Blocking Th2 Signaling Pathway Alleviates the Clinical Symptoms and Inflammation in Allergic Conjunctivitis

Abstract

Purpose: To explore the role of Th2 signaling pathway in allergic conjunctivitis (AC).

Methods: Serum Th2 cytokines IL-4 or IL-13 of patients with AC were detected using the Meso scale discovery assay to verify the correlation of Th2 immunity and AC pathogenesis. Wistar Han rats were intraperitoneally and subcutaneously injected with ovalbumin (OVA) to establish an experimental AC model and the Th2 signaling pathway was blocked by an investigational neutralizing antibody (CM310). Serum IgE and OVA-specific IgE were detected by ELISA. Conjunctivitis inflammation, infiltration of eosinophils, and mast cell degranulation were detected by histological examination. Immortalized human conjunctival epithelial cells, a conjunctival epithelial cell line, and peripheral blood mononuclear cells of patients with AC were used as the target cells to study the impact of IL-4 or IL-13 on AC progression. Finally, a STAT6 reporter gene system was constructed using immortalized human conjunctival epithelial cells to confirm whether the downstream signaling pathway activated by IL-4 or IL-13.

Results: Serum IL-4 or IL-13 were increased in patients with AC versus healthy individuals. In an OVA-induced rat experimental AC model, blocking the Th2 signaling pathway with CM310, an investigational neutralizing antibody, alleviated the conjunctival symptoms, and decreased serum IgE, suppressed infiltration of eosinophils and mast cell degranulation. Further, an in vitro model showed CM310 suppressed the secretion of inflammatory cytokine from both immune cells and epithelial cells in both patients peripheral blood mononuclear cells and cell line.

Conclusions: Blocking Th2 signaling pathway alleviates the clinical symptoms and inflammation in AC.

Allergic conjunctivitis (AC), a common ocular surface condition affecting 6% to 30% of the general population and in up to 30% in children alone, or in association with allergic rhinitis, has a significant impact on patients’ lives.¹ Typical symptoms of AC include ocular itching, redness, increased tearing, and blurred vision,² which are often accompanied by other allergic conditions such as asthma and rhinitis.³ Five clinical subtypes of AC include atopic keratoconjunctivitis, vernal keratoconjunctivitis, seasonal AC, and giant papillary conjunctivitis.⁴ In addition, the incidence of immune diseases, including various forms of AC, has been increasing annually because if the air pollution associated with industrialization and increased pollen levels caused by global warming.⁵ 

The pathogenesis of AC is attributed mainly to type I hypersensitivity which is mediated by IgE secreted from B cells that are stimulated by CD4⁺ Th2 cells.⁶ Upon re-exposure to the specific antigen, dendritic cells activate T cells, which in turn elicit adaptive immune responses.⁷ Naïve CD4⁺ T differentiation after activation is mainly influenced by the cytokine milieu, cognate antigen, and T-cell receptor affinity.⁸ Among the cytokine spectrum, Th2 cytokines, including IL-4, IL-5, and IL-13, have been shown to play an important role in stimulating IgE-mediated inflammation.⁹ It has also been reported that the levels of Th2 cytokines are increased significantly in the tears of patients with AC compared with healthy controls.^10,11 The expression levels of IL-4 and IL-13 genes are also significantly increased in the conjunctival cells of patients with AC.^12,13 In addition, IL-4 expression promotes the production of allergen-specific IgE in the plasma cells.¹⁴ Therefore, the Th2-biased immune system may be considered necessary for the pathogenesis of AC. 

