ABSTRACT
Purpose: To determine the immunologic functions of TRPA1 or TRPV1 in allergic conjunctivitis (AC).
Methods: Mice were sensitized with ovalbumin (OVA), after which TRPA1 antagonist or TRPV1 antagonist was administered before topical OVA challenge. Expression of TRPV1 or TRPA1 in AC was examined by western blotting and multicolor immunofluorescence. Clinical signs, OVAspeciic IgE, iniltration of inflammatory cells into conjunctivae (CJs), and Th2 cytokine in draining lymph nodes (LNs) were evaluated by microscopy, flow cytometry, and ELISA.
Results: TRPV1 expression was increased in CJs and LNs from AC mice, but TRPA1 expression was only increased in LNs. TRPV1 antagonist but not TRPA1 antagonist attenuated the clinical signs of AC and OVAspeciic IgEin sera. TRPV1 antagonist furthermore inhibited the iniltration of inflammatory cells into CJ and the production of Th2 cytokines in LNs.
Conclusion: TRPV1 antagonist but not TRPA1 antagonist may ameliorate AC by suppressing the Th2 response in LNs. Keywords: Allergic conjunctivitis, inflammatory cells, Th2 cytokines, TRPA1, TRPV1
Introduction
The clinical signs of allergic conjunctivitis (AC) include lid swelling, conjunctivalchemosis, conjunctival redness, and tearing.1 A compound IgEmediated and type 2 immunemediated response associated withinflammatory conditions are responsible for these symptoms,2–4 and this IgEmediated AC response is produced by a speciic conjunctiva (CJ) stimulus,5 which results in a response marked by the iniltration of inflammatory cells, such as eosinophils and mast cells.6,7 These type 2 immune reactions are dominated by the activation of CD4+ Th2 cells and the production of type 2 immune reactionrelated cytokines, such as IL4 and IL13.8
In mammals, the transient receptor potential (TRP) family of ion channels located on the plasma membrane act as receptors for stimuli. The TRP family consists of six subfamilies: TRPC (canonical: TRPC1~TRPC7); TRPV (vanilloid: TRPV1~TRPV6); TRPM (melastatin: TRPM1~TRPM8); TRPP (polycystin: TRPP2, TRPP3, TRPP5); TRPML (mucolipin: TRPML1~TRPML3); and TRPA (ankyrin: TRPA1).9 Among the TRP family, the immunologic functions of TRPA1 or TRPV1 have been researched in terms of their immunologic functions in the context of allergic disease. In allergic asthma, TRPA1 or TRPV1 yielded increased Th2 cytokine levels and induced the iniltration of eosinophils into the lungs.10,11 11 In an allergic dermatitis model, TRPA1 or TRPV1 induced the iniltration of inflammatory cells into the skin, increased Th2 cytokine levels, and provoked itching.12–14 In allergic rhinitis, TRPA1 or TRPV1 increased Th2 cytokine levels and promoted histaminemediated itching. The increased levels of Th2 cytokines also led to the iniltration of inflammatory cells.15,16
The immunologic functions of TRPA1 or TRPV1 and the mechanisms of their actions Immune function in AC, however, remain unknown. In this study, the immunologic functions of TRPA1 or TRPV1 in an ovalbumin (OVA)sensitized AC murine model were investigated using TRPA1 antagonist or TRPV1 antagonist.
MATERIALS AND METHODS
Animals
Sevenweekold BALB/c male mice were purchased from Orient Bio Inc. (Sungnam, Korea). Mice were cared for in a speciic pathogenfree environment at the animal facility, in accordance with the Catholic Institutional Animal Care and Use Committee (IACUC) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. A mixture of ketamine/xylazine suspensions (120 and 20 mg/kg administered intraperitoneally, respectively) was used for all surgical procedures.
AC Model
The murine model of AC was established as previously described.17 Briefly, BALB/c mice were immunized by a single intraperitoneal injection with 100 μgOVA in 100 μL PBS that included 1 mg aluminum hydroxide (Alum) and 300 ng pertussis toxin (all purchased from Sigma Aldrich,St. Louis, MO). The mice were sensitized for 2 weeks and then challenged by topical OVA eye drops once daily for at least 13 days.
