Alleviation of allergic conjunctivitis by (±)5(6)-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid in mice

Background: Allergic conjunctivitis (AC) is a common ophthalmologic disorder that causes symptoms that often reduces a patient’s quality of life (QOL). We investigated the effects of the eicosapentaenoic acid metabolite (±)5(6)-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid ((±)5(6)-DiHETE) on AC using a mouse model. Methods: BALB/c mice were sensitized with two injections of short ragweed pollen in alum, challenged fifth with pollen in eyedrops. The clinical signs and tear volume were evaluated at 15 min after the final challenge. Histamine-induced ocular inflammation model was prepared by instilling histamine onto the surface of the eye. Fifteen minutes after histamine application, tear volume was measured using the Schirmer tear test. Miles assay was performed to investigate vascular permeability. To cause scratching behavior 10 μg of serotonin was injected in the cheek. Results: Repeated topical application of pollen induced conjunctivitis, accompanied by eyelid edema and tearing in mice. Pollen application typically degranulates mast cells and recruits eosinophils to the conjunctiva. Intraperitoneal administration of 300 μg/kg of (±)5(6)-DiHETE significantly inhibited pollen-induced symptoms. The administration of (±)5(6)-DiHETE also attenuated mast cell degranulation and eosinophil infiltration into the conjunctiva. To assess the effects of (±)5(6)-DiHETE on the downstream pathway of mast cell activation in AC, we used a histamine-induced ocular inflammation model. Topical application of 4 μg/eye histamine caused eyelid edema and tearing and increased vascular permeability, as indicated by Evans blue dye extravasation. Intraperitoneal administration of 300 μg/kg or topical administration of 1 μg/eye (±)5(6)-DiHETE inhibited histamine-induced manifestations. Finally, we assessed the effects of (±)5(6)-DiHETE on itching. An intradermal injection of 10 μg serotonin in the cheek caused scratching behavior in mice. Intraperitoneal administration of 300 μg/kg (±)5(6)-DiHETE significantly inhibited serotonin-induced scratching. Conclusion: Thus, (±)5(6)-DiHETE treatment broadly suppressed AC pathology and could be a novel treatment option for AC.