Conjunctival epithelial cells educated by an inflammatory microenvironment also play an important role in AC progression.¹⁵ The affected conjunctival epithelial cells can secrete various cytokines, such as IL-6, IL-8, eotaxin, and TNF-α, to exacerbate the inflammatory state by promoting the proliferation, differentiation, activation, and chemotaxis of inflammatory cells in the conjunctiva and, conversely, are also involved in the immune process of the ocular surface.¹⁶ Another potential mechanism in contributing to the disease pathogenesis is that conjunctival epithelial cells upregulated the expression of adhesion molecules, such as intercellular adhesion molecule-1, which will capture more effector cells during the inflammatory response.¹⁷ 

The IL-4 receptor α (IL-4Rα), as the receptor for IL-4 and IL-13, specifically mediates Th2 immune responses and allergic diseases, and blocking this signaling pathway has been proved to have significant clinical benefits for the patients with allergy diseases.¹⁸ CM310, an investigational compound in phase III clinical trials for type 2 inflammation (NCT05265923, NCT05436275), targets human IL-4Rα and effectively blocks the interaction of the cytokines IL-4 and IL-13 with their co-receptor subunit IL-4Rα, thus simultaneously inhibiting the signaling pathway activated by IL-4 or IL-13 in type 2 inflammatory responses. In this study, we explored the further effects of the Th2 cytokines IL-4 and IL-13 on conjunctival epithelial cells and the novel mechanisms by which they induce the AC progression. First, it was found that serum IL-4 or IL-13 levels were higher in patients with AC than those in healthy individuals. Second, we found that blocking IL-4Rα with a neutralizing antibody CM310 inhibits conjunctival symptoms, level of serum IgE, and infiltration of eosinophils and mast cell degranulation in the rat model of ovalbumin (OVA)-induced AC, and also suppresses the secretion of inflammatory cytokines in both immune cells and epithelial cells, and the Th2-related STAT6 signaling pathway in vitro. Collectively, our findings contribute to the understanding of the pathogenic mechanisms of AC pathogenesis and the rational basis for new biological agents for AC treatment. 

Methods

Serum Th2 Cytokines in Patients With AC

Patients with AC and healthy control subjects were recruited with written informed consent to participate in this study, which was conducted from January to June 2021 at the Department of Ophthalmology, West China Hospital, Sichuan University. This study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of West China Hospital. All recruited patients with AC were diagnosed by clinically experienced physicians and agreed to discontinue the use of antiallergic agents for at least 1 week before enrolment. Clinical symptoms were assessed and recorded, including the degree of ocular itching, redness and conjunctival swelling, and the severity of conjunctival follicles and papillae. The clinical scores criteria of itches are as follows: 0 points = no itching; 1 point = interruptible itching; 2 points = mild persistent itching; 3 points = severe persistent itching with willing of eye rubbing; and 4 points = severe persistent itching with uncontrollable eye rubbing. The clinical scores criteria of hyperemia are as follows: 0 points = no congestion; 1 point = scarlet congestion confined to fornix conjunctiva; 2 points = carmine congestion reaching palpebral fissure; and 3 points = diffuse purple congestion. Serum cytokine levels were measured using the Meso Scale Discovery assay. 

Rat AC Model

A total of 60 Wistar Han rats (30 males and 30 females) were purchased from Shanghai Sippe-BK Laboratory Animal Co., Ltd (China) and housed in the SPF animal room of the Laboratory Animal Center of Zhejiang University. 

Sensitization and Challenge

On day 0, rats were injected abdominally and subcutaneously at multiple sites with 1 mL Al(OH)₃ gel solution containing 2 mg/mL OVA (Sigma, St. Louis, MO, USA). On days 4, 7, 11, and 14, rats were injected intraperitoneally with 0 to 5 mL Al(OH)₃ gel solution containing 4 mg/mL OVA. Rats in the control group were injected with an equal volume of Al(OH)₃ gel solution without OVA. On days 21 to 27, the eyes were challenged with a saline solution containing 120 mg/mL OVA (5 µL in each eye, twice daily for 7 days). 

Randomization and Administration

The rats were randomly divided into six groups (10 animals per group), including control group, model group, CM310 (25 mg/kg; Keymed Biosciences (Chengdu) Co., Ltd, China) group, CM310 (50 mg/kg) group, CM310 (100 mg/kg) group, and dexamethasone (DEX, 0–5 mg/kg; Shanghai Macklin Biochemical Co., Ltd, China) group. Dosing was performed once daily on days −1, 4, 9, 14, 19, 21, 23, 25, and 27, respectively. The IL-4Rα–targeting antibody CM310 (25 mg/kg, 50 mg/kg, 100 mg/kg) was injected subcutaneously, and the positive control compound DEX (0–5 mg/kg) was injected intraperitoneally into each experimental AC (EAC) model rat. 

