Topical Application of Interleukin-28A Attenuates Allergic Conjunctivitis in an Ovalbumin-Induced Mouse Model

Abstract

Purpose: Allergic conjunctivitis (AC) is an immunoglobulin E (IgE)-mediated and helper T cell 2 (Th2)--cell–mediated disease characterized by conjunctival eosinophilic infiltration. Previous study shows that IL-28A had anti-allergic activity in airway disease. In this study, we examined the effect of IL-28A on a mouse model of ovalbumin (OVA)-induced experimental allergic conjunctivitis (EAC).

Methods: Mouse EAC was induced by topical application of OVA after intraperitoneal (IP) sensitization with OVA in aluminum hydroxide (ALUM). Interleukin-28A was administered 1 hour before OVA challenge. Allergic conjunctivitis symptoms, eosinophil infiltration in the conjunctiva, antigen-specific IgE in the serum, and Th2 cytokine production by lymph node cells and splenocytes were subsequently analyzed.

Results: Topical application of IL-28A to OVA-induced EAC reduced clinical symptoms, serum OVA-specific IgE, and the infiltration of eosinophils in the conjunctiva. In addition, topical administration of IL-28A suppressed the expression of IL-4, IL-5, and IL-13 (Th2-type cytokine) but promoted the expression of IFN-γ (Th1-type cytokine) by splenocytes and cervical lymph node cells in EAC mice. Immunofluorescence staining showed decrease expression of IL-4 and IL-5 in IL-28A–treated EAC conjunctiva.

Conclusions: Interleukin-28A shows therapeutic potential for allergic conjunctival inflammation.

The incidence of allergic conjunctivitis (AC), a type I allergy that ranges from mild (seasonal AC) to severe (vernal keratoconjunctivitis, VKC),¹ has been increasing rapidly during the past 2 decades.² The combination of immunoglobulin E (IgE)-mediated and helper T cell 2 (Th2)-cell–mediated responses are involved in the immunopathogenesis of allergy.^3,4 The IgE-mediated conjunctival allergic reaction is reproduced easily by specific allergens (mainly, pollen, animal dander, and house dust mites)⁵ and causes an early reaction followed by a predominant infiltration of eosinophilic cells.⁶ The release of eosinophil granule proteins is implicated in the pathogenesis of conjunctival inflammation.⁷ As the hallmark of allergic disease, eosinophils are readily found in large numbers in tears and tissues, and the cell number is significantly correlated with the clinical severity of the allergic response.⁸ 

The Th2 CD4⁺ T cells take part in the immune response to extracellular parasites and play an important role in the induction and persistence of allergic diseases such as asthma.^9,10 It has been reported that the number of CD4⁺ T cells in total conjunctival infiltrating cells in AC patients is less than 0.1%.¹¹ However, the frequency of CD4⁺ T cells in VKC patients is significantly higher than that in AC patients.¹¹ The high frequency of conjunctival CD4⁺ IL-4–producing T cells is also observed in AC patients, especially in those with proliferative changes in the conjunctiva.¹¹ The Th2 CD4⁺ T cells are involved in the inflammatory process in experimental allergic conjunctivitis (EAC) by cytokine releasing. Experimental AC in BALB/c mice was induced by adoptive transfer of CD4⁺ but not CD8⁺ cells from EAC mice, indicating that CD4⁺ T cells are the key in the pathogenesis of EAC.¹² Some studies show that the absence of CD4⁺ T cell subsets such as natural killer T (NKT) and γδ T cells attenuated the clinical features of EAC by adoptive transfer of CD4⁺ T cells to TCR-δ^−/− mice, Jα18^−/− mice, and CD1d^−/− mice.^13,14 Although a wealth of animal data support that allergy is caused by an increase in Th2 response in combination with a decrease in Th1 response, both Th1 and Th2 cytokines are increased in the blood and airways of patients.^15,16 

