Model Of Allergic Conjunctivitis Research Articles

Evaluation methods using tear volume in a conjunctivitis mice model

Allergic conjunctival disease is an immune-mediated inflammatory disease of the conjunctiva. To develop clinically useful drugs, it is necessary to develop quantitative evaluation methods that reflect the clinical symptoms in experimental animal models. Allergic conjunctivitis model mice were systemically sensitised with ovalbumin (OVA) administered intraperitoneally and locally sensitised with OVA eye drops between day 14–28. Next, conjunctivitis induced by ocular administration of OVA solution to sensitised mice was evaluated based on tear volume. Additionally, we evaluated increase in tear volume induced by direct ocular instillation of histamine, compound 48/80, and carrageenan. An increase in antigen-induced tear volume was observed in the mice model. Additionally, direct instillation of histamine, compound 48/80, and carrageenan increased tear volume. Furthermore, levocabastine inhibited the increase in tear volume in antigen-induced allergic conjunctivitis and histamine- and compound 48/80-induced conjunctivitis models. In contrast, betamethasone suppressed carrageenan-induced tear volume but not histamine- or compound 48/80-induced tear volume. Histamine may be involved in increased tear volume in allergic conjunctivitis. Betamethasone is not directly involved in the action of histamine and is thought to suppress increase in tear volume. Evaluation of tear volume in a conjunctivitis mice model is highly quantitative; therefore, it is possible to evaluate drug efficacy. This is considered a useful index compared with conventional methods.

Epinastine Cream: A Novel Once-Daily Therapeutic Agent for Allergic Conjunctivitis.

Purpose: To investigate the in vivo efficacy of epinastine cream in type I allergic models. Methods: The dose, timing, and antiallergic effect of epinastine cream on the conjunctiva were evaluated postapplication to the eyelid skin of guinea pigs with histamine- or ovalbumin-induced allergic conjunctivitis. Additionally, we assessed its antiallergic effects on the skin postapplication to the dorsal skin of guinea pigs with ovalbumin-induced passive cutaneous anaphylaxis. Efficacy was estimated by determining the amount of dye that leaked from conjunctival or dorsal skin tissue vessels as a measure of vascular permeability, scoring the severity of allergic symptoms, and observing the scratching behaviors using clinical parameters. Results: In the histamine-induced conjunctivitis model, epinastine cream strongly inhibited conjunctival vascular permeability in a dose-dependent manner. The inhibitory effect of 0.5% epinastine cream 24 h postapplication was significantly higher than that of 0.1% epinastine hydrochloride ophthalmic solution 8 h postadministration. Additionally, the 0.5% epinastine cream inhibited conjunctival vascular permeability 15 min postapplication, and the effect was sustained over 24 h. Furthermore, the 0.5% epinastine cream effectively suppressed clinical symptom scores and exhibited ameliorated scratching bouts in conjunctival allergic reactions in the experimental allergic conjunctivitis model. Additionally, it significantly inhibited vascular permeability in skin allergic reactions in the passive cutaneous anaphylaxis model. Conclusions: The results suggest that epinastine cream is a strong, long-lasting, and skin-penetrating inhibitor of type I allergic reactions. The 0.5% epinastine cream applied once daily could be a promising, potent, and long-acting therapeutic agent for allergic conjunctivitis.

Subconjunctival injection of human umbilical cord mesenchymal stem cells alleviates experimental allergic conjunctivitis via regulating T cell response