Conjunctival congestion and edema were macroscopically observed, recorded, and scored on days 22, 24, and 26 at 24 hours after OVA challenge on days 21, 23, and 25. The scoring criteria are as follows: 0 points = no swelling; 1 point = mild swelling; 2 points = significant swelling with partial eyelid ectropion; 3 points = swelling with eyelids almost half closed; and 4 points = swelling with eyelid more than half closed.¹⁹ Rat bodies were weighed before each dose. The levels of total serum IgE and OVA-specific IgE were measured by ELISA on day 20. On day 28, the lamina propria of the tarsus, as well as the tarsus and left eyeball of the rats, were fixed in 4% paraformaldehyde solution, dehydrated, and sectioned after paraffin embedding. Eosinophil infiltration was detected by hematoxylin and eosin (H&E) staining, and mastocyte degranulation was visualized by toluidine blue staining. We scored the sections of H&E staining according to the degree of conjunctival eosinophil infiltration, edema, and epithelial damage. 

Conjunctival Epithelial Cell Line Culture

Immortalized human conjunctival epithelial cells (IOBA-NHC) were provided by Dr Li (Shanghai Jiao Tong University). The cells were cultured at 37°C in a 5% CO₂ atmosphere. The culture medium consisted of DMEM and F12 (1:1) containing 1 µg/mL bovine pancreatic insulin, 2 ng/mL rat epidermal growth factor, 0.1 µg/mL cholera toxin, 0.5 µg/mL cortisol, 10% fetal bovine serum, 50 U/mL penicillin, 50 µg/mL streptomycin, and 2.5 µg/mL amphotericin B.²⁰ 

Expression of IL-4Rα on the Surface of IOBA-NHC Cells

IOBA-NHC cells were incubated with 30 µg/mL biotin-conjugated anti-KLH and anti–IL-4Rα antibodies for 1 hour at 4°C, respectively. After being rinsed three times with 4% BCS/PBS, the cells were incubated with SA-PE (Jackson ImmunoResearch, West Grove, PA, USA) for 30 minutes. Then, the cells were rinsed and resuspended in PBS and detected using a BD Celesta flow cytometer. 

Detection of TARC/CCL17 Released From PBMCs of Patients With AC

Human peripheral blood mononuclear cells (PBMCs) were isolated from patients with AC by density gradient centrifugation according to the instructions of the manufacturer. PBMCs were incubated with IL-4 or IL-13 and CM310, and then the levels of TARC/CCL17 were detected in the cell culture supernatants. 

Detection of IL-6 and IL-8 in the Supernatants of IOBA-NHC Cells

IOBA-NHC cells were seeded in 6-well plates (5 × 10⁵ cells/well), cultured overnight to reach 80% to 90% confluence, and then starved (0% fetal bovine serum) for 9 hours. The cells were treated with the corresponding reagents (IL-4 and IL-13 at 20 ng/mL and OVA at 1 mg/mL). The concentrations of CM310 used in this study were 10 µg/mL, 30 µg/mL, and 100 µg/mL, respectively, and the concentration of control anti-KLH antibody was 100 µg/mL. After 48 hours of treatment, the levels of IL-6 and IL-8 in the supernatants were measured with ELISA. 

Detection of STAT6 Activation in IOBA-NHC Cells

The STAT6 promoter was introduced into the upstream region of the luciferase polymerase gene to construct an IOBA-NHC cell line that stably expressed STAT6-luciferase. After adding gradient concentrations of CM310 and anti-KLH antibody, a microplate reader was used to detect STAT6 activation in the presence of IL-4 (0.2 ng/mL) or IL-13 (1 ng/mL). 

