Ocular Allergy Modulation to Hi-Dose Antigen Sensitization Is a Treg-Dependent Process

Abstract

A reproducible method to inhibit allergic immune responses is accomplished with hi-dose Ag sensitization, via intraperitoneal (IP) injection. However, the role of CD4+ CD25+ FoxP3+ T regulatory cells (Treg) in this process is unknown, as is whether such modulation extends to ocular allergy. We therefore determined herein whether hi-dose sensitization modulates ocular allergy, and whether CD4+ CD25+ FoxP3+ Treg are involved. C57BL/6 mice were IP sensitized via low-dose (100 µg) versus hi-dose (1000 µg) ovalbumin (OVA), in aluminum hydroxide (1 mg) and pertussis-toxin (300 ng). Other mice received anti-CD25 Ab (PC61) to ablate Treg during sensitization. In another experiment, Treg from hi-dose sensitized mice were adoptively transferred into low-dose sensitized mice. Once daily OVA challenges were administered. Clinical signs, IgE, T cell cytokines, and eosinophils were assessed. Data revealed that hi-dose, but not low-dose, sensitization led to allergy modulation, indicated by decreased clinical signs, serum IgE levels, Th2 recall responses, and eosinophil recruitment. T cells from hi-dose sensitized mice showed a robust increase in TGF-b production, and Treg from these mice were able to efficiently suppress effector T cell proliferation in vitro. In addition, in vivo Treg ablation in hi-dose sensitized mice revoked allergy modulation. Lastly, Treg from hi-dose sensitized mice were able to adoptively transfer allergy modulation to their low-dose sensitized counterparts. Collectively, these findings indicate that modulation to hi-dose sensitization, which is extended to ocular allergy, occurs in a Treg-dependent manner. In addition, our data suggest that hi-dose sensitization may henceforth facilitate the further examination of CD4+ CD25+ FoxP3+ Treg in allergic disease.

Involvement of Corneal Lymphangiogenesis in a Mouse Model of Allergic Eye Disease

“…38 Culture supernatants were collected and assayed for IL-4, -5, and -13 via ELISA. [37][38][39] We found that T cells from VEGFR inhibitor treated mice produced significantly less levels of IL-4, -5, and -13, relative to vehicle treated AED mice (Fig. 4).…”

“…[37][38][39] Briefly, each immunization consisted of 10 lg of ovalbumin (OVA; Sigma-Aldrich Corp., St. Louis, MO, USA), 1 mg of aluminum hydroxide (Sigma-Aldrich Corp.), and 300 ng of pertussis toxin (SigmaAldrich Corp.), which were thoroughly mixed in 100 lL of sterile Hank's balanced salt solution (HBSS). Immunization was administered once via IP route, and mice were subsequently rested for 2 weeks.…”

“…[37][38][39] Briefly, scoring was performed 20 minutes post challenge and done once daily for at least 7 days. Mice were examined biomicroscopically based on four independent parameters, which include: (1) lid edema, (2) tearing/discharge, (3) chemosis, and (4) hyperemia.…”

“…Allergic eye disease is induced via systemic OVA immunization, followed 2 weeks after with administration of allergen challenge via multiple instillations of OVA eye drops, as previously described. [37][38][39] We then excised the corneas from these mice and prepared whole mounts for fluorescence microscopy following staining for lymphatic vessels (LYVE-1; Fig. 1A).…”

“…We addressed this herein using a well-established mouse model of Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 AED, which is induced by allergen sensitization and subsequent chronic allergen exposure. [37][38][39] This leads to a robust eosinophil involvement 39 and sequelae consistent with chronic allergic inflammation, such as blepharitis and corneal epitheliopathy. 37 The data presented herein reveal, for the first time, the distinct involvement for corneal LA in AED, and that prevention of corneal LA via VEGF receptor (R) inhibition diminishes AED.…”