Single-cell RNA-seq Reveals Osteoporosis-related Genes In Subsets Of PBMC In Older Woman

BackgroundOsteoporosis(OP)is a common metabolic bone disease that is not usually detectable until a fracture occurs.According to WHO,there are more than 200 million people suffering from OP worldwide.OP has become a major public health problem that threatens human life safety and health in China and worldwide.A comprehensive and in-depth study of the pathogenesis of OP to improve preventive and therapeutic measures is imperative to alleviate the burden on society’s health care system.Although hematological and immunological studies related to OP are well established,most of the existing studies are based on exploration at the cell population level,where only the average expression levels of population cells can be obtained,thus ignoring the impact of intercellular differences on OP.Single cell transcriptome sequencing technology(scRNA-seq)can obtain gene expression at the individual cell level,which is beneficial to reveal OP-related immune cell subsets and genes.PBMC subsets and the relationship between gene expression in them and OP have not been systematically evaluated by applying scRNA-seq technology.ObjectiveThe scRNA-seq technique was used to reveal the heterogeneity of peripheral blood mononuclear cells(PBMC)in different individuals and to map PBMC;to search for OPrelated cell subsets and to analyze the differentially expressed genes in OP-related PBMC subsets,so as to systematically and comprehensively explore the relationship between PBMC and OP and to enhance the understanding of OP.This study provides support for further studies on the association and mechanism of genes and OP of PBMC on the theoretical basis.MethodsIn this study,5 ml of fresh peripheral venous blood was collected from six OP patients and four healthy controls,from which PBMC were extracted for scRNA-seq.The raw data returned by sequencing were subjected to human genome matching and sample aggregation using CellRanger software to obtain cell count matrices.Downstream data analysis was performed using R software.First,the sequencing data were quality controlled to remove low-quality cells.Secondly,after sample integration and downscaling clustering of subgroups,cell annotation was performed for each cell cluster of the clusters,and trajectory analysis was used to demonstrate the differentiation trajectory among cells;differentially expressed genes among case controls in each cell subgroup were calculated.Finally,enrichment analysis of differential genes among case controls;assessment of cell state differences among case controls using gene set scoring methods;and communication analysis to obtain interactions of intercellular ligand-receptor pairs among case controls.ResultsThe single-cell sequencing dataset of 10 PBMC samples with 97 366 cells remaining after quality control was down-clustered to detect 18 clusters and annotated into 11 cell subpopulations,including:CD8+T cells,CD4+T cells,NK cells,NK cells,proliferating cells,B cells,plasma cells,monocytes,dendritic cells,plasmacytoid dendritic.The percentages of monocytes were higher in OP patients than in controls.The functional enrichment analysis of differential genes in cell subsets revealed that monocytes,B cells,NK cells,and dendritic cells were highly enriched in osteoclast differentiation.Monocytes were subdivided into three monocyte subtypes:CD 14+monocytes(also known as classical monocytes),CD 16+monocytes(or non-classical monocytes),and intermediate monocytes.The percentage of intermediate monocyte count differed between case control groups(P=0.021),indicating an elevated number of intermediate monocytes in the blood PBMC of individuals in the OP group.Intermediate monocytes were upregulated in the OP group mainly by HLA-DRB1,CST3,CD52,PTL,and RETN;and downregulated by CCL3L1,IER2,CCL4L2,TNFAIP3,CCL3,NFKBIA,CXCL8,CCL4,HLA-DQA2,DUSP1,KLF6,HLA-DRB5,NFKBIZ,JUN and TNF.These differentially expressed genes were enriched in the oxidative phosphorylation pathway and type Ⅰ interferon response pathway;in the enrichment results of GSVA,pathways such as interferon response,KRAS downregulation signaling pathway,IL2_STAT5 signaling pathway,which are closely related to OP,were upregulated in the OP group.Among the subtypes of T cells,CD4+ GZMK effector T cells highly expressed ZFP36,LYZ and CXCR4;CD8+ GZMK effector T cells highly expressed DUSP2,CTSW,MT2A and CXCR4;γδ T cells highly expressed PTGDS,CD247,DUSP1 and KLRC1;NKT cells highly expressed LAIR2 and CD7,etc.NK cells highly express LAIR2,IFI44L,MYOM2,IFITM1,IFI6 and MT2A,etc.Cytotoxicity,depletion and inflammatory response scores among all cell subsets were higher in the OP group than in the control group,except for primitive and memory T cells.In addition,both primitive and memory B cells significantly overexpressed HLA-DRB1.The total number and weight/intensity of interactions between CD8+effector T cells and CD4+effector T cells,intermediate monocytes,and CD 16+monocytes,respectively,were higher in the OP group than in control group.The interaction of MIF molecules of CD8+effector T cells with CD74 and CD44 in intermediate monocytes was enhanced in the OP group.Conclusions1.In this study,we successfully annotated 14 T and NK cell,3 monocyte and 3 B cell subtypes by scRNA-seq of PBMC,and revealed the heterogeneity of PBMC.2.T cells,NK cells and monocytes may be involved in the regulation of OP through upregulation of multiple genes and pathways closely related to the formation and differentiation of osteoclasts.3.The enhanced interaction between T cells and monocytes suggests that important upstream pathways for the differentiation of OCs are highly activated and the rate of bone resorption is increased thereby leading to OP.