Abstract
Purpose :
Ocular surface infection by human adenovirus species D (HAdV-D) causes epidemic keratoconjunctivitis (EKC). The most significant long-term complication of EKC is subepithelial infiltrate (SEI) formation, occurring in about one-third of cases. However, the mechanism of SEI formation after adenoviral infection of the cornea remains uncertain. High-mobility group box protein 1 (HMGB1) is an important late inflammatory mediator; extracellular HMGB1 variably regulates inflammatory responses and leukocyte infiltration. In this study, we aimed to define a potential role for HMGB1 in progression to SEI formation.
Methods :
Primary human corneal epithelial (PHCE) cells, hTERT-immortalized human corneal epithelial (THE) cells, and primary human corneal fibroblasts (HCFs) were infected with HAdV-D37 or HAdV-C5 at MOI = 5, and cell supernatants were collected through 48 hours post infection. Mass spectrometry (LC-MS/MS) analysis was performed on cell lysates. Immunoblotting was performed on infected cell supernatants, and on cytoplasmic and nuclear cellular fractions, using acetylated HMGB1 antibody. ELISA for secreted HMGB1 was conducted on cell-free supernatants, and HMGB1 gene expression was studied using real-time qPCR. Confocal microscopy was performed to visualize HMGB1 translocation. Cytokine expression by HCFs treated with recombinant HMGB1 was analyzed using human cytokine protein arrays.
Results :
HAdV-D37 infection resulted in HMGB1 translocation from the nucleus to the cytoplasm and then to the extracellular space in THE cells, but not in HCF. HAdV-C5, a virus not associated with EKC, did not induce secretion of HMGB1 from any of the cell types tested. Finally, treatment of HCF with recombinant HMGB1 triggered expression of pro-inflammatory mediators.
Conclusions :
Our data suggest that HMGB1 is specifically released by adenovirus infected corneal epithelial cells and also provides insights into possible mechanism of corneal SEI formation in EKC. HMGB1 expressed by adenovirus infected corneal epithelial cells could induce underlying stromal cells to express pro-inflammatory mediators, leading indirectly to the development of SEI. HMGB1 may be a viable therapeutic target for preventing the corneal stromal complications of EKC.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.