| Background:High-mobility group protein N2（HMGN2）is a class of non-histone chromatin structural proteins that are widely expressed in the nucleus of vertebrate cells.Because of its unique nucleosome binding domain,it can specifically interact with nucleosome and affect the binding of histone H1 to nucleosome,thus changing the local and global chromatin structure and epigenetic regulation of gene expression.HMGN2 plays an important role in the regulation of innate immune response.In succession in recent years,our team reported that HMGN2 could regulate micro RNAs,cell membrane receptor integrins,cytoskeleton protein molecules,MAPK and NF-κB signaling pathways,cell oxidative stress,autophagy biological behavior,thus protecting the lung and bladder epithelial cells from E.coli and other gram-negative bacteria infection.Macrophages are also one of the most important cells in innate immunity.Our recent study found that HMGN2 knockdown can affect the polarization of macrophages and the survival of non-tuberculous mycobacterium in macrophages.However,the latest literature suggests that alveolar macrophages can undergo epigenetic changes during the inflammatory period and even after inflammation subsides,affecting their immune function,indicating the complexity of macrophages’involvement in the inflammatory process.For the HMGN2 molecule concerned by our research group,the mechanism of how it regulates the bactericidal function of macrophages is far from been clarified.Based on the gene knockout cell model,transcriptome sequencing,signal pathway detection,gene knockout mouse model and other cellular and molecular biology techniques,we are trying to explore the mechanism underlying at the cellular and molecular level and the animal level to deepen our understanding of the role of HMGN2 molecule in the regulation of anti-infection defense in the body,and provide a potential candidate target for drug design.Objective:To investigate the effect of HMGN2 on the bactericidal function of macrophages and its molecular mechanism,as well as the effect of HMGN2 on the immune defense ability against microbial infection.Methods:Cell culture and treatment:Mouse mononuclear macrophages RAW264.7 cells were cultured routinely;Escherichia coli 19138,uropathogenic Escherichia coli（UPEC）and Klebsiella Pneumoniae（K.p）cells were cultured routinely.RAW264.7 cells were stimulated with LPS and Escherichia coli 19138 to detect HMGN2 expression and other related indicators.RAW264.7 cells were stimulated with Escherichia coli 19138,UPEC and K.P.pneumoniae to test their bactericidal ability.Mice were injected intraperitoneally with Escherichia coli 19138 to observe the bacterial residue in the liver,spleen,lung and abdominal cavity.RAW264.7 cells were treated with PD98059,SP600125,SB203580,T5224,SN50 and BMS-345541,respectively,and the expression of NOS2 was detected with LPS stimulation.Plasmid construction（cloning technology）:The Lenti-CRISPR-V2 plasmid stored in our laboratory was used to construct the Lenti-CRISPR-V2-HMGN2-sg RNA plasmid according to the instructions.The pGL3-CD14-promoter plasmid was constructed by using the p GL3-basic plasmid vector stored in our laboratory according to the instructions.Transfection:The constructed Lenti-CRISPR-V2-HMGN2 plasmid was transfected into RAW264.7 cells using the JETPrime?transfection reagent of Polyplus to construct the HMGN2 knockout cell line.The pGL3-basic and pGL3-CD14-promoter plasmids were transfected into wild-type and HMGN2 knockout RAW264.7 cells using the JETPrime?transfection reagent from Polyplus respectively,The transcriptional efficiency of CD14promoter was then measured under LPS stimulation.Construction of HMGN2 knockout cell line:The CRISPR-Cas9 technology was used to construct HMGN2 knockout RAW264.7cell line according to the steps described in the method,so as to detect the changes of corresponding indexes under the condition of HMGN2 deletion.By using CRISPR-Cas9 technology,HMGN2-CD14 double knockout RAW264.7cell line was constructed on the basis of HMGN2 knockout RAW264.7 cells according to the steps described in the method,and CD14’s effect on macrophage bactericidal ability,NOS2 expression and changes of inflammatory factors were detected after LPS stimulation,so as to explain the role of CD14 in this process.Macrophage phagocytosis test:The phagocytosis assay of RAW264.7 cells after HMGN2 knockout was performed using fluorescently labeled Escherichia coli biological particles.Macrophage killing assay（plate colony counting method）:The bactericidal ability of RAW264.7 cells after HMGN2 knockout was detected by plate colony counting method.At the same time,after intraperitoneal injection of Escherichia coli 19138,the survival of mice liver,spleen,lung and intraperitoneal residual Escherichia coli were detected.The NOS2 inhibitor L-NAME was applied to demonstrate the role of NOS2 in mediating the