Interleukin-28A is a cytokine that shares the same Jak/Stat signaling pathway with type I interferon (IFN) to regulate the expression of a common set of genes.¹⁷ It is produced by antigen-presenting cells upon viral infection or Toll-like receptor (TLR) ligation.^18–21 The transcription of IL-28A is mainly regulated by nuclear factor (NF)-κB, interferon regulatory factor 3 (IRF3), and IRF7 and the latter is also upregulated by IFN stimulation, providing a positive feedback signal.²² Interleukin-28A acts through a cell surface receptor composed of interferon lambda receptor 1 (IFNLR1) and interleukin-10 receptor 2 (IL10R2). Activation of the signaling pathway induces IFN-stimulated gene factor 3 (ISGF), which consists of signal transducer and activator of transcription 1 (STAT1), STAT2, and IFN-regulatory factor 9 (IRF-9), to induce the expression of downstream target genes for antiviral responses.^23,24 Recent studies showed that in vitro IL-28A exhibits antiviral activity against EMCV, IAV, HCV, HSV1, and West Nile virus,^21,25–28 but not Lassa virus replication in monocyte-derived dendritic cells or macrophages.²⁹ Furthermore, IL-28A exhibits antiproliferative activity on BW5147 T-lymphoma, neuroendocrine BON1, and glioblastoma LN319 tumor cells.^30–32 Further studies also indicated that IL-28A shows an antitumoral effect likely mediated by an action on the host rather than through its antiproliferative activity on tumor cells in vivo.^33,34 A recent study showed that IL-28A activated conventional dendritic cells (DCs) rather than T cells to express IL-12 and thereby promoted Th1-cell differentiation but inhibited the Th2 response in a mouse asthma model.^12,35 Moreover, transgenic expression of IL-28A in vivo increases the skewing of Th1 responses and the severity of ConA-induced liver injury,¹⁹ indicating that IL-28A may suppress allergic diseases. 

Based on the multivalent activities of IL-28A on immune responses, in this study, we evaluated its anti-allergic effect with an EAC model. We found that topical application of IL-28A markedly reduced the clinical symptoms, IgE production, and eosinophil infiltration and inhibited Th2-type immune responses. Thus, IL-28A may have therapeutic potential for AC. 

Materials and Methods

Animals and Reagents

All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All mice were female BALB/c mice, aged 4 to 6 weeks. Animals were kept in a specific pathogen-free facility. Animal care and use were in compliance with institutional guidelines. 

Induction of EAC by Active Immunization

Model group and IL-28A group (200 ng/mL, 1000 ng/mL, 5000 ng/mL) were sensitized intraperitoneally (IP) with 100 μg of ovalbumin (OVA, grade V; Sigma-Aldrich Corp., St. Louis, MO, USA) and 200 μL 1.5% aluminum hydroxide (ALUM; Pierce, Rockford, IL, USA), and the control group was immunized with PBS and ALUM on days 0 and 7. Then, mice were challenged with 5 mL OVA (100 mg/mL in PBS) via eye drops from day 15 to 18 (Fig. 1A).^36,37 The IL-28A group (200 ng/mL, 1000 ng/mL, 5000 ng/mL) had 5 mL IL-28A applied topically to the eye 1 hour before the OVA challenge. Meanwhile, the same volume of PBS was used as a placebo in the control group and the model group. Photographs of clinical appearance and photographs in the conjunctiva were taken within 15 and 30 minutes after administration of the OVA eye drop, and the scores were counted in a masked fashion by 2 observers. Redness, tearing, discharge, and scratching behavior, based on the criteria, are described in the Table. Scratching behavior was monitored for 30 seconds, and the frequency of scratching was counted and evaluated as follows: 1 to 3 times, mild; 4 to 6 times, moderate; and more than 7 times, severe. A score was given for each eye, and the final results show the sum of scores across both eyes of each mouse. Scores shown in Figure 1B are the average of the total points for each mouse. Blood, eyes, spleen, and cervical lymph nodes (CLN) were collected 24 hours after the final challenge. 