BackgroundT helper 2 (Th2) cells are thought to play critical roles in allergic conjunctivitis (AC). They release inflammatory cytokines to promote an allergic response in AC. Due to individual heterogeneity and long-term chronic management, current therapies do not always effectively control AC. Mesenchymal stem cells (MSCs) have been shown to be effective in treating allergy-related disorders, but it is unclear how exactly the Th2-mediated allergic response is attenuated. This study aims to elucidate the therapeutic effect and mechanism of the human umbilical cord MSCs (hUCMSCs) in a mouse model of experimental AC (EAC).MethodsA mouse EAC model was established by inoculating short ragweed (SRW) pollen. After the SRW pollen challenge, the mice received a single subconjunctival or tail vein injection of 2 × 106 hUCMSCs, or subconjunctival injection of hUCMSCs conditioned medium (hUCMSC-CM), and dexamethasone eye drops was used as positive control; subsequent scratching behavior and clinical symptoms were assessed. Immunostaining and flow cytometry were carried out to show allergic reactions and the activation of CD4 + T cell subsets in the conjunctiva and cervical lymph nodes (CLNs). Gene expression was determined by RNA-seq and further verified by qRT-PCR and Western blot. Co-culture assays were performed to explore the regulatory role of hUCMSCs in the differentiation of CD4 + naive T cells (Th0) into Th2 cells.ResultsSubconjunctival administration of hUCMSCs resulted in fewer instances of scratching and lower inflammation scores in EAC mice compared to the tail vein delivery, hUCMSC-CM and control groups. Subconjunctival administration of hUCMSCs reduced the number of activated mast cells and infiltrated eosinophils in the conjunctiva, as well as decreased the number of Th2 cells in CLNs. After pretreatment with EAC mouse serum in vitro to mimic the in vivo milieu, hUCMSCs were able to inhibit the differentiation of Th0 into Th2 cells. Further evidence demonstrated that repression of Th2 cell differentiation by hUCMSCs is mediated by CRISPLD2 through downregulation of STAT6 phosphorylation. Additionally, hUMCSCs were able to promote the differentiation of Th0 cells into regulatory T cells in CLNs of EAC mice.ConclusionsSubconjunctival injection of hUCMSCs suppressed the Th2-allergic response and alleviated clinical symptoms. This study provides not only a potential therapeutic target for the treatment of AC but also other T cell-mediated diseases.

A lacrimal duct drug delivery system based on photo-induced hydrogel for dry eye and allergic conjunctivitis therapy

To design an ocular drug delivery system is challenging due to the limited space of the ocular surface and high sensitivity of the cornea. Here, we developed a lacrimal duct drug delivery system, which was non-invasive and could cause no foreign body sensation. Based on hydrogels, our drug-loaded lacrimal plugs had ideal and adjustable properties of mechanical characteristics, swelling ratio and degradation rates. They also showed better biocompatibility and the feasibility of loading various drugs to treat different ocular diseases, compared to the commonly-used plug products. Our drug-loaded lacrimal plugs exhibited sustained and stable drug releasing profiles both in vitro and in vivo for up to three months. To test the therapeutic effect, FK506 loaded plugs and cyclosporine A loaded plugs were used to treat the rat allergic conjunctivitis model and rabbit dry eye model, respectively, which both demonstrated satisfactory long-term efficacy. This study illustrated a lacrimal plug drug delivery system with the potential application in the long-term treatments of a variety of ocular surface diseases.

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

To explore the role of Th2 signaling pathway in allergic conjunctivitis (AC). 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. 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. Blocking Th2 signaling pathway alleviates the clinical symptoms and inflammation in AC.

Production profile of lipid mediators in conjunctival lavage fluid in allergic and infectious conjunctivitis in guinea pigs.

Conjunctivitis is a major ocular disease classified into allergic or infectious. The pathological features of conjunctivitis are not fully understood despite its high morbidity rate; thus, its differentiation can be difficult. We used ovalbumin-induced allergic conjunctivitis and lipopolysaccharide-induced infectious conjunctivitis models of guinea pigs. Both models showed conjunctival swelling. Histological studies revealed that numerous eosinophils infiltrated the conjunctiva in the allergic model, whereas neutrophils infiltrated the conjunctiva in the infectious model. We collected conjunctival lavage fluid (COLF) and comprehensively analyzed lipid production using liquid chromatography-tandem mass spectrometry. COLF showed increase of 20 and 12 lipid species levels in the allergic and infectious models, respectively. Specifically, the levels of a major allergic mediator, prostaglandin D2 and its three metabolites and several cytochrome P450-catalyzed lipids increased in the allergic model. In the infectious model, the levels of prostaglandin E2 and 8-iso-prostaglandin E2 increased, indicating tissue inflammation. Moreover, the level of 12-oxo-eicosatetraenoic acid, a lipoxygenase metabolite, increased in the infectious model. These differences in lipid production in the COLF reflected the pathological features of allergic and infectious conjunctivitis.

