IL-1β Signaling Induced By All-trans-retinal Contributes To Complement Alternative Pathway Activation In Retinal Pigment Epithelium Cells

Purpose:To investigate the complement activation pathway and related molecular pathways induced by all-trans-retinal(atRAL)in retinal pigment epithelial cells(RPE).Methods:The human ARPE-19 cells were exposed to 5 μM atRAL.Primary pRPE cells were exposed to 10 μM atRAL.The BV-2 cells and RAW264.7 cells were also exposed to 5 μM atRALtreatment.MAC formation was examined by the fluorescent immunostaining of C5b-9.qRT-PCR was utilized to examine the mRNA expression of complement factors,complement regulatory proteins(CRPs)and inflammation factors in thecells challenged with atRAL.Western blots were utilized to examine the protein expression of complement factors,CRPs,inflammation factors,JNK,p38 and NF-κb signaling pathways in cells challenged with atRAL.ELISA was utilized to examine the expression and secretion of inflammation factors interleukin-1β(IL-1 β).Results:Membrane attack complexes were formed in atRAL-treated ARPE-19 cells and primary pRPE cells when incubated with normal human serum but not FB-depleted serum or heat inactivated normal human serum(HI-NHS).An increase in CFB expression as well as downregulation of CD46,CD55,CD59,and CFH were observed in RPE cells after atRAL treatment.Furthermore,IL-1β production was provoked in atRAL-treated RPE cells.N-Acetyl-L-cysteine failed to reverse the changes in the formation of MAC and expression levels of CFB,CFH,and mCRPs in ARPE-19 cells.Coincubation of RPE cells with IL1Ra and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and JNK signaling pathways.The mRNA expression of inflammatory factors and complement factors in microglia/macrophages were up-regulated after atRAL treatment,including Cfb and CRPs Cd46,Daf2 and Crry in RAW264.7 cells.as well as a slight increase of Cd46,Cd59a and Crry in BV-2 cells.Conclusion:atRAL up-regulated CFB via IL-1β through p38 and JNK signaling pathways,and atRAL down-regulated complement regulatory proteins CD46,CD55,CD59 and CFH,atRAL treatment resulted in abnormal activation of complement alternative pathway in RPE cells.atRAL may also promote the abnormal activation of complement alternative pathway in RPE cells by inducing the expression of IL-1β in microglia/macrophages,which forms a chronic pro-inflammatory microenvironment.The chronic pro-inflammatory microenvironment caused by the abnormal activation of complement alternative pathway in RPE cells induced by atRAL may be one of the causes of STGD1 and AMD.