Functional Expression Of IFNLR1 On Human Dermal Microvascular Endothelial Cells

Objective:IL-29 compared with type I interferon its side effects are lower,and also has the effect such as antiviral,anti-tumor,immune regulation,but because of its specific receptor IFNLR1 selective distribution,therefore,the clinical application of IL-29 limited scope.The aim of this study was to verify whether the IL-29 receptor IFNLR1 is expressed on human dermal microvascular endothelial cells(HDMEC)and to explore the biological effects of IL-29 binding to IFNLR1 on HDMEC.Provide new experimental basis for the clinical application of IL-29.Methods:(1)Firstly,we cultured HDMECs and then used QPCR to detect the m RNA level of specific receptor IFNLR1 on HDMEC.Then we detected the expression level of receptor IFNLR1 protein by western blot and immunofluorescence technique.(2)The HDMECs were divided into four groups:(1)blank control group(2)recombinant IL-29 treatment group(3)IFNLR1 monoclonal antibody blocking + recombinant IL-29 treated group(4)IFNLR1 monoclonal antibody homologous antibody Ig G treatment group.The levels of cytokines il-8 and il-12 were detected by ELISA in each group after 0.5,1,and 2 hours.(3)After the DENV was applied to HDMEC,the il-29 level was detected by ELISA for 24 hours,48 hours and 72 hours.(4)The HDMECs were divided into four groups:(1)blank control group(2)IFNLR1 monoclonal antibody blocking + DENV treatment group(3)DENV treatment group(4)IFNLR1 monoclonal antibody homologous antibody Ig G + DENV treatment group.A single-layer HDMEC cell model was established by Transwell to detect the cellular permeability of HDMEC in each group.The pull-down method was used to detect the activation of Rho A before and after IFNLR1 monoclonal antibody blocking after DENV treatment.Results:(1)The IFNLR1 receptor in HDMEC was detected from the gene level by R T-q PCR technology,the m RNA expression is 1.00±0.285,and the presence of IFNLR1 receptor in HDMEC was detected from the protein level by western b lot and immunofluorescence technology.(2)IL-8 had a basal secretion on HDMEC;under the stimulation of recombinant IL-29,the secretion of IL-8 was increased compared with the blank group,the level of IL-8 secretion is 72.759±1.825 pg/ml、175.402±20.731 pg/ml、278.736±19.559 pg/ml at time point 0.5 hour,1 hour,2 hour;IL-8 secretion was significantly reduced after treatment of HDMEC with IFNLR1 blocked with IL-29,the level of IL-8 secretion is 24.368±2.296 pg/ml、26.50574±12.232 pg/ml、32.36780±22.967 pg/ml;There was no significant difference in IL-8 levels between the IFNLR1 homologous antibody Ig G group and IL-29 treatment group.It was confirmed that the binding of IL-29 to IFNLR1 plays a role in regulating the secretion of IL-8 by HDMEC,and the IL-8 level is time-dependent.Under the conditions of treatment of HDMEC,IL-12 secretion was extremely low.(3)DENV can cause HDMEC to secrete IL-29,and the IL-29 level is highest at 48 hours,the secreted level of IL-29 is 90.205±1.601pg/ml、179.949±13.241 pg/ml、153.821±5.356 pg/ml at time point 24 hour,48 hour,72 hour.(4)The effect of DENV on HDMEC increased the cell permeability;blocking the IFNLR1 receptor increased the permeability of HDMEC cells more than the DENV treatment group.The maximal permeability was observed 48 hours after DENV treatment group.(5)Rho A was activated by DENV treatment on HDMEC;The activation of Rho A was most abundant in the IFNLR1 monoclonal antibody blocking + DENV treatment group.Conclusions:(1)To confirm the expression of IFNLR1 in human dermal microvascular endothelial cells.(2)IL-29 binds to IFNLR1 and can regulate the secretion of cytokine il-8 but cannot induce the secretion of il-12.(3)The absence of the IFNLR1 receptor on HDMEC has an effect on the activation of Rho A after DENV treatment,and the activation of Rho A has an effect on vascular permeability.