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

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.

Keywords: hyperhomocysteinemia; immune checkpoint; immunological gene; locus C (Ly6C) monocyte subset; lymphocyte antigen 6 complex; transcription factor.