Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus

Abstract

Neutrophil dysregulation is implicated in the pathogenesis of systemic lupus erythematosus (SLE). SLE is characterized by elevated levels of a pathogenic neutrophil subset known as low-density granulocytes (LDGs). The origin and phenotypic, functional, and pathogenic heterogeneity of LDGs remain to be systematically determined. Transcriptomics and epigenetic assessment of lupus LDGs, autologous normal-density neutrophils, and healthy control neutrophils was performed by bulk and single-cell RNA sequencing and assay for transposase-accessible chromatin sequencing. Functional readouts were compared among neutrophil subsets. SLE LDGs display significant transcriptional and epigenetic heterogeneity and comprise 2 subpopulations of intermediate-mature and immature neutrophils, with different degrees of chromatin accessibility and differences in transcription factor motif analysis. Differences in neutrophil extracellular trap (NET) formation, oxidized mitochondrial DNA release, chemotaxis, phagocytosis, degranulation, ability to harm the endothelium, and responses to type I interferon (IFN) stimulation are evident among LDG subsets. Compared with other immune cell subsets, LDGs display the highest expression of IFN-inducible genes. Distinct LDG subsets correlate with specific clinical features of lupus and with the presence and severity of coronary artery disease. Phenotypic, functional, and pathogenic neutrophil heterogeneity are prevalent in SLE and may promote immune dysregulation and prominent vascular damage characteristic of this disease.

Keywords: autoimmunity; neutrophils; systemic lupus erythematosus.

Fig. 1.

Sorted CD10⁻ LDGs have a similar transcriptional profile to LDG^imm. (A) RNA-seq was performed on sorted lupus CD10⁺ NDNs (green), autologous CD10⁻ LDGs (blue), and CD10⁺ LDGs (red) (n = 6). (B) Volcano plot of differential gene expression between CD10⁻ and CD10⁺ LDGs. Up-regulated genes with fold change ≥2 and P < 0.05 are in red, and down-regulated genes with fold change ≤2 and P < 0.05 are in blue. (C) Gene set enrichment analysis of cell cycle genes and ISGs in CD10⁻ and CD10⁺ LDGs. (D–G) IFN score (D); ISG RNA-seq analysis (E); ISG expression by qRT-PCR, normalized to GAPDH expression and reported as relative message (F); and RNA-seq analysis for cell cycle genes (G) in CD10⁺ NDN, CD10⁺ and CD10⁻ LDGs (n = 6/group for D, E, and G and n = 4/group for F). Results represent mean ± SEM from four independent experiments. *P ≤ 0.05; **P < 0.01.

Fig. 2.

Lupus neutrophil subsets differ phenotypically and functionally. (A) Representative images of Giemsa-stained sorted lupus CD10⁺ NDNs, CD10⁺ LDGs, and CD10⁻ LDGs (n = 4); original magnification 60×. (B) RNA-seq analysis of CD10⁺ NDNs, CD10⁺ LDGs, and CD10⁻ LDGs (n = 6/group) for TFs involved in myeloid development. (C and D) Representative images of cells undergoing NET formation in unstimulated sorted CD10⁺ LDGs and CD10⁻ LDGs after a 2-h incubation (n = 5/group). MPO is in red and DNA is in blue; original magnification 40×. (E) MPO ELISA of culture supernatants of unstimulated CD10⁺ NDNs, CD10⁺ LDGs, and CD10⁻ LDGs (n = 4/group) after a 2-h incubation. (F) RNA-seq analysis for genes involved in chemotaxis in lupus neutrophil subsets. (G) fMLP-induced chemotactic index in lupus neutrophil subsets after a 2-h incubation (n = 4 for CD10⁺ NDNs; n = 8 for CD10⁺ and CD10⁻ LDGs). (H) Expression of phagocytosis genes by RNA-seq analysis. (I) Phagocytosis of S. aureus bioparticles for lupus neutrophil subsets (n = 5). Results for all measurements are mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 3.

Immature and mature LDGs are identified in lupus PBMCs using single-cell RNA-seq and differ in ISG expression. (A) a t-SNE plot representing gene expression in single cells from SLE PBMCs (n = 3) identifying 12 unique cell clusters. (B) IFN score in each cell cluster. (C) ISGs are highly expressed in LDGs. (D) ISG heatmap showing a high IFN response in LDGs relative to other cell clusters. (E and F) Neutrophils were filtered from PBMCs based on their expression of FCGR3B and ELANE. The t-SNE plot shows 2 transcriptionally distinct clusters based on FCGR3B or ELANE expression, identified as immature (Imm) and mature (Mat), respectively. (G) Pathway analysis indicating that mature neutrophils are activated and respond to type I IFNs.