| Background and purposeThrombospondin-1(TSP-1)is a stromal cell glycoprotein that was first discovered in activated platelets.It is expressed in human macrophages,monocytes,endothelial cells,fibroblasts,and adipocytes.TSP-1 can regulate the occurrence of tissue inflammation by inhibiting or enhancing the secretion of multiple cytokines.The thymus is a central immune organ for T cells to differentiate and mature.Thymic stromal cells,including thymic epithelial cells(TEC)and thymic mesenchymal stem cells(tMSC),co-incubate and develop thymocytes into mature T cells to the periphery;at the same time,thymic stromal cells also participate in immune regulation through the production of various cytokines.Myasthenia Gravis(MG)is an organ-specific chronic autoimmune disease.Most patients develop antibodies against the auto-acetylcholine receptor(AChR).Previous studies have found that thymic inflammation and other lesions in patients with AChR+MG are closely related to the pathogenesis of MG,and that thymic inflammation in MG patients is driven by the secretion of proinflammatory cytokines IL-6,IFN-?,TNF? and IL-17.The research team analyzed the expression of MG thymus tissue inflammation-related factors in the early stage by gene chip,and found that in addition to the abnormal expression of chemokines(CCL-2,CCL-3,CXCL-10)and pro-inflammatory factors(IL-6,IFN-?),the expression of TSP-1 in MG thymus tissue was increased by 20 times,suggesting that TSP-1 may be related to the formation of MG thymus inflammation microenvironment.In this study,immunohistochemical staining showed that TSP-1 positive cells were mainly distributed in the thymic medullary region and the lobular space in thymus tissue.In order to investigate the relationship between the up-regulation of TSP-1 expression and the inflammatory environment of thymus.In this study,the effects of TSP-1 expressed by exosome-transferred MSCs on macrophage activation and CD4+T cell differentiation were analyzed by interfering with the expression of TSP-1 in thymic mesenchymal stem cells.The effect of cell differentiation provides experimental evidence for clarifying the way TSP-1 participates in tissue inflammation.Methods1.Expression and distribution of TSP-1 in thymus:Collect normal thymus specimens from thymus and congenital heart of MG-related patients,prepare paraffin sections of thymus,and stain TSP-1 immunohistochemically to determine the expression and distribution of TSP-1 in different pathological thymus.2.Effect of LPS on TSP-1 expression in tMSC:LPS stimulated tMSC,qRT-PCR and WB to observe the change of TSP-1 expression.3.Preparation of TSP-1 interference lentivirus and tMSC infection:Construct TSP-1 interference vector plasmids shRNAl and shRNA2,and package the virus with 293 T cells.tMSC cells were infected with shl or sh2 lentiviruses for 72 h.qRT-PCR and WB detected changes in TSP-1 expression.4.Extraction and identification of exosomes secreted by tMSC:Use exosomes isolation kit to extract normal MSCs,LPS stimulated tMSCs,and TSP-1 to interfere with exosomes in culture supernatants after lentivirus infection,Exosome surface markers CD63,CD9,CD81 were detected by WB,flow cytometry,and electron microscope was used to detect the morphology of exosomes.Flow cytometry was used to detect TSP-1 expression in exosomes.5.Induction and identification of macrophages from different sources:Purchase of THP1 monocyte cell line,PMA induces its differentiation into M0,INF-?/LPS is known to induce M0 to differentiate into M1-type pro-inflammatory macrophages,and IL-4/IL-13 induces M0 to differentiate into M2 type anti-inflammatory macrophages.Alternatively,human PBMCs were isolated and GM-CSF induced macrophages.Morphological changes of macrophages were observed after co-culture with exosomes.6.Effects of TSP-1 on macrophage activation:exosomes,cell culture supernatant(CM),exosomes-removed cell culture supernatants were co-cultured with THP1-derived macrophages for 24 h,and RT-PCR was used to detect RNA expression of IL-6,IL-1?,TNF-?.LPS stimulated tMSC exosomes(hereinafter referred to as MEXLPS)and TSP-1 interfered exosomes LPS stimulated tMSCs based on LPS stimulated tMSCs(hereinafter referred to as MEXLPS-TSP1sh)co-culture with THP-1 derived macrophages or human PBMC derived macrophages,RT-PCR and flow cytometry were used to detect changes of inflammatory factors secreted of macrophages.7.Effect of TSP-1 on the differentiation and proliferation of CD4+T cells:CD4+T lymphocytes in human PBMCs were sorted by immunomagnetic beads,co-cultured with MEXLPS,MEXLPS-TSP1sh,and inflammatory factors in the cell culture supernatant were detected by flow cytometry And the differentiation and proliferation of CD4+T cells.Results1.Distribution of TSP-1 in thymus tissue of different MG-related cases:Immunohistochemical results showed that the cortical pulp structure of normal thymic tissue in children with cardiomyopathy was obvious,and TSP-1 expression was less,mainly distributed