Abstract
Purpose :
Nuclear factor activated in T cells (NFAT) has been known to regulate the expression of a wide variety of inflammatory cytokines. The NFAT family of proteins possess five different isoforms (NFAT1-5). NFAT1-4 isoforms are dephosphorylated and activated by calcineurin. Activated NFAT proteins translocate to the nucleus and enhance the expression of several inflammatory cytokines including interferon gamma (IFNγ), interleukin (IL)-2, IL-4, and IL-17, granulocyte-macrophage colony-stimulating factor (GM- CSF). We investigated the involvement of NFAT proteins on lipopolysaccharide (LPS)-mediated expression of inflammatory cytokines in the retinal pigment epithelial cells (RPE).
Methods :
We treated ARPE-19 cells with NFAT inhibitor, MAGPHPVIVITGPHEE (VIVIT), for 4-6 hrs followed by 10 µM LPS for 24 hrs to analyze whether NFAT is involved in the regulation of LPS-induced expression of inflammatory cytokines in the RPE. Human cytokine antibody array was used to examine several cytokines expression in the cells treated with LPS and LPS in combination with the NFAT inhibitor, VIVIT. Quantitative real time PCR (qRT-PCR) analysis was performed to evaluate the expression of LPS-induced cytokines in the RPE.
Results :
In ARPE-19 cells treated with 10 μM LPS, human cytokine antibody array showed an induction in the expression of several cytokines including IL-6, IL-8, IL-7, Monocyte Chemoattractant Protein-1 (MCP-1), GM-CSF, growth-related oncogene (GRO) and growth-related oncogene alpha (GRO alpha). The cytokine array revealed that LPS-induced expression of IL-6, IL-8 and growth-related oncogene alpha (GRO alpha) was decreased in the presence of 10 and 20 μM of the NFAT inhibitor, VIVIT. qRT-PCR analysis showed an induction in the expression of IL-7, CCL3, GM-CSF, and MCP-1 upon LPS treatment and LPS-induced expression of MCP-1 and GM- CSF was decreased in the presence of the NFAT inhibitor, VIVIT. qRT-PCR analysis also revealed that the expression of tumour necrosis factor alpha (TNF- α) was decreased upon treatment with LPS and VIVIT.
Conclusions :
These results suggest NFAT proteins play an important role in regulation of inflammatory cytokine expression in the RPE. Furthermore, these studies suggest that suppression of NFAT activity by a peptide inhibitor can inhibit inflammatory cytokine expression
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.