Figure 1

 

Clinical symptoms in EAC mice. (A) Experimental protocol. BALB/c mice were injected IP with 100 μg OVA and 200 μL 1.5% ALUM, with control group immunized with PBS and ALUM on days 0 and 7. Interleukin-28A (200 ng/mL, 1000 ng/mL, 5000 ng/mL) was topically administered in IL-28A group, and PBS was used in control group and model group 1 hour before OVA challenge into conjunctival sac on days 15, 16, 17, and 18. (B) Eye appearance and clinical scores (mean ± SD) of the EAC mice, 15 to 30 minutes after challenge (n = 6 in each mouse group, 3 independent experiments).

Table

 

Grading Table for Clinical Symptoms

 

Evaluation of Eosinophilic Infiltration

The eyes, including eyelids and conjunctivae, were exenterated. After harvest, the samples were fixed in 10% buffered formalin, cut into horizontal 5-μm–thick sections, and subjected to hematoxylin and eosin (H&E) staining for detection of eosinophils.^36,37 Ovalbumin-specific AC in this model develops in an eosinophil-dependent manner.^37,38 The cell identity in H&E-stained tissue sections was determined by cytoplasm staining and nucleus morphology of the cells under light microscope. Eosinophils contain red granules in the cytoplasm, and nuclei are segmented. Lymphocytes have a round or slightly indented heterochromatic nucleus that almost fills the entire cell and a thin rim of basophilic cytoplasm that contains few granules. In each section, infiltrating cells in the lamina propria mucosae of the tarsal and bulbar conjunctivas were counted in a masked fashion by 2 observers.^36,37 The total number in 5 representative fields of 6 sections from each group was counted. The data are presented as mean ± SD per slide. 

ELISA for OVA-Specific IgE Antibody in Serum

Twenty-four hours after the final OVA challenge, mouse blood was collected, serum was prepared, and antigen-specific IgE was measured by ELISA. Briefly, the immuno plates (Nalge Nunc International, Naperville, IL, USA) were coated with OVA (1 mg/mL) overnight at 4°C. After blocking with 1% BSA in PBS for 1 hour at room temperature, serial dilutions of serum samples were added and incubated for 4 hours at room temperature. The plates were then washed with PBS plus 0.05% Tween (PBS/T) and incubated for 2 hours at room temperature with horseradish peroxidase (HRP)–conjugated rat anti-mouse IgE antibody (Southern Biotech, Birmingham, AL, USA). After a wash with PBS/T, color reaction was developed with 3,3′,5,5′-tetramethyl-benzidine (Moss, Inc., Pasadena, CA, USA) and stopped with 0.1 N HCl.^37,38 The absorbance in each well was determined by spectrophotometry at the dual wavelengths of 570 and 630 nm on a microplate reader (Pharmacia, Uppsala, Sweden). 

Lymphoid Cell Culture and Cytokine Analysis

Spleens and CLN were harvested, and a single cell suspension was cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 mM 2- mercaptopurine, and 10% heat-inactivated fetal calf serum (all from Invitrogen Life Technologies, Carlsbad, CA, USA). Cells were cultured at 5×10⁶ cells/mL with 1 mg/mL OVA for 96 hours in 96-well plates (Nunc, Rochester, NY, USA). The Th2 cytokine levels (including IL-4, IL-5, and IL-13) and Th1 cytokine levels (including IFN-γ) of culture supernatants were assayed using a Cytometric Bead Array Mouse Th1/Th2 Cytokine Kit (BD Pharmingen, San Diego, CA, USA). 

