Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

MODIC CHANGE BONE MARROW NEUTROPHILS ARE ACTIVATED AND ACTIVATED NEUTROPHILS DEGRADE CARTILAGE ENDPLATES (#MP-15b)

Irina Heggli 1 2 , Nick Herger 1 2 , Tamara Mengis 1 2 , Mazda Farshad 3 , Mohamed Habib 4 , Christopher P Ames 5 , Oliver Distler 1 2 , Aaron Fields 4 , Stefan Dudli 1 2
  1. Center of Experimental Rheumatology, University of Zurich, Zurich, Switzerland
  2. Department of Physical Medicine and Rheumatology, Balgrist University Hospital, Balgrist Campus, University of Zurich, Zurich, Switzerland, Zurich
  3. Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich, Switzerland, Zurich
  4. Department of Orthopaedic Surgery, University of California San Francisco, San Francisco, CA, USA, San Francisco
  5. Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA, San Francisco

 

 

INTRODUCTION: Bone marrow (BM) inflammation is a hallmark of painful vertebral BM lesions called Modic changes (MC). MC occur adjacent to damaged cartilage endplates (CEPs). Neutrophilic infiltrates and dysregulated granulopoiesis are indicative for neutrophil involvement in inflammatory MC processes. Clarification of their role in inflammatory MC processes could help for the development of currently non-existing disease-modifying MC treatments. We hypothesized that i) activated blood neutrophils degrade CEPs, ii) MC BM neutrophils are activated and iii) that MC BM neutrophils release CEP destroying enzymes.

METHODS: To investigate if activated neutrophils damage CEPs, we exposed circular halves of lumbar CEP biopsies for 18h at 37°C to PMA stimulated vs. unstimulated neutrophil supernatants or positive (collagenase P) vs. negative (HBSS) controls respectively. Relative sulphated glycosaminoglycan (sGAG) release was quantified with dimethylmethylene blue assay and relative release (stimulated vs. non stimulated) was tested against null hypothesis (μ0=100%) using one sample t-test. To assess MC BM neutrophil activation and releases, we isolated BM neutrophils from low back pain patients with MC undergoing lumbar spondylodesis. From each patient, a BM aspirate from a MC lesion and from an unaffected vertebral body (intra-patient control) was collected. First, we performed bulk RNA sequencing of CD45+CD66b+ BM neutrophils (n=7+7) and identified dysregulated pathways by overrepresentation analysis (ORA). Second, we analyzed neutrophil activation (CD66b intensity) (n=10+10) and proportions of mature (CD66b+CD10+CD11bhigh) and immature band (CD66b+CD10-CD11bhigh) neutrophils (n=7+7) with flow cytometry and compared it between MC and controls with paired t-tests. Third, we assessed the supernatant of 3h cultivated BM neutrophils for neutrophil elastase (NE) activity with a fluorogenic NE substrate and the relative activity (MC vs. control) was tested against null hypothesis (μ0=100%) using one sample t-test.

RESULTS: Exposure of CEPs to neutrophil supernatants caused significant release of sGAG from the CEP tissues in a dose-dependent manner (25 mio neutrophils/ml: 380.1%±177, p=0.01; 12.5 mio/ml: 123.7%±22.3, p=0.05, positive control: 545.0%±302.8, p=0.02) (Figure 1a), showing that activated neutrophils degrade CEPs. Transcriptomic analysis of MC BM neutrophils revealed upregulated processes associated with inflammation and neutrophil activation (Figure 1b). Further upregulated were calcium signaling processes (not shown), supporting neutrophil activation in MC, and indicating increased neutrophil degranulation. Significantly higher CD66b intensity on MC neutrophils (ΔMC-control=1475±1801 RFI, p=0.03) supported neutrophil activation.  Moreover, in six out of seven patients, we found increased fractions of pro-inflammatory immature band neutrophils (ΔMC-control=6.69±8.00%, p=0.07) (Figure 1c). Finally, we found higher NE activity in MC BM neutrophil supernatants (212.9%±148.0, p=0.16) in four out of five patients (Figure 1d), an enzyme shown to degrade articular cartilage1.

DISCUSSION: Here we show that activated blood neutrophils degrade CEPs and that MC BM neutrophils are activated, have higher number of pro-inflammatory band neutrophils, and release more of potential CEP damaging NE. Therefore, it is plausible that activated MC BM neutrophils contribute to inflammatory MC pathomechanisms and potentially contribute to CEP damage. CEP damage promotes a pro-inflammatory disc/BM crosstalk and coincides with increased nerve fiber density. Hence, these findings could have implications for treatment strategies to mitigate inflammation and CEP damage in MC.

 

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  1. 1 Carmona-Rivera C, Carlucci PM, Goel RR, James E, Brooks SR, Rims C, Hoffmann V, Fox DA, Buckner JH, Kaplan MJ. Neutrophil extracellular traps mediate articular cartilage damage and enhance cartilage component immunogenicity in rheumatoid arthritis. JCI Insight. 2020 Jul 9;5(13):e139388.