Abstract
Purpose :
Goblet cell functions especially secretion of mucins are relatively well studied owing to their protective role on the ocular surface; however, little attention is given to the other secretory products, such as extracellular vesicles (EVs). The purpose of this study is to examine the function of goblet cell secreted EVs, especially their secretagogue activity, and test whether EVs produced by vehicle (health) or inflammatory stimuli (disease) will cause distinct actions on recipient goblet cells.
Methods :
Serum starved primary human conjunctival goblet cells (HCGCs) grown from conjunctiva explants were incubated for 4 h either with the allergic mediator histamine (His, 10-5 M) to induce inflammation or HBSS (non-treated control) to indicate normal cells. EVs isolated from collected media using Total Exosome Isolation Reagent (Invitrogen) were denoted as EVs-His and EVs-NT, respectively. To examine secretagogue activity, first passage cells (trypsinized and passaged primary cells) were treated with EVs diluted to 1, 10, 100, 1000 ng/mL) for 4 h in HBSS. The amount of secreted high molecular weight proteins (HMWP) in the cell culture supernatants was measured using an enzyme-linked lectin assay (ELLA).
Results :
HCGCs stimulated with His secreted 4-fold higher amount of total EV proteins into the media compared to that of the untreated cells, which confirmed that the primary cells are effectively stimulated with His. For secretagogue activity of EVs, addition of 10 ng/mL EVs-His to HCGCs induced secretion of HMWP by 4.8-fold. The same concentration of EVs-NT caused a 1.6-fold increase. For both EV-His and EV-NT, an increase of 1.0~1.9-fold was observed at the other four concentrations; however, all lower than the activity seen at 10 ng/mL. Fold-change of secretion in His treated (positive cntl) and untreated cells (negative cntl) was 2.2 and 1.0, respectively.
Conclusions :
We conclude that HCGCs EVs have differential secretagogue activity when stimulated with inflammatory mediator compared to under control (healthy). EVs have an indispensable role in regulating goblet cell secretion at the extracellular level, which warrants in-depth investigation into their molecular properties and mechanism of action.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.