RIG-I Contributes To DsDNA-induced Innate Immune Activation In Human Brain Microvascular Endothelial Cells

Objective: Human Brain Microvascular Endothelial Cells(HBMEC)are a kind of cells located in the inner wall of human Brain microvessels,which is an important part of human Blood Brain Barrier(BBB).It closely combines with smooth muscle cells of cerebral vessels to form microvascular walls.HBMEC have several important physiological roles,including maintaining the integrity of the BBB,controlling capillary permeability,maintaining neuronal health,and regulating blood flow.In addition,HBMEC recognize foreign substances and regulate immune responses by producing cytokines and neurotransmitters that regulate interactions with neurons.BBB is the protective barrier between human blood and brain tissue cells,which is mainly composed of HBMEC,basement membrane and astrocytes.It can restrict the entry of external substances into brain tissue,and is an effective physiological barrier to prevent the invasion of exogenous pathogens and protect brain tissue from pathogens.Therefore,the integrity and stability of blood-brain barrier is of great significance for human physiological health.However,some pathogens,such as Double-stranded DNA(ds DNA)viruses,would have escaped the immune response of HBMEC and crossed the BBB,resulting in severe viral neurological diseases,and there was no much report on the immune response mechanism of stranded DNA.Therefore,the study of HBMEC is of great significance for the study of neurological diseases and the delivery and effect of drugs in the brain.Vesicles Simplex Virus type 1(HSV-1),the most common ds DNA virus,evades the immune action of HBMEC in some specific way.Herpes Simplex Encephalitis(HSE)was caused by crossing the human BBB.The main symptoms of HSE were hemiplegia,quadriplegia and vision loss.At the same time,HSV-1 infection can lead to a series of neurological sequelae and even death,which poses a serious threat to human body,and is one of the major diseases that seriously affect the world’s public health.Therefore,an in-depth study of its immune response mechanism is helpful to provide theoretical support for the subsequent treatment of viral encephalitis.This study focuses on the regulatory mechanism of exogenous ds DNA recognition by HBMEC and explores the crucial recognition receptors or immune factors required for ds DNA-induced innate immune activation of HBMEC.The elaboration of these mechanisms will not only helpful to understand the mechanism of immune response in HBMEC,but also is expected to provide a new intervention target for the prevention or treatment of viral encephalitis.Methods: In this study,dsDNA simulator poly(dA:dT)was used to simulate exogenous substance stimulation,and a variety of immune signaling factors expressed by HBMEC were detected to explore ds DNA-mediated signaling pathways.HBMECs were transfected with increasing concentrations of poly(d A:d T),cellular RNA and total proteins were extracted,and the cell-free supernatant was collected.Real-Time quantitative PCR was used to detect the m RNA level of multiple immunoregulatory genes.ELISA assay was used to determine the IFNs expression.Protein expression of related factors within HBMEC was detected using Western Blot.CRISPR-Cas9 technology was used to construct c GAS or IFI16 gene knockout cell lines to identify their roles in immune response.Silencing of RIG-I using siRNA to determine its role in regulating immune signaling pathways in HBMEC.Results: Poly(dA:dT)transfection of HBMEC induced significant upregulation of a series of antiviral factors,including IFN-β,IFN-λ1 and Interferon-stimulated genes(ISGs).In addition,activation of HBMEC was found to effectively inhibit HSV-1replication.Combined with CRISPR-Cas9 technology and si RNA transfection for relevant cellular receptors silencing,we observed that RIG-I had a key role in the recognition of poly(d A:d T)compared to c GAS and IFI16,the two conventional DNA sensors.Specifically,the expression of various immune factors induced by ds DNA was significantly inhibited by RIG-I silencing,and the poly(d A:d T)-induced anti-HSV-1response was significantly weakened accordingly.In summary,this study concludes that RIG-I plays an important role in dsDNA-induced innate immune activation of HBMEC.