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

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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.

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Data availability

All data generated or analyzed during this study are included in this paper and its supplementary information files.

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Acknowledgements

Schematic images were generated using biorender.com.

Funding

This work was supported in part by awards from the Department of Veteran’s Affairs MERIT Award BX002286, CX002565, IK6BX006474, the National Institutes of Health NIH R01 AI167155, NIH R41 AI147697, and the Innovative Research Award from the Rheumatology Research Foundation (RRF, no number assigned).

Author information

Authors and Affiliations

  1. Jesse Brown VA Medical Center, Chicago, IL, USA

    Stephanie R. Zack, Anja Meyer, Neha Satoeya & Shiva Shahrara

  2. Department of Medicine, Division of Rheumatology, The University of Illinois at Chicago, Chicago, IL, USA

    Stephanie R. Zack, Anja Meyer, Sania Deen, Neha Satoeya, Nadera Sweiss & Shiva Shahrara

  3. Department of Microbiology and Immunology, Midwestern University, Downers Grove, IL, USA

    Brian Zanotti & Michael V. Volin

  4. Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK

    Myles J. Lewis & Costantino Pitzalis

  5. Centre for Experimental Medicine & Rheumatology, William Harvey Research Institute, Queen Mary University of London and Barts NIHR BRC & NHS Trust, London, UK

    Myles J. Lewis & Costantino Pitzalis

  6. Department of Biomedical Sciences, Humanitas University, and Humanitas Research Hospital, Milan, Italy

    Costantino Pitzalis

  7. Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, IL, 60612, USA

    Jan K. Kitajewski

  8. University of Illinois Cancer Center, University of Illinois Chicago, Chicago, IL, 60612, USA

    Jan K. Kitajewski

Authors
  1. Stephanie R. Zack
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  2. Anja Meyer
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Contributions

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Shahrara had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design: SRZ and SS. Acquisition of data: SRZ, AM, BZ, MVV, SD, and SS. Analysis and interpretation of data: SRZ, AM, BZ, MVV, SD, NS, MJL, CP, JKK, and SS. Providing crucial reagents: NS

Corresponding author

Correspondence to Shiva Shahrara.

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Conflict of interest

The authors declare that they have no competing interests.

Research ethics approval

RA patients were collected according to the protocol approved by the University of Illinois at Chicago Institutional Ethics Review Board. To ensure a robust and unbiased experimental design, samples were obtained from RA patients of both genders. Rigor and reproducibility were maintained through well-powered studies and multiple distinct approaches to confirm the results.

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Supplementary Information

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10456_2023_9897_MOESM1_ESM.tif

Supplementary file1 (TIF 392 KB) Supplemental Figure 1. JAG2, DLL1, and Notch1/3 expression was unaffected by DMARDs or anti-IL6R Ab. A) Relative expression of JAG2 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. B) 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 JAG2 expression. C) RA STs from patients with fibroid pathotype (n=8) that were non-responders, moderate responders, or good responders to anti-TNF therapy were assessed by RNA-seq for JAG2 expression [15]. D) 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 JAG2. E) RA STs were fluorescently stained for JAG2 expression on Vimentin+ RA FLS (n=3, original magnification x20). F) Relative expression of DLL1 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 DLL1 expression. H) RA STs from patients with fibroid pathotype (n=8) that were non-responders, moderate responders, or good responders to anti-TNF therapy were assessed by RNA-seq for DLL1 expression [15]. I) 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 DLL1. J) RA STs were fluorescently stained for DLL1 expression on Vimentin+ RA FLS (n=3, original magnification x20). K) Relative expression of Notch1 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. L) 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 Notch1 expression. M) RA STs from patients with fibroid pathotype (n=8) that were non-responders, moderate responders, or good responders to anti-TNF therapy were assessed by RNA-seq for Notch1 expression [15]. N) Relative expression of Notch3 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. O) 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 Notch3 expression. P) RA STs from patients with fibroid pathotype (n=8) that were non-responders, moderate responders, or good responders to anti-TNF therapy were assessed by RNA-seq for Notch3 expression [15]. Data are presented as mean ± SEM; differences were determined by one-way ANOVA. The p-value was adjusted for multiple comparisons by FDR. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

10456_2023_9897_MOESM2_ESM.tif

Supplementary file2 (TIF 583 KB) Supplemental Figure 2. Notch ligands and receptors are expressed on CD14+ RA MΦs. A-E) RA STs were fluorescently stained for A) JAG1, B) JAG2, C) DLL1, D) Notch1, and E) Notch3 expression on CD14+ RA MΦs (n=3, original magnification x20). F-K) RA MΦs were stimulated with 100 ng/ml IL-1β (6h) and transcription of F) JAG1, G) JAG2, H) DLL1, I) DLL4, J) Notch1, and K) Notch3 was assessed by qRT-PCR (n=6-7). Data are presented as mean ± SEM; differences were determined by the Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

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Zack, S.R., Meyer, A., Zanotti, B. et al. Notch ligands are biomarkers of anti-TNF response in RA patients. Angiogenesis 27, 273–283 (2024). https://doi.org/10.1007/s10456-023-09897-2

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