Statistical Analysis

All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, NY, USA). Data obtained from animal experiments are presented in mean value ± standard error, and data obtained from IOBA-NHC cell experiments are expressed as a mean value ± standard deviation. A homogeneity test for variance was performed for each set of data. For equal variances (P > 0.05), one-way analysis of variance was performed, and Dunnett's test was used to compare each dose group and control group. If equal variances were not assumed (P ≤ 0.05), a nonparametric test was performed, and the Mann–Whitney U test was applied to compare each dose group and control group. When two groups were compared, an independent samples t test was performed. A P value of less than 0.05 was considered statistically significant for all tests. 

Results

Serum IL-4 Levels Were Significantly Increased in Patients With AC

To determine the IL-4 or IL-13 levels in the serum of patients with AC, a total of 26 patients with AC (16 females and 10 males) with typical symptoms of AC, such as conjunctival hyperemia and eye itching, were included in this clinical study (Table). Among them, 11 patients had conjunctival follicles and 18 patients had conjunctival papillae. Fifteen healthy individuals were enrolled as control subjects. The serum level of IL-4 was significantly increased in patients with AC than healthy individuals (P = 0.026), whereas the serum IL-13 were increased only in some patients with AC (Fig. 1). In addition, there was a significant correlation between gender and serum level of IL-4 (P = 0.005, rs = 0.555), with higher level observed in females (P = 0.011). However, no significant difference in serum level of IL-13 was observed between males and females (P = 0.341), and there was no correlation between IL-13 level and gender (P = 0.503, rs = 0.144). The levels of other cytokines in the serum did not show significant differences or were below the lower limit of detection when comparing patients with AC and healthy individuals (data not shown). These data suggested that systematic Th2 cytokines IL-4 or IL-13 played a role in the pathogenesis of AC. 

Table.

 

Demographic Characteristic and Clinical Presentations of the Enrolled Patients With AC

 

Figure 1.

 

Serum IL-4 or IL-13 levels in patients with AC and healthy individuals.

Blocking Th2 Signaling Pathway Alleviates the Clinical Symptoms and Inflammation in an EAC Model

We established a rat model of EAC induced by OVA to investigate the role of Th2 immunity in AC pathogenesis (Fig. 2A). There was no significant change in the body weight of rats in the CM310 groups compared with the model rats, although the body weight of rats in the DEX group were slightly decreased, but without reaching significance (Fig. 2B). The severity of conjunctivitis symptoms in the model group was significantly elevated, however, treatment with CM310 and DEX showed a decrease in the symptoms (Fig. 2C). On day 26, the scores of conjunctivitis symptoms was decreased by 38.89%, 27.78%, and 27.78% in the CM310 25 mg/kg, 50 mg/kg, and 100 mg/kg groups, respectively, and 44.44% in DEX group (Fig. 2D). 

Figure 2.

 

Blocking the Th2 signaling pathway alleviates the clinical symptoms and inflammation in experiment AC (EAC) model. (A) Experimental protocol. OVA was used to establish the EAC model and CM310 was applied to block the Th2 signaling pathway. (B) Body weight changes with different treatments. (C) Demonstrative images of ocular allergic symptoms in the specified investigational group. (D) Statistical analysis of clinical score of AC symptoms. (E) Serum IgE (left) and OVA-specific IgE (right) levels were measured by ELISA. (F). H&E staining of conjunctival samples from mice in the specified investigational group that showed more eosinophil infiltration in the conjunctiva of the model group that compared with the control group and the treated groups. (G) Statistical analysis of H&E score for conjunctiva eosinophil infiltration. (H) Toluidine blue staining showed mastocytes (MCs) degranulation in the rat conjunctiva that normal MCs were identified based on their regular shape in control group and activated MCs were identified based on their irregular shape in model group. (I) Statistical analysis of t mastocyte degranulation rate. Statistical data are expressed as the mean value ± SEM (n = 10), *P < 0.05, **P < 0.01, ***P < 0.001.

