Htra1-Induced Matrix Fragmentation Drives Pro-Inflammatory Mechanisms in Modic Type 1 Discs

Vertebral bone marrow lesions, known as Modic type 1 changes (MC1), are associated with accelerated disc degeneration and endplate damage. While disc degeneration is necessary for MC1 development, not all degenerated discs exhibit MC1, suggesting unique characteristics in MC1 discs. This study aimed to identify MC1-specific mechanisms of disc degeneration. We hypothesize that MC1 discs contain more cleaved extracellular matrix (ECM) fragments, generated through MC1-specific mechanisms, that may act as pro-inflammatory mediators. Twenty-three degenerated nonMC discs and 30 MC1 discs from lumbar fusion patients were analyzed using N-terminal Amine Isotopic Labeling of Substrates (N-TAILS) mass spectrometry to identify differentially abundant ECM fragments (degradome). Shotgun proteomics was employed to identify proteins, including proteases, differentially abundant in MC1 discs. The protease of interest was tested on a Pfirrmann grade 2 disc to determine the degradome it generated. Recombinant ECM proteins were cleaved in vitro to verify the protease's activity. Cleavage was evaluated using SDS-PAGE, Coomassie staining, and N-TAILS. The pro-inflammatory potential of the protease-generated fragments was determined using NF-κB SEAP THP1 reporter cells. MC1 discs showed greater abundance, but not different types, of ECM fragments compared to nonMC discs (Figure 1A/B). Fibronectin (FN), type 1 collagen alpha-1 (COL1A1), cartilage intermediate layer protein 1 (CILP1), and cartilage oligomeric matrix protein (COMP) had the greatest number of significantly increased fragments (Figure 1C). Pre-TAILS identified 77 proteins in both groups; 70 were increased in MC1 discs, including the protease high-temperature serine protease 1 (HTRA1) (Figure 1D). Digestion of a Pfirrmann grade 2 disc by HTRA1 produced 94 ECM fragments, 43 of which were identified in the previously measured degradome and were significantly more abundant in MC1 discs compared to nonMC discs (Figure 1E). HTRA1 cleaved recombinant CILP1, COMP, and FN but not COL1A1, generating 20 N-terminal peptides, with 9 significantly more abundant in MC1 discs (p<0.001). HTRA1-generated CILP1 fragments strongly activated NF-κB signaling, while COMP and FN also induced NF-κB activation, regardless of cleavage (Figure 2A/B/C). HTRA1 cleavage of the ECM was more pronounced in MC1 discs, producing pro-inflammatory fragments of CILP1, COMP, and FN. This suggests a cascade of degenerative and inflammatory events in MC1 discs. This study identifies HTRA1 as a driver of pro-inflammatory mechanisms in MC1 discs. Targeting HTRA1 or its pro-inflammatory ECM fragments may mitigate degeneration and inflammation in MC1 discs. For any figures or tables, please contact the authors directly.