The Effect And Related Mechanism Of Thalidomide On Neutrophil Extracellular Trap Formation In Systemic Lupus Erythematosus

Objective:Systemic lupus erythematosus（SLE）is a common systemic autoimmune disease characterized by a range of autoantibodies directed against autoantigens production.Nowadays,it has been noted that over a million of patients have suffered from SLE in China which topped the global list.But the pathogenesis and therapeutic targets are not clear.Thus,understanding the pathogenesis of this disease and finding therapeutic targets have become a important medical research.Neutrophil extracellular trap（NET）is a web-like chromatin structure released from neutrophils.Recent researches found NET plays an essential role in the pathogenesis of SLE.Inhibiting NET may become a new target of SLE treatment.Thalidomide（Thd）can effectively improve skin manifestations and other symptoms in SLE.NET participates in the development of SLE with skin damage.However,it is unclear that whether thalidomide affects NET formation.Hence,this study examined whether thalidomide has abilities to affect NET formation and NET-induced angiogenesis in SLE,in order to decipher the underlying mechanism of thalidomide treatment of SLE.Our research is divided into the following sections:PART1:The effect of thalidomide on NET formationObjective:To investigate whether thalidomide could affect inflammatory stimuli induced NET formation and SLE spontaneous NET formation.Methods:a.The effect of thalidomide on inflammatory stimuli-induced NET formation of healthy neutrophils:1.Neutrophils were separated from peripheral venous blood samples of healthy volunteer.2.Neutrophils were purified and confirmed by flow cytometry.Cell viability was tested via Cell counting Kit-8 to test the effect of different concentrations of thalidomide on neutrophils.3.Neutrophils were pretreated with PBS or different concentrations of thalidomide（50ug/ml,100ug/ml）for 1 hour.Then,neutrophils were stimulated with medium,PMA 20n M,250ug/ml of purified SLE IgG,or 1%SLE serum for 2 hours at 37℃,5%CO₂.NET formation was qualified by immunofluorescence and ELISA.b.The effect of thalidomide on spontaneous NET formation of SLE neutrophils:1.Neutrophils were separated from peripheral venous blood samples of healthy volunteer and SLE patients.2.Neutrophils were pretreated with PBS or different concentrations of thalidomide（50ug/ml,100ug/ml）for 3 hours at 37℃,5%CO₂.NET formation was qualified by immunofluorescence and ELISA.Results:1.Flow cytometry results showed purity of neutrophils was above 90%.2.CCK-8 results showed thalidomide（≤150ug/ml）did not affect cell viability of neutrophils.3.Immunofluorescence results showed NET formation was increasing in SLE serum group（P<0.001）and SLE IgG group（P<0.001）compared with control group.And NET formation was increasing in SLE serum group compared with SLE IgG group（P<0.001）.4.Immunofluorescence and ELISA results showed NET formation was decreased in PMA+Thd50ug/ml group（P<0.001）and PMA+Thd100ug/ml group（P<0.001）compared with PMA group.And NET formation was decreased in PMA+Thd100ug/ml group compared with PMA+Thd50ug/ml group（P<0.001）.5.Immunofluorescence results showed NET formation was decreased in SLE serum+Thd50ug/ml group（P<0.001）and SLE serum+Thd100ug/ml group（P<0.001）compared with SLE serum group.And NET formation was decreased in SLE serum+Thd100ug/ml group compared with SLE serum+Thd50ug/ml group（P<0.001）.6.Immunofluorescence and ELISA results showed SLE neutrophils were more likely to undergo spontaneous NET formation than healthy neutrophils（SLE polymorphonuclear group vs healthy polymorphonuclear group,P<0.001）.Thalidomide could potently inhibit spontaneous NET formation of SLE neutrophils（SLE polymorphonuclear+Thd group vs SLE polymorphonuclear group,P<0.001）.Conclusion:Except IgG,other factors in SLE serum also participated in NET formation.Thalidomide inhibited either inflammatory stimuli（PMA or SLE serum）induced NET formation of healthy neutrophils or spontaneous NET formation of SLE neutrophils.PART2:Signaling pathway mechanisms of thalidomide inhibited NET formation Objective:MAPK signaling pathway has a key role in NET formation.The activities of p38 and ERK1/2 