NF-κB/TWIST1 Mediates Macrophages Migration And Phagocytosis In The Implant-associated Staphylococcus Aureus Osteomyelitis Mice Model

Background and objective:Osteomyelitis(OM)is an inflammatory process in bone caused by infection by a suppurative organism such as bacteria,fungi,or mycobacterium.This is a common problem in the pediatric population.According to statistics,the incidence of osteomyelitis in children was about 5-8/10000 in 2014,and the incidence of chronic hematopoietic osteomyelitis in children was 21.6%in developing countries,especially in Africa.Acute and subacute hematopoietic osteomyelitis is common in childhood,and 85%of cases have been reported in several literatures in children under 17 years old.The major aetiology of osteomyelitis can be haematogenous,tracking from adjacent foci of infection,and direct inoculation from trauma or surgery.When the duration of symptoms is less than 2 weeks,it is an acute process.Acute haemorrhagic osteomyelitis often involves children’s bone,growth long epiphyseal vasculature is rich,endothelial leakage,blood flow is slow,often is the main place of infection.Delayed diagnosis and inappropriate treatment can lead to sepsis,a strong inflammatory response,destruction of bone structure,longitudinal growth stagnation or bone defects,and even worsening outcomes of multiple organ failure and death.Staphylococcus aureus(S.aureus)infection is the most common pathogenic bacteria of osteomyelitis,it through the secretion of virulence factors and the recognition of its structural components,can cause a strong inflammatory response,resulting in a large number of bone loss during osteomyelitis.Peripheral blood mononuclear cells(PBMCs)are the precursors of dendritic cells,osteoclasts and macrophages and are key molecules for recruitment.They have an immediate and effective immune response to staphylococcus aureus infection and play an important role in inflammatory bone loss.Suppressed monocytes/macrophages may increase susceptibility to staphylococcus aureus infection,while early accumulation of inflammatory monocytes/macrophages may serve as intracellular hosts for staphylococcus aureus survival,thereby promoting bacterial resistance to antibiotic treatment.However,the mechanisms of macrophage migration and immune response in the acute phase of intra-osseous infection induced by S.aureus remain unclear.Identifying the response of macrophage migration to staphylococcus aureus infection is critical to identifying targets for the treatment of osteomyelitis caused by staphylococcus aureus infection.TWIST1(twist family bHLH transcription factor 1),which codes for a basic helical-cyclo-helical(bHLH)transcription factor,plays an important role in embryonic development.The encoded proteins form homodimers and heterodimers that bind to the DNA e-box sequence and regulate gene transcription involved in craniosystalline closure during skull development.The protein may also regulate neural tube closure,limb development,and brown fat metabolism.The gene is hypermethylated and overexpressed in a variety of human cancers,the protein of TWIST1 can promote the invasion and metastasis of tumor cells.Through the bioinformatics analysis of the chip GSE16129,we carried out that TWIST1 in mononuclear cells increased,and is associated with bone mineralization regulation.We demonstrate that TWIST 1 increased within the marrow macrophages in the implant-related S.aureus osteomyelitis mice model,and this research mainly from three main respects the animal model,cell experiment and bacteria,demonstrated the molecular mechanism that TWIST1 high expressed in macrophages could accelerate macrophage migration and enhance the phagocytosis of macrophage.Methods:1.We downloaded GSE16129 chip database data from GEO public database and conducted bioinformatics analysis based on three platforms(GPL96,GPL97 and GPL6106).Since there was only one staphylococcus aureus-infected osteomyelitis sample on the GPL6106 platform,we did not include it in this study.Differential genes associated with osteomyelitis(OMRGs)were obtained by bioinformatics.2.DAVID database(http://david.abcc.ncifcrf.gov)was used to analyse the OMRGs GO functional annotations and KEGG pathway enrichment analysis.3.STRING database was used to analyze the interacting proteins in OMRGs,and MCODE plug-in of Cytoscape software was used to analyze the modularizations of the interacting protein networks by setting threshold values such as degree cutoff=2,node score cutoff=0.2,k-core>=2,and Max.Depth=100.4.S.aureus was isolated and identified from patients with osteomyelitis in Nanfang hospital,and then cultured,quantified and diluted for application.5.Eight weeks old C57BL/6 mice were used to construct the osteomyelitis model.We created the infection model by placing a 2mm stainless steel intramedullary needle(0.3mm thick)in the right femoral bone marrow and injected 2 microliters of 1×106 CFU/ml of S.aureus diluent into the bone marrow.Animal experiments were conducted in strict accordance with the guidelines and policies of Nanfang hospital for animal surgery.6.The concentration of macrophages was detected through the F4/80 moleclue immunohistochemistry in marrow and the expression level of TWIST 1 in marrow tissues after infection in paraffin section specimens of the implant related osteomyelitis