Regulation Of Polarization Of SLE-DAH Macrophages By HUCMSCs-exo

Objective To identify key molecules and pathways that significantly regulate SLE-DAH cell polarization in mice by constructing a SLE-associated diffuse alveolar hemorrhage(SLE-DAH)model mouse and transcriptome sequencing technology,and then to explore the regulatory effects of human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSCs-exo)on these key molecules and pathways and on We then investigated the effects of hUCMSCs-exo on these key molecules and pathways and on the polarization of SLE-DAH cells,so as to systematically elucidate the regulatory mechanisms of hUCMSCs-exo on the polarization of SLE-DAH macrophages.Methods(1)Mice were injected intraperitoneally with hypocretin to establish the SLE-DAH model,and the presence of macrophage M1-type polarization was experimentally assessed in model mice;(2)Transcriptomic techniques were used to screen differentially expressed genes in lung tissues of SLE-DAH mice,and their possible pathogenesis and relationship with macrophage polarization were explored by clustering and functional enrichment;(3)hUCMSCs-exo was extracted and identified exo and investigated its regulatory effect on macrophage polarization in SLE-DAH;(4)mouse experiments and cellular experiments to further validate the molecular mechanism of hUCMSCs-exo on the regulation of macrophage polarization in SLE-DAH.Results(1)Compared with the control group,intraperitoneal injection of hypocretin in mice caused different degrees of pulmonary hemorrhage with neutrophil vasculitis,and a large infiltration of iron-containing macrophages was seen,consistent with the pathological changes of SLE-DAH.lung macrophage M1/M2 ratio was increased in SLE-DAH mice(P < 0.05),and iNOS,IL-6,TNF-α,IL-1β genes expression was upregulated(P < 0.05),Arg-1,IL-10,TGF-β,chi3l3 gene expression was downregulated(P<0.05),IL-6 and TNF-αwere elevated(P < 0.05),and IL-10 and TGF-β were decreased(P < 0.05),showing an overall M1 macrophage polarization.(2)The transcriptome of SLE-DAH mice was sequenced,and the differentially expressed genes were clustered and functionally enriched,and the enriched pathways were found to be mainly related to biological processes such as immunity and inflammation.Among them,signaling molecules such as TLR4 and NOTCH and their pathways are key factors of macrophage polarization and may be involved in the regulation of macrophage polarization in SLE-DAH;(3)After human umbilical cord MSCs were cultured and passaged,microvesicles were isolated by ultracentrifugation,and were seen under fluoroscopy as typical The average particle size of microvesicles was 99.5 nm and the average concentration was1.01×10~9 particles/ml by nanoparticle tracking analyzer.The above proved that the extracted microvesicles were hUCMSCs-exo.After 14 days of hUCMSCs-exo intervention,the lung tissue hemorrhage in SLE-DAH mice was significantly alleviated,the ratio of M1/M2 of lung macrophages decreased(P<0.05),and the expression of iNOS,IL-6,TNF-α,IL-1β genes decreased(P<0.05),Arg1,IL-10,TGF-β,and chi3l3 gene expression increased(P<0.05),IL-6 and TNF-α levels decreased(P < 0.05),and IL-10 and TGF-β levels increased(P<0.05),showing an overall polarization of macrophages from type M1 to type M2.(4)Compared with the control group,TLR4,NOTCH1,IL-1β, and iNOS were highly expressed in the lung tissue of SLE-DAH mice(P<0.05),while CD206,Arg-1,and IL-10 were lowly expressed(P<0.05).These results were reversed when hUCMSCs-exo treatment was given(P < 0.05).Further,plasmids carrying TLR4 or NOTCH1 were transfected into SLE-DAH mice,respectively,and hUCMSCs-exo treatment was administered simultaneously.The results revealed that TLR4,NOTCH1,IL-1β,iNOS were highly expressed in the lung tissues of TLR4 or NOTCH1 plasmid-transfected SLE-DAH mice compared with the empty vector group(P<0.05),and CD206,Arg-1 