JAK2V617F Mutation In MPN Promotes NETs Formation By Upregulating Neutrophil NAP Expression

Objective:Thromboembolism is the most common complication and one of the important causes of death in patients with MPN.Clinical data show that the JAK2V617F mutation is an independent risk factor for MPN thrombosis.Our group has previously found that JAK2V617F can significantly up-regulate neutrophil alkaline phosphatase（NAP）expression in MPNS and promote the formation of neutrophil extracellular traps（NETs）,which act as platelet adhesion scaffolds to induce thrombosis.Therefore,we propose a scientific hypothesis that JAK2V617F promotes the formation of NETs by upregulating NAP expression in neutrophils,thereby increasing the risk of MPN thrombosis.The aim of this study is to construct various knock-in and knock-out cell models to explain whether JAK2V617F mutation,NAP high expression and NETs formation are sequentially regulated from multiple perspectives,and to demonstrate the proposed scientific hypothesis.Methods:1.NAP gene knockout NB4 cell model was constructed using CRISPR-cas9systemThe g RNA sequence of human NAP gene（ALP）was searched from the Zhang Feng Lab database.The g RNA sequence of human NAP was constructed into the lentiviral recombinant CRISP-Cas9 expression vector Lenti CRISPV2 by enzyme digestion,ligation and transformation.The NAP-g RNA lentivirus plasmid and packaging plasmid were co-transfected into 293T cells（cell line within 15 generations）by lipo2000 in a certain proportion.The lentivirus suspension was collected for 48hours and infected with NB4 cells.The infected cell lines were expanded and the positive monoclonal cells were selected and expanded.DNA was extracted,and the corresponding primers were designed and synthesized.The sequences of NAP in the monoclonal cells were identified by sequencing.The constructed NAP knockout cell model was named NAP-KO-NB4.2.NB4 cell model with NAP overexpression and control gene was constructed by lentivirus transduction systemThe c DNA sequence of human NAP gene（NM＿000478.6）was chemically synthesized and inserted into the overexpression lentiviral overexpression vector EX-NEG-Lv156.The NAP expression plasmid and its empty vector were co-transduced into 293T cells with the packaging plasmid,respectively.The lentiviral suspension was collected for 48 hours and infected with NB4 cells,and the positive infected cells were screened by puromycin.The expression of NAP in each group was identified by p-nitrophenyl phosphate（p NPP）method.The constructed NAP overexpression and Empty vector cell models were named NAP-OE-NB4 and empty vecter-NB4,respectively.3.Lentivirus transduction system was used to construct a NAP knockout cell model expressing JAK2V617F mutationThe JAK2V617F mutant and JAK2-WT wild-type plasmids were co-transduced into 293T cells by lipofectamine.The lentivirus suspension was collected for 48 hours and infected with NAB-KO-NB4 cells.GFP positive cells were sorted by flow cytometry.The expression of JAK2 protein in each group was detected by Western blot.The constructed JAK2V617F mutant and wild-type cell models were named JAK2V617/NAP^-/--NB4 and JAK2-WT/NAP^-/--NB4.4.NAP,NETs and ROS induction protocol in NB4 cell line（1）NAP etection induction protocol:The NB4 cell model was induced by ATRA（10μmol/l）and G-CSF（10ng/ml）for 72 hours.（2）NETs induction protocol:The NB4 cell model was induced by ATRA（10μmol/l）and G-CSF（10ng/ml）for 72 hours,then stimulated with PMA（500n M）for 3hours.（3）ROS detection induction protocol:The NB4 cell model was induced by ATRA（10μmol/l）and G-CSF（10ng/ml）for 72 hours,then stimulated by PMA（500n M）for 1 hour.5.NAP detection methodThe expression of NAP was detected by p-nitrobenzene phosphate（p NPP）method:The induced NB4 cells were collected from each well,centrifuged,discarded supernatant and washed with PBS,and 5×10⁵ cells were collected to detect the expression of NAP.6.NETs detection method（1）Cell membrane MPO/DNA was detected by flow cytometry:Cells in each group were collected,and appropriate amount of MPO-PE and SYTOX Blue antibodies were added into the cells and incubated against light.Cells were collected and detected by flow cytometry.（2）Cell membrane NE was detected by microplate substrate color development method:Cells in each group were collected,NETs supernatant was extracted,and the absorbance at 405nm was detected by enzyme-labeled instrument.