Immunohistochemical Analysis

The eyes, including eyelids and conjunctivae, were fixed in 10% buffered formalin and cut into horizontal 5-μm–thick sections. The 5-μm–thick sections were dewaxed by immersion in xylene (twice for 5 minutes each time) and hydrated by serial immersion in 100%, 90%, 80%, and 70% ethanol and PBS. Antigen retrieval was performed by microwaving sections for 20 minutes in Target Retrieval Solution (DAKO, Carpinteria, CA, USA). Sections were washed with PBS (twice for 10 minutes each time), and blocking buffer (10% BSA in PBS) was added for 1 hour. Sections were incubated with primary antibodies in blocking buffer overnight at 4°C. The primary antibodies used for this study include rat anti-mouse IL-4 (clone 11B11) from BD Pharmingen and rat anti-mouse IL-5 (clone TRFK5) from BioLegend (San Diego, CA, USA). After extensive washing, the sections were incubated with AlexaFluor 488–labeled goat anti-mouse antibody (1:200; Molecular Probes, Eugene, OR, USA) for 1 hour. The sections were washed with 0.01 M PBS 3 times and mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The sections were viewed using fluorescence microscopy (DMI3000 B; Leica, Wetzlar, Germany). Staining intensity of IL-4– and IL-5–expressing cells in the entire area was evaluated using Image-Pro plus 6.0 software (Media Cybernetics, USA). The expression intensity of IL-4 and IL-5 was determined based on the integrated optical density (IOD). 

Reproducibility and Statistical Analysis

Experiments were repeated at least 3 times. Results were highly reproducible. Representative results are shown in the figures. The means and SEM were calculated on all parameters determined in the study. Data were analyzed statistically using 1-way ANOVA or 2-tailed Student's t-test. A value of P < 0.05 was accepted as statistically significant. 

Results

Reduction in Clinical Symptoms of EAC by Topical Application of IL-28A

To investigate the topical effect of IL-28A on AC, BALB/c mice were sensitized by IP injection with OVA and ALUM on days 0 and 7. Five microliters IL-28A (200 ng/mL, 1000 ng/mL, and 2000 ng/mL) were topically applied to the eye 1 hour before the OVA challenge on days 15, 16, 17, and 18 (Fig. 1A). The clinical symptoms in EAC were scored immediately after the administration of eye drops. Experimental AC mice displayed clear clinical symptoms of conjunctivitis (Fig. 1B), as obvious hyperemia and edema in the conjunctiva were observed compared with the control mice. Figure 1B shows that topical treatment of IL-28A on EAC mice reduced the clinical signs and EAC scores of conjunctivitis in comparison with model group. Thus, IL-28A on a dose-dependent basis inhibits OVA-induced EAC. 

Topical Application of IL-28A Reduced OVA-Induced Allergic Conjunctival Inflammation

Since eosinophils are essential in allergic inflammation and changes in the conjunctival mucosa, we next examined the infiltration of eosinophils in the EAC mice. Histopathologic study showed significant infiltration of eosinophils in the conjunctiva after OVA challenge in the control group (Fig. 2). However, topical application of IL-28A markedly reduced the infiltration of eosinophils in conjunctiva in EAC mice. Eosinophil counts in the IL-28A–treated group were significantly reduced in a dose-dependent manner (200 ng/mL, 1000 ng/mL, 2000 ng/mL; Fig. 2). These results indicate an inhibitory effect of IL-28A on allergic inflammation in OVA-induced EAC. 

Figure 2

 

Interleukin-28A attenuates the allergic conjunctival inflammation. Corresponding paraffin sections from IL-28A–treated conjunctival tissues were stained with H&E (original magnification ×400). Images were taken and eosinophil infiltration quantified. Data represent the mean ± SD from 3 independent experiments. *P < 0.05 compared with mice in the control group (n = 6 in each group; Scale bars: 20 μm).

Reduced Serum Level of OVA-Specific IgE and Eotaxin in IL-28A–Treated EAC Mice

Immunoglobulin E plays a major role in allergic diseases. We determined the level of OVA-specific IgE antibody in IL-28A–treated mice. A combination of systemic priming and local boosting with OVA resulted in significantly higher levels of OVA-specific IgE antibody in the serum (Fig. 3A). Significantly lower OVA-specific IgE level was observed in the IL-28A–treated group (Fig. 3A). Eotaxin is a chemoattractant for eosinophils, and the eosinophil counts in the IL-28A–treated group were reduced. Similarly, the eotaxin level in serum was upregulated in the model group (Fig. 3B). However, a lower eotaxin level was seen in the IL-28A–treated group (Fig. 3B). These data suggest that IL-28A suppresses allergen-specific IgE production and the recruitment of eosinophils in OVA-induced EAC. 