and IL-10 were lowly expressed(P<0.05).In addition,the results of cellular experiments showed that LPS-induced M1 macrophages expressed TLR4,MyD88,NF-κB,and iNOS proteins(P<0.05),TLR4,MyD88,and iNOS genes(P<0.05),and TNF-α,IL-6,and NF-κB levels in cell culture supernatants(P < 0.05)were increased;meanwhile Arg-1,CD206 protein expression(P<0.05),Arg-1 gene expression(P < 0.05),IL-10,TGF-β levels(P < 0.05)in the cell culture supernatant were decreased.And when LPS-induced M1-type macrophages were co-cultured with TAK-242 or hUCMSCs-exo,respectively,both significantly reversed the above indices when compared with the LPS-induced group(P<0.05).Conclusion(1)There is macrophage M1-type polarization in hypocretin-induced SLE-DAH mice.(2)TLR4,NOTCH signaling molecules and their pathways may be involved in the regulation of macrophage polarization in SLE-DAH.(3)hUCMSCs-exo can regulate macrophage polarization from M1 to M2 type thereby alleviating SLE-DAH.(4)hUCMSCs-exo can target and regulate macrophage polarization from M1 to M2 type through TLR4 and NOTCH1 receptors thereby alleviating SLE-DAH.In addition,TLR4-Myd88-NF-κB signaling pathway may The TLR4-Myd88-NF-κB signaling pathway may be involved in the regulation of macrophage polarization in SLE-DAH.Part 1 Establishment and study of mouse SLE-DAH model induced by PristineObjective To establish a mouse model and explore its pathogenesis.Methods(1)C57BL/6J mice were divided into SLE-DAH and normal control groups.A single intraperitoneal injection of 0.5 ml of Pristane was given in the SLE-DAH group mice for modeling,while 0.5 ml of PBS was given intraperitoneally in the normal control group.(2)After 14 days of modeling,mice were executed by decortication,and lung tissue was taken for H&E staining and Prussian blue staining to assess the success of modeling.(3)Immunofluorescence homology double-labeling assay to detect the proportion of M1-type macrophages(iNOS-labeled)and M2-type macrophages(Arg-1-labeled)in mouse lung tissue.(4)Alveolar macrophages were isolated and purified from the bronchoalveolar lavage fluid of mice,and the M1macrophages(F4/80~+CD11b~+CD86~+CD206~-labeled)and M2 macrophages(F4/80~+CD11b~+CD86~-CD206~+ labeled)were detected by flow cytometry.(5)qRT-PCR was performed to detect the expression of M1 macrophage markers(iNOS,IL-6,TNF-α and IL-1β)and M2 macrophage markers(Arg-1,IL-10,TGF-β and chi3l3)genes.(6)ELISA for the determination of pro-inflammatory cytokines(IL-6,TNF-α)and anti-inflammatory cytokines(IL-10,TGF-β)in the supernatant of mouse alveolar lavage fluid.Results(1)Compared with the normal control group,H&E staining of lung tissue in the SLE-DAH group showed different degrees of hemorrhage with neutrophil vasculitis,and Prussian blue staining showed a large number of iron-containing hemocyanin cells.(2)Immunofluorescence homology double-labeling assay showed that the percentage of M1-type macrophages was increased and the percentage of M2-type macrophages was decreased in the lung tissue of mice in the DAH group compared with normal controls.(3)Flow cytometry results showed that the percentage of M1-type macrophages in alveolar lavage fluid of mice in the SLE-DAH group increased(P<0.01)and the percentage of M2-type macrophages decreased(P < 0.01)compared with that of normal controls.(4)qRT-PCR results showed that the gene expressions of iNOS,IL-6,TNF-α and IL-1β were upregulated in the lung tissues of mice in the SLE-DAH group compared with the normal control group(P<0.05),while the gene expressions of Arg1,IL-10,TGF-β and chi3l3 were downregulated(P<0.05).(5)ELISA results showed that IL-6 and TNF-α were elevated in the alveolar lavage fluid of mice in the SLE-DAH group compared with the normal control group(P<0.01),while IL-10 and TGF-β were decreased(P<0.01).Conclusion(1)Intraperitoneal injection of Pristane induces pathological changes of SLE-DAH in C57BL/6J mice.