The clinical evaluation of AC, which involved checking for immediate hypersensitivity responses 10 min posttopical OVA challenge, was conducted in a masked fashion by two independent observers. Clinical scoring consisted of four parameters: lid swelling, conjunctivalchemosis, conjunctival redness, and tearing. Each limitation was scored on a scale from 0 (absence of signs) to 3 (maximal), and the sum of the scores for the four parameters yielded the total score for each animal.18,19
Topical Administration of TRPA1 Antagonist or TRPV1 Antagonist
The TRPA1 antagonist, HC030031 (Abcam, Cambridge, MA), was diluted to 10 mM in 1% dimethyl sulfoxide (DMSO) and PBS. The TRPV1 antagonist, Capsazepine (R&D Systems, Minneapolis, MN), was diluted to 10 mM in 1% DMSO and PBS. The vehicle control consisted of 1% DMSO in PBS. TRPA1 antagonist, TRPV1 antagonist, or vehicle eye drops were administered 20 min before topical OVA eye drops (500 μg/mL) once daily to prevent the dilution of TRPA1 antagonist, TRPV1 antagonist, or vehicle and OVA.
ELISA for OVAspeciic IgE in Serum
Following topical challenges for 13 days, blood was collected from mice euthanized by cardiac puncture. Sera were isolated via coagulation and centrifugation before being analyzed using an ELISA kit for OVAspeciic IgE according to the manufacturer’s instructions (AbD Serotec, Raleigh, NC).
Western Blot Analysis
Following topical challenges for 13 days, CJs and ipsilateral cervical lymph nodes (CLNs) were collected from naive (untreated) mice or AC mice. Proteins were extracted from each tissue using RIPA buffer and were quantiied using a bovine serum albumin (BSA) standard curve, after which concentrations were adjusted to allow for equal loading. Together with appropriate markers, the proteins were subjected to 10% SDS PAGE and were then transferred onto PVDF membrane. The membranes were blocked for 1 h with 3% BSA (in PBST; PBS containing 0.5% Tween 20) before being incubated overnight at 4ºC with antiTRPA1 or antiTRPV1 (1:500) antibody diluted in the 3% BSA solution. After being washed with PBST, the membranes were incubated with antirabbit IgGHRP (1:1000 dilution in 3% BSA) at room temperature for 1 h. Finally, speciic protein bands were visualized using chemiluminescent HRP substrate solution.
Immunoluorescence
Following topical challenges for 13 days, ipsilateral whole eyes including CJs and CLNs were collected from naive (untreated) mice or AC mice. Cryosections (7 μm) were air dried for 30 min and washed with PBS ive times (3 min/ wash). The CJs and CLNs were blocked with 1% BSA in PBS at room temperature for 1 h. The CJs were incubated in 1% BSA in PBS with mixtures of antiTRPA1 antibody (or antiTRPV1 antibody) and Alexa Fluor 488conjugated antimouse CD4 antibody (BioLegend, San Diego, CA) overnight at 4ºC. The CLNs were incubated in 1% BSA in PBS with mixtures of antiTRPA1 antibody (or antiTRPV1 antibody), Alexa Fluor 488conjugated antimouse CD4 antibody and Paciic Blueconjugated antimouse/ human CD45R/B220 antibody (BioLegend) overnight at 4ºC. The CJs and CLNs were incubated in BSA in PBS with mixtures of secondary antibodies (Dylight 594conjugated goat antirabbit polyclonal antibody) for 1 h at room temperature.
