Subnormal cytokine profile in the tear fluid of keratoconus patients

Abstract

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.

Figure 1. Cytokine measurements in tear fluids by a multiplex immuno-bead assay.

Each cytokine was measured in individual subjects. The horizontal bar indicates the mean value for each group. After correcting for age and contact lens use, IL-12, TNF-α, and RANTES/CCL5 were significantly lower in KC than in controls. Statistical analyses of the data are shown in Tables 2 and 3.

Figure 2. Decrease in IL-4 measured by standard ELISA.

Samples shown in Table S2 were pooled into 13 control and 10 keratoconus samples (Methods). Each ELISA value is an average of two measurements. The decrease in IL-4 in the keratoconus pool (7.6±5 pg/ml), compared to controls (14.2±8.6 pg/ml) was statistically significant (p≤0.05, Mann-Whitney test). The horizontal bar indicates the mean value for each group.

Figure 3. Increase in IL-17 measured by standard ELISA.

Five pools of keratoconus and control subjects (Table S2) were used. Each ELISA value is an average of two measurements. The increase in IL-17 in keratoconus samples (72.5±59.2 pg/ml) compared to controls (18.5±7.8 pg/ml), was statistically significant (p≤0.05, Mann-Whitney test). The horizontal bar indicates the mean value for each group.