Figure 3

 

Reduction in serum OVA-IgE and eotaxin in IL-28A–treated EAC mice. Interleukin-28A or PBS was topically applied 1 hour before OVA challenge in EAC mice. OVA-specific IgE and eotaxin in serum were analyzed by ELISA. Data represent the mean ± SD from 3 independent experiments. *P < 0.05 compared with mice in the control group (n = 6 in each group).

The Effect of IL-28A on Th2 Type Cytokine Responses in EAC Mice

To determine the mechanisms of IL-28A on EAC inhibition, we analyzed Th1-type and Th2-type cytokines in IL-28A–treated EAC mice. Immunostaining data of the conjunctival stroma showed that infiltration of IL-4– and IL-5–producing cells was increased after OVA challenge in the model group (Fig. 4). However, topical treatment of IL-28A attenuated infiltration of IL-4– and IL-5–producing cells in OVA-induced AC (Fig. 4). Similar to the expression of conjunctiva, the level of OVA-specific Th2-type cytokines (IL-4, IL-5, and IL-13) was increased, but the level of Th1-type cytokine (IFN-γ) was decreased in the model group (Fig. 5). However, the expression of Th2-type cytokines was decreased in IL-28A–treated EAC mice (Fig. 5). These data demonstrate that topical application of IL-28A inhibits Th2-type response to alleviate OVA-induced AC. 

Figure 4

 

Interleukin-28A administration (5000 ng/mL) decreases the infiltration of IL-4– and IL-5–producing cells in EAC mice. Corresponding paraffin sections from treated conjunctival tissues are immunostained with anti-IL-4 or anti-IL-5 Ab (original magnification ×100). Insets show higher magnification of the IL-4– and IL-5–producing cells. Staining intensity of IL-4– and IL-5–expressing cells in the entire area was evaluated using Image-Pro plus 6.0 software. The expression of IL-4 and IL-5 was determined based on the IOD. Data represent the mean ± SD from 3 independent experiments. Scale bars: 40 μm.

Figure 5

 

Interleukin-28A reduces Th2-type immune responses in OVA-induced EAC mice. Splenocytes and CLN were harvested on day 19 from EAC mice and cultured with 1 mg/mL OVA in vitro. Culture supernatants were collected at 48 hours and cytokine concentration was determined using the Cytometric Bead Array Cytokine kit. Data represent the mean ± SD from 3 independent experiments. *P < 0.05 (n = 6 in each group).

Discussion

Although AC is considered the most benign condition of all ocular allergic diseases, it may lower the quality of life of patients and increase considerably their economic expense.³⁹ Treatment with mast-cell stabilizers and corticosteroids have sufficient efficacy but leads to unpleasant side effects. Thus, many therapies with immune-modulators and antimediator-release have been tried, but none of them has demonstrated satisfaction for AC.¹² Recent studies showed that CC chemokine receptors (CCRs) play crucial roles in the development of allergic conjunctival inflammation in the EAC model. The absence of CCR6 inhibited IgE secretion and allergic conjunctival inflammation in EAC mice.⁴⁰ In addition, blockade of CCR7 attenuated allergic reaction in EAC mice.⁴¹ The absence of CCRs resulted in the inhibition of Th2 reactivity to attenuate AC in EAC mice. Our present study provided evidence that IL-28A has an anti-allergic effect on OVA-induced mouse EAC by reducing Th2-driven allergic conjunctival inflammation and production of antigen-specific IgE antibody, as well as recruitment of eosinophils. 

Increased levels of serum IgE antibody and infiltration of eosinophils into the conjunctiva are the main pathologic changes in the conjunctival allergic reaction.⁴² The IgE binds to the membrane of conjunctival mast cells in the early phase response (EPR) when a new contact with the allergen occurs. It then triggers an immediate allergic response by releasing mediators such as IL-4, IL-5, and IL-13.³⁹ The late-phase response (LPR) is dependent primarily on the recruitment and activation of eosinophils and T-lymphocytes.^12,37 The increase in the IgE antibody level in serum and the infiltration of eosinophils into the conjunctiva in EAC mice are thought to be mediated by Th2-type cells, which preferentially produce IgE-enhancing cytokines such as IL-4 and IL-5.^4,12 However, the mechanisms of the LPR are not fully understood. 

