MODIC CHANGE BONE MARROW NEUTROPHILS ARE ACTIVATED AND ACTIVATED NEUTROPHILS DEGRADE CARTILAGE ENDPLATES

Modic type 1 changes (MC1) are vertebral endplate bone marrow lesions and are a source of low back pain. Concomitant cartilage endplate (CEP) damage and severity of MC1 suggest a causal relationship. Dysregulated neutrophil maturation has been reported in MC1, and in rheumatoid arthritis, neutrophils damage joint cartilage. We hypothesize that i) MC1 bone marrow neutrophils are activated, ii) activated neutrophils degrade CEP and iii) MC1 bone marrow neutrophils release CEP degrading enzymes. From MC1 patients undergoing lumbar spondylodesis, CEP tissue was collected, and bone marrow neutrophils were isolated from aspirates from MC1 and an intra-patient control vertebra. First, RNA of CD45 + CD66b + neutrophils ( n = 7+7) was sequenced and dysregulated pathways identified. Expression of activation marker CD66b ( n = 10+10) and the proportion of immature band neutrophils (CD66b + CD10 - CD11b high )( n = 7+7) was measured with flow cytometry. MC1 data was compared to intra-patient controls using paired t-tests. Second, CEP biopsies were incubated with conditioned media from PMA-activated neutrophils and compared to unstimulated neutrophils, media only, and collagenase P exposure (positive control). Release of sulphated glycosaminoglycan (sGAG) was quantified with alcian blue assay and compared against media control (100%). Third, neutrophil elastase (NE) activity - an cartilage degrading enzyme [3]- was compared in media conditioned for 3h with neutrophils from MC1 vs. intra-patient control bone marrow using a fluorogenic NE substrate (paired t-test). Transcriptomic analysis of MC1 neutrophils revealed upregulated processes associated with inflammation and neutrophil activation. Upregulation of calcium signaling (not shown) supports neutrophil activation, indicating enhanced degranulation. Higher CD66b intensity on MC1 neutrophils supports neutrophil activation. 6/7 patients had increased fractions of pro-inflammatory immature band neutrophils (Fig.1b). Exposure of CEPs to neutrophil supernatants caused dose-dependent release of sGAG from the CEP tissues, showing that activated neutrophils degrade CEPs. NE activity was higher in conditioned media from MC1 vs. intra-patient control bone marrow neutrophils in 4/5 patients. Neutrophils are activated in MC1, shifted towards a pro-inflammatory band neutrophil population, and can degrade CEP, possibly through release of NE. Hence, neutrophils may contribute to the inflammatory pathomechanisms in MC1 and to CEP damaging. 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.