P < 0.0001, ω-6 vs. ω-3; ††P < 0.01, †††P < 0.001, ††††P < 0.0001, PBS vs. RW.
Figure 7
Figure 7
mRNA expression levels of fatty acid metabolizing enzymes. mRNA expression levels of eicosanoid biosynthetic enzymes, cyclooxygenase1/2 (Cox1/2), hematopoietic PGD synthase (Hpgds), PGE synthase 1/2/3 (Ptges1/2/3), PGF synthase (Fam213b and Akr1b3), PGI synthase (Ptgis), TXA synthase 1 (Txas1), LTA4 hydrolase (Lta4h), and LTC4 synthase (Ltc4s), were evaluated by real-time PCR (n = 3). Data represent means ± se, analyzed by 1-way ANOVA and Tukey’s multiple-comparison test. †P < 0.05, ††P < 0.01, †††P < 0.001, control vs. RW.
Figure 8
Figure 8
Mechanisms of AC suppression by ω-3 diet. A) The control diet: Th2 cells produce IL-4 and -13, which activate B cells. These activated B cells produce IgE, which activates mast cells. In the activated mast cells, free AA is produced from the phospholipid membrane by PLA2, and proinflammatory lipid mediators are synthesized by enzymes such as oxygenases and terminal prostanoid synthases. These proinflammatory lipid mediators elicit allergic symptoms in the early phase such as conjunctival redness, discharge, and itch. These lipid mediators such as PGD2 and LTB4 also play a role in attracting eosinophils. Infiltrated eosinophils produce inflammatory mediators and cause the late-phase allergic symptoms. B) The ω-3 diet: dietary ω-3 fatty acids do not alter the Th2 immune response, but increase the EPA content in the membrane phospholipids. Free EPA liberated by PLA2 competes with AA, and reduces the production of AA-derived mediators, resulting in the reduction of proinflammatory lipid mediators. Furthermore, lipid mediators derived from EPA function as anti-allergic or proresolving mediators. This altered lipid profile leads to the alleviation of AC in both the early and late phases.
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