| Part Ⅰ:Single-cell transcriptome profiling of the GM-CSF-induced Macrophages and their abnormalities in patients with Pulmonary hypertensionBackground:Single-cell RNA sequencing(scRNA-seq)is a type of high-throughput sequencing technology at the single-cell level,which can reveal gene expression profiles at the single cell level Pulmonary hypertension(PH)is a clinical and pathophysiological syndrome characterized by marked remodeling of the pulmonary vascular system and progressive elevation of pulmonary vascular load due to multiple heterogeneous diseases(etiology)and different pathogenetic mechanisms,which in turn leads to right ventricular hypertrophy and remodeling or even death.The pathogenesis of PH is complex and still not fully elucidated.Many studies have shown that the fuction of monocyte-derived macrophage is markedly abnormal and play a key role in the inflammatory response and vascular remodelling in PH.However,what is the mechanism of macrophage abnormality in patients with PH?Are the abnormalities present at the time of differentiation from monocytes into macrophages,or are they due to the microenvironment of PH?Given the complexity of macrophage subpopulations,whether there is an altered ratio of cell subpopulations and their altered function in macrophage abnormalities in PH patients,and other questions have not been reported so far.Objective:To construct a single-cell transcriptional profile of monocyte-derived macrophages from PH patients and the types of cell subpopulations by making full use of the discriminatory power of scRNA-seq at the single-cell level.To identify alterations in the macrophage subpopulations and their functional genes in PH,reveal the types and functional abnormalities of monocyte-derived macrophages in PH patients,and provide valuable clues for understanding the role of macrophages and immune inflammation in the development of PH.Materials and Methods:We collected blood from three patients with pulmonary hypertension and three healthy individuals.After extracting PBMCs,PBMCs were stimulated with granulocyte macrophage colony-stimulating factor(GM-CSF)to produce GM-CSF-derived macrophages,and the induced macrophages were extracted for scRNA-seq,the induced macrophages were extracted for scRNA-seq.The obtained single-cell sequencing data were analyzed by bioinformatics methods,and the differences in gene expression of macrophages in the disease and normal groups were compared.Peripheral blood was first collected from three patients with pulmonary hypertension and three healthy individuals.Peripheral blood mononuclear cells(PBMC)were extracted from Peripheral blood and treated with granulocyte macrophage colony-stimulating factor(GM-CSF)to differentiate them into macrophages.Then single-cell transcriptome sequencing was performed.The data obtained were analysed using a combination of bioinformatics methods to compare the abnormalities in macrophage types and functional genes between PH patients and healthy individuals.Results:1.monocyte-derived macrophages from PH patients have five subpopulations:C1 with high expression of CD 14,C2 with high expression of deoxyribonuclease 2 Beta(DNASE2B),C3 with high expression of S100 calcium-binding protein A9(S100A9)and peroxiredoxin 1(PRDX1),and high expression of growth differentiation factor-15(GDF15)with solute carrier family Of these,C1,C2,C4 and C5 express both CD86 and MRC1/CD206,while C3 expresses neither.The genes highly expressed in C2 were mainly associated with cell adhesion and tissue growth;the genes highly expressed in C3 were mainly associated with precursor metabolites,energy production and cellular respiration;the genes highly expressed in C4 were mainly associated with negative regulation of cell growth;and the genes highly expressed in C5 were mainly associated with ion binding and metabolic processes.2.Single cell transcriptome data showed a significant decrease in the proportion of GDF15high macrophages in PH.High expression of GDF15 in this cell subpopulation may be associated with a negative regulation of macrophage growth.We also confirmed the existence of GDF15high macrophages that express both CD80,CD 163,and MRC1/CD206 by immunofluorescence assays and flow cytometric sorting,and analysis of published data on single cell transcriptome sequencing of lung tissue also indicated the existence of this macrophage subpopulation in lung tissue.These results suggest that GDF15high macrophages are highly associated with PH.3.The functional enrichment analysis of differentially expressed genes(DEGs)showed that upregulated DEGs in various cell populations of macrophages from PH patients were mainly involved in the production of cytokines,chronic inflammatory response,cell chemotaxis,interferon gamma(IFN-γ)response,tumour necrosis factor(TNF)and growth factor receptor binding;downregulated DEGs were mainly involved in extracellular matrix organization,cell junction assembly,cell substrate adhesion,etc.4.The proportion of C3,C4 and C5 macrophage