The Effect And Mechanism Of ELR~+ CXC Chemokines Induced Nets

Background and ObjectiveNeutrophils play a pivotal role in host innate immune response.The release of neutrophil extracellular traps(NETs)is an unique and robust antimicrobial strategy employed by neutrophils.However,increasing evidences also suggest the harmful effects of NETs in inflammatory and autoimmune diseases.Thus it is important to understand the underlying mechanisms of NETs formation.Chemokines are the direct modulators of migration,activation and other aspects of neutrophil biology but their roles in NETs formation have not been well understood.CXC chemokines bearing the glutamic acid-leucine-arginine(ELR)motif are crucially involved in the migration and activation of neutrophils by interaction with membrane CXC chemokine receptor(CXCR)1 and CXCR2.However,it is not known whether ELR~+ CXC chemokines induce neutrophil extracellular traps(NETs).The purpose of this study is to elucidate the role of ELR~+ CXC chemokine in inducing the formation of extracellular trap(NETs)and its mechanism.Methods1.We compared the NETs triggering ability among representative ELR~+ CXC chemokines,ELR~-CXC chemokines and non chemotactic cytokines via confocal microscopy and extracellular MPO ELISA.2.Confocalmicroscopy,scanningelectronmicroscopeand immunofluorescence were applied to characterize NETs formation induced by CXCL8.3.The effect of inhibitors on CXCL8 induced NETs was detected via confocal microscopy,the effect of inhibitors on CXCL8 induced ROS,mtROS and calcium level were measured by flowcytometry.qRT-PCR and immunofluorescence were also used to research the mechanism of CXCL8induced NETs.Results1.Our results demonstrated that ELR~+ CXC chemokines are most competent to induce NETs formation in neutrophils.Then we characterized the properties of NETs release and explored its underlying mechanisms by using CXCL8 as a representative ELR~+ CXC chemokine.2.In our study,NETs formation by CXCL8 was relatively slow in progress,lytic NETosis dependent and requiring activation of MPO and PAD4.Moreover,CXCL8 elicited NETs required the binding and internalization of CXCR1 instead of CXCR2.3.Indeed,CXCR1 dependent ERK phosphorylation was triggered by CXCL8 stimulation and thus promoted NOX2 activation and mtROS generation,both of which essentially mediated NETs formation.4.We further verified that CXCL8 induced calcium influx and calcium dependent autophagy to enhance ROS generation and promote NETs release in neutrophils.ConlusionsIn this study,we provide new evidences that CXC chemokines bearing the glutamic acid-leucine-arginine motif(ELR~+ CXC chemokines)are more competent in inducing NETs,compared to ELR~-CXC chemokines and non chemotactic cytokines.We also characterize the requirement of CXCR1 and several intracellular mechanisms that mediate the NETs release induced by ELR~+ CXC chemokines.Our work highlights a new role of ELR~+ CXC chemokines which helps to understand the neutrophil biology under physiological and pathological conditions.