Abstract
Purpose :
Systemic autoimmune disease such as Stevens Johnson Syndrome have ocular manifestations including dry eye, ocular surface inflammation and in severe cases corneal stem cell deficiency. Several proinflammatory cytokines such as TNF-α, IFN-¥, IL-6 and IL-8 have been shown to play a role in the pathogenesis of these conditions. Symptomatic management of dry eye disease include artificial lubricants, warm compresses, punctal occlusion, etc. It is hypothesized that autologous serum tears influence IL-8 expression in human corneal epithelial cells.
Methods :
HCECs were subcultured in cell media. 150,000 cells per well were trypsinized and plated in a 12-well plate with a coverslip and cultured for 24 hours. The next day cells were exposed to TNF-α at concentrations of 0, 1, 10 and 100 ng/ml or AST for 16 hours, then incubated with IL-8 primary antibody in blocking solution for 1 hour. Subsequently, the cells were washed 3 times with 1XPBS and incubated with fluorescent secondary antibody in blocking solutions for 1 hour. Next, the cells were mounted onto DAPI mounting medium and allowed to cure overnight. Coverslips were imaged within 2-3 days after mounting. For quantitative PCR 300,000 cells per well were trypsinized, plated in a 12-well plate and cultured for 24 hours. Cells were then exposed to TNF-α at concentrations of 0, 1, 10, and 100 ng/ml or AST for 16 hours. Cells were lysed and Total RNA was extracted following the Nucleospin RNA plus kit. Subsequently, cDNA was created following the promega cDNA kit. qPCR was performed following the iTaq universal SYBR green protocol.
Results :
TNF-α induced IL-8 expression in HCEC in a dose-dependent manner. This effect was not seen when IL-8 expression was associated with immunofluorescence microscopy. Autologous serum tears down regulated IL-8 expression in HCEC. This effect was independent of whether patient had autoimmune disease as a cause of their dry eye disease. There was no significant difference between HCEC treated with autologous serum tears derived from patients with systemic immune disease and autologous serum tears derived from normal.
Conclusions :
Autologous serum tears can down regulate IL-8 expression in HCEC. Proinflammatory cytokine downregulation of inflammatory cytokines could be one mechanism of how autologous serum tears reduce signs and symptoms of patients with dry eyes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.