Topical interferon-gamma neutralization prevents conjunctival goblet cell loss in experimental murine dry eye

Highlights

• Interferon gamma (IFN-γ) inhibits goblet cell differentiation.

• IFN-γ stimulates apoptosis and inhibits IL-13 signaling in the conjunctival epithelium.

• Topically applied anti-IFN-γ inhibits apoptosis and maintains IL-13 signaling.

Abstract

Evidence suggests that the cytokine interferon (IFN)-γ released by natural killer and CD4⁺ T cells contributes to the conjunctival goblet cell (GC) loss in dry eye. The purpose of this study was to investigate if topical neutralization of IFN-γ prevents or alleviates GC loss in an experimental desiccating stress (DS) model of dry eye. In this study, we found that topical IFN-γ neutralization significantly decreased DS-induced conjunctival GC loss. This was accompanied by decreased epithelial apoptosis, and increased IL-13 and decreased FoxA2 expression in the forniceal conjunctiva. To establish that IFN-γ produced by pathogenic CD4⁺ T cells contributes to DS-induced GC loss, adoptive transfer of CD4⁺ T cells isolated from DS exposed donors to naïve RAG-1^−/− recipient mice was performed. Similar to the donor mice, topical IFN-γ neutralization decreased conjunctival GC loss, suppressed apoptosis and increased IL-13 expression in adoptive transfer recipients. In summary, this study demonstrated that topical neutralization of IFN-γ prevents GC loss via modulating apoptosis and maintaining IL-13 signaling.

Introduction

Dry eye is one of the most prevalent eye diseases, affecting tens of millions of people worldwide. The pathogenesis of keratoconjunctivitis (KCS), the ocular surface disease of dry eye is a multifactorial process that includes activation of stress pathways in the ocular surface epithelia by desiccation, the hyperosmolar tear film and inflammatory cytokines, such as interleukin (IL)-17 and interferon gamma (IFN-γ) that are produced by resident intraepithelial lymphocytes and infiltrating CD4⁺ T cells (De Paiva et al., 2007, De Paiva et al., 2009, De Paiva et al., 2010, Lam et al., 2009, Niederkorn et al., 2006). Conjunctival goblet cells (GCs) are simple columnar epithelial cells that secrete the gel-forming mucin MUC5AC that stabilizes the tear film and protects the cornea. GC loss in dry eyes is often associated with a poorly protected and irregular cornea and may lead to sight-threatening corneal ulceration and perforation (Murube and Rivas, 2003, Pflugfelder et al., 1997, Stern et al., 1998a, Stern et al., 1998b). The mechanisms responsible for GC loss in KCS are not completely understood; however, there is increasing evidence indicating that an altered balance of T helper cell (Th) cytokines has a prominent role this process. We previously demonstrated that CD4⁺ T cells activated by exposure to desiccating stress (DS), when adoptively transferred to naïve T-cell-deficient nude mice, were sufficient to elicit autoimmune KCS with pronounced conjunctival GC loss (Niederkorn et al., 2006, Zhang et al., 2011b).

Th1 and Th2 cytokines have been found to have opposing effects on conjunctival goblet cell development. The Th2 cytokine IL-13 has been found to induce GC hyperplasia in nonocular mucosa, such as the gut and respiratory tracts (Atherton et al., 2003, Kanoh et al., 2011, Marillier et al., 2008). We have observed that IL-13 produced by resident NKT cells has a homeostatic function in promoting conjunctival epithelial goblet cell differentiation and mucus production (De Paiva et al., 2011). IL-13 signals through STAT6 and has been found to stimulate production of GC mucin MUC5AC directly or indirectly by suppressing production of the forkhead transcription factor FoxA2, a MUC5AC repressor (Kim et al., 2008, Oh et al., 2010, Rogers, 2003). In contrast, experimental desiccating stress increased the number of cells staining positively for the Th1 cytokine IFN-γ⁺ in the goblet cell zones of the conjunctiva and increased the concentration of IFN-γ in tears (De Paiva et al., 2007). This was accompanied by a decrease in IL-13/IFN-γ ratio and progressive GC loss. No change in conjunctival GC density was noted in IFN-γ-knockout mice subjected to desiccating stress; however, loss of GCs similar to wild type was observed following subconjunctival injection of IFN-γ in these mice (De Paiva et al., 2007). These findings suggest that strategies to neutralize IFN-γ may prevent dry eye induced GC loss.

One mechanism for IFN-γ induced GC loss in KCS is induction of conjunctival epithelial apoptosis. IFN-γ-knockout mice were found to be resistant to DS-induced conjunctival apoptosis; however, exogenous administration of IFN-γ to this strain significantly increased apoptosis after DS (Zhang et al., 2011a). Meaningfully, apoptosis was greatest in the goblet cell area, and MUC5AC expression was inversely associated with level of apoptosis in experimental murine dry eye, suggesting that IFN-γ may cause GC loss in DS by promoting apoptosis under DS (Zhang et al., 2011a).

The purpose of this study was to investigate if topical neutralization of IFN-γ would alleviate or prevent GC loss by maintaining IL-13 expression and modulating apoptosis using a murine DS model with features similar to human KCS.