Introduction
Allergic conjunctivitis (AC), a common ophthalmologic disorder affecting up to 40% of the general population (Singh K et al., 2010;Miyazaki et al., 2020), causes several symptoms, such as conjunctival hyperemia, lacrimation and ocular itching.During the period of antigen exposure, these symptoms significantly reduce the patient's quality of life (QOL).There are two main types of AC: seasonal and perennial.During seasonal AC, symptoms are observed at a fixed time every year.Most seasonal cases of AC are caused by pollen.Perennial AC is not seasonal and tends to be chronic.Mites and house dust are the major causative antigens of perennial AC.
AC includes two phases of the inflammatory response.In the early phase of AC, an immunoglobulin E (IgE)-dependent inflammatory response occurs within approximately 15 min after allergen exposure.Mast cells activated by the allergen-IgE complex release inflammatory mediators such as histamine and serotonin.These mediators can cause conjunctival hyperemia, lacrimation, eyelid swelling and ocular itching.The late phase of AC usually occurs 12-24 h after allergen exposure.In this phase, eosinophils and T-helper 2 cells infiltrate the conjunctiva and release cytokines, including IL-4, IL-5 and IL-13, which exacerbate and prolong the symptoms of AC (Magone MT et al., 1998;Hirakata et al., 2019a).
First-line AC treatment consists of topical anti-histamines and mast cell stabilizers.However, these treatments sometimes fail to provide satisfactory relief, particularly in the chronic phase.Although anti-inflammatory steroids are another option for the treatment of AC (Sacta et al., 2016), they have side effects, including increased intraocular pressure and infection (Miyazaki et al., 2020).Therefore, a more effective and safer treatment with a novel mechanism of action is required, especially for the control of chronic and severe AC.
Lipid mediators are small molecules derived mainly from ω-6 or ω-3 polyunsaturated fatty acids (PUFAs) via enzymatic or nonenzymatic oxidation processes.The lipid mediators produced regulate the progression and resolution of inflammation.The intake or treatment of ω-6 PUFA and its derivatives, lipid mediators such as prostaglandins and leukotrienes often represent pro-inflammatory or anti-inflammatory reactions in various types of diseases.Recent studies have focused on the anti-inflammatory effects of ω-3 PUFAs including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and their derivatives.Indeed, several studies have reported that dietary ω-3 fatty acids alleviate AC in mouse models (Hirakata et al., 2019b;Hamazaki et al., 2019).Hirakata et al. (2019a) showed that a ω-3 fatty acid diet reduced the levels of various inflammatory lipid mediators in the conjunctival tissue of AC mice.The DHA derivative resolvin D1 attenuates murine AC induced by an excretory-secretory protein of Acanthamoeba (Kang MS et al., 2021).However, the mechanism underlying the anti-inflammatory effects of ω-3 PUFAs and their derivatives remains unclear.
CYP450 monooxygenases catalyzes the epoxygenation of EPA.Epoxygenase products are then metabolized via soluble epoxide hydrolase (sEH) to form dihydroxy FAs such as DiHETE (Konkel and Schunck, 2011).Previously, our group found that the level of (±) 5( 6)-DiHETE increased in the colon of dextran sulfate sodium (DSS)-induced colitis mice and that post-administration of (±) 5(6)-DiHETE promoted recovery from colitis (Hamabata et al., 2018a;Hamabata et al., 2018b;Kobayashi et al., 2021a).We also found that treatment with (±)5(6)-DiHETE attenuated histamineinduced vascular hyperpermeability, at least partially, by inhibiting the activation of transient receptor potential vanilloid (TRPV) 4 channels (Hamabata et al., 2018a;Kobayashi et al., 2021a).TRPV4 is a non-selective cation channel responsible for sensing osmotic and mechanical signals and regulating calcium signaling across cell membranes.Accumulating evidence shows that TRPV4 activation is involved in various types of cells, including mast cells, vascular endothelial cells and nerve cells.These observations allowed us to hypothesize that the EPA derivative (±)5( 6)-DiHETE may attenuate the symptoms of AC.In this study, we aimed to investigate the therapeutic potential of (±) 5( 6)-DiHETE in pollen-or histamine-induced AC in mice.

Animals
Male BALB/c mice were purchased from CLEA Japan (Tokyo, Japan).Mice were maintained in isolated cages under a 12-h light/ 12-h dark photoperiod.Water and food were provided ad libitum.All experiments were approved by the Institutional Animal Care and Use Committee of the University of Tokyo.

Pollen-induced conjunctivitis model
Allergic conjunctivitis model was generated as described previously (Stern et al., 2005;Tanaka et al., 2015).Briefly, pollen from short ragweed (Polysciences, PA, United States) was emulsified with Imject Alum Adjuvant (Thermo Fisher Scientific, Tokyo, Japan).6-8 weeks old wild-type male BALB/c mice were sensitized on day 0 by applying 50 μL of pollen (50 μg pollen with 50 μL alum) into the left hind foot.Five days later (day 5), a second sensitization was performed by injecting the reagents into the left hind foot.On days 10-14, the eyes of the mice were topically challenged daily with pollen in saline (2 mg per 10 μL/eye).On day 14, 15 min after the final eye drop challenge, the tear volume was measured using the Schirmer tear test.Fifteen minutes after the Schirmer test, the eyeballs (with lids and conjunctival tissue) were collected.

May-Grunwald Giemsa staining
Dissected eyeballs (with lids and conjunctival tissue) were fixed in 4% paraformaldehyde (PFA) for 24 h and embedded in paraffin.Tissue sections (4 μm) were prepared and stained with May-Grunwald Giemsa.Briefly, the sections were immersed in May-Grunwald stain solution (FUJIFILM Wako, 1:20 in 1/150 M phosphate-buffer, pH6.7) for 5 min and then in diluted Giemsa solution (FUJIFILM Wako, 1:20 in H 2 O) for 5 min.May-Grunwald Giemsa-positive mast cells and eosinophils were counted in five randomly selected fields (×400 magnification) per section.