participate in NET formation.Here,we investigate whether thalidomide affects the activities of p38 and ERK1/2 so that thalidomide inhibits NET formation.Methods:1.Thalidomide affects the activities of p38 and ERK1/2 leading to thalidomide-inhibited NET formation of healthy neutrophils induced by PMA or SLE serum:Neutrophils were separated from peripheral venous blood samples of healthy volunteer.Neutrophils were pretreated with PBS or 100ug/ml thalidomide at 37℃,5%CO₂.Then,neutrophils were stimulated with PBS,PMA 20n M,250ug/ml of purified SLE IgG,or 1%SLE serum.Expression of phosphor-p38（p-p38）,phosphor-ERK1/2（p-ERK1/2）,and GAPDH was analyzed by western blotting.2.Thalidomide affects the activities of p38 and ERK1/2 leading to thalidomide-inhibited NET formation of SLE neutrophils:Neutrophils were separated from peripheral venous blood samples of SLE patients and healthy volunteer（control）.Neutrophils were pretreated with PBS or 100ug/ml thalidomide at 37℃,5%CO₂.Expression of p-p38,p-ERK1/2,and GAPDH was analyzed by western blotting.Results:1.Compared with corresponding control group,our results showed the relative phosphorylation levels of p38 and ERK1/2 in PMA group and SLE serum group were higher（P<0.01）.And in corresponding thalidomide group the relative phosphorylation levels of p38 and ERK1/2 were lower than that in PMA group or SLE serum group（P<0.01）.2.SLE neutrophils had increased phosphorylation of p38 and ERK1/2 compared with healthy neutrophils（P<0.01）.In thalidomide treatment group the phosphorylation of p38 and ERK1/2 was decreased compared with SLE neutrophils group（P<0.01）.Conclusion:MAPK signaling pathway is more active in SLE neutrophils than healthy volunteers’neutrophils.MAPK signaling pathway is involved in NET formation.Thalidomide inhibits NET formation through the suppression of MAPK signaling pathway.PART3:Identification of the effect of thalidomide on NET related inflammatory angiogenesisObjective:Research has demonstrated that the inflammatory angiogenic activities in SLE patients are increasing.Through observing endothelial cell migration and tube formation,this study investigates spontaneous NET of SLE neutrophils whether affect inflammatory angiogenesis and the effect of thalidomide on it.Methods:1.The effect of SLE spontaneous NET on inflammatory angiogenesis:Human umbilical vein endothelial Cells（HUVEC）were used between passages 3and 5.Spontaneous NET formation of SLE neutrophils were collected and quantified by Pico Green ds DNA Assay.Endothelial cell migration and tube formation were measured by transwell assay.The bottom chamber was filled with complete medium.HUVEC with different treatments（PBS or NET）were seeded onto top chamber,cultured for 24 hours and observed results.For tube formation,HUVEC were divided to 2 groups with treatments（PBS or NET）and cultured for 4 hours and recorded the results.2.The effect of thalidomide on SLE spontaneous NET-related inflammatory angiogenesis:HUVEC were used between passages 3 and 5.Cell viability was tested via Cell counting Kit-8 to test the effect of different concentrations of thalidomide on HUVEC.Endothelial cell migration were measured by transwell assay.The bottom chamber was filled with complete medium.HUVEC with different treatments（PBS,NET,or NET+thd100ug/ml）were seeded onto top chamber,cultured for 24 hours and observed results..For tube formation,HUVEC were divided into 3 groups with treatments（PBS,NET and NET+thd100ug/ml）and cultured for 4 hours and recorded the results.Results:1.CCK-8 results showed thalidomide（≤150ug/ml）did not affect cell viability of HUVEC.2.Transwell results showed HUVEC migration was increasing in NET group compared with control group（P<0.01）.Tube formation results showed tube length,the number of loops and branch points in NET group was increasing compared with control group（P<0.01）.3.Transwell results showed HUVEC migration was decreasing in thalidomide group compared with NET group（P<0.01）.Tube formation results showed tube length,the number of loops and branch points in thalidomide group was decreasing compared with NET group（P<0.01）.Conclusion:Inflammatory angiogenesis can be excited by SLE spontaneous NET which can be inhibited by thalidomide.