Quantitation of Conjunctival Mast Cells and Eosinophils
As described previously,17,19 mice were euthanized 20 min after the challenge and CJs were collected. The conjunctival tissues were digested with 2 mg/mL collagenase (Roche, Mannheim, Germany) and 0.05 mg/ mL DNase I (Roche). The suspensions were iltered through a 70μm cell strainer and washed carefully. The cells were resuspended in 0.5% FBS buffer and treated with antiFcR (CD16/CD32, BioLegend) antibodies to block Fcmediated reactions, as per the manufacturer’s instructions. The cells were subsequently stained with an Alexa Fluor 488conjugated antiCD45 antibody (BioLegend), a PECy7conjugated antickit (BioLegend) antibody, and a PEconjugated sialic acidbinding immunoglobulintype lectinF (SiglecF; BD Biosciences, East Rutherford, NJ) antibody. The isotype control was stained with appropriate matched antibodies (BioLegend). Additionally, all samples were analyzed using an LSRFortessa flow cytometer (BD Biosciences).
Measurement of Tcell Responses to Recall Allergen Stimulation
Single cells fromCLNs collectedfromeuthanizedmice17,19 were plated on 96 wells, as indicated above. Following 72 h of OVA stimulation,the cultures were restimulated with phorbol myristate acetate/ionomycin (Sigma Aldrich, St. Louis, MO) for up to 6 h. The IL4 and IL13 cytokines in the collected supernatant were quantiied by ELISA as per the manufacturer’s instructions (Readysetgo ELISA kit, eBioscience, San Diego, CA).
Statistical Analyses
Data are expressed as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Differences between groups were analyzed by analysis of variance (ANOVA) or twotailed Student’s ttest as indicated. Differences with p values <0.05 were considered statistically signiicant. RESULTS TRPV1 Antagonist Reduced Clinical Signs of Allergic Reaction First, we aimed to clarify whether topical application of the TRPA1 antagonist or TRPV1 antagonist modulates immune reactions in AC. In a previous study, we demonstrated AC reactions to topical OVA eye drops in mice that were sensitized by intraperitoneal injection of OVA.17,19 In this study, topical TRPA1 antagonist, TRPV1 antagonist, or vehicle eye drops were applied with topical OVA challenge once daily to OVAsensitized mice for 13 days. To evaluate AC Cathodic photoelectrochemical biosensor responses, clinical signs of AC were scored about 10min postchallenge.18,19 The results from this experiment demonstrated that the topical TRPA1 antagonisttreated mice did not have improved clinical AC symptoms compared with the vehicletreated mice, while topical TRPV1 antagonisttreated mice showed an improvement in clinical AC symptoms compared with the vehicletreated mice (Figure 1). The TRPV1 antagonist but not the TRPA1 agonist was thus shown to suppress the clinical signs of AC.
TRPV1 Antagonist Suppresses Serum OVAspeciic IgE Levels in AC
Next, the OVAspeciic IgE levels in the sera of mice were assessed (Figure 2) and it was shown that OVAspeciic IgE levels in TRPV1 antagonisttreated mice were signiicantly lower than those in the vehicletreated mice (p=0.0017 vs vehicle). The levels of OVAspeciic IgE were furthermore shown to be lower in TRPV1 antagonisttreated mice than in TRPA1 antagonisttreated mice (p=0.0016 vs TRPA1 antagonist).
Expression of TRPA1 or TRPV1 in CJs and Lymph Nodes (LNs)
In this study, the expression levels of TRPA1 or TRPV1 were assessed in naive (untreated) mice and in AC mice (topical OVA in OVAsensitized mice). Western blotting showed that the expression of TRPA1 was signiicantly higher in the LNs of AC mice than in the LNs of naive mice (p=0.011). TRPV1 expression was, moreover, found to be signiicantly higher in the CJs and LNs of AC mice compared with the CJs (p=0.019) and LNs (p=0.007) of naive mice (Figure 3). To demonstrate the localization of the expression of TRPA1 or TRPV1 and CD4+ T cells in the CJs and LNs of AC mice and naive mice, multicolor immunofluorescence staining was performed (Figure 4). The immunofluorescence staining revealed that the expression level of TRPA1 or TRPV1 was increased in the CJs and LNs of AC mice. Interestingly, TRPV1 exhibited colocalization with the CD4+ T cell marker in the CJs and LNs of AC mice.
