Generation Of Bone Marrow-Derived Macrophages And The Effect Of Histone Methylation Modification On The Release Of IL-6 And TNF-? By Macrophages

The monocyte-macrophage includes premonocyte of the bone marrow mononuclear cells,mononuclear cells of peripheral blood mononuclear cells,and macrophages within the organization.Macrophage derived from the mononuclear cells of the blood,and mononuclear cells derived from bone marrow of precursor cells.Monocyte-macrophage system is importance of body's immune system,macrophages also are the body's immune cells,they play a important biological role of anti-infection,anti-tumor and immune regulation.In the body's nonspecific immune defense,Macrophages are important cells who nonspecifically phagocytose and kill a variety of pathogenic microorganisms during anti-infection.It can be presenting antigen,starting the body's immune response,also play an important role in innate immune.After macrophage phagocytosing and processing,the body's most TD antigen(thymus dependent antigen)form MHC antigen peptide complexes with major histocompatibility antigens on the surface(MHC)which express in the cell membrane surface during the specific immune response.The complex submit to T cells,and then start the body's innate immune response.As is known to all,at the begininig of immune response,there are a lot of adhesion molecules on the surface of macrophages which can be combined with coordinated stimulus molecular receptors on the surface of the T cells,and then produce synergistic stimulation signal,so as to induce T cell activation.The anti-tumor mechanism of macrophage is mainly due to the macrophages are some cytokines such as IFN-? after activation can effectively kill tumor cells,so they are involved in important effector cells of the immune surveillance.The immune regulating function of macrophages performed on secreting a variety of inflammatory cytokines and participating in immune regulation in the specific immune response.It is given priority to with proinflammatory cytokines release,main secretion of proinflammatory cytokines IL-1,IL-6,IL-12,TNF-?,etc.At the same time those function also are affected by the local microenvironment.Macrophage is one of the most commonly used cells in laboratory,especially application research of the direction of the inflammation and immunity.There are many kinds of macrophages,but cell lines and the generation of macrophages are commonly used in laboratory.Accurately,cell lines and primary cell are different,and not every organization has a corresponding source sex cells cell lines,some cells must keep the original generation.Cell line is derived from the original generation cells,which go into cell lines after importing the virus proliferation so that cell lines with the characteristics of cancerous cells.Cell lines of biological genetic material have been changed,and no longer have contact growth inhibition phenomenon.Those shape and state is not match for primary cells,they also change easily.Compared with cell lines,primary cell growth is limited.What can correctly reflect the body's physiological and pathological state are primary cell receptors,signal transduction,signal system,protein biology characteristics of biology,DNA,RNA biology and genetics,reaction of outside world,the test in vitro,and the toxicology and teratogenic reaction,screening and individualized therapy of drug.Like the morphological structure and function of organization in vivo,primary cell is the best experiment object of test and drug therapy.Cell lines include THP-1(the human macrophage cell line),Raw264.7(murine peritoneal macrophage cell line),MHS(murine alveolar macrophage cell line),and primary macrophages include peritoneal macrophage(PM),alveolar macrophage(AM)and bone marrow-derived macrophages(BMDM),etc.Due to the primary macrophage comes from organization and it is more stable than cell lines,therefore,it can reflect the state of the body which recive the favour of researchers.In primary cell,the access to the limited number of PM and AM,for example,in the absence of induction can only obtain(1.5?3)× 106 per mouse macrophage cell,which can't meet the demand of some experiments,such as Western blot,IP(immunoprecipitation),etc,so BMDM is often used in laboratory.Currently,there are two methods to obtain BMDM,one is using recombinant M-CSF induced differentiation of mouse bone marrow,another is to use of L929 cell culture supernatant.There are little reports that telling us what different between L929 cell culture supernatant which induce BMDM differentiation with simply using the M-CSF,and what different between BMDM induced by in vitro with mature abdominal macrophage.In the process of inflammation,macrophages secrete a variety of inflammatory cytokines.Especially the proinflammatory factors affect the body's state or cause disease.Pathogenic microorganisms activate macrophages by pattern recognition receptors and signaling pathways,which can induce the expression of a variety of cytokines,like proinflammatory cytokines,anti-inflammatory cytokines,chemokines,metabolic regulation factor,tissue repair factor,antimicrobial proteins and acquired immune regulatory factors.Due to the different function,these products may increase inflammation and kill pathogenic microorganisms in different microenvironment.Proinflammatory cytokines,for example,is crucial for protecting and activating immune response,however,persistent inflammation of stimulation may induce systemic inflammatory response syndrome and shock.In order to improve the infection of pathogenic bacteria and inhibit excessive inflammatory reaction process,we need to understand the regulation of inflammation gene expression.In macrophages,TLR4 receptor and its regulation of signaling pathways mediate these different kinds of inflammatory genes,therefore these downstream level are different so that expression or silence the same function of inflammatory genes selectively.This adjustment does not affect activation or inhibition under the same other genes which induced by TLR4 receptor,and it is a kind of specific genes regulate.The specificity of the gene regulation is mainly implemented in epigenetics,which including histone modification,DNA methylation and the influence of some non-coding RNA,and histone modification is an important part of regulating gene specificity.There are many forms of histone modification means,such as lysine and arginine