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Ageing and interferon gamma response drive the phenotype of neutrophils in the inflamed joint
  1. Ricardo Grieshaber-Bouyer1,2,3,4,
  2. Tarik Exner1,2,
  3. Nicolaj S Hackert1,2,
  4. Felix A Radtke1,2,4,
  5. Scott A Jelinsky5,
  6. Olha Halyabar6,
  7. Alexandra Wactor4,
  8. Elham Karimizadeh4,
  9. Joseph Brennan5,
  10. Jorge Schettini5,
  11. Helena Jonsson4,
  12. Deepak A Rao4,
  13. Lauren A Henderson6,
  14. Carsten Müller-Tidow3,7,
  15. Hanns-Martin Lorenz1,
  16. Guido Wabnitz2,
  17. James A Lederer8,
  18. Angela Hadjipanayis5,
  19. Peter A Nigrovic4,6
  1. 1 Division of Rheumatology, Department of Medicine V (Hematology, Oncology and Rheumatology), Heidelberg University Hospital, Heidelberg, Germany
  2. 2 Institute for Immunology, Heidelberg University Hospital, Heidelberg, Germany
  3. 3 Molecular Medicine Partnership Unit, European Molecular Biology Laboratory (EMBL), University of Heidelberg, Heidelberg, Germany
  4. 4 Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
  5. 5 Computational Systems Immunology, Worldwide Research & Development, Pfizer Inc, Cambridge, Massachusetts, USA
  6. 6 Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA
  7. 7 Department of Medicine V (Hematology Oncology Rheumatology), Heidelberg University Hospital, Heidelberg, Germany
  8. 8 Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
  1. Correspondence to Dr Ricardo Grieshaber-Bouyer, Division of Rheumatology, Department of Medicine V (Hematology, Oncology and Rheumatology), Heidelberg University Hospital, Heidelberg, Germany; Ricardo.GrieshaberBouyer{at}med.uni-heidelberg.de; Dr Peter A Nigrovic, Division of Immunology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA; peter.nigrovic{at}childrens.harvard.edu

Abstract

Objective Neutrophils are typically the most abundant leucocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint.

Methods We performed RNA sequencing of neutrophils from healthy human blood, arthritic blood and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils.

Results Blood neutrophils from healthy donors and patients with active arthritis showed largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFN-γ), as well as to tumour necrosis factor, interleukin-6 and hypoxia, in both humans and mice. Mass cytometry confirmed that healthy and arthritic donor blood neutrophils are largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFN-γ and prolonged culture.

Conclusions Circulating neutrophils from patients with arthritis resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFN-γ response and ageing as complementary drivers of the synovial fluid neutrophil phenotype.

  • arthritis
  • rheumatoid
  • arthritis
  • juvenile
  • arthritis
  • synovial fluid

Data availability statement

Data are available in a public, open access repository. All data relevant to the study are included in the article. All relevant data are included in the article and online supplemental information. RNA-sequencing data were deposited in GEO (accession number GSE193117).59 Cytometry by time of flight data were deposited in flow repository (accession number FR-FCM-Z4TF).60

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Data availability statement

Data are available in a public, open access repository. All data relevant to the study are included in the article. All relevant data are included in the article and online supplemental information. RNA-sequencing data were deposited in GEO (accession number GSE193117).59 Cytometry by time of flight data were deposited in flow repository (accession number FR-FCM-Z4TF).60

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Footnotes

  • Handling editor Josef S Smolen

  • Twitter @grieshaber, @radtke_f, @deepakarao

  • Contributors RG-B conceptualised the study; acquired patient samples and healthy donor samples; performed neutrophil isolation, cell sorting, RNA extraction, cryopreservation of fresh neutrophils; designed the mass cytometry panel; performed and guided RNA sequencing (RNAseq) and mass cytometry data analysis; designed the neutrophil ex vivo culture system; guided the validation experiments; created the figures; and wrote the manuscript. TE performed in vitro validation experiments on prospectively collected healthy donor neutrophils and analysed mass cytometry data. NSH performed RNAseq data analysis and improved visualisation techniques. FAR performed RNAseq data analysis. SAJ performed RNAseq and mass cytometry data analysis. OH acquired patient samples and healthy donor samples; performed neutrophil isolation, cell sorting, cryopreservation of fresh neutrophils and designed the mass cytometry panel. AW performed neutrophil isolation, cell sorting and cryopreservation of fresh neutrophils. EK analysed the data. JB and JS performed RNAseq and mass cytometry data analysis. HJ, LAH and DAR acquired patient samples. CM-T and H-ML analysed data. GW designed the neutrophil ex vivo culture system, provided critical input for validation experiments and analysed the data. JAL performed mass cytometry analysis. AH performed and guided RNAseq and mass cytometry data analysis. PAN conceptualised the study, acquired patient samples and healthy donor samples, designed the mass cytometry panel, guided data analysis, guided the validation experiments and wrote the manuscript. Guarantors: RG-B, PAN.

  • Funding This work was supported by funds from the state of Baden-Wuerttemberg within the Centres for Personalised Medicine Baden-Wuerttemberg (ZPM), an MD fellowship from Boehringer Ingelheim Fonds, a physician-scientist development grant from the Medical Faculty Heidelberg, and a research grant from the German Society for Rheumatology (DGRh), and a Gilead grant (to RG-B). TE was funded by the MD/PhD Programme of Heidelberg Faculty of Medicine. FAR was supported by an MD fellowship from Boehringer Ingelheim Fonds. PAN was supported by NIH/NIAMS awards 2R01AR065538, R01AR075906, R01AR073201, R21AR076630, 2P30AR070253, R56AR065538, NIH/NHLBI R21HL150575, the Fundación Bechara, the Samara Jan Turkel Centre for Paediatric Autoimmune Diseases at Boston Children’s Hospital and the Arbuckle Family Fund for Arthritis Research. Some data reported were collected with the support of an investigator-initiated research grant to PAN from Pfizer, but final decision about data analysis and publication remained with PAN.

  • Competing interests RG-B received research support from Gilead. SAJ, JB, JS and AH are employees of Pfizer. H-ML received research grants from Abbvie, Pfizer, Novartis, Sobi, Roche/Chugai, Gilead, Galapagos, GSK, UCB, MSD and BMS. PAN received investigator-initiated research grants from AbbVie, BMS, Novartis, Pfizer and Sobi; consulting fees from BMS, Cerecor, Miach Orthopedics, Novartis, Pfizer, Quench Bio, Sigilon, Simcere, Sobi, XBiotech and Exo Therapeutics; royalties from UpToDate and the American Academy of Pediatrics; and salary support from the Childhood Arthritis and Rheumatology Research Alliance.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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