bactericidal ability of macrophages.RT-qPCR:RT-qPCR was used to detect the expression levels of HMGN2（wild type）,CD14,NOS2,COX-2,IL-6,IL-1β,IL-18,IL-10,IL-13,IL-4,IL-12,TNFα,IFNγ,TGFβand chemokines CCL2,CCL 3,CCL 7,CCL 17,CCL 5 and CCL 22 in wild-type and HMGN2 knockout RAW264.7 cells after LPS stimulation to determine the effect of HMGN2 on these genes.The expression changes of NOS2,COX-2,IL-6,IL-1βand TNFαwere detected in HMGN2-CD14 double knockout cell,so as to illustrate the effect of CD14 on the process.Some results of transcriptome sequencing were also verified.Western Blot:The expression of HMGN2,NOS2 and CD14 in wild-type and HMGN2 knockout RAW264.7 cells was detected by Western Blot after LPS stimulation（or non-stimulation）.The changes of ERK,phosphorylated ERK,P38,phosphorylated P38,JNK and phosphorylated JNK under the same conditions were detected to verify the role of MAPK signaling pathway in this process.The expression of P65,phosphorylated P65,IκBα,and phosphorylated IκBαwere detected to prove the role of NF-κB signaling pathway in this process.The expression changes of histone H3,H3K4me3,H3K9Ac,H3K27Ac and H3K27me3 were detected to try to explain the role of histone modification in the promoter region in this process.Immunofluorescence microscopy:The expression of HMGN2 in wild-type RAW264.7 cell after LPS stimulation was detected by immunofluorescence assay.The phagocytosis of HMGN2 knockout RAW264.7 cell was also detected.Flow cytometry:The phagocytosis of wild-type and HMGN2 knockout RAW264.7 cells to fluorescent labeled Escherichia coli biological particles was detected by flow cytometry.In addition,flow cytometry was also used to detect the changes of HMGN2 in mice macrophage infected with Escherichia coli.The changes of macrophages and total cell numbers in peritoneal rinse fluid of wild-type and HMGN2 knockout mice were detected when they were infected with Escherichia coli.Chromosome immunoprecipitation（ChIP）:Chromatin immunoprecipitation was used to detect the enrichment of H3K4me3,H3K9Ac,H3K27Ac,H3K27me3 and transcription factor CREB in the CD14promoter region of wild-type and HMGN2 knockout RAW264.7 cells stimulated by LPS,in order to demonstrate the influence of HMGN2 to CD14.Dual Luciferase Reporter Gene assay:CD14 promoter mediated bioluminescence expression in wild-type and HMGN2knockout RAW264.7 cells after LPS stimulation was detected by dual luciferase reporter gene assay,indicating the regulatory role of HMGN2 on CD14 promoter.ELISA:IL-6,IL-1βand CD14 levels of wild-type and HMGN2-knockout RAW264.7 cells in cell culture supernatant were detected after LPS stimulation using IL-6,IL-1βand CD14 ELISA kits.The changes of plasm CD14 in wild-type and HMGN2knockout mice after intraperitoneal injection of LPS were also detected.Transcriptome sequencing and analysis:The wild-type and HMGN2 knockout RAW264.7 cells were stimulated by LPS,and the RNA was extracted and sequenced by Illumina,which was then analyzed and counted by informatics.Statistical analysis:SPSS 18.0 statistical software was used for statistical analysis of all experimental data.Results:1.The expression level of HMGN2 was decreased in macrophages infected by pathogenic microorganisms.By screening the NCBI GEO database,the trends of HMGN2 expression level in macrophages infected by different pathogenic microorganisms（bacteria,fungi and viruses）were analyzed.The results indicated that the expression level of HMGN2in both human and mouse macrophages showed a downward trend after microbial infection.RT-qPCR results showed that after 24,48 and 72 hours of LPS stimulation of wild-type RAW264.7 cells at 1,10,100 and 1000ng/m L,the m RNA level of HMGN2 decreased by more than 50%compared with the unstimulated group.Western Blot results and immunofluorescence result also showed a similar trend.At the whole animal level,flow cytometry results showed that the expression level of HMGN2 in macrophages in the lung tissues of the model mice infected with bacterial was reduced by about 40%compared with that in the normal saline group.2.HMGN2 knockout can significantly enhance the phagocytosis and bactericidal ability of macrophages.CRISPR-Cas9 technology was used to construct HMGN2 knockout RAW264.7macrophage cell line.The phagocytosis assay showed that HMGN2 knockout RAW264.7 cells were more efficient than wild-type（WT）macrophages in phagocytosis of Escherichia coli fluorescent granules.Escherichia coli standard strain 19138,UPEC and K.P.pneumoniae（MOI=0.1,0.5,1.0,5.0）were incubated with WT and HMGN2 knockout RAW264.7 cells for 2,4,6,8 hours,respectively.The results of plate colony counting assay showed that compared with WT,HMGN2knockout RAW264.7 cells had relatively stronger bactericidal ability under different infection times and different MOI.In addition,with the increase of bacterial/cell co-incubation time,the bactericidal ability of HMGN2 knockout cells