Histamine-induced conjunctivitis model
Histamine-induced conjunctivitis was induced as described previously, with some modifications (Mochizuki et al., 2022).Briefly, histamine (4 μg in 4 μL/eye, FUJIFILM Wako) was dropped onto the surface of the eye.Fifteen minutes after histamine application, tear volume was measured using the Schirmer tear test.To investigate vascular permeability, Evans blue (SIGMA, Tokyo, Japan, 10 mg/mL, 0.1 mL) was injected intravenously 5 min after histamine challenge.The eyeballs (with lids and conjunctival tissue) were harvested 15 min after the histamine challenge, dried at 55 °C and weighed.

Measurement of Evans blue concentration in the conjunctiva by Miles assay
The extravasated Evans blue present in the harvested eyeball was extracted for 18 h by placing the dissected eye tissues in 1 mL of 1 N KOH at 40 °C.After neutralization, the dye was extracted with acetone (7 mL).After drying in vacuo, the resulting residue was reconstituted in 25 μL DMSO.The amount of dye was estimated by measuring the absorbance at 620 nm.

Serotonin-induced scratching
The mice were acclimatized to the recording room for at least 1 h before the experiments.The scratching behavior was observed as described previously (Shimada and LaMotte, 2008;Kobayashi et al., 2021b).Briefly, 10 μg serotonin in 10 μL saline was injected intradermally (i.d.) into the mouse cheek.Mouse behavior was recorded for 30 min to assess scratching.One bout of scratching was defined as an episode in which a mouse lifted its hind paw and scratched continuously for any length of time until the paw returned to the floor or mouth for licking.We evaluated scratching behavior by counting the number of paw episodes brought to the cheek.HC-067047 (HC) was dissolved in 10% DMSO and 10% tween20 in saline.(±)5(6)-DiHETE (300 μg/kg) or HC (20 mg/kg) was intraperitoneally injected into mice immediately before serotonin injection.

Statistics
Statistical analyses were performed using Bell Curve for Excel software (Social Survey Research Information Co., Ltd.Tokyo, Japan).Statistical differences between the control and test groups were analyzed using Student's t-test for two-group comparisons and one-way ANOVA with Tukey's test for multiple-group comparisons.Data are expressed as the mean ± SEM.Statistical significance was defined as *p < 0.05, **p < 0.01.

Attenuation of allergic symptoms by (±) 5(6)-DiHETE
In the AC mouse model, early phase allergic symptoms occurred approximately 20 min after the antigen challenge (Magone MT et al., 1998).We first generated a murine model of AC induced by the repeated topical application of ragweed pollen to the eye.Mice were sensitized twice on days 0 and 5 by subcutaneously injecting pollen, then challenged five more times with eye drops containing pollen once daily from days 10-14 (Figure 1A). Figure 1B shows typical images of mouse eyes 20 min after the 5th pollen application.Pollen application induced conjunctivitis accompanied by eyelid edema, conjunctiva redness and tearing.Some mice (3 in 6) closed their eyes after pollen application, likely because of pain and itching.To evaluate the severity of AC symptoms, we compared clinical scores based on eyelid edema, redness, and tearing was scored (Figure 1C).The application of pollen significantly increased the score on day 14 (vehicle, 0.0 ± 0.0; pollen, 4.8 ± 0.4).Intraperitoneal administration of 300 μg/kg (±)5(6)-DiHETE or 2 mg/kg of DEX from day 10 to day 14 decreased increased clinical scores by 62.8% or 75.2%, respectively [(±) 5(6)-DiHETE, 1.8 ± 0.6; DEX, 1.2 ± 0.4].These treatments ameliorated pollen-induced eye closing.Schirmer's test was performed to evaluate the tear volume on day 14, at 15 min after the final pollen eye drop applications.We also confirmed that the administration of (±)5(6)-DiHETE did not change the appearance or behavior of mice 30 min after administration.The pollen application increased tearing by 190% compared with the vehicle application (vehicle, 1.65 ± 0.22 mm; pollen, 3.14 ± 0.14 mm) and intraperitoneal administration of (±) 5( 6)-DiHETE or DEX attenuated this increment by 64.8% or 110%, respectively [(±)5(6)-DiHETE, 2.18 ± 0.19 mm or DEX, 1.51 ± 0.17 mm] (Figure 1D).No significant differences between (±)5( 6)-DiHETE and DEX were observed in clinical score and tearing.These results suggest that (±)5( 6)-DiHETE suppressed a variety of allergic symptoms that occur in the early phases of AC.