A mouse model of allergic conjunctivitis permitting tear eosinophil quantification

IntroductionAllergic conjunctivitis is an immune-mediated inflammatory disease of the conjunctiva that is induced by antigens. Allergic conjunctivitis can cause various symptoms such as ocular itching, hyperemia and edema. Developing experimental animal models that show clinical symptoms and methods for quantitative and objective evaluation is important for understanding allergic conjunctivitis. Therefore, this study aimed to develop an ovalbumin (OVA)-induced mouse model of allergic conjunctivitis and a useful method for evaluating symptoms of allergic conjunctivitis. MethodsICR mice were sensitized by an intraperitoneal injection of OVA in PBS containing alum on days 0 and 5. Subsequently, local sensitization was then performed once daily from days 14 to 28, by instilling OVA in PBS into the both eyes. Drug treatment was administered once daily from days 14 to 28. Mice were randomly assigned topical treatment groups: Group 1, 0.1% betamethasone; Group 2, 0.025% levocabastine; Group 3 PBS (control). ResultsMice showed marked eye scratching behavior, hyperemia, edema, infiltration of eosinophils into tears and increased antigen-specific immunoglobulin E antibody levels in tears and the serum. These symptoms were inhibited by instillation of levocabastine and betamethasone, which are used clinically for the treatment of allergic conjunctivitis. DiscussionThis method may be useful for evaluation of the symptoms of allergic conjunctivitis in experimental and clinical settings. In particular, the developed method, which measures the number of eosinophils in tears collected with phenol red threads, may enable the quantitative, objective, and noninvasive evaluation of the severity of allergic conjunctivitis.

Characteristics of histamine H4 receptor agonist-induced allergic conjunctivitis model in Guinea pigs

Histamine is strongly associated with the onset of allergic conjunctivitis. The most recent cloned histamine H4 receptor antagonist is highly expected as a new therapeutic drug candidate. As a model for a therapeutic drug targeting the histamine H4 receptor, a mouse model in which conjunctivitis symptoms are induced by instilling 4-methylhistamine, a histamine H4 receptor agonist, has been reported. However, the affinity of the H4 receptor for histamine varies in species, and it is known that the histamine binding affinity for the guinea pig H4 receptor is closer to that for human receptor than mice receptor. In this paper, we investigated a possibility that a guinea pig model would become a drug efficacy evaluation model with higher evaluation accuracy than the mouse model.As a result, hyperemia was observed in the conjunctivae and iris of guinea pigs after instillation of 4-methylhistamine and specifically suppressed by the histamine H4 receptor antagonist. Unlikely to the previously reported mouse model, however, none of edema, increased vascular permeability or scratching behavior was observed, suggesting that there may be differences between mice and guinea pigs not only in the binding affinity of histamine to the H4 receptor but also in the biological reaction to 4-methylhistamine. Although the symptoms of allergic conjunctivitis do not appear comprehensively in the guinea pig model, results of this study indicated a possibility that this model can be used as a simple screening model in the early stages of drug development.

The Protective Effects of KAT5 Inhibition on Ocular Inflammation by Mediating the PI3K/AKT Pathway in a Murine Model of Allergic Conjunctivitis.

PurposeWe aimed to explore the effect of lysine acetyltransferase KAT5 on allergic conjunctivitis (AC).MethodsThe effect of KAT5 on inflammatory response during AC progression was analyzed in the experimental allergic conjunctivitis (EAC) mouse model.ResultsThe clinical score, permeability, total IgE, ovalbumin (OVA)-specific IgE, and IgG1/IgG2a were induced in the EAC mice, in which the overexpression of KAT5 could further enhance but KAT5 inhibitor NU9056 reduce the phenotypes. The eosinophilic infiltration was induced in EAC mice, in which the overexpression of KAT5 was able to further promote but NU9056 attenuate the phenotype. The expression of Eotaxin and RANTES and the inflammatory factors were upregulated in EAC mice and KAT5 overexpression increased, but NU9056 decreased the expression in the model. Significantly, the CD11c+ dendritic cells and CD4+ T cells infiltration in the conjunctiva was enhanced in EAC mice, whereas KAT5 overexpression induced but NU9056 suppressed the effect in the model. Mechanically, the phosphorylation of PI3K and Akt and the levels of histone H3 lysine 27 acetylation (H3K27ac) were enhanced in EAC mice, whereas the overexpression of KAT5 promoted and NU9056 repressed the phenotype in the mice. The enrichment of KAT5 and H3K27ac on PI3K promoter was increased in EAC mice, and the overexpression of KAT5 further enhanced the enrichment in the mice. Significantly, we observed similar results in the KAT5 knockout mice as well. Moreover, PI3K/AKT signaling inhibitor LY294002 reversed KAT5 overexpression-mediated phenotypes and inflammatory response after induction AC in vivo.ConclusionsTherefore we concluded that KAT5 inhibition protected against ocular inflammation by mediating the PI3K/AKT pathway in EAC mouse model.