gradually increased,and the maximum bactericidal ability difference was shown in8 hours after infection.3.The effect of HMGN2 on the gene expression profile of LPS-induced RAW264.7 cells was analyzed by whole gene transcriptome sequencing.WT and HMGN2 knockout RAW264.7 cells were treated with LPS（1μg/m L）for24 h.Meanwhile,the untreated group was set as the control group.Total RNA was extracted and performed transcriptome sequencing.Transcriptome sequencing results showed that there were significant differences in gene expression between WT and HMGN2 knockout RAW264.7 cells,regardless of LPS stimulation.In RAW264.7 cells unstimulated by LPS,HMGN2 knockout cells showed significant changes in phagocytosis,antigen presentation,cell adhesion molecules,and Staphylococcus aureus infection compared with WT.In LPS-stimulated RAW264.7cells,HMGN2 knockout showed significant changes in the signaling pathways related to amino acid synthesis in cells compared with WT.4.HMGN2 knockout enhances the bactericidal ability of macrophages by promoting the expression of CD14.Transcriptome sequencing showed that CD14 expression increased significantly in a series of receptors associated with phagocytosis.At the same time,Western Blot and RT-q PCR results also showed that after LPS stimulation,CD14 expression in HMGN2 knockout RAW264.7 cells was significantly increased compared with WT cells,which was about 2.3 times and 1.5 times higher.Furthermore,compared with WT cells,the mebrane CD14 and free CD14 in the culture supernatant of HMGN2knockout cell were increased.Based on the HMGN2 knockout RAW264.7 cell line,the HMGN2-CD14 double knockout cell line was constructed.Compared with HMGN2 knockout,the HMGN2-CD14 double knockout reversed the enhanced bactericidal capacity induced by HMGN2,suggesting that CD14 plays an important role in the HMGN2-mediated bactericidal capacity of macrophages.5.HMGN2 knockout enhances the recruitment of transcription factors and histone H3 methylation and acetylation modifiers in the CD14 promoter region.The CD14 promoter sequence was inserted into the luciferase reporter plasmid by cloning technique.Dual luciferase reporter assay results showed that HMGN2knockout cells stimulated higher luciferin expression than WT cells under LPS stimulation.Ch IP-PCR results showed that after chromatin immunoprecipitation using transcription factor CREB antibody,the enrichment of CREB in the CD14promoter region in HMGN2 knockout cells was significantly increased about five-fold compared with WT.Western Blot results showed that there was no significant difference in the overall protein expression of H3K4me3,H3K9Ac,H3K27Ac,and H3K27me3 in WT and HMGN2-knockout macrophages under LPS stimulation.Ch IP-PCR results showed that in the CD14 promoter region of HMGN2 knockout macrophages,compared with WT group,H3K4me3 was enriched in multiple sites of the CD14 promoter.The enrichment of H3K9Ac at-50 and-900 sites increased by 1.5 and 2.8 times,respectively.The enrichment of H3K27Ac at-900 site increased by 3.13 times.However,no increase in H3K27me3 enrichment occurred at any sites.6.HMGN2 knockout may enhance the bactericidal ability of macrophages by promoting CD14-mediated NO production.Transcriptome sequencing showed that the expression of nitric oxide synthase 2（NOS2）was significantly increased in a number of bactericidal related molecules.Western Blot and RT-q PCR results also showed that after LPS stimulation,the expression of NOS2 in HMGN2 knockout RAW264.7 cells significantly increased about 3 times and 18.5 times compared with WT.Nitric oxide（NO）detection results also showed that after LPS stimulation,there was about 1.8 times increase of NO in HMGN2 knockout RAW264.7 cell supernatant than WT cell supernatant.Compared with HMGN2 knockout macrophages,the expression level of NOS2induced by LPS in HMGN2-CD14 double knockout macrophages was inhibited,suggesting that HMGN2 may regulate the expression of NOS2 through CD14.The results of plate colony counting assay showed that after NOS2 inhibitor was used to treat WT and HMGN2 knockout RAW264.7 cells,the enhanced bactericidal ability induced by HMGN2 knockout was inhibited,and the bactericidal ability of WT and HMGN2 knockout macrophages was almost at the same level.It is suggested that NO plays a key role in the regulation of bactericidal ability of macrophage mediated by HMGN2.7.MAPK and NF-κB signaling pathways are involved in HMGN2-mediated NO production.Western Blot results showed that the levels of phosphorylated ERK,P38 and JNK in RAW264.7 cells were increased in WT and HMGN2 knockdown cells at 0,7.5,15,30,60 and 120min after LPS stimulation.Moreover,compared with WT cells at the same time point,the levels of phospho-ERK,P38 and JNK were higher in HMGN2 knockout cells.It suggested that the activation of MAPK signaling pathway was enhanced in HMGN2 knockout macrophages.At the same time,Western