HARVARD DEPARTMENT OF OPHTHALMOLOGY
Category : Basic and Translational Research Candidate : Amrita Saha Vadher Poster #: B22
Potential Role of High Mobility Group Box 1 in Formation of Corneal Subepithelial Infiltrates in Adenovirus Keratitis
Amrita Saha , Mohammad Mirazul Islam , Ashrafali Mohamed Ismail , Xiaohong Zhou , James Chodosh , Jaya Rajaiya
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 realtime 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 andalso 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 proinflammatory mediators , leading indirectly to the development of SEI . HMGB1 may be a viable therapeutic target for preventing the corneal stromal complications of EKC .