at the junction of thymic cortex and pulp and in the lobular space;Non-tumor MG patients have significantly smaller thymus tissue,unclear borders of the cortex and pulp,more messy cells and vacuoles,TSP1+cells increased and scattered.Thymoma without MG patients had unclear borders of thymic tissue and cortex and pulp,and TSP-1+cells were not numerous and scattered.The tissue of MG with thymoma cannot be accurately located,but the tissue destruction is more serious,and the distribution of TSP-1+cells is not obvious.2.Effect of LPS on the expression of TSP-1 in tMSCs:After tMSCs were stimulated with LPS for 24h,48h,and 72h,the WB results showed that with the increase of the stimulation time,the expression of TSP-1 increased and the expression was highest at 72h(P<0.0001);Real-time quantitative PCR showed the same trend(P<0.05).3.TSP-1 interferes with lentivirus infection and tMSC causes the expression of TSP1 to decrease:TSP-1 interferes with the lentivirus shl and sh2 to infect tMSC 72h,and WB results show that the expression of TSP-1 in the shl and sh2 groups is significantly reduced(P<0.01);real-time PCR also showed the same trend(P<0.001).4.Extraction and identification of exosomes secreted by tMSC:WB results showed that CD63 and CD9 were expressed in the extracted exosomes,and CD63 and CD81 were positively expressed in flow cytometry.Under the electron microscope,the exosomes have a double-layer membrane structure with a diameter of 40-120nm.TSP-1 interferes with the decrease in fluorescence intensity of TSP-1 expression in exosomes secreted by tMSC after lentivirus infection.5.Induction culture and identification of macrophages:THP1 is induced to differentiate into M0 by PMA for 24 hours.It is known that M0 is induced to differentiate into M1 type macrophages with the inducing factor INF-y/LPS,and IL-4/IL-13 induces M0 to differentiate into M2.Macrophages.After co-culture of MEXLPS and MEXLPS-TSP1sh with M0 for 12 hours,M1 type macrophages were labeled with CD86 and HLA-DR,and M2 type macrophages were labeled with CD 163.The expression of CD86 and HLA-DR was increased(P<0.05),and CD 163 There was no significant difference in expression(P>0.05).6.Effect of TSP-1 on THP1-derived macrophage activation:MEXLPS and MEXLPS-TSP1sh were co-cultured with THP1-derived M0 for 12 h,respectively.The qRT-PCR results showed that compared with the control,MEXLPS stimulated M0-related cytokines IL-6(P<0.001),IL-1?(P<0.001),and TNF-?(P<0.01)had significantly increased RNA expression.IL-6(P<0.05),TNF-?(P<0.01),The secretion of MCP-1(P<0.01)and IL-18(P<0.05)were significantly increased.Compared with the MEXLPS-stimulated group,the RNA expression of M1-type cytokines IL-6(P<0.01),IL-1?(P<0.01),and TNF-?(P<0.05)after MEXLPS-TSP1sh stimulation of M0 was significant.Decreased,the secretion of IL-6(P<0.05),IL-1?(P<0.05),TNF-?(P<0.01),MCP-1(P<0.01),and IL-18(P<0.05)was significantly reduced.7.Effect of TSP-1 on the activation of human PBMC-derived macrophages:MEXLPS and MEXLPS-TSP1sh were co-cultured with human PBMC-derived M0 for 12 h.The qRT-PCR results showed that compared with controls,MEXLPS stimulated M0-related cytokines RNA expression of IL-6(P<0.01),IL-1?(P<0.01),and TNF-?(P<0.01)all increased significantly.IL-6(P<0.001),IL-1?(P<0.01)),TNF-?(P<0.0001),IFN-y(P<0.001),IL-10(P<0.001),IL-17a(P<0.01),IL-18(P<0.01),IL-23(P<0.01),IL-33(P<0.01)secretion increased.Compared with the MEXLPS stimulated group,RNA expression of M1-type cytokines IL-6(P<0.01),IL-1?(P<0.01),and TNF-?(P<0.01)was significantly reduced after MEXLPS-TSp1sh stimulation of M0.,IL-6(P<0.001),IL-1?(P<0.01),TNF-?(P<0.0001),IFN-?(P<0.001),IL-10(P<0.001),IL-17a(P<0.01),IL-18(P<0.01),IL-23(P<0.05),and IL-33(P<0.05)all decreased secretion.8.Effects of TSP-1 on the differentiation and proliferation of CD4+T cells:The flow cytometry results after co-culture with MEXLPS and MEXLPS-TSP1sh for 12 hours with activated CD4+T cells,respectively,showed that compared with controls,Th1,Th2,Th17,Treg cells There was no significant change in the ratio,T cell proliferation was significantly reduced(P<0.05),and IL-6(P=0.01)and TNF-?(P<0.001)in the cell culture supernatant were significantly increased.Compared with the MEXLPS stimulated group,the proportion of Th1(P<0.05)and Th17(P<0.05)cells significantly decreased after 12 hours of activation of CD4+T cells stimulated by MEXLPS-TSP1sh,and the proportion of Treg(P<0.05)cells increased,Th2 The proportion of cells did not change significantly,and T cell proliferation increased significantly(P<0.05).IL-6(P<0.05)and TNF-?(P<0.001)were significantly reduced in the cell culture supernatant.ConclusionsTSP-1 expressed by exosomes transfers tMSCs can induce macrophages to undergo M1 polarization,CD4+T cells to differentiate into Th1,Th17,and promote the secretion of inflammatory factors such as IL-6 and TNF-?,thereby participating in the thymic inflammation.Regulation and formation. |
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