Attenuation of the infiltration of eosinophils into the conjunctiva by (±)5(6)-DiHETE
In the early phase of onset of AC, mast cell degranulation releases inflammatory mediators, leading to the infiltration of eosinophils into the conjunctiva.Infiltrated eosinophils promote late-phase allergic symptoms by releasing pro-inflammatory mediators (Stone et al., 2010).The pollen applications increased the number of eosinophils in the conjunctiva by 309% on day 14 at 30 min after the 5th pollen application (vehicle, 24.4 ± 3.4 cells/mm 2 ; pollen, 75.3 ± 8.7 cells/mm 2 ) (Figure 2D).Intraperitoneal administration of (±)5(6)-DiHETE or DEX reduced the number of infiltrated eosinophils by 85.0% or 82.3%, respectively [(±)5(6)-DiHETE, 32.0 ± 3.6 cells/mm 2 or DEX, 33.4 ± 1.5 cells/mm 2 ].These results suggest that (±)5(6)-DiHETE inhibited mast cell degranulation, resulting in the reduction of eosinophil infiltration into the conjunctiva and suppression of late-phase symptoms of AC.These results suggest that the reduction in eosinophil infiltration was at least partially due to the inhibition of mast cell degranulation by (±)5(6)-DiHETE.
Activated mast cells produce large amounts of inflammatory mediators and promote eosinophil infiltration.The administration of (±)5(6)-DiHETE inhibited mast cell degranulation.The suppression of eosinophil infiltration observed at the same time is attributed, at least in part, to the suppression of mast cell degranulation by (±)5(6)-DiHETE.On the other hand, it is possible that (±)5(6)-DiHETE directly suppresses eosinophil activity and infiltration, and further studies are needed.
Although anti-histamines or mast cell stabilizers are often used to treat AC, their effects are sometimes insufficient to control AC symptoms, especially in the chronic phase.In addition, allergic contact dermatitis has been reported as an adverse event caused by the anti-histamine drug ketotifen fumarate contained in eye drops (Romita et al., 2020).Steroid drugs are also used to treat severe AC symptoms, but they have side effects, including elevation of intraocular pressure and infection (Miyazaki et al., 2020).Since (±) 5( 6)-DiHETE inhibited mast cell degranulation, vascular hyperpermeability and itching, it can be a new option to manage AC.Although many antigens are known to cause AC, we evaluated the effect of (±)5( 6)-DiHETE in a pollen-induced AC model.However, (±)5( 6)-DiHETE has proven beneficial for AC induced by antigens other than pollen, considering its therapeutic action mechanisms.
Supplementation with ω-3 PUFAs has been reported to be beneficial in conjunctivitis.However, a relatively high concentration of ω-3 PUFAs must be ingested over several months, leading to these therapeutic effects.Silva et al. (2018) reported that oral treatment with ω-3 PUFAs containing EPA and DHA (1 capsule of 500 mg/7 kg/day for 6 months) enhanced the effectiveness of topical tacrolimus for the treatment of keratoconjunctivitis sicca in dogs.Hirakata et al. (2019a) reported that a ω-3 diet containing 4% linseed oil (containing 60% linolenic acid) for 2 months alleviated allergic conjunctivitis induced by pollen.In this study, 300 μg/kg (±)5(6)-DiHETE attenuated the pollen-induced AC symptoms.Thus, (±)5( 6)-DiHETE may be superior to ω-3 fatty acids in that it can exhibit stronger and more immediate therapeutic effects.