subsets was altered in the PH patient group,with the most significant alteration in the C4 subset.Further analysis showed that the functional alterations in each of the five macrophage subpopulations in PH patients were also distinctive:the expression of genes related to lymph node development and trans-epithelial transport was significantly upregulated in the C1 subpopulation,suggesting an enhancement of the corresponding functions;while the expression of genes related to the negative regulation of protein localization,DNA cytosine deoxygenation and cytidine catabolic processes was significantly downregulated,suggesting a decrease of the corresponding functions;the expression of genes related to IFN-γ production was significantly downregulated in the C2 subpopulation,suggesting a decrease in the corresponding functions.The expression of genes associated with lymphocyte differentiation and activation was significantly upregulated in subpopulation C3,while genes associated with extracellular matrix organization were significantly downregulated.Genes related to interleukin-6(IL-6)production and macrophage activation were significantly up-regulated in the C5 subpopulation,while genes related to cellular oxygen transport and other functions were significantly down-regulated..5.Protein-Protein Interaction Networks(PPI)of up-regulated genes and down-regulated genes were constructed respectively:genes of upregulated key modules include Fas Ligand(FASLG),Chemokine(C-C Motif)Ligand 15(CCL15),Tumor Necrosis Factor Receptor Superfamily,Member 25(TNFRSF25),Colony Stimulating Factor 2),LTA(Lymphotoxin Alpha(CSF2),Interleukin-32(IL32),CD7,Interleukin 18 Receptor Accessory Protein(IL18RAP),HLA-DQA2,Interferon Induced Transmembrane Protein 1(IFITM1),Interleukin-26(IL26),while genes of downregulated key modules include Phosphatidic Acid Phosphatase Type 2 Domain-Containing Protein 1A(PPAP2C),Transthyretin(TTR),Apolipoprotein A1(APOA1),Glypican 1(GPC1),Apolipoprotein A1(APOA2),Carboxyl Ester Lipase(CEL),Phosphatidic Acid Phosphatase Type 2 Domain-Containing Protein 1A(PPAPDC1A),Pancreatic Lipase Related Protein 3(PNLPRP3).They are associated with interferon,cytokine production and the Janus kinase-signal transducer and activator of transcription(JAK-STAT)pathway as well as the metabolism of various compounds,cholesterol transport,and LDL particle remodelling,respectively.They are associated with interferon,cytokine production and the Janus kinase-signal transducer and activator of transcription(JAK-STAT)pathway as well as the metabolism of various compounds,cholesterol transport,and LDL particle remodelling,respectively.6.In the Intercellular Communications of macrophages in PH,the communication probability of receptor-ligand pairs associated with the VISFATIN,COMPLEMENT,and GALECTIN signalling pathways is upregulated.These signaling pathways are associated with inflammation,macrophage activation,and macrophage polarization.7.Analysis of transcription factor regulatory networks in macrophages in PH revealed a large overlap of functions regulated by transcription factors shifted in major macrophage subpopulations,including Wnt signaling pathway,lymphocyte differentiation,cell adhesion,and cytokine production,suggesting a consistency of altered functions across cell subpopulations in PH.Also the activity of transcription factors associated with response to interferon gamma,and oxidative stress showed specific upregulation in C2.Conclusions:We constructed a single-cell transcriptional profile of GM-CSF-induced monocyte-derived macrophages from patients with pulmonary hypertension and found that some GM-CSF-induced monocyte-derived macrophages expressed both CD86 and MRC1/CD206.The GDF15high macrophages may be closely associated with PH,and GSEA enrichment revealed that their function was mainly related with negative regulation of cell growth.The Functional enrichment analysis of DEGs indicated enhanced cytokine production and cell chemotaxis in various subpopulations of macrophages from PH patients.Seven genes,LTA,CD7,IL18RAP,IFITM1,IL26,CCL15 and TNFRSF25,are key genes in the upregulated PPI network.The endolipin(VISFATIN),complement(COMPLEMENT),and galactose lectin(GALECTIN)signalling pathways were upregulated in the communication probability of receptor-ligand pairs.Transcription factors associated with Wnt signalling pathway,cytokine production and other functions were generally up-regulated in PH cell subpopulations.PART Ⅱ:Single-cell landscape of peripheral blood mononuclear cells of patients with Graves’ diseaseBackground:Single-cell sequencing technologies is a method for high-throughput sequencing at single cell level,and can be used to reveal Gene Expression Variability at the single cell level.Graves’ disease(GD)is a classic organ-specific autoimmune disease and is the main type of hyperthyroidism.Similar to other autoimmune diseases,the pathogenesis of GD is complex and still not fully elucidated.A number of studies using scRNA-seq have revealed