Immunological Feature And Transcriptome Signaling Of Ly6c Monocyte Subsets From RNA Sequencing Analysis In Wild-type And Hyperhomocysteinemic Mice

Background:Monocytes（MC）are bone marrow derived mononuclear phagocytes that play an important role in innate immune response and are the major immune cell population in chronic tissue inflammatory.Human MC were initially divided into three subsets based on the cell surface expression of CD14 and CD16,including CD14⁺⁺CD16^-classical MC,CD14⁺⁺CD16⁺intermediate MC and CD14⁺CD16⁺⁺non-classical MC.Murine MC are divided into three subsets based on surface expression of lymphocyte antigen 6 complex,locus C（Ly6C）,including Ly6C^high,Ly6C^middle and Ly6C^lowMC.Murine Ly6C^high and Ly6C^middle MC subsets perform pro-inflammatory functions,which are considered the counterpart of human CD14⁺⁺CD16⁺intermediate MC.Murine Ly6C^lowMC perform patrolling and anti-inflammatory function,similar to human CD14⁺CD16⁺⁺non-classical and CD14⁺⁺CD16^-classical MC.However,the molecular mechanism underlying MC subset differentiation and transcriptional regulation remain to be elucidated.Ly6C^high MC displays developmental plasticity and are recruited to tissues to complement M（37）and DC on demand.The molecular mechanism underlying MC plasticity and subset differentiation remain unclear.Hyperhomocysteinemia（HHcy）,an independent risk factor for cardiovascular,diabetic and Alzheimer’s disease,induced Ly6C^highinflammatory MC subsets differentiation,which contributed to tissue inflammatory and accelerated arteriosclerosis and chronic kidney disease.The effect of HHcy on MC subset differentiation in patient would be an interesting topic for future clinical research.Discover of regulatory mechanisms mediating HHcy-induced MC subset differentiation may lead to the discovery of novel therapeutic target.Objective:This study aims to systemically examine m RNA expression profiles of key immunological genes in Ly6C^high and Ly6C^low MC subsets by intensive bioinformatic analysis and to develop models of molecule pathways and transcriptional regulatory signaling for subset differentiation.Methods:RNA sequencing was performed in Ly6C^high and Ly6C^lowMC isolated by flow cytometry sorting from peripheral blood of wild type and HHcy human cystathionineβ-synthase gene（CBS）transgenic Cbs-deficient(Tg-h CBS⁺Cbs^-/-)mice.Transcriptome data were analyzed by comparing Ly6C^high vs Ly6C^lowin wild-type mice,Ly6C^highvs Ly6C^lowin Tg-h CBS⁺Cbs^-/-mice,Tg-h CBS⁺Cbs^-/-Ly6C^highvs wild-type Ly6C^highMC and Tg-h CBS⁺Cbs^-/-Ly6C^low vs wild-type Ly6C^low MC by using various bioinformatic strategies.We analyzed transcription factors（TF）and 4sets of immunological related gene sets（secreted proteome,cytokines,surface markers and immune checkpoints）and 3 sets of immune cell lineage-specific genes（newly suggested signature genes of immune cells recognized by single-cell RNA sequencing,lineage-specific transcription factors and surface markers）expression patterns to further explore the immunological characteristics and transcriptional regulation mechanisms of Ly6C MC subset.Results:Part 1:（1）A total of 3,064 significantly differentially expressed genes were identified in wild-type mice in Ly6C^high vs Ly6C^lowMC in wild-type mice.（2）Ly6C^high MC activated the inflammatory signal pathway in wild-type mice,while Ly6C^low MC showed up-regulation of lymphocyte immune signal pathway.（3）Nine up-regulated transcription factors（Cebpa,Cebpd,Cebpe,Spi1,Ifi16,Irf5/7 and Stat1/2）and 4 down-regulated transcription factors（Tbx21,Pax5,Sp110and Ikzf3）were identified in the wild-type mice Ly6C^high MC subset matched with immunological genes.（4）A molecular signaling model of MC subset was established,and the transcriptional regulation mechanism of Ly6C^high and Ly6C^low MC subset differentiation was described.Part 2:（1）A total of 3,605 significantly differentially expressed genes were selected from the Ly6C^highvs Ly6C^lowin HHcy mice.（2）Ly6C^high MC activated abundant inflammatory signal pathways in both control mice and HHcy mice,while Ly6C^low MC activated lymphocyte signal pathways.（3）HHcy mice Ly6C^high MC subset identified 4 up-regulated TF（Cebpa,Cebpe,Irf7 and Trps1）and 6 down-regulated TF（Egr2,Foxm1,Myb,Pax5,Spib and Tbx21）matched with immunological genes.（4）Ly6C^high MC down-regulated costimulatory receptors（CD2,GITR and TIM1）to induce the proliferation of immune cells and up-regulate costimulatory ligands（LIGHT and SEMA4A）in both mice to induce antigen priming and differentiation.（5）Ly6C^high MC highly expressed macrophage lineage-special TF and surface markers in both mice,while Ly6C^low MC highly expressed lymphocyte lineage-special TF and surface markers.Part 3:（1）HHcy induces 691 significantly differentially expressed genes in HHcy Ly6C^high MC vs wild-type Ly6C^highMC,and 567 significant differential expression genes in HHcy Ly6C^low vs wild-type Ly6C^low MC.（2）HHcy inhibits some metabolic signaling pathways in Ly6C^low MC,and activates natural killer cell signaling pathways in Ly6C^high MC.（3）HHcy up-regulates transcription factors Ets1 and Tbx21 in Ly6C^high MC and upregulates transcription factors Irf7 and Fos in Ly6C^low MC.（4）HHcy down-regulates Ly6C^high MC co-inhibitory ligand PD-L1/2 and up-regulates Ly6C^low MC co-stimulatory receptors DR3 and ICOS.（5）HHcy enhances Ly6C^low MC to lymphocyte functional adaptation by increasing the expression of CD3,DR3 and ICOS.Conclusions:Ly6C^highMC displayed enriched inflammatory pathways,regulated immune checkpoint molecules to suppress proliferation and increase antigens priming,and favored to be differentiated into macrophage and osteoclasts.Ly6C^lowMC manifested activated T-cell signaling pathways and potentially can adapt the function of lymphocytes.HHcy reinforced inflammatory feature in Ly6C^high MC and strengthened lymphocytes functional adaptation in Ly6C^lowMC in Tg-h CBS⁺Cbs^-/-mice.