High-salt Enhances The Inflammatory Response By RPE Cells Following LPS Stimulation

BackgroundThe retinal pigment epithelium(RPE), situated on the outer side of the retina, is a monolayer of cells connected by tight junctions with important functions for the visual system. ARPE-19, a spontaneously arisen cell line of RPE, has been extensively used in the past decades to investigate the role of this cell layer in the pathogenesis of a number of diseases including age related macular degeneration(AMD), vitreoretinopathies and uveitis. The major inflammatory cytokines produced by ARPE-19 in response to various stimuli are interleukin-6(IL-6), interleukin-8(CXCL8; IL-8) and monocyte chemoattractant protein-1(CCL2; MCP-1). IL-6 is a pro-inflammatory cytokine that plays an important role in intensifying the intraocular immune and inflammatory response. IL-8 and MCP-1 are important chemoattractants of neutrophils, lymphocytes and monocytes, causing these cells to infiltrate into intraocular tissues during inflammatory retinal disease.Both genetic as well as environmental factors are considered to play a role in the pathogenesis of intraocular inflammation. The role of environmental factors has mainly focused on a role of infections in the development of autoimmune or autoinflammatory uveitis and as yet little attention has been paid to dietary factors. Evidence is now emerging that a high-salt diet may be involved in the pathogenesis of autoimmune disease. Recent studies have shown that high-salt could aggravate the severity of experimental autoimmune encephalomyelitis(EAE) by inducing a Th17 cell immune response, whereby the effect of high-salt on Th17 cell polarization was mediated by activating the p38/MAPK pathway via nuclear factor of activated T cells 5(NFAT5) and serum/glucocorticoid-regulated kinase 1(SGK1). Case-control studies showed that high-salt intake in smokers was associated with the risk of rheumatoid arthritis and a higher salt intake is also associated with increased clinical and radiological disease activity in patients with multiple sclerosis. Whether high-salt affects autoimmune diseases of the eye such as uveitis is not yet known.Since RPE cells play an important role as cellular mediators of the intraocular inflammatory response we decided to investigate the effect of high-salt on the cytokine release by these cells. PurposeHigh-salt(sodium chloride; NaCl) has been shown to play a role in the pathogenesis of autoimmune disease. However, if high-salt affects intraocular inflammatory diseases, especially autoimmune diseases of the eye such as uveitis is not yet known. In this study, we investigated the effect of high-salt on the production of inflammatory mediators by ARPE-19 cells and the possible mechanisms involved in. MethodsARPE-19 cells were cultured with lipopolysaccharide(LPS) in a culture medium to which extra NaCl had been added(20 mM and 40 mM). The apoptosis of ARPE-19 cells was detected by flow cytometry, and the proliferation was measured by using CCK-8. ELISA was used to detect the concentration of IL-6, IL-8, and monocyte chemoattractant protein-1(MCP-1) in culture supernatants. Flow cytometry and Real-time PCR were used to detect the possible signaling pathways involved in the NaCl-mediated inflammatory response. Results(1) RPE cells were stimulated with LPS in the absence or presence of extra additions of NaCl(20 mM, 40 mM) to the culture medium. Cells were stained with AnnexinV and PI for FACS analysis. The data are expressed that there were no significant differences on cell apoptosis between the groups.(2) RPE cells were stimulated with LPS in the absence or presence of extra additions of NaCl(20 mM, 40 mM) to the culture medium.for 24 h, 48 h and 72 h. The results showed that the extra addition of NaCl did not affect the proliferation of the cells at any given point in time.(3) RPE cells were stimulated with LPS in the absence or presence of extra additions of NaCl(20 mM, 40 mM) to the culture medium.for 24 h. ELISA was used to detect the cytokines in cell culture supernatants. The results showed that NaCl could significantly induce IL-6 and MCP-1 secretion at 40 mM, and that it only significantly promote the production of MCP-1 at 20 mM. However, NaCl had no influence on IL-8 secretion at both concentrations used.(4) RPE cells were stimulated with LPS in the absence or presence of extra additions of 80 mM mannitol and 40 mM NaCl to the culture medium.for 24 h. After the cytokines in cell culture supernatants were detected, the results showed that mannitol had no effect on the production of IL-6, IL-8 and MCP-1. However, NaCl at 40 mM could upregulate the production of IL-6 and MCP-1 which accorded with result 3. These results suggest that osmotic stress alone did not mediate inflammatory process.(5) RPE cells stimulated with LPS were incubated with or without high-salt(20 mM or 40 mM). The cells were then harvested to measure the level of phosphorylated cell signal pathways by FACS. The results showed that NaCl significantly induced the phosphorylation of P38, AKT and NF-κB at 40 mM. It had no effect on the level of phosphorylated JNK and ERK-1/2 as compared to controls.(6) The down-stream signal pathway was detected. The data showed that 40 mM NaCl increased the gene expression of NFAT5 and SGK1 in RPE cells, and 20 mM NaCl could only upregualte the expression of NFAT5, but not SGK1. ConclusionThese results suggest that local high-salt may contribute to the intraocular inflammatory response by promoting pro-inflammatory cytokine production. is associated with activation of the p38 MAPK-NFAT-SGK1 pathways, Akt and NF-κB pathways.