abnormalities in multiple immune cells in a variety of autoimmune diseases.Peripheral blood mononuclear cells(PBMC)contain a variety of immune cells that play an important role in the regulation of immune inflammation in the body.However,until now,no studies have been reported on the single-cell transcriptome profiles of PBMCs from patients with GD,and a comprehensive and systematic understanding of the abnormalities of PBMC cell types and functional genes in patients with GD is still lacking.Objective:This study aimed to construct a single-cell landscape of PBMC from patients with Graves’ disease.It also attempts to explore the altered gene function of each cell type in Graves’ disease,the changes in crosstalk between cell populations,and the transcription factors involved in the disease process.To construct a single-cell transcriptome landscape of PBMC from patients with GD;and further to systematically reveal the abnormalities in cell types/clusters,functional genes,transcriptional regulation of each cell population,and intercellular regulation of PBMC from patients with GD using various bioinformatics tools.Materials and Methods:PBMCs were isolated from the blood of 8 Graves’ disease patients and 12 healthy controls(HC)to create single cell suspensions,after which each sample was sequenced using 10x Genomics’ standard workflow for single cell sequencing under full quality control.Then,the single-cell sequencing data of all samples were analyzed using bioinformatics methods.For example,Gene ontology(GO)enrichment analysis was used to analyze the genes enriched in each cell subpopulation in PBMCs and their functions;Cellchat and SCENIC were used to analyse the inter-regulation between cells and the alteration of transcription factors in each cell population..Results:1.PBMCs in both GD patients and healthy individualshave 4 large cell populations,which can be subdivided into 16 subpopulations.2.Compared to healthy controls,the cell clusters in Graves’ disease showed a universally conserved activation of interferon gamma(IFN-y)response,tumor necrosis factor(TNF)response,antigen presentation,as well as the inhibition of negative regulation of immune processes and reactive oxygen species response.3.Cellchat analysis categorised the intergroup variation in receptor-ligand pairs into individual signalling pathways,and identified eight important signalling pathways in various cell subpopulations of PBMC,mainly including B-cell activating factor(BAFF),proliferation-inducing ligand(APRIL),interleukin 1(IL-1),interleukin 6(IL-6),CD80,CD86,erythropoietin hepatocyte receptor A receptor(EPHA),CD48 and other signalling pathways.Among them,IL-1,CD86,CD48 and EPHA signalling pathways were significantly enhanced in GD.In particular,CD48 and EPHA signalling pathways were only present in GD patients.4.The top 20 active transcription factors in PBMCs of GD patients,i.e.the top 20 transcription factors in terms of AUC values calculated by SCENIC,were mainly concentrated in monocytes and associated with TNF,Toll-like receptor(TLR)signalling pathway and myeloid differentiation.The top 20 active transcription factors in healthy human PBMCs were concentrated in B cells and associated with response to metal ions and circadian rhythms.5.348 autoimmune disease-related genes revealed by genome-wide association studies(GWAS)were significantly altered in expression in PBMC of GD patients.These genes were associated with functions such as cell adhesion and activation,IFN-γproduction,lymphocyte activation and differentiation,and TNF,TLR,IL-17,mitogen-activated protein kinase(MAPK)and nuclear factor kappa B(NF-κB)pathways.the distribution of GWAS-associated DEGs in cell types of PBMC was significantly cell-specific.The distribution intensity of GWAS-related differential genes was highest in CD16 monocytes and regulatory T cells(Treg).6.T cells included nine subpopulations,with granzyme B(GZMB)-positive CD8+effector memory T cells(Tem),granzyme K(GZMK)-positive CD8+ Tem,natural killer T cells(NKT),CD4+central memory T cells(Tcm),a significant increase in the relative content of CD8+naive T cells,and a significant decrease in the relative content of CD4+naive T cells and γδT cells.In GD patients,CD8+Tem cells showed activation of chronic inflammation and IL-1,interleukin 4(IL-4)response function.significant upregulation of Wnt and NF-κB pathway-related genes in Treg suggested enhanced signalling,while the OX40 signalling pathway-related TNF receptor superfamily member 4(TNFRSF4)gene in Treg and its associated gene Basic Leucine Zipper ATF-Like Transcription Factor(BATF)was significantly up-regulated,along with a decrease in the expression of genes such as Tuberous Sclerosis Gene 1(TSC1)and Tuberous Tclerosis Gene 2(TSC2)associated with the Target of Rapamycin(TOR)signalling pathway.Among the cell subpopulations,there was a significant increase in the communication probability of IL-4 signalling-related receptor-ligand pairs between CD4 naive T cells and other cell