NEK7 Amplifies NLRP3 Inflammasome Pro-inflammatory Signaling In Microglia/Macrophages After Spinal Cord Injury In Mice

Objective:NIMA-related kinase-7(NEK7)is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis.It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3(NLRP3)inflammasome.However,the role of NEK7 in microglia/macrophages post-spinal cord injury(SCI)is not well defined.Methods:(1)Expression profiles of NEK7 and NLRP3 inflammasomes in mice post-SCI by double-immunofluorescence staining,qRT-PCR and Western blot;(2)Expression of NEK7 and NLRP3 inflammasomes in B V-2 microglia cells stimulated by LPS and ATP using CCK8 assays,qRT-PCR and Western blot;(3)The role of NEK7 down-regulation playing in NLRP3 inflammasomes activation in the inflammatory model of BV-2 microglia cells by ELISA,qRT-PCR and Western blot;(4)NEK7 interacts with NLRP3 and regulates inflammasomes activation in the inflammatory model of BV-2 microglia cells and post-SCI by double-immunofluorescence staining and Western blot;(5)Western-blot,ELISA,qRT-PCR and BMS score were used to elucidate the effect of NEK7 down-regulation on the local inflammatory response and lower limb motor function in mice post-SCI;(6)Double-immunofluorescence staining was used to investigate the effects of NEK7 down-regulation on the activation of NLRP3 inflammasomes in microglia/macrophages from the injured spinal cord in mice.Results:(1)Double-immunofluorescence staining indicated elevated immunofluorescence intensities of NEK7 and NLRP3 in microglia/macrophages after SCI.Furthermore,quantification of mRNA and protein expression levels revealed a significant increase in NEK7,NLRP3,Pro-caspase-1,Caspase-1 p20 and ASC proteins within 72 hours.On the other hand,qRT-PCR results showed a gradual increase in NEK7 and NLRP3 mRNA levels within 72 hours,with a significant increase in IL-1β and IL-18 within the same period;(2)qRT-PCR results indicated that LPS+ATP treatment increased the expression levels of NLRP3,NEK7,ASC,Caspase-1,IL-1β,and IL-18,whereas Western blot analysis indicated increased levels of NLRP3,NEK7,ASC,and Caspase-1 p20 proteins;(3)NEK7-siRNA reduced the expression of NEK7 and Caspase-1 p20 in LPS+ATP-treated BV-2 microglia cells.qRT-PCR and ELISA results revealed a significant reduction in mRNA levels and secretion of IL-1β and IL-18,respectively,following NEK7-siRNA transfection;(4)IP analysis showed exposure to LPS+ATP triggered the interaction between NEK7 and NLRP3.Subsequently,down-regulating NEK7 attenuated its binding with NLRP3.Double-immunofluorescence staining showed that LPS+ATP triggered the interaction between NEK7 and NLRP3,which was also enhanced post-SCI;(5)Western blot,ELISA and qRT-PCR results showed NEK7-siRNA treatment suppressed the expression of NEK7 and Caspase-1 p20,finally reduced the secretion of IL-1β and IL-18.However,BMS score showed inhibiting NEK7 did not significantly improve motor function post-SCI in mice;(6)Double-immunofluorescence staining showed NEK7-siRNA could block the activation of NLRP3 inflammasomes in microglia/macrophages of spinal cord injured mice.Conclusions:We first confirmed that NEK7 participated in the inflammatory response mediated by NLRP3 inflammasomes through its interaction with NLRP3 in microglia/macrophages after SCI.Therefore,agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.