Abstract
Purpose :
Non-infectious uveitis (NIU) is a significant cause of blindness. It is an immune mediated disease however the exact pathogenesis remains elusive. Considerable evidence exists on the immunomodulatory effects of EVs in various diseases; although very little is known regarding EVs in uveitis patients. We have previously shown that cytokine stimulated ARPE19 cells, release Extracellular Vesicles (EVs); these EVs are capable of inhibiting T cell proliferation, inducing pro-inflammatory cytokines from monocytes as well as inducing monocyte cell death, in PBMCs from NIU patients. The purpose of this study is to establish a reliable protocol to isolate EVs from uveitis patients and to determine their immune-modulatory potential in-vitro.
Methods :
EVs were isolated from the serum of NIU patients using Size Exclusion Chromatography (SEC), in 500ul fractions. Appropriate fractions were then pooled and concentrated via filtration. Various markers on the surface of isolated EVs were analyzed using the MACSPlex Exosome kit. The EVs were set up in culture with PBMCs or ARPE19 cells; after stimulation the cells were stained to determine cell activation and cytokine production.
Results :
The first six SEC fractions contained EVs of appropriate size with very low protein content along with the presence of exosome associated markers CD9, CD63 and CD81. A higher number of EVs were isolated from the sera of patients, as compared to healthy controls. Additionally, EVs from patients expressed increased levels of CD8, CD29, CD44, and importantly HLA-DR/DP/DQ, indicating their potential to activate cells in-vivo. An upregulation of CD69 and CD25 on T cells was observed when the cells were cultured with EVs from patients. Culturing PBMCs, with EVs from patients lead to an increase in IFNg production from CD4+ T cells as compared to those cultured with EVs from healthy controls. Additionally, incubating ARPE19 cells with the EVs from patients, increased the expression of MCP1 and IL8 in ARPE19 cells.
Conclusions :
We observed that SEC, along with filtration, is a reliable and fast method for isolating EVs from low volume samples. Preliminary data indicates that EVs isolated from uveitis patients can induce a robust activation of various cells in-vitro. Future experiments are designed to understand the pro-inflammatory nature of the EVs isolated from NIU patients as well as to further substantiate these results.
This is a 2021 ARVO Annual Meeting abstract.