The Role And Mechanism Of Different Subtype Of Macrophage In Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis is a demyelinating disease in the central nervous system.It is an autoimmune disease.It is characterized by chronic inflammation of central nervous system,demyelination of white matter,axonal and loss ofneuronal.Experimental autoimmune encephalomyelitis is a common animal model for MS,which is widely used in the basic research of MS.At present,MS treatment include corticosteroids,interferon beta,but the effects were not significant and only part of the patients effectively,immunosuppressive agents may have significant effect,but due to the severely side effects limits its clinical application,so MS therapy has always been a hot research all over the world.Macrophages are very important immune cells,which play an important role in the occurrence and development of multiple sclerosis and experimental autoimmune encephalomyelitis.Macrophages can be classified into M1 and M2 macrophages.M1 is proinflammatory macrophages,releasing proinflammatory cytokines,leading to injury of nervous system tissues.M2 is anti-inflammatory macrophages,which has anti-inflammatory effect and promotes tissue repair.It has been found that the inflammatory cells in the brain of patients with MS are mainly M1 cells,rather than M2 cells.Therefore,M1 is the main inflammatory cell causing inflammatory lesions in MS brain.MRI monitoring tracers labeled M1 and M2 cells in mouse brain showed that in severe recombined EAE mice,M1 could promote the recurrence of EAE,while M2 enhanced immunoregulation,which was beneficial to the alleviation of EAE.Therefore,inhibiting M1 and promoting the transformation of macrophages into M2 may contribute to the treatment of MS.Objective:A EAE model was established to observe(1)The role of M1 and M2 cells in EAE;(2)Inducing M2 cell differentiation,adoptive transfer M2,and observing the therapeutic effect of M2 on the M2 mice.(3)The role of intracellular signal transduction molecules NFkappa B in the transformation of M1 to M2.Methods:(1)MOG35-55 plus complete Freund's adjuvant(FCA)mixed emulsion was injected subcutaneously into C57BL/6 mice,and the EAE model was established by intraperitoneal injection of pertussis toxin(PTX).(2)To observe the effect of M1 and M2 cells in EAE:(1)Incidence in different period were EAE mice by spinal cord pathological staining(HE and myelin staining),EAE mice spinal cord inflammation and demyelination of the situation;(2)Separation of spleen in EAE mice by selecting macrophages by flow cytometry(F4/80,CD11b)were determined M1,M2(M1 phenotype detection of i NOS,CD40,M2 detection of Arg-1,CD206 to identify M1 and M2);(3)Through the CBA method for the detection of EAE different onset period of M1/M2 cells to secrete inflammatory / anti-inflammatory cytokines;(3)To induce the differentiation of M2 cells,adoptive transfer of M2,observe the treatment effect of M2 on EAE mice: the mice with bone marrow stem cells in vitro by adding IL-4/M-CSF stimulated bone marrow stem cells toward M2 transformation;(2)The possession of more than 90% confirmed M2 cells by flow cytometry;(3)The EAE mice just when infusion of M2 cells by observing the clinical symptoms;the treatment effect of macrophages on EAE 1-2 weeks after the death of animal,mice spinal cord pathology(HE staining and myelin staining),macrophages were isolated from spleen,macrophage phenotype;(4)Through detection of M2 cells transfusion group and EAE group inflammatory cytokines by CBA method.(4)The role of intracellular signal transduction molecule NF-kappa B for conversion of M1 to M2:(1)EAE mice,M1/M2 lesion detection in different periods in the spleen of the phenotype and function of,and by Western blotting(Western blot)to detect the expression of signal transduction molecule EAE in mouse spinal cord NF-kappa B;(2)Application of EAE in mice NF-kappa B inhibitor for the prevention and treatment,detected the expression level of signal transduction molecules in the mouse spinal cord NF-kappa B after treatment,at the same time,the mouse spinal cord pathology(HE staining and myelin staining),and spleen M1 and M2 phenotype and function determination,to understand the impact of NFkappa B on macrophage differentiation the lack of signal transduction molecules in order to determine whether the macrophage activity in the cells in an intermediate state,which can further differentiate into M1 or M2.(3)The changes of inflammatory and anti-inflammatory cytokines secreted by M1/M2 cells in the NF-kappa B blockage group and EAE were detected by CBA method.(5)All tests were repeated at least three times,using the data obtained by standard deviation,measurement data between the two groups were compared with t test,whether the difference between the group EAE and the different periods and between the treatment groups was statistically significant,P < 0.05,the difference was statistically significant.Result:(1)The subcutaneous injection of MOG35-55 and CFA can effectively induce C57BL/6 mice to become the EAE model,and the onset of the disease on the 10 th day after immunization,and the peak in the 20 th day.(2)The early onset of macrophages in EAE mice increased(35.56 + /-1.659%)and a higher percentage of M1(73.37 + /-1.451%),low proportion M2(26.63 + /-1.451%)along with the progression of M1 proportion reduced(56.16 + /-1.297%),M2 is higher(43.84 + /-1.297%),rose to the highest peaks,recovery M2 decline.Cytokines TNF,il-6,il-17 a were increased in the early stage of onset and decreased during peak period.(3)Using IL-4 and M-CSF to induce bone marrow stem cell differentiation of M2 macrophages can reach over 90%.(4)The M2 celltreatment group havelower score than EAE group(3.208 + /-0.1145 VS0.7500 + /-0.3184),spleen macrophages decreased,the M2 macrophages reduce obviously,cytokine IL-6,IL – 17 A and TNF in EAE group than in M2 cells back to lose.(5)The activity of NF-kappa B was decreased in the spinal cord of mice after the application of NF-kappa B blocker.The symptoms of the NF-kappa B group and the NFkappa B treatment group were less than that of the EAE group.The macrophages production and consumption were reduced in the spleen of mice after blocking.Conclusion:(1)Mog35-55 mixed with FCA emulsion can successfully induce EAE model of C57BL/6 mice.(2)Macrophages are involved in the pathogenesis of EAE,and M1 has anti-inflammatory effect,aggravating EAE symptoms,M2 has anti-inflammatory effect and alleviates EAE symptoms.(3)IL-4 and M-CSF could successfully induce M2 macrophage on mouse bone marrow stem cells.(4)M2 macrophage retransfusion can effectively treat EAE by reducing splenic macrophages and pro-inflammatory cytokines in mice.(5)NF-?b blocker Bay-11-7082 can effectively inhibit the phosphorylation of NF-?b 65,and inhibit the production and consumption of macrophages.(6)Blocking the NF-?b pathway can reduce the clinical symptoms of EAE.