Iniltration of Inlammatory Cells in CJ
To determine the functions of the TRPA1 antagonist or TRPV1 antagonist in the conjunctivalinflammatory reaction in AC, CJs were collected and the CD45+ cells (Figure 5A), eosinophils (CD45+ SiglecF+; Figure 5B), and mast cells (CD45+ ckit+; Figure 5C) in the CJs were counted using flow cytometry. TRPA1 antagonisttreated mice showed signiicantly reduced iniltration of mast cells (p=0.019 vs vehicle) into CJs relative to the vehicletreated mice (Figure 5D). TRPV1 antagonisttreated mice exhibited signiicantly reduced iniltration of CD45+ cells (p<0.0001 vs vehicle), eosinophils (p=0.001 vs vehicle), and mast cells (p=0.015 vs vehicle) into CJs relative to the vehicletreated mice (Figure 5D). The TRPV1 antagonisttreated mice furthermore displayed signiicantly reduced iniltration of CD45+ (p<0.0001 vs TRPA1 antagonist) cells and eosinophils (p=0.007 vs TRPA1 antagonist) into CJs relative to the TRPA1 antagonisttreated mice (Figure 5D). These indings indicate that the TRPA1 antagonist did not inhibit the iniltration of eosinophils in the conjunctivalinflammatory reaction in AC, but that the TRPV1 antagonist inhibited the iniltration of inflammatory cells in the conjunctivalinflammatory reaction in AC. Regulating the Level of Th2 Cytokines in LNs To determine whether the TRPA1 antagonist or TRPV1 antagonist led to allergic response regulation in AC, drained LNs were collected 13 days after daily TRPA1 antagonist or TRPV1 antagonist and OVA administration, and puriied T cells were stimulated with OVA in vitro. Secretions of Th2 cytokines, such as IL4 and IL13 into the supernatants were analyzed by ELISA (Figure 6). The level of IL4 in TRPA1 antagonisttreated mice was not signiicantly reduced compared with that in the vehicletreated mice, while the level of IL13 was signiicantly lower in TRPA1 antagonisttreated mice compared with vehicletreated mice (p=0.0095 vs vehicle). In TRPV1 antagonisttreated mice, the levels of Th2 cytokines such as IL4 (p=0.0003 vs vehicle) and IL13 (p<0.0001 vs vehicle) were signiicantly reduced compared with those in the vehicletreated mice. The levels of Th2 cytokines such as IL4 (p=0.0004 vs TRPA1 antagonist) and IL13 (p=0.0083 vs TRPA1 antagonist) were furthermore signiicantly lower in TRPV1 antagonisttreated mice than in TRPA1 antagonisttreated mice. DISCUSSION Allergic conjunctivitis (AC) responses involve augmented levels of IgE in sera and the iniltration of eosinophils and mast cells into CJ, mediated by Th2 cytokines.20,21 The TRPA1 or TRPV1 has been studied in terms of their immunologic functions in allergic diseases. In allergy, increased calcium ion levels were shown to activate TRPA1 or TRPV1.16 The activated TRPA1 or TRPV1 have been shown to induce the iniltration of inflammatory cells into tissues and to increase Th2 cytokine levels.10,12 In this study, the immunologic functions of TRPA1 or TRPV1 were assessed using TRPA1 antagonist or TRPV1 antagonist in an OVAsensitized AC murine model. The topical application of the TRPA1 antagonist did not relieve the clinical symptoms of AC in this murine model and did not decrease the serum levels of OVAspeciic IgE. In contrast, the topical application of the TRPV1 antagonist relieved the clinical symptoms of AC in this murine model and decreased the serum levels of OVAspeciic IgE. In a recent study on the sensory neural mechanisms of AC, particularly on whether the TRPA1 antagonist or TRPV1 antagonist is associated with the clinical symptoms of AC, TRPA1 antagonisttreated rats showed reduced blinking without changes in tearing, while the TRPV1 antagonisttreated rats showed a reduction in both blinking and tearing.22 Together with the present indings, this indicates that TRPV1 plays a more important role in regulating the clinical symptoms of AC compared with TRPA1. Expression levels and protein localization in the CJs and CLNs of naive mice or AC mice were assessed and it was found that, compared with the expression levels in naive mice, TRPA1 expression was elevated in the CLNs of AC mice and TRPV1 expression was elevated in