Itching is a major complaint of patients in several allergic diseases.This greatly reduces the QOL of patients.Scratching exacerbates and prolongs allergic diseases by disrupting the epithelial barrier of the conjunctiva, nasal mucosa and the skin.Furthermore, we expect that this anti-itch effect can be applied in diseases other than AC.Most seasonal AC are caused by pollen and approximately 40% of seasonal AC patients develop allergic rhinitis (Yoshisue et al., 2022).Since blueback fish intestines contain (±) 5(6)-DiHETE abundantly (Kida et al., 2020), patients could manage allergic rhinitis by ingesting them in some form.
TRPV4, responsible for sensing osmotic and mechanical signals, is broadly expressed in various tissues; its activation increases intracellular Ca 2+ levels (Toft-Bertelsen and MacAulay, 2021).Ocular system including conjunctiva (Mergler S et al., 2012;Guarino BD et al., 2020), vascular endothelial cells (Köhler et al., 2006;Earley et al., 2009;Bagher et al., 2012), mast cells (Mascarenhas et al., 2017) and some neurons (Chen et al., 2013) express TRPV4.Some studies have suggested an association between TRPV4 expression and AC symptoms.TRPV4 inhibition reduced C48/80 induced-degranulation of human mast cells (Mascarenhas et al., 2017).Its inhibition also reduces eosinophilia in a toluene diisocyanate (TDI)-induced occupational asthma model (Yao et al., 2019).TRPV4 gene deficiency reduced carbachol-induced tear secretion in mice (Derouiche et al., 2018).It also scratched less after an intradermal injection of pruritogens such as histamine (Chen et al., 2016), serotonin (Akiyama et al., 2016) and C48/ 80 (Chen et al., 2016).Thus, TRPV4 is involved in various cell activation processes during the onset and progression of AC.It is reasonable to assume that the beneficial effects of (±)5(6)-DiHETE were mediated by the inhibition of TRPV4.Due to the multifactorial causes of AC, a combination of treatment is sometimes used to treat the symptoms of AC (Labib and Chigbu, 2022).(±)5( 6)-DiHETE may have possibility to act synergistically with other anti-inflammatory compounds.However, we cannot exclude the possibility of other mechanisms of action of (±) 5(6)-DiHETE.Further studies are required to confirm this hypothesis.
In summary, this study demonstrated that the administration of the EPA metabolite (±)5(6)-DiHETE is useful for suppressing the symptoms of allergic conjunctivitis in a mouse model.This lipid is abundant in fish organs (Kida et al., 2020).Therefore, it is necessary to study the administration of (±)5(6)-DiHETE including its extraction and utilization.
NN and TM wrote the manuscript.All authors contributed to the article and approved the submitted version.

FIGURE 1
FIGURE 1 Improvement of allergic symptoms by (±)5(6)-DiHETE (A) Protocol for pollen-induced AC. (B) Representative pictures of mouse eyes on day 14, at 15 min after the final pollen application.(C) The clinical scores at 20 min after the final pollen application.The total score consisted of the sum of scores for 3 factors (eyelid edema, redness and tearing) each scored from 0 to 3 points, depending on the severity (n = 5-6).Intraperitoneal administration of (±)5(6)-DiHETE (300 μg/kg) or dexamethasone (DEX, 2 mg/kg) from day 10 to day 14 reduced clinical scores.(D) Schirmer's test was performed to evaluate the tear volume at 15 min after the final pollen application (n = 5-7).Intraperitoneal administration of (±)5(6)-DiHETE (300 μg/kg) or DEX (2 mg/kg) from day 10 to day 14 reduced tear volume.Data are presented as a mean ± SEM. **p < 0.01.