Impact of the Host Microbiota on the Development of Murine Desiccating Stress-Induced Dry Eye Disease

Abstract

Purpose: Recently, alterations in the host microbiota have been shown to impact the development of various autoimmune diseases. To begin to understand the contribution of commensal enteric bacteria on the development of Dry Eye disease, C57BL/6 mice were treated with a broad spectrum antibiotic cocktail, and then exposed to desiccating stress (DS) to induce experimental Dry Eye disease.

Methods: Dry Eye disease was induced by exposing female C57BL/6 mice to desiccating stress (DS: subcutaneous scopolamine, 0.1mg/day; humidity 20%; sustained airflow). Three weeks prior to inducing Dry Eye, commensal flora were depleted by treating mice with an antibiotic cocktail consisting of the antifungal amphotericin-B (1 mg/kg, oral gavage BID), ampicillin (1 mg/ml, ad libitum via drinking water), and oral gavage of vancomycin (50 mg/kg), neomycin (100 mg/kg), metronidazol (100 mg/kg) and amphotericin-B (1 mg/kg) administered every 12 hours. To ensure the antibiotic cocktail was effective at depleting commensal flora, fresh fecal samples were collected and monitored for bacterial load. Subsequently, mice were evaluated for markers of inflammation and corneal epithelial cell damage. In addition, the effect of microbiota depletion on pathogenic T cell activation was addressed with CD4+ T cell adoptive transfer studies into T cell-deficient nude recipient mice.

Results: Microbiota-depleted C57BL/6 mice exposed to DS displayed a significant increase (p≤0.001) in the average number of CD4+ T cells in the conjunctiva (10.3±1.4), with a trend towards increased counts in the lids (25.8±6.1) when compared to DS mice with unaltered microbiota (conjunctiva: 7.5±0.9; lid: 15.7±3.7). Elevated CD4+ T cell infiltration in microbiota-depleted mice was associated with a significant increase (p≤0.001) in corneal fluorescein staining on Day 9 of DS (8.0±2.1) relative to staining in DS mice with normal flora (5.8±2.1).

Conclusions: These data suggest that alteration of the host microbiota increases the severity of DS-induced Dry Eye disease in mice.

Keywords: 486 cornea: tears/tear film/dry eye • 557 inflammation • 422 antibiotics/antifungals/antiparasitics