The Role Of Single Cell Rna Sequencing In The Study Of Immune Cell Heterogeneity And TCR Repertoire After Aneurysmal Subarachnoid Hemorrhage

Objective Aneurysmal subarachnoid hemorrhage(a SAH)is a stroke caused by the rupture of a intracranial aneurysm.Although a SAH only accounts for 5-10% of stroke,it is characterized by high disability rate and high mortality rate.Therefore,a SAH seriously affects the quality of life of patients and their families,and imposes a heavy medical burden on society.After a SAH,red blood cells produce a large number of metabolites in subarachnoid space.The metabolites of red blood cells in the subarachnoid space,such as oxyhaemoglobin and heme,become antigens to activate various immune cells and inflammatory pathways,leading to a severe neuroinflammation.Neuroinflammation after a SAH can lead to brain damage,impaired neurological function and deteriorate the clinical prognosis.Previous studies on neuroinflammation after a SAH have focused on immune cells and inflammatory factors.And it is not clear how the gene transcriptomics of immune cells in peripheral blood(PB)and cerebrospinal fluid(CSF)are different.In this study,we performed single cell RNA sequencing(sc RNA-seq)on the immune cells of a SAH patients,aiming to analyze the heterogeneity and gene transcriptome characteristics of immune cells from CSF and PB and explore the pathophysiology and pathogenesis of neuroinflammation.Method In this study,CSF and PB samples were collected from aSAH patients,and mononuclear cells were extracted by density-gradient centrifugation.In the first part of the study,mononuclear cells from CSF and PB samples were sequenced using 10 x Genomics sc RNA-seq.In addition,the single-cell sequencing data of CSF from healthy controls were downloaded from Gene Expression Omnibus and compared with that of a SAH patients,to analyze the pathophysiology and pathogenesis of neuroinflammation.Various R packages were performed to make bioinformatics analysis on the gene expression matrices,exploring the gene transcriptome and cellular heterogeneity of immune cells.Seurat package was used for cell quality control,dimensionality reduction,clustering and results visualization.Monocle was used to construct the cell differentiation trajectory and explore the gene transcription mechanism that regulates cell development.Cellchat was used for the communication interaction of immune cells.GSVA was used to explore the activation of inflammatory pathways in immune cells from healthy controls and a SAH patients.In the second part of the study,single-cell TCR repertoire sequencing was performed on T cells extracted from CSF and PB to analyze the TCR repertoire for a SAH patients.Result Part I The role of single-cell RNA sequencing in the study of immune cell heterogeneity after aneurysmal subarachnoid hemorrhage.(1)A total of 17242 cells were obtained in this study,of which 7224 cells were obtained from CSF and 10018 cells were obtained from PB.Monocytes,T cells,B cells and NK cells were identified in these cells.(2)The differentiation trajectories of monocytes and T cells were also constructed.It was found that gene modules differentially expressed along with cell differentiation,which regulated the differentiation process and cell function.(3)Compared with PB,the activation of TLR 、 JAK-STAT and other immune pathways in CSF intermediate monocytes significantly increased.Moreover,compared with healthy controls,the activation of pathways such as TLR and MAPK significantly increased in CSF intermediate monocytes of a SAH patients.The biological processes related to neuroinflammation such as leukocyte recruitment and immune response regulation were enriched in CSF intermediate monocytes of a SAH patients.(4)The strength and number of interaction communication between monocytes in CSF were higher than those in PB.Part II The role of single-cell TCR sequencing in the study of TCR repertoire after aneurysmal subarachnoid hemorrhage.(1)Paired CSF and PB samples from a SAH patients were included and sequenced;a total of 2959 and 2049 T cells were obtained from CSF and PB,respectively.(2)In CSF,2222 T cell subtypes were detected,of which 234 were clonetypes containing 971 clonal T cells.In PB,1691 T cell subtypes were detected,of which 88 were clonetypes containing 446 clonal T cells.(3)The proportion of clonal T cells and T cell clonetypes in CSF was significantly higher than that in PB(33% vs 22% and 11% vs 5%,respectively;P<0.05).(4)TCR in CSF and PB showed similar CDR3 feature,V and J gene use preference and V-J gene pair combination pattern.Conclusions(1)This study analyzed the single-cell transcriptome atlas and immune microenvironment characterization of immune cells from CSF and PB,and identified the cellular heterogeneity.(2)The differentiation trajectories of monocytes and T cells were constructed,and it was preliminarily confirmed that gene transcription mechanisms may control the differentiation process and biological functions.(3)Intermediate monocytes of CSF may play a key role in neuroinflammation after a SAH through mediating TLR and other inflammatory pathways.