MiR-146a upregulates FOXP3 and suppresses inflammation by targeting HIPK3/STAT3 in allergic conjunctivitis.

BackgroundAllergic conjunctivitis (AC) is an inflammation caused by a hypersensitive immune reaction of conjunctiva to external allergens. The microRNA (miRNA) miR-146a has been reported to suppress the exacerbation of inflammation. However, the underlying influence and mechanism of miR-146a in AC has not been completely elucidated.MethodsWe first successfully established an AC mouse model and AC cell model. After each model was treated based on the experimental purposes, miR-146a, FOXP3, and homeodomain-interacting protein kinases 3 (HIPK3) expressions were estimated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The levels of immunoglobulin E (IgE), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), interleukin-4 (IL-4), and transforming growth factor-β (TGF-β) were assessed using enzyme-linked immunosorbent assay (ELISA) kits; the related proteins were analyzed by western blot, immunofluorescence, or immunohistochemistry (IHC) assays; the interaction between miR-146a and HIPK3 were validated by a dual-luciferase reporter gene assay; and the inflammatory infiltration was certified by hematoxylin and eosin (H&E) staining.ResultsOur results indicated that miR-146a and FOXP3 were downregulated in AC model mice. Meanwhile, miR-146a overexpression could upregulate FOXP3 and inhibit inflammatory response in TGF-β-induced thymocytes. Besides, our results testified that HIPK3, as a target gene of miR-146a, could reverse miR-146a-mediated FOXP3 upregulation and inflammation inhibition. Moreover, we discovered that miR-146a could downregulate p-STAT3 by targeting HIPK3, and activation of STAT3 also could reverse miR-146a-mediated inflammation suppression in TGF-β-induced thymocytes. More importantly, miR-146a could ameliorate inflammatory infiltration and downregulate HIPK3 and p-STAT3 in AC model mice.ConclusionsWe demonstrated a possible protective mechanism by the miR-146a/HIPK3/STAT3 axis, by which decrease of miR-146a could aggravate the inflammation of AC.

Neuronal Fc\u03b5RI\u03b1 directly mediates ocular itch via IgE-immune complex in a mouse model of allergic conjunctivitis

BackgroundClassical understanding of allergic conjunctivitis (ACJ) suggests that ocular itch results from a mast cell-dependent inflammatory process. However, treatments that target inflammatory mediators or immune cells are often unsatisfying in relieving the stubborn itch symptom. This suggests that additional mechanisms are responsible for ocular itch in ACJ. In this study, we aim to determine the role of neuronal FcεRIa in allergic ocular itch.MethodsCalcium imaging was applied to observe the effect of IgE-immune complex in trigeminal neurons. Genomic FcεRIa knockout mice and adeno-associated virus (AAV) mediated sensory neuron FcεRIa knockdown mice were used in conjunction with behavioral tests to determine ocular itch. In addition, immunohistochemistry, Western blot and quantitative RT-PCR were used for in vitro experiments.ResultsWe found that FcεRIα was expressed in a subpopulation of conjunctiva sensory neurons. IgE-IC directly activated trigeminal neurons and evoked acute ocular itch without detectible conjunctival inflammation. These effects were attenuated in both a global FcεRIa-knockout mice and after sensory neuronal-specific FcεRIa-knockdown in the mouse trigeminal ganglion. In an ovalbumin (OVA) induced murine ACJ model, FcεRIα was found upregulated in conjunctiva-innervating CGRP+ sensory neurons. Sensory neuronal-specific knockdown of FcεRIa significantly alleviated ocular itch in the ACJ mice without affecting the immune cell infiltration and mast cell activation in conjunctiva. Although FcεRIα mRNA expression was not increased by IgE in dissociated trigeminal ganglion neurons, FcεRIα protein level was enhanced by IgE in a cycloheximide-resistance manner, with concordant enhancement of neuronal responses to IgE-IC. In addition, incremental sensitization gradually enhanced the expression of FcεRIα in small-sized trigeminal neurons and aggravated OVA induced ocular itch.ConclusionsOur study demonstrates that FcεRIα in pruriceptive neurons directly mediates IgE-IC evoked itch and plays an important role in ocular itch in a mouse model of ACJ. These findings reveal another axis of neuroimmune interaction in allergic itch condition independent to the classical IgE-mast cell pathway, and might suggest novel therapeutic strategies for the treatment of pruritus in ACJ and other immune-related disorders.

