Conjunctival immunological tolerance in a murine evaporative dry eye model

Abstract

Purpose: To assess the state of conjunctival tolerance in a murine model of evaporative dry eye and to evaluate the effect of topical nuclear factor kappa B (NFkB) modulation on disease progression.

Methods: A previously reported murine model of evaporative dry eye (Niederkorn JY et al. J Immunol. 2006 Apr 1;176(7):3950-7) was used with 8-week-old Balb/c mice (3 animals per treatment group). Ovalbumin (OVA) was used as a model antigen to assess immunological tolerance. In brief, OVA was instilled daily starting on days 1 and 4 in both eyes of mice under escopolamine- and air draft-induced desiccating stress for 5 days (DS5). In other experiment, topical NFkB inhibitors or vehicle were instilled 3 times a day during DS5. OVA specific T cell responses, as well as tear production and histopathological markers of disease, were evaluated. Conjunctival tolerance to OVA is expressed relative to the systemic T cell response of non-tolerant immunized mice.

Results: In the evaporative dry eye model (3 independent experiments), conjunctival tolerance to OVA was observed when antigen was instilled from day 1 (53±3%, p<0.05) but not when started on day 4 (110±20%). Mice treated with topical NFkB inhibitors while under DS5 exhibited greater tear production after DS5 than vehicle-treated mice (n=3, p=0.02) and recovered pretreatment levels of tear secretion before their untreated cagemates. Remarkably, hyperosmolarity did not induce NFkB activation in vitro in an epithelial cell line, and consistently, it did not increase epithelial expression of major histocompatibility complex II in cocultures with T cells.

Conclusions: Conjunctival tolerance is affected in mice under desiccating stress but only after 3 days of exposure, suggesting that ocular surface damage must reach a threshold in order to affect the immune outcome. As topical NFkB inhibitors during DS5 favor recovery of tear production afterwards, this signaling pathway, and conjunctival tolerance indirectly, must be implicated in disease progression. However, hyperosmolarity does not activate this signaling pathway in vitro, indicating that a more complex cellular & molecular interplay is involved in this model. Altogether these results highlight the therapeutic potential of conjunctival tolerance modulation in ocular surface disease.

Keywords: 486 cornea: tears/tear film/dry eye • 553 immune tolerance/privilege • 480 cornea: basic science