(4)The proportion of clonal T cells and T cell clonetypes in CSF was significantly higher than that in PB,showing that the activation of T cells in CSF may be higher than that in PB.Part I The Role of Single-cell RNA Sequencing in the Study of Immune Cell Heterogeneity after Aneurysmal Subarachnoid HemorrhageObjective Aneurysmal subarachnoid hemorrhage(a SAH)is a stroke caused by the rupture of intracranial aneurysm.The breakdown products of red blood cells cause severe neuroinflammation in the subarachnoid space,leading to brain damage and impaired neurological function.Previous studies on neuroinflammation after a SAH have focused on immune cells and inflammatory factors.But Transcriptome differences in immune cells are still unclear between peripheral blood(PB)and cerebrospinal fluid(CSF).In this study,single cell RNA sequencing(sc RNA-seq)was performed on immune cells from CSF and PB of a SAH patients to study the transcriptome characteristics and heterogeneity of immune cells,and to explore the mechanism and pathophysiology of neuroinflammation.Method In this study,CSF and PB samples were collected from aSAH patients,and mononuclear cells were extracted by density-gradient centrifugation.Mononuclear cells from CSF and PB samples were then sequenced using 10 x Genomics sc RNA-seq.In addition,the CSF single-cell sequencing data from healthy controls were downloaded from Gene expression Omnibus(GEO)database and compared with that of a SAH patients,to analyze the pathophysiology and mechanism of neuroinflammation.Various R packages were performed to make bioinformatics analysis on the gene expression matrices,to explore the gene transcriptome characteristics and cellular heterogeneity of immune cells.Seurat package was used for cell quality control,dimensionality reduction,clustering and results visualization.Monocle was used to construct the cell differentiation trajectory and explore the gene transcription mechanism that regulates cell development.Cellchat was used for the communication interaction of immune cells.GSVA was used to explore the activation of inflammatory pathways in immune cells from healthy controls and a SAH patients.Results(1)A total of 17242 cells were obtained in this study,of which 7224 cells were obtained from CSF and 10018 cells were obtained from PB.Monocytes,T cells,B cells and NK cells were identified in these cells.(2)The differentiation trajectories of monocytes and T cells were also constructed.It was found that gene modules differentially expressed along with cell differentiation,which regulated the differentiation process and cell function.(3)Compared with PB,the activation of TLR 、 JAK-STAT and other immune pathways in CSF intermediate monocytes significantly increased.Moreover,compared with healthy controls,the activation of inflammatory pathways such as TLR and MAPK significantly increased in CSF intermediate monocytes of a SAH patients.The biological processes related to neuroinflammation such as leukocyte recruitment and immune response regulation were also enriched in CSF intermediate monocytes of a SAH patients.(4)The strength and number of interaction communication between monocytes in CSF were higher than those in PB.Conclusions(1)This study analyzed the single-cell transcriptome atlas and immune microenvironment characterization of immune cells from CSF and PB,and identified the cellular heterogeneity.(2)The differentiation trajectories of monocytes and T cells were constructed,and it was preliminarily confirmed that gene transcription mechanisms may control the differentiation process and biological functions.(3)Intermediate monocytes of CSF may play a key role in neuroinflammation after a SAH through mediating TLR and other inflammatory pathways.Part II The Role of Single-cell TCR Sequencing in the Study of TCR Repertoire after Aneurysmal Subarachnoid HemorrhageObjective The breakdown products of red blood cells after aneurysmal subarachnoid hemorrhage can cause severe neuroinflammation,resulting in neurological impairment.Previous studies have found that T cells infiltrated into the subarachnoid space to participate in post-a SAH neuroinflammation.However,it is still unclear how T cells are activated to participate in neuroinflammation after a SAH.In this study,single-cell TCR sequencing were performed on Tcells from PB and CSF of a SAH patients to analyze the T cell receptor(TCR)and explore the pathophysiological mechanism of neuroinflammation.Method Single-cell RNA sequencing was performed on T cells extracted from CSF and PB to analyze the TCR repertoire for a SAH patients.Result(1)Paired CSF and PB samples from two aSAH patients were included and sequenced;a total of 2959 and 2049 T cells were obtained from CSF and PB,respectively.(2)In CSF,2222 T cell subtypes were detected,of which 234 were clonetypes containing 971 clonal T cells.In PB,1691 T cell subtypes were detected,of which 88 were clonetypes containing 446 clonal T cells.(3)The proportion of clonal T cells and T cell clonetypes in CSF was significantly higher than that in PB(33% vs 22% and 11% vs 5%,respectively;P<0.05).(4)TCR in CSF and PB showed similar CDR3 feature,V and J gene use preference and V-J gene pair combination pattern.Conclusion In this study,single-cell TCR sequencing was performed to analyze the characteristics of TCR repertoire in CSF and PB of a SAH patients.The proportion of clonal T cells and T cell clonetypes in CSF was significantly higher than that in PB,showing that the activation of T cells in CSF may be higher than that in PB.