（3）Cell membrane double-stranded DNA（ds DNA）assay:Cells of each group were collected,NETs supernatant was extracted,and instructions were followed.Finally,fluorescence was detected by excitation light at 500nm and emission light at530nm using CYT3MF multifunctional enzyme marker（Bio Tek,USA）.7.ROS detection methodsThe induced cells of each group were collected and added with DCFH-DA probe after proportional dilution.The cells were incubated for 30min in the dark,cleaned with serum-free medium for 3 times,and then detected by flow cytometry.Results:1.Identification of NB4 cell models in each groupIdentification results of NAP knockout NB4 cell model:The results of cell sequencing showed that the base deletion in exon 4 of NAP caused a frameshift mutation in NB4 cells.The results of p NPP assay showed that NAP expression in NAP-KO-NB4 cells was significantly lower than that in NB4 cells（P<0.05）.NAP overexpression NB4 cell model identification results:The results of QPCR showed that compared with NB4 cells,the relative expression of NAP RNA in empty-vecter-NB4 cells had no significant difference（P>0.05）,while the relative expression of NAP RNA in NAP-OE-NB4 cells was significantly increased（P<0.05）.The results of p NPP assay showed that compared with NB4 cells,the expression of NAP in empty-vective-NB4 cells was significantly decreased（P<0.05）,while the expression of NAP in NAP-OE-NB4 cells was significantly increased（P<0.05）.Identification of JAK2V617F mutant and wild-type NAP-KO-NB4 cell models:The positive rates of GFP expression detected by flow cytometry were 94.0%and 98.7%,respectively.Western-bolt results showed that JAK2 protein expression in JAK2-WT/NAP^-/--NB4 cells and JAK2V617/NAP^-/--NB4 cells was higher than that in NAP-KO-NB4 cells.The expression of JAK2 protein in JAK2V617/NAP^-/--NB4 cells was higher than JAK2-WT/NAP^-/--NB4 cells,suggesting that the cell model was successfully constructed.2.Analysis of NETs results in NB4 cell models stably expressing different driver genesThe levels of NETs in NB4-related cell models constructed in the laboratory,namely JAK2V617F-NB4,JAK2-WT-NB4,MPL515L-NB4,MPL-NB4,CALR-type1-NB4,CALR-NB4 and NB4 cells,showed that:Compared with NB4cells in the control group,NETs in JAK2V617F-NB4 cells were significantly increased（P<0.05）,while NETs in MPL515L-NB4 and CALR-type1-NB4 cells were significantly decreased（P<0.05）.The levels of NETs in JAK2V617F-WT-NB4,MPL-WT-NB4 and CALR-type1-NB4 wild-type cells were not significantly different（P>0.05）.3.Analysis of NETs results in the NAP knockout NB4 cell modelThe NETs formation levels of NAP knockout NB4 model（NAP-KO-NB4）after different induction regimens were detected and compared,and the results showed that:Compared with NB4 cells,the level of NETs in NAP-KO-NB4 cells was significantly decreased（P<0.05）,and there was no significant difference in the level of NETs in Empty vctor-NB4 cells（P>0.05）,while the level of NETs in NAP-OE-NB4 cells was significantly increased（P<0.05）.4.Analysis of NETs results in NB4 cell model with NAP overexpressionThe NETs formation levels of NAP overexpressed NB4 cell model（NAP-KO-NB4）and the control cell model（Empty vecter-NB4）after culture under different induction regimens were detected and compared,and the results showed:Compared with NB4 cells,the level of NETs in NAP-KO-NB4 cells was significantly decreased（P<0.05）,and there was no significant difference in the level of NETs in Empty vctor-NB4 cells（P>0.05）,while the level of NETs in NAP-OE-NB4 cells was significantly increased（P<0.05）.5.Analysis of NETs from NAP knockout cells stably expressing the JAK2V617F mutationThe levels of NETs in NAP-KO-NB4 cells stably expressing JAK2V617F mutant gene,JAK2-WT wild-type gene and control cells,namely JAK2V617/NAP^-/--NB4,JAK2-WT/NAP^-/--NB4,NAP-KO-NB4 and NB4 cells,showed that:Compared with NAP-KO-NB4 cells,the levels of NETs in JAK2-WT/NAP^-/--NB4 and JAK2V617/NAP^-/--NB4 cells were not significantly different（P>0.05）,while the levels of NETs in NB4 cells were significantly increased（P<0.05）.6.Comparative analysis of ROS levels in NAP knockdown and NAP overexpression cell modelsThe ROS expression levels of NB4,Empty vector-NB4,NAP-KO-NB4 and NAP-OE-NB4 cells were detected,and the results showed that:Compared with NB4cells,the MFI value of ROS in NAP-KO-NB4 cells was significantly decreased（P<0.05）,and the MFI value of Empty vctor NB4 cells was not significantly different（P>0.05）,while the MFI value of NAP-OE-NB4 cells was significantly increased（P<0.05）.Conclusion:By comparing the formation of NETs in NB4 cell models with different driver genes,it was confirmed that JAK2V617F mutation could significantly promote the formation of NETs,while MPL and CALR mutations could significantly inhibit the formation of NETs,which was highly consistent with the expression of NAP.By observing the changes in the formation of NETs in NB4 cell models with NAP knockout and NAP overexpression,it was confirmed that high expression of NAP promoted the formation of NETs,while NAP knockout significantly inhibited the formation of NETs,which further suggested that NAP could regulate the formation of NETs.By recreating the formation of NETs after JAK2V617F mutation in the NAP knockout model,it was confirmed that JAK2V617F mutation induced NETs formation was mediated by NAP.Taken together,these results confirmed that JAK2V617F mutation,NAP overexpression and NETs formation were sequentially regulated.