ACTIVATED NEUTROPHILS DEGRADE CARTILAGE ENDPLATES

I. Heggli, M. Habib, J. Scheer, N. Herger, T. Mengis, C. Laux, F. Wanivenhaus, J. M. Spirig, M. Betz, M. Farshad, O. Distler, A. Fields, S. DudliUniversity of Zurich, Center of Experimental Rheumatology, Balgrist Campus, University Hospital Zurich and Balgrist University Hospital, University of Zurich, Switzerland, Zurich, Switzerland University of California San Francisco, Department of Orthopaedic Surgery, University of California San Francisco, San Francisco, CA, USA, San Francisco, United States of America University of California San Francisco, Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA, San Francisco, United States of America University of Zurich, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Switzerland, Zurich, Switzerland  Background Modic type 1 changes (MC1) are painful vertebral bone marrow lesions often found in patients with chronic low back pain. Damage of the endplate associates with MC1, is a risk factor for MC1 progression, and associates with pain. Recent evidence suggests a role of MC bone marrow neutrophils in endplate damage. Objectives The aim of this study was 1) to show that MC1 bone marrow neutrophils are activated and 2) to show that activated blood neutrophils degrade cartilage endplates (CEPs). Methods From MC1 patients undergoing lumbar spinal fusion, MC1 and intra-patient control bone marrow neutrophils (CD45CD66b) were isolated from bone marrow aspirates by cell sorting. Bulk RNA sequencing was performed (n = 5 + 5), differentially expressed genes (DEGs) were identified, and enrichment analysis was performed. In addition, neutrophil activation was assessed as CD66b expression by flow cytometry (n = 3 + 3). To assess the effect of activated neutrophils on CEP degradation, a neutrophil-mediated CEP damage model was established. Blood neutrophils were isolated from one donor and exposed to lumbar CEPs. A lumbar CEP was collected from six patients undergoing spinal fusion. From each CEP, three punch biopsies (ø=4-mm) were taken and halved. The six CEP biopsy halves per patient (1A,1B; 2A, 2B; 3A, 3B) were exposed for 18 h at 37 °C to: 1A) 0.75 U/ml collagenase P (positive control) and 1B) Hanks balanced salt solution (negative control); 2A) conditioned medium from 25 mio/ml activated neutrophils (100 nM PMA, 3 h, 37 °C) and 2B) conditioned medium from 25 mio/ml non-activated neutrophils; 3A) conditioned medium from 12.5 mio/ml activated neutrophils (100 nM PMA, 3 h, 37 °C) and 3B) conditioned medium from 12.5 mio/ml non-activated neutrophils. Exposure supernatant and CEP tissues were assayed for sulphated glycosaminoglycans (sGAG) and hydroxyproline (as measure for total collagen). Release of sGAG and hydroxyproline from condition 1A, 2A, and 3A was normalized to condition 1B, 2B and 3B, respectively. Relative release was tested against null hypothesis (µ0 = 100%) using a one sample t-test. Results 185 genes were differentially expressed between MC1 and control vertebral bone marrow neutrophils. Enrichment analysis revealed an activated pro-inflammatory transcriptome (Figure 1a). Flow cytometric analysis confirmed neutrophil activation on protein level (measured as % of CD66) in MC1 (control: 43.1% ± 15.7%, MC1: 54.1% ± 16.7%, p = 0.018) (Figure 1b). Exposure of CEP tissues to conditioned medium from activated neutrophils significantly increased the release of sGAG from the CEP tissues in a dose-dependent manner (25 mio/ml: 380.1% ± 177%, p = 0.012; 12.5 mio/ml: 123.7% ± 22.3%, p = 0.048, positive control: 545.0 % ± 302.8 %, p = 0.016) (Figure 1c) but there was no significant hydroxyproline release (not shown). Our data shows that neutrophils in MC1 bone marrow are activated and activated blood neutrophils degrade CEPs. Hence, neutrophils in MC1 bone marrow might promote and exacerbate CEP damage. Therefore, MC1 are not just reactive changes but can drive CEP damage that enhances a vicious inflammatory crosstalk with the adjacent disc. Image/graph:Figure 1. (a) Top 5 upregulated biological processes in MC1 bone marrow neutrophils (b) Representative flow cytometry image c) Relative sGAG released from CEP tissues. * P < 0.05. Conclusion We identified neutrophils as a potentially important player in MC1. Therefore, the present findings could have implications for treatment strategies to mitigate CEP damage in MC1. REFERENCES: NIL. Acknowledgements: We thank the FOREUM Foundation for supporting this project. Disclosure of Interests Irina Heggli: None declared, Mohamed Habib: None declared, Justin Scheer: None declared, Nick Herger: None declared, Tamara Mengis: None declared, Christoph Laux: None declared, Florian Wanivenhaus: None declared, Jose Miguel Spirig: None declared, Michael Betz: None declared, Mazda Farshad: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant Siences, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Pfizer, Prometheus, Redxpharna, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Aaron Fields: None declared, Stefan Dudli: None declared. Keywords: Cartilage, -omics, Innate immunity DOI: 10.1136/annrheumdis-2023-eular.197Citation: , volume 82, supplement 1, year 2023, page 464Session: Spine, mechanical musculoskeletal problems, local soft tissue disorders (Poster View)