Abstract
Abstract: :
Purpose: To maintain a healthy cornea, the outer-most corneal epithelial layers undergo cell turnover/death and initiate cellular immune defence. Apoptosis is thought to be involved in both of these processes, in particular to dampen viral infections. Herpes Simplex virus type 1 (HSV1), a significant causative agent of microbial keratitis and blindness, can infect the eye exogenously altering apoptosis in corneal keratocytes. How the virus interacts with the epithelium is unclear. HSV1 has been observed in liver epithelium to alter apoptosis, possibly prolonging cell survival. Our aim is to determine if HSV1 alters (suppresses) early corneal epithelial apoptosis therefore maximizing infection. Method: Virulent HSV1 (McIntyre strain; Ultraviolet (UV)-irradiated or non-UV irradiated), avirulent HSV1, and media alone were incubated with cultured human corneal epithelial cells (HCEs) or primary corneal epithelial cells. A time course was run (0.5, 1, 2, and 4 hours). Harvested cells were analyzed using early apoptotic stains (Annexin V [detects phosphatidylserine (PS) flipping] and M30 CytoDeath [Caspase dependent]). UV experiments were utilized to determine whether attachment/entry of the virus was initiating early apoptosis. Results: We observed a significant increase and decrease with Annexin V staining at 1 hour with McIntyre-infected HCEs (15% versus 2.9%, 0.8%, p<0.05). Also, a significant increase and decrease in M30 CytoDeath staining was observed at 2 hours with McIntyre-infected HCEs (6% versus 2%, 1.2%, p<0.05). Only with Annexin V staining did McIntyre-infected primary cells have the same significant increase and decrease at 2 hours (10% versus 1%, 0%, p<0.05). UV irradiated McIntyre virus did not initiate Annexin staining in contrast to the non-UV irradiated McIntyre in HCEs at 1 hour (18% versus 88% out of total observed Annexin V positive cells for both conditions, p<0.05). Conclusion: There was an early initiation and suppression in the apoptotic event of PS flipping which transiently occurred between 30 minutes and 2 hours in virulent HSV1-infected cultured and primary human corneal epithelial cells. This alteration manifested just prior to HSV immediate-early gene synthesis. However, cultured and primary cells did not share the same significant initiation and suppression of the caspase dependent pathway. Furthermore, the UV irradiated virulent HSV1 infection did not evoke an apoptotic response suggesting that PS flipping transpired between entry of the virus and immediate-early gene synthesis.
Keywords: 425 herpes simplex virus • 372 cornea: epithelium • 341 cell death/apoptosis