Blot results also showed that under LPS stimulation,ERK,P38,JNK and its downstream transcription factor AP-1 inhibitors were used to treat WT and HMGN2 knockout cells,and the expression of NOS2,which was originally increased by HMGN2 knockout was inhibited.These results suggest that MAPK signaling pathway may be involved in the HMGN2-mediated NO production regulation.In addition,Western Blot results showed that the expression of IκBαand P65 in RAW264.7 cells were increased after 0,7.5,15,30,60,and 120min of LPS stimulation.Compared with WT cells at the same time point,the expression levels of phosphorylated IκBαand P65 were higher in HMGN2 knockout RAW264.7 cells.It suggested that the activation of NF-κB signaling pathway was enhanced in HMGN2 knockout macrophages.However,RAW264.7 cells treated with NF-κB signaling pathway inhibitors BMS-345541 and SN50 showed no significant inhibitory effect on HMGN2-induced elevated NOS2.8.HMGN2 knockout mice showed stronger bacterial clearing ability and survival ability than wild-type mice.The mice were intraperitoneally injected with Escherichia coli standard strain 19138to establish the mice abdominal infection model.The results of the plate colony counting assay showed that the remaining live bacteria in the liver,spleen,lung and peritoneal fluid of HMGN2 knockout C57BL/6 mice significantly reduced compared with that of the wild type after intraperitoneal injection of Escherichia coli 19138 for 24 h.It suggested that HMGN2 knockout mice had stronger ability to eliminate bacteria and resist bacterial infection than WT mice.The mice abdominal infection model was established by intraperitoneal injection of Escherichia coli standard strain.The death of mice was observed in real time and the survival curve was plotted.The results showed that HMGN2 knockout mice showed stronger survival ability than WT mice.After 24 hours of infection,all WT mice died in 24 hours,and 50%of the HMGN2 knockout mice were still alive until the symptoms recovered.9.HMGN2 knockout can promote the production of CD14 and NO in mice.The sepsis model was established by intraperitoneal injection of LPS into WT and HMGN2 knockout mice.Plasma levels of CD14 and NO were measured 24 hours later.The results showed that the plasma levels of CD14 and NO in HMGN2knockout mice were significantly increased by 1.45-fold and 1.3-fold,respectively compared with WT.In addition,LPS-induced sepsis model was used to detect the effect of HMGN2knockout on the survival ability of mice.The results showed that HMGN2 knockout mice in the sepsis model showed fewer deaths compared to WT.The death of WT mice began at 28h,and all WT mice died within 50h,while 50%of the HMGN2knockout mice survived and fully recovered from the symptoms of infection.10.HMGN2 knockout can promote the expression of chemokines in macrophages and promote the local recruitment of macrophages.RT-qPCR results showed that after LPS stimulation,the expression of CCL2,CCL3 and CCL 7 in HMGN2 knockout RAW264.7 cells significantly increased compared with WT cells,which were about 11-fold,1.6-fold and 5.3-fold increased,respectively.The model of bacterial infection in mice was established by intraperitoneal injection of standard strains of Escherichia coli into WT and HMGN2 knockout mice.After 12 hours,the peritoneal flushing fluid was collected and the number of macrophages collected from the peritoneal cavity was measured by flow cytometry.The results showed that compared with WT,the peritoneal cavity of HMGN2 knockout mice showed a nearly two-fold increase in macrophage recruitment.11.HMGN2 knockout also has a regulatory effect on inflammatory cytokines in macrophages.RT-qPCR results showed that the expression of IL-6,IL-1βand COX-2 in RAW264.7 cells after LPS stimulation was significantly decreased compared with WT cells,which were about 20-fold,2.2-fold and 3.7-fold,respectively,while the expression of TNFαwas increased by about 2-fold.ELISA results also showed that after LPS stimulation,IL-6 and IL-1βwere detected in the supernatant of HMGN2knockout RAW264.7 cells,which decreased by 4.8-fold and 2.1-fold.In LPS-induced sepsis mice,lung tissue of HMGN2 knockout mice showed significantly less inflammatory damage and inflammatory cell infiltration than lung tissue of WT mice.Conclusion:In pathogenic bacteria and their products infected macrophages,HMGN2expression decreased.By raising the promoter region of histone H3K4me3,H3K9Ac,H3K27Ac enrichment level,the decreased hmgn2 expression promoted the expression of receptor CD14,then activated MAPK/NF-κB signaling pathways,inducing macrophage NO and chemokine CCL2,CCL3,CCL7 release,thus enhanceing the macrophages collect and sterilization effect.Besides,HMGN2knockout can reduce the release of inflammatory factors such as IL-6,and may play a protective role to host inflammatory injury. |
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