Effects of rAmb a 1-Loaded PLGA-PEG Nanoparticles in a Murine Model of Allergic Conjunctivitis.

Ambrosia artemisiifolia (Amb a) contains many allergens. Allergic conjunctivitis caused by Ambrosia artemisiifolia and its related allergen-specific immunotherapy (AIT) are seldom studied at present. poly(DL-lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) is a very good nano-carrier, which has been applied in the medical field. In this context, we studied the immunotherapy effect and potential mechanism of recombinant Amb a 1 (rAmb a 1)-loaded PLGA-PEG nanoparticles. A mouse allergic conjunctivitis model was established with Ambrosia artemisiifolia crude extract, and the nanoparticles were used for AIT through direct observation of conjunctival tissue, degranulation of mast cells in conjunctival tissue, serum-specific antibodies, cytokines and other assessment models. The treatment of nanoparticles enhanced the secretion of T-helper 1 (Th1) cytokine Interferon-gama (IFN-γ) and the production of immunoglobulin G (IgG)2a (IgG2a), inhibited the secretion of T-helper 2 (Th2) cytokine Interleukin (IL)-13 and IL-4 and the level of IgE. Especially, degranulation of mast cells and expression of mast cell protease-1 (MCP-1) in conjunctival tissue was reduced significantly. In this study, we proved that the nanoparticles prepared by rAmb a 1 and PLGA-PEG have an immunotherapy effect on allergic conjunctivitis in mice.

Optimization of a histamine-induced allergic conjunctivitis model in Guinea pigs

Allergic conjunctivitis is one of the most common immune diseases in the field of ophthalmology. The number of patients suffering from allergic conjunctivitis has been increasing, and there is still a strong need for development of therapeutic agents for this disease. In drug development, the utmost important point to improve the success probability is to accurately single out good compounds in the early stage of drug development. Therefore, drug efficacy evaluations in the nonclinical stage should be conducted with high reliability and accuracy. However, there are no literatures investigating the preparation and evaluation methods of animal models of conjunctivitis in details nor the standardized criteria.In this study, we verified the reproducibility of an animal model in the previous report and made improvements in test methods focusing on a guinea pig model of histamine-induced allergic conjunctivitis. Furthermore, the drug efficacy evaluation was conducted using a commercially available antihistamine drug, levocabastine hydrochloride, to judge the suitability of the improved model.As a result, the dose level of histamine needed to be increased to use the existing model for drug efficacy evaluation, but allergic-like symptoms were induced very easily and stably in this model. For observations of symptoms of conjunctivitis, we eliminated ambiguity of evaluation by adopting the Draize scale and ensured a higher objectivity on the evaluation method. The drug efficacy evaluation of levocabastine hydrochloride in the prepared model revealed that drug efficacy of the antihistamine drug was captured according to the standardized test method and highly-reproducible results were obtained.

FAK inhibitor PF-431396 suppresses IgE-mediated mast cell activation and allergic inflammation in mice

Mast cells (MCs) initiate and maintain allergic inflammation. Upon being stimulated with immunoglobulin (Ig)E and antigen (Ag), MCs exhibit FcεRI (high-affinity IgE) receptor-mediated degranulation, cytokine secretion, and increased focal adhesion kinase (FAK) activity. The aims of this study were to examine mechanisms of FAK regulation in IgE-mediated MC activation and the effects of FAK inhibition on MC-mediated allergic responses. FAK activity was manipulated with short hairpin RNA (shRNA) knockdown, FAK overexpression, and the FAK inhibitor PF-431396 (PF). Gene expression and kinase activation were analyzed with quantitative molecular biology assays. PF effects were tested in the passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and allergic conjunctivitis (AC) mouse models. Our results showed that FAK overexpression increased IgE-mediated degranulation and reduced the dexamethasone inhibitory effect on MCs activation. The FAK inhibitor PF diminished MC release of β-hexosaminidase (β-hex), histamine, and inflammatory cytokines, via a mechanism that involves MAPK and NF-κB signaling pathways. CaMKII was identified as a robust FAK-associating protein. Inhibition of CaMKII activation by KN-93 suppressed FAK activity and its downstream pathway. PF attenuated inflammatory responses in our PCA and ASA models, and relieved signs of allergic disease in AC model mice. In conclusions, MC degranulation and production of inflammatory mediators in allergic disease may be consequent to FcεRI crosslinking inducing CaMKII-mediated activation of FAK activity. FAK inhibition may represent a new MC-suppressing treatment strategy for the treatment of allergic diseases.