(2)Presence of lung macrophage polarization toward M1 type in SLE-DAH mice.Part 2 Transcriptome sequencing-based screening of signaling molecules and pathways related to the regulation of polarization in SLE-DAH macrophagesObjective To screen the signaling molecules and pathways related to the regulation of polarization in SLE-DAH macrophages based on transcriptome sequencing.Methods(1)Experimental modeling and grouping were as in Part I.(2)Transcriptomic techniques were applied to sequence m RNAs from lung tissues of SLE-DAH and normal control mice to screen out differentially expressed genes.(3)Bioinformatics analyses such as cluster analysis,GO annotation and KEGG pathway enrichment were performed for differentially expressed genes.Results(1)Compared with the normal control group,a total of 690 significantly differentially expressed genes were obtained in the SLE-DAH group,but up-regulated expression genes were predominant,with 503 genes and187 genes down-regulated.(2)Cluster analysis showed that differentially genes were predominantly highly expressed in the SLE-DAH group mice.(3)GO annotation results showed that: in terms of biological processes,the up-regulated genes in SLE-DAH group mice were mainly associated with immunity and inflammation;in terms of cellular components,they were mainly associated with immunoglobulin complexes and nucleosomes;while in terms of molecular functions,they were mainly associated with antigen binding,immunoglobulin receptor binding,TLR4 receptor binding,NOTCH receptor binding,etc.(4)KEGG pathway enrichment analysis showed that the upregulated genes in SLE-DAH group mice were mainly associated with NOTCH signaling pathway,Toll-like receptor signaling pathway.Conclusion Based on the transcriptome sequencing results,the development of SLE-DAH may be significantly associated with biological properties such as immunity and inflammation,especially macrophage polarization-related molecules and pathways such as TLR4 and NOTCH are significantly altered,suggesting that they may be involved in the regulation of macrophage polarization in SLE-DAH.Part 3 Study of the effect of hUCMSCs-exo on the regulation of polarization of SLE-DAH macrophages in miceObjective Preliminary investigation of the efficacy and possible mechanism of hUCMSCs-exo on SLE-DAH in mice.Methods(1)Extraction and identification of hUCMSCs-exo: 1.Human umbilical cord MSCs were cultured and passaged.2.Exosomes secreted by human umbilical cord MSCs were extracted by gradient ultracentrifugation.3.hUCMSCs-exo morphology was observed by transmission electron microscopy,and the size and concentration of hUCMSCs-exo were measured by nanoparticle tracking analyzer.4.Western Blot The expression of CD9,CD63 and Calnexin was detected to identify hUCMSCs-exo.(2)Experimental grouping: mice were divided into normal control group,DAH group,DAH + hUCMSCs-exo group and DAH + methylprednisolone group.(3)To evaluate the effect of hUCMSCs-exo in treating SLE-DAH mice: 1.H&E staining to assess alveolar hemorrhage in each group of mice.2.Immunofluorescence homology double-labeling assay to detect the ratio of M1/M2 type lung macrophages in lung tissue of each group of mice.3.Flow cytometry to detect the ratio of M1/M2 type lung macrophages in alveolar lavage fluid of each group of mice.4.qRT-PCR to detect the expression of iNOS,IL-6,TNF-α,IL-1β,Arg-1,IL-10,TGF-β,chi3l3 genes in lung tissue of each group of mice.5.ELISA was performed to detect the levels of IL-6,TNF-α,IL-10 and TGF-β in the supernatant of alveolar lavage fluid of mice in each group.6.Fluorescent microsphere assay was performed to detect the phagocytic function of alveolar macrophages in mice in each group.Results(1)hUCMSCss were cultured and passaged,and microvesicles were isolated by ultracentrifugation,and a typical "Teatro " structure was seen under fluoroscopy;the average particle size of microvesicles was 99.5 nm and the average concentration was 1.01×10~9 particles/ml by nanoparticle tracking analyzer.Western Blot assay showed that the isolated microvesicles expressed CD9,CD63 positive and Calnexin negative.The above proved that the microvesicles were hUCMSCs-exo.