methylation and acetylation of serine phosphorylation,ubiquitin,glycosylation,etc.These modifications mainly involved in regulation of a variety of cell biological processes,like gene expression and silence,mitosis,cell differentiation,X chromosome inactivation and genetic imprinting.Histone modification includes histone modification of histone methyltransferase,methylation enzyme,acetyltransferase and acetylation enzyme and the enzyme related protein complexes.Histone modification changes by these enzymes and its related protein complexes which influence the highly ordered spatial structure change of chromatin and transcription regulation of cytokines.This adjustment mechanism mainly are follows:(1)The close degree of histone and DNA is changed through acetylation/deaceylation to change the charge of histone acetylation;(2)Form of high affinity sites which recruit of the effector of non-histone proteins and complex that possess local reconstruction of chromatin.Previous study found that the released of inflammatory cytokines are closely associated with histone methylation modification,especially the IL-6 and TNF-?.Due to some genes have specific regulation,so some inflammatory disease drug treatment effect is not ideal,so it is very important explore its mechanism about the treatment of diseases.Early,many researchers think that signaling pathways are solutions which curb excessive release of inflammatory cytokines in the pathogenesis.Excessive release of inflammatory cytokines can be reduced by inhibiting the signal pathways,however,it not only suppress antimicrobial peptides or product proteins,but also increase susceptibility of infection,and due to inhibition of other protective reaction(metabolism and tissue repair)result in a great side effects.Study the role of histone modification in gene regulation specificity,not only elucidate the pathogenesis of endotoxic shock and related inflammatory diseases,but it is also a important guidance for seeking more effective and reasonable anti-inflammatory drugs.Based on the above understanding,in order to studying what method can induce BMDM,we first by comparing the morphology,purity and function between reorganization of the M-CSF induced BMDM and L929 cell culture supernatant induced BMDM,and then studying histone specific regulation of inflammation genes IL-6 and TNF-? with BMDM.Preliminary research results found that histone methylation modification are closely associated with the release of inflammatory cytokines,so the research of regulation of macrophage inflammatory cytokine release is of great significance.Therefore,the main purpose of this study is the preparation of the macrophage,thereby we explore histone modification mechanism of IL-6 and TNF-a transcription.On this basis,this experiment is divided into two parts:The first part:comparing morphology,purity and function between M-CSF induced BMDM and L929 cell culture supernatant induced BMDM respectively,and study what method can induce BMDM.The second part:studying histone specific regulation of inflammation genes IL-6 and TNF-a with BMDM.The experimental results are as follows:1.M-CSF and L929 culture supernatant induced mouse bone marrow macrophage are irregular type or spindle type in the seventh day which are observed by the Zeiss microscope,and it is similar with matured abdominal macrophage shape.We take the seventh day of differentiation of bone marrow of mice macrophage,then we identify with flow cytometry that the M-CSF induced macrophage in the number of F4/80 marked positive cells was 98.6%,and L929 induced macrophage was 99.6%,abdominal macrophage was 98.8%,there was no significant difference between the three.2.We observe fluorescence by Zeiss fluorescence microscopy and find there are no different between M-CSF and L929 induced mature BMDM and PM.In order to be more objective reflect the phagocytosis of macrophage,we lysis cell with FITC-E.coli in it which incubate together for 30 min,then test the average fluorescent intensity by the Microplate reader(M5)in cell.The results are the M-CSF group of fluorescence intensity was 21.31 ± 5.83 on average,L929 group was 26.10 ± 6.11,there is no significant statistical difference(t = 0.745,P = 0.745,P>0.05,t test)between the two.PM group was 37.61 ± 4.21,compared with the M-CSF and L929,P>0.05(t test),there was no significant difference.BMDM with living E.coli were wash extracellular bacteria after incubation for 30 min,and we continue to cultivate cells in the incubator for 30 min or 60 min.At 30 min and 60 min time points,there is no statistic difference between M-CSF induced BMDM and L929 induced BMDM of ability of killing bacteria(P>0.05,t test),two ways of inducing BMDM and PM sterilization function has no statistical difference(P>0.05,t test).3.M-CSF and L929 after LPS stimulation on the macrophages release IL-6 in each testing point in time without significant difference(P>0.05,t test),the release of TNF-? 24 h after LPS stimulation,L929 group is higher than the M-CSF group(P<0.05,t test);Compared with peritoneal macrophages in vivo matured,both L929 group and M-CSF group in addition to 6 hours after LPS stimulation,the release of IL-6 is low,but other time point without significant difference(P>0.05,t test).4.The results show that there are statistic difference(P<0.05,t test)possessing in IL-6 mRNA level between protein inhibitors MTA group(65.073 ± 47.671)and don't give inhibitors MTA group(2737.977 ± 1308.751)after LPS stimulation with 2 hours;there are statistic difference(P<0.05,t test)possessing in TNF-a mRNA level between protein inhibitors MTA group(116.709 ± 54.134)and don't give inhibitors MTA group(373.557±108.76)after LPS stimulation with 1 hour.5.The results show that there are statistic difference(P<0.05,t test)possessing in IL-6 and TNF-a protein level between protein inhibitors MTA group and don't give inhibitors MTA group after LPS stimulation with 6 h or 12 h hours.According to these results,the following preliminary conclusions are as follows:1.we conclude that both the M-CSF and L929 cell culture supernatant induced macrophage in vitro can be used instead of differentiation of mature macrophages in vivo;We should consider the purpose of experiment when select the induced way of BMDM.2.Histone modification of H3K4me2 involved in the inflammatory process which regulate release and transcription of macrophage related inflammatory cytokine IL-6 and TNF-?.