Development of allergic conjunctivitis induced by Acanthamoeba excretory-secretory protein and the effect of resolvin D1 on treatment

ABSTRACT Purpose To evaluate whether allergic conjunctivitis (AC) could be induced by Acanthamoeba excretory-secretory protein (ESP) and analyze the therapeutic effect of resolvin (Rv) D1 and antiallergic agents. Methods Human conjunctival epithelial cells (HCVCs) were treated with 10 µg/well of ESP, and Th2 cytokines were measured using real-time PCR. C57BL/6 mice were treated with 10 µg/5 µL of ESP after sensitization, and conjunctivas isolated from the mice were stained with hematoxylin and eosin (H&E) for the analysis of eosinophils and periodic acid–Schiff (PAS) for the analysis of goblet cells. Cytokine levels in the eye-draining lymph nodes (dLNs) and spleens were measured using the enzyme-linked immunosorbent assay (ELISA). Then, the treatment effects of RvD1 and the antiallergic agents (olopatadine, bepotastine, and alcaftadine) on the HCVCs, mouse conjunctivas, dLNs, and spleens were assessed. Results Th2 cytokines were increased in the ESP-treated conjunctival cells. Mouse conjunctivas treated with ESP showed significant infiltration of eosinophils and goblet cells, and the dLN and spleen exhibited increased IL-4, IL-5 and IL-13 levels. All findings were significantly decreased upon treatment with RvD1 and the antiallergic agents. Conclusions Acanthamoeba could be used to establish an animal model of AC, which could be effectively treated with RvD1 or topical antiallergic agents.

Experimental Mouse Models of Ragweed- and Papain-Induced Allergic Conjunctivitis.

Mouse models of allergic conjunctivitis mimic various aspects of human allergic conjunctivitis. They are useful as acute models of allergic conjunctivitis to study immunological aspects of this condition. In this chapter, we will describe ragweed-pollen-induced experimental allergic conjunctivitis (mostly driven by adaptive immunity), and papain-soaked contact lens-induced experimental allergic conjunctivitis (mostly driven by innate immunity). Giemsa staining of histological sections is used for quantification of the number of infiltrating eosinophils, which is useful to evaluate the severity of the allergic inflammation. Immunohistochemical staining and quantitative PCR are used to clarify spatiotemporal expression of proinflammatory molecules in the conjunctival tissue. Flow cytometric analysis of conjunctival tissue is used for the detection of innate lymphoid cell type 2 (ILC2) in the ocular surface tissues.

Roles of Type 2 Immune Response-Initiating Cytokines and Detection of Type 2 Innate Lymphoid Cells in Mouse Models of Allergic Conjunctivitis.

Allergic conjunctivitis is one the most common global diseases and affects many people worldwide. It has been reported that 15% to 20% of the total population in Japan suffers from allergic conjunctival disease. Although TH2 cytokines suchs as interleukin (IL)-4, IL-5, and IL-13 have long been known as causes of allergic conjunctivitis, new cytokines involved in allergic diseases have been identified since 2000. The discovery of type 2 immune response-initiating cytokines, such as IL-25, IL-33, and thymic stromal lymphopoietin, and type 2 innate lymphoid cells has suggested that allergic diseases can arise from not only T cells but also barrier function disruption. In this article, we summarize the results of experiments in mouse models of ragweed-induced experimental allergic conjunctivitis and papain-soaked contact lens-induced conjunctivitis.