The levels of serum total IgE and OVA-specific IgE were significantly higher in the model group compared to the control group on day 20 (Fig. 2E). Meanwhile, CM310 effectively decreased the total serum IgE levels in a dose-dependent manner, with decreases of 27.27%, 40.15%, and 51.93% in the 25 mg/kg, 50 mg/kg, and 100 mg/kg groups, respectively. The decrease in the 100 mg/kg group was similar to that in the DEX group (49.59%). CM310 also significantly decreased the serum OVA-specific IgE levels in a dose-dependent manner, with decreases of 28.83%, 35.31%, and 52.81% in the 25 mg/kg, 50 mg/kg, and 100 mg/kg groups, respectively. Furthermore, we examined eosinophil infiltration and mastocyte degranulation in the rat EAC model. H&E and toluidine blue staining revealed that the eosinophil infiltration and mastocyte degranulation rate were significantly increased in the rat conjunctiva in the model group, but no significant changes were observed in the tarsus lamina propria. Compared with the model group, eosinophil infiltration was significantly reduced in the 100 mg/kg CM310- and 0.5 mg/kg DEX-treated groups (Figs. 2F, G). Meanwhile, mastocyte degranulation rate was significantly decreased only in the 100 mg/kg CM310-treated group with an inhibition rate of 28.98% (Figs. 2H, I). Thus, our results from the rat model showed that CM310 alleviated the symptoms of EAC in vivo. 

Blocking Th2 Signaling Pathway Inhibits IL-6 or IL-8 Secretion From Conjunctival Epithelial Cells

IOBA-NHC, a conjunctival epithelial cell line, was used as the target cells to study the impact of IL-4 or IL-13 on AC progression. The results showed that the IL-4Rα receptor, which is responsible for responding to IL-4/IL-13, was expressed in high levels on the surface of IOBA-NHC cells (Fig. 3A). Then we evaluated the regulatory role of IL-4 or IL-13 on the secretion of various cytokines (IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-10, TNF-α, INF-γ, and eotaxin) in IOBA-NHC cells. It was found that IL-4/IL-13 significantly induced the secretion of IL-6 and IL-8 in the IOBA-NHC cells, which was subsequently inhibited by CM310 (Figs. 3B and 3C). Additionally, CM310 at a concentration of 100 µg/mL, significantly inhibited OVA-induced IL-6 secretion in IOBA-NHC cells (Fig. 3D). 

Figure 3.

 

Blocking the Th2 signaling pathway inhibits IL-6 or IL-8 secretion from conjunctival epithelial cells. (A) The expression of IL-4Rα on the surface of IOBA-NHC cells. (B) IL-4 (left) and IL-13 (right) induced the secretion of IL-6 in IOBA-NHC cells. (C) IL-4 (left) and IL-13 (right) induced the secretion of IL-8 in IOBA-NHC cells. (D) OVA induced the secretion of IL-6 in IOBA-NHC cells. Statistical data are expressed as a mean value ± SD, n = 3. * P < 0.05, **P < 0.01, ***P < 0.001.

Blocking Th2 Signaling Pathway Reduces TARC/CCL17 Release From Patients With AC PBMC

To investigate the impact of blocking Th2 signaling pathway in effector cells from patients with AC, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with IL-4 or IL-13 and incubated with CM310. The levels of TARC/CCL17 released in the supernatant were then measured to assess the effect (Fig. 4). The findings showed that CM310 inhibited TARC/CCL17 release in a dose-dependent manner. 

Figure 4.

 

Blocking the Th2 signaling pathway reduces TARC/CCL17 release from PBMCs of patients with AC.

Blocking Th2 Signaling Pathway Suppresses the STAT6 Signaling Activated by IL-4 or IL-13

To confirm whether the downstream signaling pathway activated by IL-4 or IL-13 was also inhibited by the neutralizing antibody CM310 in conjunctival epithelial cells, a STAT6 reporter gene system was constructed using IOBA-NHC cells. It was found that IL-4 or IL-13 activated the STAT6 signaling pathway, while CM310 attenuated this activation (Fig. 5). These results suggested that the inflammatory signaling pathways involved in AC progression are also active in target cells, such as conjunctival epithelial cells. 