model.7.The expression level of TWIST 1 in macrophages around the infected sites was detected by co-localization of immunofluorescence in frozen section specimens of our osteomyelitis model.8.Transient transfection technique was used to transfect with specific small interfering RNA(si-TWIST1-RNA)for downregulating TWIST 1 at the gene level.9.RT-Q-PCR was used to detect(1)S.aureus bacteria concentrations co-cultured with the macrophage cell line Raw264.7 withl hour,and then removed extracellular bacteria with gentamicin.Following,continue culturing 24 hours and then tested the livability of Raw264.7 macrophage cells,and also the TWIST1,NANOG,ERBB2 mRNA and protein level changes,according to the three gene mRNA and protein level changes,got the most suitable infection MOI(infection bacteria abundance ratio and number of Raw264.7).(2)S.aureus was co-cultured with Raw264.7 cells with the most suitable MOI for 1 hour,after which the extracellular bacteria were removed,the genes related to macrophage polarization were detected.(3)The interference efficiency of small interfering RNA(siRNAs)after TWIST 1 interference.10.Western-blot was used to detect(1)The changes of TWIST1 protein level in the femoral bone marrow of the mouse model of plant-related staphylococcus aureus osteomyelitis.(2)The protein levels of TWIST1,MMP9,MMP13,p-P65 and P65 in Raw 264.7 after co-cultured with S.aureus.(3)TWIST1,MMP9 and MMP13 protein level changes after p-P65 was down-regulated by the p-P65 specific inhibitor BAY11-7082(BAY).(4)The changes of MMP9 and MMP13 protein levels after TWIST 1 was interfered with by small interfering RNA(siRNAs)under infection conditions.11.The influence of migration to Raw264.7 after infection by S.aureus and downregulation of TWIST 1 were evaluated using wound-healing assay and transwell-based migration assay.12.The phagocytosis of S.aureus was tested in Raw264.7 cells after TWIST1 down-regulated through cell phagocytosis experiment,bacterial clearance experiment,bacterial culture and colony count.Results:1.Analysis of differential genes related to osteomyelitis(OMRGs)from GSE16129 dataset.GSE16129 dataset was downloaded from GEO database and 5 7 OMRGs were obtained under GPL96 platform,including 29 up-regulated OMRGs and 28 down-regulated OMRGs.25 OMRGs were obtained under GPL97 platform,of which 21 were up-regulated OMRGs and 4 down-regulated OMRGs.2.GO functional enrichment,KEGG pathway and protein to protein interaction network(PPI)were used to enrich the biological functions of OMRGs.The above 82 OMRGs were enriched in GO enrichment database,which were mainly enriched in transcriptional positive regulation with RNA polymerase II promoters,bone mineralization,immune response.Among which the genes enriched at bone mineralization were S1PR1,TWIST 1 and PHOSPHO1.KEGG pathways mainly concentrate on cytotoxicity mediated by natural killer cells and antigen processing and presentation,especially those related to antigen processing and presentation.In PPI network,TWIST1,NANOG and ERBB2 were found to be at the center of protein interaction network.Combined with GO enrichment,KEGG pathway enrichment and PPI network,it was concluded that TWIST 1 may play an important role in the mononuclear/macrophage system in osteomyelitis model.3.TWIST1 is highly expressed in macrophages around the focus of infection.The levels of mRNA and protein of TWIST1 in marrow were high regulated after infection detected by RT-Q-PCR and Western Bolt,and the high expression of TWIST1 around infection sites was found by tissue immunohistochemistry.Through the co-localization with immunofluorescence,we found TWIST 1 high expressed in macrophages around the infected sites.4.TWIST1 migh t be related to the polarization of macrophages after infection by S.aureus.The polarization melocules of macrophage were also changed after TWIST 1 down-regulated under infection conditions from the result of RT-Q-PCR.5.TWIST1 might regulate the migration and phagocytosis of macrophage.As the wound-healing assay and transwell-based migration assays results that the Raw 264.7 cells infected by S.aureus migrated faster than control cells treated with vehicle,the down-regulation of TWIST 1 blocked the migration of Raw 264.7 cells in response to S.aureus.In additional,knocking-down TWIST 1 significantly reduced the phagocytosis rate and killing rate of Raw 264.7 in response to S.aureus infection.6.S.aureus promote macrophage migration through P65/TWIST1/MMP9/MMP13 axis.Raw264.7 cells were pre-treated with P65 specific inhibitor BAY11-7082(BAY)and co-cultured with staphylococcus aureus,then Western Blot analysis showed that TWIST1,MMP9 and MMP13 were down-regulated compared with the control group.In addition,MMP9 and MMP13 were also down-regulated when TWIST1 was down-interfered in Raw264.7 cells.Conclusion:In this study,we identified the differentially expressed genes(DEGs)of osteomyelitis caused by S.aureus infection based on publicly available transcriptional dataset GSE16129 using bioinformatic analysis.Further,our in vivo and in vitro data demonstrate that NF-κB/TWIST1 are necessary for S.aureus infection-induced macrophage migration and phagocytosis.Our study highlights the essential role of P65/TWIST1/MMP9/MMP13 axis in the early stages of the innate immune response to S.aureus infection in bone.