Efficacy of Alcaftadine 0.25% (AGN-229666) for Once-daily Prevention of Cedar-Pollen Allergic Conjunctivitis: A Phase 3 Randomized Study

ABSTRACT Purpose: This study evaluated the efficacy and safety of once-daily Alcaftadine 0.25% (AGN-229666) for prevention of signs and symptoms of Japanese cedar-pollen allergic conjunctivitis. Methods: This was a single-center, placebo-, and comparator-controlled study using the Ora-CAC® model of allergic conjunctivitis. The primary endpoint was ocular itching 16 hours after Alcaftadine 0.25% instillation; efficacy at 16 hours was compared with 0.1% Olopatadine, 4 hours after instillation. Secondary endpoints included conjunctival hyperemia. Results: 263 Japanese subjects were enrolled; 224 completed the trial. Alcaftadine 0.25% was statistically superior to vehicle for relief of ocular itching at 16 hours (p < .0001). Alcaftadine 0.25% at 16 hours was non-inferior to Olopatadine at 4 hours. Alcaftadine 0.25% was significantly better than vehicle for relief of conjunctival hyperemia. All treatments showed a low frequency of ocular adverse events. Conclusion: Once-daily Alcaftadine 0.25% is safe and effective in preventing signs and symptoms of Japanese cedar-pollen allergic conjunctivitis.

IL-33/ST2/IL-9/IL-9R signaling disrupts ocular surface barrier in allergic inflammation.

This study was to explore a novel IL-33/ST2/IL-9/IL-9R signaling pathway that disrupts ocular surface barrier and amplifies allergic inflammation. Two murine models of experimental allergic conjunctivitis (EAC) and IL-9 topical challenge in wild type Balb/c and ST2−/− mice, and two culture models of primarily human corneal epithelial cells (HCECs) and mouse CD4+ T cells were performed. Clinical manifestations, Oregon Green Dextran (OGD) staining, the apical junction complexes (AJCs), IL-33/ST2 and IL-9/IL-9R signaling molecules were evaluated in ocular surface and its draining cervical lymph nodes (CLNs) by RT-qPCR, immunostaining and ELISA. The typical allergic signs, enhanced OGD staining intensity, disrupted morphology of AJCs, including ZO-1, claudin 1, occludin and E-cadherin, and the stimulated signaling of IL-33/ST2 and IL-9/IL-9R were observed in ocular mucosa and draining CLNs in EAC-Balb/c mice, but significantly reduced or eliminated in EAC-ST2−/− mice. Topical challenge of IL-9 resulted in the obvious OGD staining and disrupted ocular surface AJCs in Balb/c mice and in HCECs in vitro. IL-9 production was found to be stimulated by IL-33 in CD4+ cells from Balb/c mice in vitro. Our findings uncovered a novel phenomenon and mechanism by which ocular surface barrier integrity is disrupted in allergic conjunctivitis by IL-33/ST2/IL-9/IL-9R signaling pathway, which may amplify the allergic inflammation.

The Potential Inhibitory Effects of miR-19b on Ocular Inflammation are Mediated Upstream of the JAK/STAT Pathway in a Murine Model of Allergic Conjunctivitis.

PurposeThymic stromal lymphopoietin (TSLP) is a pro-allergic cytokine that initiates allergic inflammatory reaction between epithelial and dendritic cells (DCs). miR-19b was reported to suppress TSLP expression. The present study aimed to examine miR-19b expression, regulation, and function in allergic conjunctivitis (AC).MethodsA murine model of experimental AC was induced in BALB/c mice by short ragweed pollen. The serum, eye balls, conjunctiva, and cervical lymph nodes (CLN) were used for the study. Gene expression was determined by RT-PCR, whereas protein production and activation were evaluated by immunostaining, ELISA, and Western blotting.ResultsIn the murine AC model, miR-19b was aberrantly downregulated, whereas the levels of TSLP and p-STAT3, as well as the number of CD11c+ pSTAT3+ DCs were increased. Moreover, Th2 inflammatory cytokine expression was significantly increased. These severe phenotypes could be counteracted by either applying exogenous miR-19b mimic microRNAs or the JAK/STAT inhibitor CYT387. Moreover, overexpression of miR-19b repressed p-STAT3 expression and the number of CD11c+ cells in AC eye and CLN tissues.ConclusionsThese findings suggested that miR-19b reduced ocular surface inflammation by inhibiting Stat3 signaling via TSLP downregulation in a murine AC model. Moreover, the present study further demonstrated the clinical potential of applying miR-19b and anti-JAK/STAT therapies in the treatment of AC.