Abstract
BACKGROUND: Murine monocytes (MC) are classified into Ly6C(high) and Ly6C(low) MC. Ly6C(high) 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. METHODS: RNA-seq was performed in blood Ly6C(high) and Ly6C(low) MC sorted by flow cytometry from control and HHcy cystathionine β-synthase gene-deficient (Cbs(-/-)) mice. Transcriptome data were analyzed by comparing Ly6C(high) vs. Ly6C(low) in control mice, Ly6C(high) vs. Ly6C(low) in Cbs(-/-) mice, Cbs(-/-) Ly6C(high) vs. control Ly6C(high) MC and Cbs(-/-) Ly6C(low) vs. control Ly6C(low) 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. RESULTS: A total of 7,928 SDE genes and 46 canonical pathways derived from it were identified. Ly6C(high) MC exhibited activated neutrophil degranulation, lysosome, cytokine production/receptor interaction and myeloid cell activation pathways, and Ly6C(low) 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(high) 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(high) MC expressed higher levels of macrophage (MΦ) markers, whereas, Ly6C(low) MC highly expressed lymphocyte markers in both mice. HHcy in Cbs(-/-) mice reinforced inflammatory features in Ly6C(high) MC by upregulating inflammatory TFs (Ets1 and Tbx21) and strengthened lymphocytes functional adaptation in Ly6C(low) MC by increased expression of CD3, DR3, ICOS, and Fos. Finally, we established 3 groups of transcriptional models to describe Ly6C(high) to Ly6C(low) MC subset differentiation, immune checkpoint regulation, Ly6C(high) MC to MΦ subset differentiation and Ly6C(low) MC to lymphocyte functional adaptation. CONCLUSIONS: Ly6C(high) MC displayed enriched inflammatory pathways and favored to be differentiated into MΦ. Ly6C(low) MC 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(low) MC.