Figure 5.

 

Blocking the Th2 signaling pathway suppresses the STAT6 signaling activated by IL-4 or IL-13. Statistical data are presented as a mean value ± SD, n = 3.

Discussion

Although AC is not a life-threatening disease, it can have a significant impact on patients' quality of life and cause great suffering.² In some severe cases, such as AC accompanied by keratopathy, the disease might even lead to vision loss. Current treatment options for AC mainly include antigen avoidance, artificial tears for symptom relief, and various antiallergic agents, such as antihistamines and mastocyte stabilizers.²¹ However, these agents require a high frequency of use and provide only short-term relief of symptoms. Corticosteroids and immunosuppressive therapies are also available for recurrent AC that is severely symptomatic or unresponsive to conventional antiallergic therapy. However, both treatments are associated with serious adverse effects, including delayed wound healing, secondary infection, and ocular hypertension.^22,23 Therefore, there is an urgent need to develop new and effective treatments for AC. 

IL-4 or IL-13 in tears has been reported increased in patients with AC,¹² but the serum level is not known. In this study, we first found that the systemic levels of IL-4 or IL-13 were also increased compared with healthy subjects, indicating that AC is a systemic atopic disease and not just a local disease. The serum level of IL-4 increased correlatively with gender, and was significantly higher in female patients. Although there is some evidence suggesting that gender may play a role in the development of allergic diseases, there is currently limited information available on the specific relationship,^24,25 and there is also research showing contrary results,²⁶ which requires more exploration. 

In this study, we used CM310, a humanized anti–IL-4Rα monoclonal antibody cross-reactive with human, cynomolgus monkey, and rat, to investigate the role of IL-4 and IL-13 in both effector cell PBMC and target conjunctival epithelial cells in the progression of AC. Accumulating evidence suggests that conjunctival epithelial cells play an important role in the progression of ocular surface inflammatory diseases.^27,28 This study reported for the first time that IL-4Rα, the mediator of the Th2 pathway, is also expressed on conjunctival epithelial cells and that blocking with CM310 inhibited the secretion of the inflammatory cytokines IL-6 or IL-8.²⁹ Also, the prominent inhibitory effect of CM310 in EAC rats presented as a decrease in the clinical AC symptoms supported the important role of Th2 immunity in AC pathogenesis. In addition, we measured the weight of rats to avoid the occurrence of severe systemic inflammatory response, which may cause excessive weight changes and introduce error into the analysis. Clinically, TARC/CCL17 has recently shown promise as a useful biomarker for Th2-associated diseases such as atopic dermatitis severity, as well as efficacy of treatment.^30,31 CM310 also suppressed the secretion of TARC/CCL17 from patients with AC PBMC. These results proved that Th2 cytokines promote AC progression involved with both immune cells and epithelial cells. 

Somewhat contradictory to previous reports were the observations from the dupilumab clinical trials that the incidence of conjunctivitis was increased with dupilumab (8.6%–22.1%) versus placebo (2.1%–11.1%).³² However, the CHRONOS and CAFÉ clinical trials provided evidence that patients with better treatment efficacy, including those with higher Investigator's Global Assessment 0/1 or Eczema Area and Severity Index–75 responses, rarely developed conjunctivitis. Therefore, the conjunctivitis side effect observed in clinical trials with dupilumab has been attributed to insufficient inhibition of IL-4Rα or activation of Th2 inflammation via alternative pathways. Therefore, the therapeutic effect of targeting IL-4Rα in conjunctivitis remains to be validated in future clinical trials. 

Acknowledgments

Funding provided by Project of Sichuan Department of Science and Technology (2023NSFSC1667). 

Disclosure: Z. Tao, None; W. Liu, None; Q. Chen, None; L. Zhang, None; K. She, None; G. Zhao, None; L. Liang, None; X. Chen, None; Y. Yang, None; Q. Song, None; F. Lu, None