(2)Compared with DAH mice,tail vein injection of hUCMSCs-exo alleviated alveolar hemorrhage in mice;down-regulated the elevated ratio of macrophage M1/M2 in alveoli and lung tissues(P<0.01);down-regulated iNOS,IL-6,TNF-α,IL-1β gene expression in lung tissues(P < 0.01)and up-regulated Arg1,IL-10,TGF-β,chi3l3 gene expression(P<0.01);it could down-regulate IL-6,TNF-α levels(P<0.01)and up-regulate inflammatory cytokine IL-10,TGF-β levels(P<0.01),showing an overall polarization from M1 to M2 type of macrophages.In addition the proportion of alveolar macrophages that phagocytosed fluorescent microspheres was significantly higher in the DAH+hUCMSCs-exo group compared with the DAH group(P<0.01).Conclusion hUCMSCs-exo attenuates alveolar hemorrhage and inflammatory response and upregulates phagocytosis of alveolar macrophages by regulating lung macrophage polarization from M1 to M2 in SLE-DAH mice.Part 4 Exploring the mechanism of hUCMSCs-exo regulating macrophage polarization in SLE-DAH based on TLR4 and NOTCH1 receptorsObjective Exploring the mechanism of hUCMSCs-exo regulating macrophage polarization in SLE-DAH based on TLR4 and NOTCH1 receptors.Methods(1)Validation of TLR4 / NOTCH1 regulatory targets: 1.Mice were divided into DAH group,DAH(exo)group,DAH(exo)+ pcDNA3.1group,DAH(exo)+ pcDNA3.1-TLR4/NOTCH1 group.2.H&E staining was performed to compare alveolar hemorrhage in each group.3.qRT-PCR was performed to detect the expression of TLR4,NOTCH1,IL-1β,iNOS,CD206,Arg-1,IL-10 gene expression.4.Western blot to detect TLR4,NOTCH1,IL-1β,iNOS,CD206,Arg-1,IL-10 protein expression.(2)TLR4-Myd88-NF-κB signaling pathway: 1.Mouse macrophages RAW264.7 were divided into LPS-induced group,LPS + TAK-242 group,LPS+exo group,and control group.2.Westernblot to detect the protein expression of TLR4,MyD88,NF-κB,iNOS,Arg-1,CD206.3.qRT-PCR to detect the gene expression of TLR4,MyD88,iNOS,Arg-1.3.ELISA to detect the level of IL-6,TNF-α,NF-κB,IL-10,TGF-β.Results(1)In the TLR4/NOTCH1 target validation experiment,TLR4,NOTCH1,IL-1β,and iNOS were highly expressed in the lung tissue of mice in the SLE-DAH group compared with the control group(P<0.05),while CD206,Arg-1,and IL-10 were lowly expressed(P<0.05).These results were reversed when hUCMSCs-exo treatment was administered(P < 0.05).The plasmids carrying TLR4 or NOTCH1 were further transfected into SLE-DAH mice and treated with hUCMSCs-exo,respectively.The results revealed that TLR4,NOTCH1,IL-1β,and iNOS were highly expressed in the lung tissues of TLR4 or NOTCH1 plasmid-transfected SLE-DAH mice compared with the empty vector group(P<0.05),and CD206,Arg-1,and IL-10 were lowly expressed(P< 0.05).(2)The results of in vitro experiments showed that LPS-induced M1-type macrophages had higher TLR4,MyD88,NF-κB,and iNOS protein expression(P<0.05),TLR4,MyD88,and iNOS gene expression(P<0.05),and TNF-α,IL-6,and NF-κB levels(P<0.05)in the cell culture supernatant than control macrophages increased;meanwhile Arg-1,CD206 protein expression(P<0.05),Arg-1 gene expression(P<0.05),IL-10,TGF-β levels(P<0.05)in cell culture supernatant were decreased.And when LPS-induced M1-type macrophages were co-cultured with TAK-242 or hUCMSCs-exo,respectively,both significantly reversed the above indices compared with the LPS-induced group(P<0.05).Conclusion(1)hUCMSCs-exo can target and regulate macrophage polarization from M1 to M2 type through TLR4 and NOTCH1 receptors thereby alleviating SLE-DAH.(2)TLR4-Myd88-NF-κB signaling pathway may The TLR4-Myd88-NF-κB signaling pathway may be involved in the regulation of macrophage polarization in SLE-DAH.