Interleukin-4, IL-5, and IL-13 are Th2-type cytokines that are involved in many aspects of allergic pathophysiology. Interleukin-4–induced STAT6 upregulates the expression of the master regulator GATA3 (GATA-binding protein)⁴³ and Th2 differentiation, selective proliferation of Th2 cells through recruitment of Gfi-1 and inhibition of Th1-cell differentiation presumably by interacting with T-box transcription factor (T-bet).⁴⁴ Also, IL-4 is essential for IgE production.⁴⁵ Interleukin-5 is required for the attraction of eosinophil granulocytes,⁴⁶ and IL-13 regulates the airway response in asthma.⁴⁷ Our study shows that topical treatment with IL-28A attenuates the infiltration of IL-4– and IL-5–producing cells in OVA-induced allergic conjunctiva. Although it is not clear whether T cells or eosinophils expressed IL-4 and IL-5 in the conjunctival tissues in our study, recent studies have implicated both T cells and eosinophils in IL-4 and IL-5 production in LPR of AC. Eosinophils secreted IL-4, IL-5, IFN-γ, and other cytokines through the regulation of vesicular-based process from intracellular stores of preformed cytokines.⁴⁸ Furthermore, allergen-specific Th2-type lymphocytes play significant roles in the immune-pathophysiology of allergy because of the ability to produce IL-4, IL-5, and IL-13. Thus, Th2 cytokines are involved in IgE production and eosinophil activation to promote the allergic response.³⁹ 

Interleukin-28A suppresses the allergic response by shifting a Th2 to a Th1 cytokine profile and induction of IFN-γ.³⁵ However, IL-28A–induced immune shifting to a Th1 cytokine profile was regulated by conventional DC rather than by its direct effect on T cells.³⁵ Dendritic cells upregulate the expression of IL-28RA and respond to IL-28A treatment to promote the expression of IL-28 target genes and IL-12.³⁵ Interleukin-12 is a potent inducer of Th1 cytokine, which directly inhibits Th2 responses and promotes the production of IFN-γ.^12,35 T cell–derived IFN-γ is essential for IL-28A–mediated inhibition of Th2 responses and the allergic response by suppressing Th2 cytokine production, eosinophilia, neutrophilia, goblet cell metaplasia, and airway hyperresponsiveness (ARH).^35,49 Interferon-γ is the critical cytokine for developing Th1 cells via T-bet,⁵⁰ which was found to be dependent on STAT1 activated by IFN-γ.^51,52 T-bet is defined not only by its ability to activate a set of genes to promote differentiation of Th1 but also by being able to suppress the development of Th2 and Th17 responses.^52,53 Our data similarly show that IL-28A inhibited the expression of IL-4, IL-5, and IL-13 but enhanced the expression of IFN-γ in OVA-induced EAC (Figs. 4, 5). Our study thus indicates that IL-28A promotes the differentiation of Th1 but suppresses the development of Th2 to inhibit OVA-induced AC. 

In conclusion, topical application of IL-28A attenuates allergic conjunctival inflammation by reducing eosinophil infiltration and serum OVA-specific IgE antibody in OVA-induced EAC. Interleukin-28A promotes the differentiation of Th1 but suppresses the development of Th2 in EAC. Thus, IL-28A may have therapeutic potential for AC. 

Acknowledgments

The authors thank Ji Ming Wang, National Cancer Institute, National Institutes of Health for his critique of the manuscript. 

Supported in part by Grant 31471122 from the National Natural Science Foundation of China and Grant 2014A030313047 from the Science and Technology Planning Project of Guangdong Province, China. 

The authors alone are responsible for the content and writing of the paper. 

Disclosure: J. Chen, None; J. Zhang, None; R. Zhao, None; J. Jin, None; Y. Yu, None; W. Li, None; W. Wang, None; H. Zhou, None; S.B. Su, None