the CJs and CLNs of AC mice. It was furthermore revealed that TRPV1 colocalized with the CD4+ T cell in the CJs and LNs of AC mice. In agreement with these indings, Bertin et al. showed that TRPV1 is functionally expressed in CD4+ T cells and plays a crucial role of in the activation and acquirement of inflammatory character in CD4+ T cells.23 The topical application of TRPA1 antagonist resulted in the inhibited iniltration of mast cells into the CJ, but did not block the iniltration of eosinophils into the CJ. The topical application of TRPV1 antagonist, on the other hand, blocked the iniltration of eosinophils and mast cells into the CJ. In previous studies, the TRPA1 and TRPV1 receptors were shown to be expressed in mast cells. Chen and Hackos showed that the TRPA1 antagonist inhibits the iniltration of mast cells and attenuates itchscratching behavior,24 and van Diest et al. showed that the TRPV1 antagonist prevents the iniltration of mast HOpic ic50 cells and pain responses.25 Jian et al. furthermore showed that the H4 receptor was activated by TRPV1, but not by TRPA1, and that the H4 receptor in terms of histaminedependent itching was expressed mainly in eosinophils.26 Previous studies demonstrated that both the iniltration into tissues and inflammatory functions of mast cells were mediated by IL13 in allergy model and IL4 participated in eosinophil iniltration in a murine allergy model.27,28 In the present study, the TRPA1 antagonist was shown to decrease IL13 levels and to suppress the iniltration of mast cells, as previously reported. TRPA1 antagonist, however, did not suppress IL4 levels in the T cells of LNs and eosinophil iniltration in CJs.
TRPV1 antagonist not only inhibited the infiltration of eosinophils and mast cells, but also attenuated IL4 and IL13 in the T cells of CLN in this study. Rehman et al. reported that the siRNAmediated knockdown of TRPV1 attenuated allergic inflammation in IL13,29 and Biro et al. showed that the TRPV1 antagonist inhibited the induction of IL4 production in murine mast cells.30 Ramachandran et al. reported that TRPV1 antagonisttreated mice exhibited decreased allergic inflammation and decreased Th2 cytokine production, demonstrating that TRPV1 inhibition reduced allergic inflammation.31
The CJ is supplied by capillaries and lymphatics32 and the administration of drugs for AC may thus be cleared through blood and lymph. The conjunctival blood vessels do not form a tight junction barrier,33 which means that small molecule drugs can penetrate the blood circulation by pinocytosis and/or convective transport through paracellular pores in the vascular endothelial layer. The conjunctival lymphatics act as an efflux system for efficient elimination from the CJ. In a previous study, at least 10% of a small molecule (sodium fluorescein) compound administered in the subconjunctiva was shown to be eliminated into LNs and into the systemic circulation via the lymphatics within the irst hour in rat eyes.34 The topical administration of β1,3glucan was furthermore shown to suppress the systemic immune responses including serum IgE and the production of Th2 cytokines by T cells in LNs in a murine model of AC.19 Drugs transported by lymphatics in conjunction with the elimination by blood circulation may therefore contribute to systemic exposure via the interstitial fluid returned to the systemic circulation after iltration through LN.
In this study, the immunologic function of TRPV1 was assessed using a TRPV1 antagonist, which was shown to inhibit the iniltration of eosinophils and mast cells by inhibiting Th2 response, speciically by decreasing the levels of IL4 and IL13 in CLNs. The TRPV1 antagonist, and not the TRPA1 antagonist, was thus shown to suppress allergic responses in the OVAsensitized AC murine model. Although further studies are required to understand the mechanisms of TRPA1 or TRPV1 in AC, the indings reported here suggest that topical TRPV1 antagonist eye drops may be a novel therapeutic drug for regulating Th2 immune reactions, including the iniltration of eosinophils and mast cells into the CJ.