Adaptive Immune Response Signaling Is Suppressed in Ly6Chigh Monocyte but Upregulated in Monocyte Subsets of ApoE-/- Mice - Functional Implication in Atherosclerosis.

Pingping Yang,Hong Wang,Yong Ji,Xiaofeng Yang,Lu Liu,Qinghua Wu,Xuebin Qin,Joon-Young Park,Lizhe Sun,Pu Fang

doi:10.3389/fimmu.2021.809208

Abstract

RationaleInflammatory monocyte (MC) subset differentiation is a major feature in tissue inflammatory and atherosclerosis. The underlying molecular mechanism remains unclear.ObjectiveThis study aims to explore molecule targets and signaling which determinate immunological features in MC subsets.Methods and ResultsBlood Ly6Chigh and Ly6Clow MC subsets from control and ApoE -/- mice were isolated by flow cytometry sorting and subjected for bulk high-throughput RNA-sequencing. Intensive bioinformatic studies were performed by analyzing transcriptome through four pairs of comparisons: A) Ly6Chigh vs Ly6Clow in control mice; B) Ly6Chigh vs Ly6Clow in ApoE-/- mice; C) ApoE-/- Ly6Chigh vs control Ly6Chigh MC; D) ApoE-/- Ly6Clow vs control Ly6Clow MC. A total of 80 canonical pathways and 16 enriched pathways were recognized by top-down analysis using IPA and GSEA software, and further used for overlapping analysis. Immunological features and signaling were assessed on four selected functional groups, including MHCII, immune checkpoint, cytokine, and transcription factor (TF). Among the total 14578 significantly differentially expressed (SDE) genes identified though above four comparison, 1051 TF and 348 immunological genes were discovered. SDE immunological genes were matched with corresponding upstream SDE TF by IPA upstream analysis. Fourteen potential transcriptional axes were recognized to modulate immunological features in the Ly6C MC subset. Based on an intensive literature search, we found that the identified SDE immune checkpoint genes in Ly6Chigh MC are associated with pro-inflammatory/atherogenic balance function. Immune checkpoint genes GITR, CTLA4, and CD96 were upregulated in Ly6Clow MC from all mice and presented anti-inflammatory/atherogenic features. Six cytokine genes, including Ccl2, Tnfsf14, Il1rn, Cxcl10, Ccl9, and Cxcl2, were upregulated in Ly6Chigh MC from all mice and associated with pro-inflammatory/atherogenic feature. Cytokine receptor gene Il12rb2, Il1r1, Il27ra, Il5ra, Ngfr, Ccr7, and Cxcr5 were upregulated in Ly6Clow MC from all mice and presented anti-inflammatory/atherogenic features. MHCII genes (H2-Oa, H2-DMb2, H2-Ob, H2-Eb2, H2-Eb1, H2-Aa, and Cd74) were elevated in Ly6Clow MC from all mice. ApoE -/- augmented pro-atherogenic/inflammatory and antigen-presenting cells (APC) feature in both subsets due to elevated expression of cytokine genes (Cxcl11, Cntf, Il24, Xcl, Ccr5, Mpl, and Acvr2a) and MHCII gene (H2-Aa and H2-Ea-ps). Finally, we modeled immunological gene expression changes and functional implications in MC differentiation and adaptive immune response for MC subsets from control and ApoE-/- mice.ConclusionsLy6Chigh MC presented pro-inflammatory/atherogenic features and lower APC potential. Ly6Clow MC displayed anti-inflammatory/atherogenic features and higher APC potential. ApoE -/- confers upon both subsets with augmented pro-atherogenic/inflammatory function and APC potential.

Highlights

Atherosclerosis is a chronic inflammatory disease of blood vessels and the essential pathological cause of cardiovascular disease, the leading cause of mortality worldwide [1, 2]
Through transcriptome analysis in sorted mouse Ly6C MC subset, we reported that the Ly6Chigh MC displayed enriched inflammatory pathways and favored to be differentiated into MF and osteoclast [13]
When comparing Ly6Chigh MC with Ly6Clow MC in the ApoE-/mice, we found that antigen-presenting cells (APC) potential was elevated, but other ligand/receptor-determined immunological features were similar as that in CT mice in both MC subsets (Figure 8B)

Summary

Introduction

Atherosclerosis is a chronic inflammatory disease of blood vessels and the essential pathological cause of cardiovascular disease, the leading cause of mortality worldwide [1, 2]. Innate immune cell monocyte (MC) and macrophage (MF) is the major cellular components in the advanced atherosclerosis lesion, which is correlated with increased inflammatory MC differentiation [3,4,5]. The molecular mechanism underlying inflammatory MC differentiation, especially under hyperlipidemia (HL) conditions, and related adaptive immune response remains unknown. MC circulates in the blood and migrates to inflammatory tissues, but their functions can be either detrimental or beneficial, determined by their subsets. Human CD14++CD16+ intermediate and CD14+ CD40+ MC are considered as inflammatory MC subsets, similar to murine Ly6Chigh and Ly6Cmiddle MC [7, 8]. Human CD14+CD16++ nonclassical, CD14++CD16- classical, and CD14+CD40- MC are antiinflammatory MC, similar as mouse Ly6Clow MC [7, 8]

Results

Discussion

Conclusion