Notch ligands are biomarkers of anti-TNF response in RA patients

Abstract

Notch and its ligands play a critical role in rheumatoid arthritis (RA) pathogenesis. Hence, studies were conducted to delineate the functional significance of the Notch pathway in RA synovial tissue (ST) cells and the influence of RA therapies on their expression. Morphological studies reveal that JAG1, DLL4, and Notch1 are highly enriched in RA ST lining and sublining CD68⁺CD14⁺ MΦs. JAG1 and DLL4 transcription is jointly upregulated in RA MΦs reprogrammed by TLR4/5 ligation and TNF, whereas Syntenin-1 exposure expands JAG1, DLL4, and Notch1 expression levels in these cells. Single-cell RNA-seq data exhibit that JAG1 and Notch3 are overexpressed on all fibroblast-like synoviocyte (FLS) subpopulations, in parallel, JAG2, DLL1, and Notch1 expression levels are modest on RA FLS and are predominately potentiated by TLR4 ligation. Intriguingly, JAG1, DLL1/4, and Notch1/3 are presented on RA endothelial cells, and their expression is mutually reconfigured by TLR4/5 ligation in the endothelium. Synovial JAG1/JAG2/DLL1 or Notch1/3 transcriptomes were unchanged in patients who received disease-modifying anti-rheumatic drugs (DMARDs) or IL-6R Ab therapy regardless of disease activity score. Uniquely, RA MΦs and endothelial cells rewired by IL-6 displayed DLL4 transcriptional upregulation, and IL-6R antibody treatment disrupted RA ST DLL4 transcription in good responders compared to non-responders or moderate responders. Nevertheless, the JAG1/JAG2/DLL1/DLL4 transcriptome was diminished in anti-TNF good responders with myeloid pathotype and was unaltered in the fibroid pathotype except for DLL4. Taken together, our findings suggest that RA myeloid Notch ligands can serve as markers for anti-TNF responsiveness and trans-activate Notch receptors expressed on RA FLS and/or endothelial cells.

Keywords: DLL4; Endothelial cells; JAG1; Notch1; RA FLS; RA macrophages.

Figure 1.. Synovial JAG1 expression was suppressed in anti-TNF good responders.

A) Graphical summary of Notch ligand/receptor expression induced by TLR-signaling or cytokines in RA MΦs, FLS, or endothelial cells. B) Synovial tissue sections from NL, OA, and RA were stained for JAG1 (original magnification x10) and the staining was scored on a 0–5 scale (n = 4). C) Relative expression of JAG1 was determined by RNA-seq [17] in ST biopsies from RA non-responsive (ΔDAS28 ≤ 0.6, n = 23), moderate (ΔDAS28 ≤ 1.2 & > 0.6, n = 29) and good responders (ΔDAS28 > 1.2, n = 29) to DMARD therapies. D) RA STs from patients (n = 81) that were either non-responders, moderate responders, or good responders to anti-IL6R Ab (Tocilizumab) therapy were assessed by RNA-seq [19] for JAG1 expression. E-G) RA STs from patients (n = 46) that were non-responders, moderate responders, or good responders to anti-TNF therapy were assessed by RNA-seq [15] for differences in JAG1 expression between groups. RA STs from patients with F) myeloid pathotype (n=21) or G) fibroid pathotype (n=8) were further delineated based on response to therapy and JAG1 expression. H) RA STs were fluorescently stained for JAG1 expression on CD68⁺ RA MΦs, Vimentin⁺ RA FLS, and VWF⁺ endothelial cells (n=3, original magnification x20). I) RA MΦs were stimulated with 100 ng/ml inflammatory factors or 1000 ng/ml Syntenin-1 (SYN1) (6h) and transcription of JAG1 was assessed by qRT-PCR (n=5–13). J) scRNA-seq analysis was performed on RA ST (n = 18) [20]. Clustering revealed 4 fibroblast subpopulations [three types of THY1⁺ sublining fibroblasts (F1-CD34⁺, F2-HLA-DRA^hi, and F3-DKK3⁺) and CD55⁺ lining fibroblasts (F4)] and 4 MΦ subpopulations (M1-IL1B⁺, M2-NUPR1⁺, M3-C1QA⁺, and M4-IFN-activated). Distinct patterns of expression are displayed amongst cell types and subpopulations for JAG1. K) Normalized expression levels of JAG1 are displayed for RA lining and sublining FLS and endothelial cells based on scRNA-seq data from Wei et al. [7]. L) Endothelial cells were stimulated with 100 ng/ml inflammatory factors (6h) and transcription of JAG1 was assessed by qRT-PCR (n=6). Data are presented as mean ± SEM; differences were determined by the Mann-Whitney test, one-way ANOVA with either Tukey post-test, or the p-value was adjusted for multiple comparisons by FDR. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 2.. JAG2 and DLL1 expression on synovial tissue acts as a biomarker for anti-TNF good responders.

