Immunological Feature and Transcriptional Signaling of Ly6C Monocyte Subsets From Transcriptome Analysis in Control and Hyperhomocysteinemic Mice

Murine monocytes (MC) are classified into Ly6C and Ly6C MC. Ly6C MC is the pro-inflammatory subset and the counterpart of human CD14 CD16 intermediate MC which contributes to systemic and tissue inflammation in various metabolic disorders, including hyperhomocysteinemia (HHcy). This study aims to explore molecule signaling mediating MC subset differentiation in HHcy and control mice. RNA-seq was performed in blood Ly6C and Ly6C MC sorted by flow cytometry from control and HHcy cystathionine β-synthase gene-deficient ( ) mice. Transcriptome data were analyzed by comparing Ly6C vs. Ly6C in control mice, Ly6C vs. Ly6C in mice, Ly6C vs. control Ly6C MC and Ly6C vs. control Ly6C MC by using intensive bioinformatic strategies. Significantly differentially expressed (SDE) immunological genes and transcription factor (TF) were selected for functional pathways and transcriptional signaling identification. A total of 7,928 SDE genes and 46 canonical pathways derived from it were identified. Ly6C MC exhibited activated neutrophil degranulation, lysosome, cytokine production/receptor interaction and myeloid cell activation pathways, and Ly6C MC presented features of lymphocyte immunity pathways in both mice. Twenty-four potential transcriptional regulatory pathways were identified based on SDE TFs matched with their corresponding SDE immunological genes. Ly6C MC presented downregulated co-stimulatory receptors (CD2, GITR, and TIM1) which direct immune cell proliferation, and upregulated co-stimulatory ligands (LIGHT and SEMA4A) which trigger antigen priming and differentiation. Ly6C MC expressed higher levels of macrophage (MΦ) markers, whereas, Ly6C MC highly expressed lymphocyte markers in both mice. HHcy in mice reinforced inflammatory features in Ly6C MC by upregulating inflammatory TFs ( and ) and strengthened lymphocytes functional adaptation in Ly6C MC by increased expression of CD3, DR3, ICOS, and . Finally, we established 3 groups of transcriptional models to describe Ly6C to Ly6C MC subset differentiation, immune checkpoint regulation, Ly6C MC to MΦ subset differentiation and Ly6C MC to lymphocyte functional adaptation. Ly6C MC displayed enriched inflammatory pathways and favored to be differentiated into MΦ. Ly6C MC manifested activated T-cell signaling pathways and potentially can adapt the function of lymphocytes. HHcy reinforced inflammatory feature in Ly6C MC and strengthened lymphocytes functional adaptation in Ly6C MC.