Establishment And Significance Of A Single-cell Transcriptome Atlas Of Adult Human And Mouse Normal Adrenal Tissues

BackgroudsThe adrenal gland is an important and complex endocrine organ that consists of various cell types with diverse functions.Two embryonically different tissues coexist in the adrenal gland: mesodermally derived,corticosteroid-producing adrenal cortex and ectodermally derived,catecholamine-producing adrenal medulla.The adult human cortex is composed of three histologically and functionally distinct layers: the outermost zona glomerulosa(z G),the middle zona fasciculata(z F)and the inner zona reticularis(z R).Adrenomedullary chromaffin cells secrete two products: norepinephrine and epinephrine.Survival of human depends on maintenance of a steady state of homeostasis,which process relies on its ability to react and adapt to different physiological and pathological threats.Activation of the stress system,which is composed of the hypothalamic–pituitary–adrenal axis and the sympathetic–adrenal medullary system,is crucial.In many critical ill patients,both the levels of corticotropin-releasing hormone and adrenocorticotropin are suppressed.However,levels of glucocorticoids remain normal or are elevated,suggesting a shift from central to local intra-adrenal regulation of adrenal stress response.Among mechanisms potentially involved in this progression,the intra-adrenal microenvironment plays an important role.As a major effector organ of the stress system,there is a coordinated interaction of various cell types within the adrenal microenvironment that is involved in maintenance of body homeostasis during stress-free and stress-related conditions.In the stage of inflammatory stress,the regulation of synthesis and secretion of adrenal hormones is mainly mediated by activating of the pituitary independent mechanism(the intra-adrenal microenvironment).More and more studies support the crucial role of the activating of intra-adrenal microenvironment in maintaining the homeostasis of internal environment.Within the microenvironment,the immune-adrenocortical and immune-adrenomedullary crosstalk,adrenocortical-medullary cell interactions and adrenal vascular system exert crucial functions.There is a complex interplay among numerous cells,including adrenocortical,chromaffin,endothelial,immune and others.These various cells influence each other through secretion of biologically active products directly or in a paracrine manner.Alterations in the adrenal gland microenvironment are known to affect the progression of various diseases including diabetes,depression,cardiovascular disease and inflammation,and also be influenced by these diseases.However,the current understanding of the specific cell type characteristics within the adrenal microenvironment is limited.Many details about the exact cell subgroup are still unknown.Single-cell RNA sequencing(s RNA-seq)is a new technique for high-throughput amplification and sequencing of whole transcriptome RNA at the single-cell level to establish a cell map.Using the advantages of 10 X sc RNA-seq,this study explored the cell distribution and characteristics of normal adrenal tissues in humans and mice,and compared the differences between human and mouse normal adrenal glands at the single-cell level,with the aim of establishing normal adrenal glands in humans and mice.Single-cell atlas.Methods(1)The adrenal single cells were separated from human normal adrenal tissues by enzymatic digestion.A c DNA library that meet the requirements of the machine was tested for sequencing.The softwares including cell ranger,seurat and monocle were analysized for quality control,cell clustering and subtype analysis,quasi-temporal analysis,cell cycle analysis,differential gene expression analysis of adrenal sequencing results data.(2)Comparison of expression profiles between the left and right adrenal gland at single-cell resolution.According to our sc RNA-sequencing data,we explored the expression of rhythmic genes like CLOCK,NPAS2,ARNTL,ARNTL2,PER1,PER2,CRY1,CRY2 and RORA between the left and right adrenal glands.We also performed q PCR analysis to find the difference of expression of RORA,CLOCK,ARNTL and PER1.(3)The mouse adrenal single cells were separated from normal adrenal tissue from 8-12 week old C57 / BL6 mice by enzymatic digestion.A c DNA library that meet the requirements of the machine was tested for sequencing.The softwares including cell ranger and seurat were analysized for quality control,cell clustering and subtype analysis,quasi-temporal analysis,cell cycle analysis,differential gene expression analysis of adrenal sequencing results data.(4)We first pre-performed the mouse adrenal sequencing dataset.We transferred the mouse gene names into human homologous gene names with the ENSEMBL R package Bio Mart(version 2.40.4).We discarded genes with no gene names and retained identical names between the human and mouse adrenal datasets.We used the average expression of these homologous genes for comparison between human and mouse cell types and to calculate the coefficients of correlation between two species.We also identified the human special,mouse special and their shared differentially expressed genes(DEGs)in four main cell types(adrenocortical cells(human(z G cells and z F cells),mouse(z G cells,z F cells1 and z F cells2)),adrenomedullary cells,endothelial cells and fibroblasts)with the Seurat R package(version 3.1.0).Results(1)23 cell clusters were revealed from human normal adrenal tissues.We identified clusters 0-22 corresponded to macrophages1,effector CD8+T cells1,z F cells1,endothelial cells1,effector CD8+T cells2,effector CD8+T cells3,memory CD4/8-T cells,neutrophil,NK cells,plasma cells,z R cells,macrophages cells2,z G cells,effector CD4/8-T cells,macrophages cells3,macrophages cells3,endothelial cells2,endothelial cells3,vascular smooth muscle cells(VSMC),fibroblasts cells,mature B cells,rare cells1,adrenomedullary cells,rare cells2.(2)Various types of immune cells were identified in adrenal microenvironment.Within the immune cell clusters of our sequencing data,we identified T cells,macrophages,natural killer(NK)cells,neutrophil and B cells.T cells and macrophages were the main components among these immune cells.(3)Pseudotime analysis showed that adrenocortical cells arise in the outer part of the gland and migrate centripetally from z G through z F to z R,with changes in the phenotype.(4)The finding indicated that the expression level of most genes was relatively well correlated between the left and right adrenal.However,some variations were also observed.Unfortunately,no significant statistical difference was observed via q PCR as the small sample sizes.(5)18 cell clusters were revealed from mouse normal adrenal tissues.We identified the 18 cell clusters corresponded to EC1,macrophages1,z G cells,CD8+ T cells1,NK,z F cells1,z F cells2,macrophages2,neutrophil,fibroblasts,B cells,macrophages3,RBCs,adrenomedullary cells,EC2,macrophages4,adipocytes,CD8+ T cells2.(6)According to the average expression of these homologous genes,the correlation coefficient between total human and mouse adrenal cells was 0.86.We also identified the shared and species-specific DEGs between humans and mice.ConclusionsWith the help of 10 x single cell sequencing platform,we constructed a single cell map of normal adrenal tissues of human and mouse,and analyzed the correlation and difference between them at the single cell level.The single-cell description of the adrenal gland contributes to the understanding of the cellular basis of human adrenal function and provides the baseline for the next functional research.Additionally,it provides clues for the development of novel cell-based approaches to prevent and treat adrenal disease.