Abstract
Purpose :
RPE cells representing the outer blood-retinal barrier (BRB) express the transcription factor Foxp3 and marker of regulatory T cells in a stressed state, potentially enabling them to suppress immune responses. Here we investigated the effect of various additional stressors on FoxP3 expression in vitro, its posttranslational modification (phosphorylation) and the expression of the chemokine receptor CXCR4 that is regulated by Foxp3.
Methods :
The RPE cell line ARPE-19 was cultured in DMEM/F12 medium with 10% FBS and treated with either LPS (1 or 10µg/ml) for 6 and 24h or by a scratch disrupting the call layer 24h prior to immunostaining for FoxP3 and CXCR4 with and without phosphorylation (FoxP3: phosphorylation at Ser19P or Ser418P; CXCR4 and CXCR4-P). Fluorescence intensity was determined with Image J.
Results :
Cultured RPE cells are in a constitutively stressed state expressing FoxP3 that could be altered or enhanced by scratching the cell layer (BRB injury) or stimulation with LPS (imitates bacterial infection). After scratching or treatment with LPS FoxP3 was increased in both, cytoplasm (scratch: 3.5fold; LPS: 2.3fold) and nucleus (scratch: 2.6fold; LPS: 1.5fold). In the cytoplasm phosphorylated FoxP3-Ser19P was only expressed after scratch and LPS-treatment, while in the nucleus it increased only weakly from baseline (scratch: 1.8fold, LPS: 1.49fold). Also in the cytoplasm FoxP3-Ser418P was 10.5fold elevated after scratch and only 2.3fold after LPS-treatment , while nuclear expression was unaltered. CXCR4 was mainly expressed in the cytoplasm, decreasing after scratch, and increasing 2.5fold after LPS-treatment with unaltered nuclear expression. However, CXCR4-P was 3.7fold elevated in the cytoplasm and 2.3fold in the nucleus in scratched cultures and only slight enhanced after LPS-treatment (1.9fold cytoplasm, 1.2fold nucleus).
Conclusions :
Cell destruction and LPS stressed ARPE-19 cells differently. FoxP3, FoxP3-Ser19P, FoxP3-Ser418P and CXCR4-P were preferentially induced by scratch, while LPS upregulated FoxP3-Ser418P and CXCR4. The expression of posttranslationally modified FoxP3 increased in stressed ARPE-19 cells in vitro, probably supporting RPE to control inflammation. CXCR4 leads to migration of epithelial cells, and increased expression of CXCR4-P after cell destruction (scratch) might be important to maintain the integrity of the BRB after damage.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.