Study On The Signal Pathway Of Macrophage Polarization Induced By The TB-specific Polypeptide E7

Backgroud Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis infection.Mycobacterium tuberculosis mainly invades the host’s macrophages when they entering huanman body.Macrophages not only have the function of phagocytosis and presentation of antigens,but also secrete various cytokines or tumor necrosis factors to mediate inflammatory responses and regulate immune responses.In addition,macrophages can also exhibit polarization.Common types of polarization are classified into classical activated macrophages(M1-type)and alternative activated macrophages(M2-type).The links are antagonistic to each other and thus exert different functions and roles.M1 macrophages function primarily to kill pathogens by promoting the release of inflammatory responses and proinflammatory cytokines.M2 macrophages can promote blood vessel formation,matrix remodeling,and tissue fibrosis by inhibiting the secretion of pro-inflammatory cytokines and counteracting inflammatory responses.This polarization of macrophages can be regulated by multiple factors and multiple pathways.Current studies generally believe that JAK/STAT signaling pathway、JNK signaling pathway、PI3K/Akt signaling pathway and Notch signaling pathway may be involved in macrophage In the process.In tuberculosis,there are currently many studies that suggest that M1 is the main macrophage in tuberculosis patients in the early and active stage of Mycobacterium tuberculosis infection,and M2 is the main type in the process of chronic infection of tuberculosis.ESAT-6 and CFP-10 are two small proteins secreted by Mycobacterium tuberculosis.They can be used as the main antigenic peptide to stimulate the release of IFN-γ in the peripheral blood of patients infected with Mycobacterium tuberculosis,which can be used to diagnose the patients infected with Mycobacterium tuberculosis.E6 、 E7 and C14 are polypeptide fragments selected from the ESAT-6 and CFP-10 proteins in our laboratory.Their molecular weights are smaller than those of ESAT-6 and CFP-10,and their tuberculosis may have stronger immune response specificity.Previous experiments have shown that TB-specific peptides can affect the macrophage polarization,but there is no further discussion on the signaling pathways that induce macrophage differentiation by the tuberculosis-specific polypeptides.Objectives 1.To investigate the effect of TB specific polypeptides on macrophage polarization type and cytokine expression.2.To investigate the signaling pathway of macrophage polarization type induced by tuberculosis-specific polypeptide E7.Part Ⅰ Effect of TB Specific Polypeptides on Macrophage Polarization一、Methods 1.Resuscitate and culture THP-1 cells.PMA stimulates cell adherent growth.2.The adherent mononuclear-macrophage cells were divided into blank control group、M1 positive stimulation group、M2 positive stimulation group、E6、E7 and C14 polypeptide stimulation groups.Each group was added with the corresponding stimulant,and cell culture was performed for 12h、18h、24h 、36h and 48 h to collect cells.3.Total cellular RNA was extracted and expression of M1 type markers(CD16、IL-6 and TNF-α)and M2 type markers(CD163、CD206 and IL-10)were detected by RT-PCR.二、Results 1.Tuberculosis-specific peptide E6 mainly stimulated monocyte-macrophage secretion of M2 type markers/cytokines,which IL-10 was mainly increased.2.Tuberculosis-specific peptide C14 stimulated monocyte-macrophage secretion of M2-type markers/cytokines,mainly as elevated CD163.3.M1 and M2 markers/cytokines were increased after TB-specific peptide E7 stimulated monocytes-macrophages.Among them,TNF-α in M1 markers was highest at 18 h and M2 markers.The expression of CD163 was highest after 24 hours of stimulation.Part Ⅱ Study on the polarization signal pathway induced by TB-specific peptides E7 in macrophages一、Methods 1.Resuscitate and culture THP-1 cells.PMA stimulates cell adherent growth..2.The mononuclear cells isolated from pleural effusions were sorted with CD4+ T cells using magnetic bead sorting methods,and cultured and stimulated to adhere to the cells.3.The adherently grown THP-1/thoracic mononuclear macrophages were divided into blank control group,M1 positive stimulation group and E7 polypeptide stimulation group,and added with NF-kB blocking agent(PDTC)and JNK blocker(SP600125),respectively,and stimulated by western blot method.The expression of NF-kB p65、p-NF-kB p65 和 p-JNK protein was detected,and the expression of TNF-α was detected by RT-PCR.4.The adherently grown THP-1/thoracic mononuclear macrophages were divided into blank control group,M2 positive stimulation group and E7 polypeptide stimulation group,respectively added PPARγ blocker(T0070907)for stimulation,and PPARγ protein expression was detected by western blot method,while RT-PCR was used.Methods The expression of CD163 was detected.5.Statistical analysis was performed using SPSS 16.0 software;Graph Pad Prism 5 software was used for mapping.二、Results 1.Blocking NF-kB signaling pathway has no significant effect on the differentiation of THP-1/Pleurary mononuclear-macrophage into M1 type by tuberculosis-specific peptide E7 stimulation;2.Blocking JNK signaling pathway can affect tuberculosis-specific peptide E7 stimulation of THP-1/Hypochondism mononuclear-macrophages differentiate into M1 type.(The JNK signaling pathway may be involved in the process of differentiation of a macrophage into an M1 type by the tuberculosis-specific polypeptide E7.)3.Blocking PPARγ signaling pathway can affect the differentiation of THP-1/Pleurotus monocyte-macrophages into M2 type induced by the tuberculosis-specific peptide E7.(The JAK-STAT signaling pathway may be involved in the process of differentiation of a macrophage into a M2 type by the tuberculosis-specific polypeptide E7).Conclusions 1.The tuberculosis-specific polypeptide E6 mainly stimulated the differentiation of monocytes-macrophages into M2 type,and mainly elevated IL-10.2.The tuberculosis-specific polypeptide C14 mainly stimulated the differentiation of monocytes-macrophages into M2 type,and mainly increased as CD163.3.In the process of differentiation of monocyte-macrophages stimulated by tuberculosis-specific polypeptide E7,the M1 type represented by high expression of TNF-α was predominant in the early stage,and M2 type represented by high expression of CD163 was mainly in the late stage.4.Tuberculosis-specific peptide E7 stimulates mononuclear-macrophage differentiation to TNF-α when it differentiates into M1 type.The signaling pathway involved in this process is mainly JNK.5.When tuberculosis-specific polypeptide E7 stimulated the differentiation of monocytes-macrophages to M2 type,the increase of CD163 was predominant.The signaling pathway involved in this process was mainly JAK-STAT.