A) Synovial tissue sections from NL, OA, and RA were stained for JAG2 (original magnification x10), and B) the staining was scored on a 0–5 scale (n = 4). C) RA STs were fluorescently stained for JAG2 expression on CD68⁺ RA MΦs and VWF⁺ endothelial cells (n=3, original magnification x20). D) Synovial tissue sections from NL, OA, and RA were stained for DLL1 (original magnification x10), and E) the staining was scored on a 0–5 scale (n = 4). F) RA STs were fluorescently stained for DLL1 expression on CD68⁺ RA MΦs and VWF⁺ endothelial cells (n=3, original magnification x20). G-H) Normalized expression levels of G) JAG2 and H) DLL1 are displayed for RA lining and sublining FLS and endothelial cells based on scRNA-seq data from Wei et al. [7]. I-L) RA STs from patients (n = 46) that were non-responders, moderate responders, or good responders to anti-TNF therapy were assessed by RNA-seq [15] for differences in I) JAG2 and K) DLL1 expression between groups. RA STs from patients with myeloid pathotype (n=21) were further assessed based on response to therapy and I) JAG2 and L) DLL1 expression. M) RA MΦs and N) RA FLS were stimulated with 100 ng/ml inflammatory factors (6h) and transcription of JAG2 was assessed by qRT-PCR (n=5–10). O) RA MΦs, P) RA FLS, or Q) endothelial cells were stimulated with 100 ng/ml inflammatory factors or 1000 ng/mL Syntenin-1 (SYN1) (6h), and transcription of DLL1 was measured by qRT-PCR (n=4–6). Data are presented as mean ± SEM; differences were determined by the Mann-Whitney test, one-way ANOVA with either Tukey post-test, or the p-value was adjusted for multiple comparisons by FDR. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 3.. DLL4 expression is uniquely mitigated in anti-IL6R Ab and anti-TNF good responders.

A) Synovial tissue sections from NL, OA, and RA were stained for DLL4 (original magnification x10) and the staining was scored on a 0–5 scale (n = 4). B) RA STs were fluorescently stained for DLL4 expression on CD68⁺ RA MΦs, Vimentin⁺ RA FLS, and VWF⁺ endothelial cells (n=3, original magnification x20). C) Normalized expression levels of DLL4 are displayed for RA lining and sublining FLS and endothelial cells based on scRNA-seq data from Wei et al. [7]. D) RA MΦs and E) endothelial cells were stimulated with 100 ng/ml inflammatory factors or 1000 ng/ml Syntenin-1 (SYN1) (6h) and transcription of DLL4 was assessed by qRT-PCR (n=5–8). F) Relative expression of DLL4 was determined by RNA-seq [17] in ST biopsies from RA non-responsive (ΔDAS28 ≤ 0.6, n = 23), moderate (ΔDAS28 ≤ 1.2 & > 0.6, n = 29) and good responders (ΔDAS28 > 1.2, n = 29) to DMARD therapies. G) RA STs from patients (n = 81) that were either non-responders, moderate responders, or good responders to anti-IL6R Ab (Tocilizumab) therapy were assessed by RNA-seq [19] for DLL4 expression. H-J) RA STs from patients (n = 46) that were non-responders, moderate responders, or good responders to anti-TNF therapy were assessed by RNA-seq [15] for differences in H) DLL4 expression between groups. RA STs from patients with I) myeloid pathotype (n=21) or J) fibroid pathotype (n=8) were further delineated based on response to therapy and DLL4 expression. Data are presented as mean ± SEM; differences were determined by the Mann-Whitney test, one-way ANOVA with either Tukey post-test, or the p-value was adjusted for multiple comparisons by FDR. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Figure 4.. Synovial Notch1 and Notch3 receptor expression was unaffected by anti-TNF therapy.

A-D) Synovial tissue sections from NL, OA, and RA were stained for A) Notch1 and C) Notch3 (original magnification x10), and the B) Notch1 and D) Notch3 staining was scored on a 0–5 scale (n = 4–6). E) scRNA-seq analysis was performed on RA ST (n = 18) [20]. Clustering revealed 4 fibroblast subpopulations [three types of THY1⁺ sublining fibroblasts (F1-CD34⁺, F2-HLA-DRA^hi, and F3-DKK3⁺) and CD55⁺ lining fibroblasts (F4)] and 4 MΦ subpopulations (M1-IL1B⁺, M2-NUPR1⁺, M3-C1QA⁺, and M4-IFN-activated). Distinct patterns of expression are displayed amongst cell types and subpopulations for Notch1. F) Normalized expression levels of Notch1 are displayed for RA lining and sublining FLS and endothelial cells based on scRNA-seq data from Wei et al. [7]. G) scRNA-seq analysis was performed on RA ST (n = 18) [20]. Distinct patterns of expression are displayed amongst cell types and fibroblast and MΦ subpopulations for Notch3. H) Normalized expression levels of Notch3 are displayed for RA lining and sublining FLS and endothelial cells based on scRNA-seq data from Wei et al. [7]. I-J) RA STs were fluorescently stained for I) Notch1 and J) Notch3 expression on CD68⁺ RA MΦs, Vimentin⁺ RA FLS, and VWF⁺ endothelial cells (n=3, original magnification x20). K) RA MΦs, L) RA FLS, and M) endothelial cells were stimulated with 100 ng/mL inflammatory factors or 1000 ng/ml Syntenin-1 (SYN1) (6h), and transcription of Notch1 was assessed by qRT-PCR (n=5–6). N) RA MΦs, O) RA FLS, and P) endothelial cells were stimulated with 100 ng/ml inflammatory factors (6h) and transcription of Notch3 was assessed by qRT-PCR (n=6). Q-T) RA STs from patients (n = 46) that were non-responders, moderate responders, or good responders to anti-TNF therapy were assessed by RNA-seq [15] for differences in Q) Notch1 and S) Notch3 expression between groups. RA STs from patients with myeloid pathotype (n=21) were further assessed based on response to therapy and R) Notch1 and T) Notch3 expression. Data are presented as mean ± SEM; differences were determined by the Mann-Whitney test, one-way ANOVA with either Tukey post-test, or the p-value was adjusted for multiple comparisons by FDR. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001