subpopulations.two subpopulations of CD8+effector T cells also showed a significant increase in the communication probability of IL-1 and CD48-related receptor-ligand pairs with other cell subpopulations.In addition,the transcription factors T-Box Transcription Factor 21(TBX21),RUNX Family Transcription Factor 3(RUNX3),X-Box Binding Protein 1(XBP1),Negative Elongation Factor Complex Member E(NELFE)were increased in the GZMK+CD8Tem cell population,and the transcription factors Histone Deacetylase 2(HDAC2),Interferon Regulatory Factor 2(IRF2),Activating Transcription Factor 1(ATF1)were increased in GZMB+Tem cell population,transcription factors Promyelocytic Leukaemia Protein(PML),XBP1,BATF were increased in Treg.7.The proportion of memory B cells was significantly increased and the proportion of naive B cells was decreased in B cells from GD patients.Genes associated with immunoglobulin and NF-κB pathways were significantly upregulated in B cells.suggesting enhanced related functions.Crosstalk between B cell subsets and other cell subsets mainly involved B And T Lymphocyte Attenuator(BTLA),IL-1,IL4,and Insulin-Like Growth Factor-I(IGF-1)related receptor-ligand pairs.IL-1B related receptor-ligand pairs were present in both healthy and GD patient groups,while IL-4 and IL-1A related receptor-ligand pairs were mainly in the GD patient group.The communication probability of IGF-1-associated receptor ligand pairs was present in each cell population communicating with memory B cells.The communication probability of BTLA-associated receptor ligand pairs was elevated in the crosstalk of each cell to naive B cells and decreased in the crosstalk of GZMB+CD8Tem and γδT to memory B cells.The activity of Friend Leukemia Virus Integration 1(FLI1),Tata-Box Binding Protein Associated Factor 7(TAF7),Interferon Regulatory Factor 3(IRF3),Lymphoid Enhancer-Binding Factor 1(LEF1),Splicing Factor 1(SF1),and Chromodomain Helicase Dna Binding Protein 2(CHD2)were specifically upregulated in memory B cells.8.The proportion of CD16+ natural killer(NK)cells in peripheral blood was significantly increased in GD patients,whereas the proportion of CD56+NK cells was significantly decreased.immune responses in NK cells of GD patients activated cell surface receptor signaling,coagulation,cell killing and NK cell-mediated immune-related pathways were activated.Negative regulation of NF-κB,MAPK,IFN-γ and TLR3 pathways were diminished.Crosstalk between other cell subsets and NK cell subsets mainly involved Integrin Subunit Beta 2(ITGB2),CCL3,CD48 and IL4 related receptor-ligand pairs,Chemokine(C-C Motif)Ligand 3(CCL3)signaling pathway related receptor-ligand pairs are mainly present in the crosstalk from CD 14 monocytes to NK cells.The activity of ATF1 and ATF6 transcripts are enhanced in NK cells from GD patients.9.The proportion of monocytes in the peripheral blood of GD patients was not significantly altered.functions related to antigen presentation,cell chemotaxis,cytokine production,and NF-κB pathway were activated in monocytes from GD patients,whereas functions related to translation elongation,response to IFN-γ,and intrinsic apoptosis were reduced.The crosstalk between monocyte subclusters and other cell subclusters mainly involved IL1A/1B,CCL3,BTLA and IL4 signaling pathways related receptor-ligand pairs.the CCL3 pathway related receptor-ligand pairs was mainly present in the communication of CD14+monocytes with other cells in GD patients.The activity of E74-Like Factor 2(ELF2),HDAC2,Spi-B Transcription Factor(SPIB),Pou Class 2 Homeobox Associating Factor 1(POU2AF1),IRF2 and BATF transcription factors are significantly upregulated in CD16+monocytes of GD patients.Conclusions:We used single-cell transcriptome sequencing to reveal the single-cell profiles of PBMCs from GD patients and health controls.We found that the composition and function of T cell populations,including natural Killer T(NKT),memory T and other subpopulations,were most significantly altered in PBMC from GD patients compared to healthy subjects.The expression of inflammation-related genes was generally upregulated in each cell population,while genes related to negative regulation of immune process were downregulated,suggesting an enhanced inflammatory response.In particular,genes of NF-kB and MAPK signalling pathways were commonly upregulated in all cell populations of PBMC from GD patients.In addition,autoimmune disease-related genes have been reported to be expressed mainly in CD16+monocytes and Treg cells,suggesting their important role in the pathogenesis of GD,particularly the upregulation of OX40 and TOR signalling pathways in Treg cells.We found that CD14+monocytes communicate with various cell populations in PBMC via CCL3 chemokines.In addition,abnormalities in immune-related transcription factors such as IRF2,IRF3,XBP1 and RUNX3 may be important in the dysfunction of these cell populations.These findings provide insights into the overall understanding of the pathogenesis of GD. |
PDF Full Text Request