Intervertebral disc (IVD) degeneration (DD) is associated with low back pain, the leading cause of disability worldwide. Damage‐associated molecular patterns (DAMPs) that contribute to inflammation and trigger DD have not been well characterized. Extracellular high mobility group box‐1 (HMGB1) protein has been implicated as a potent DAMP and pro‐inflammatory stimulus in the immune system. In this study, we show that HMGB1 and IL‐6 levels increase in patients with advanced DD in comparison to early DD. This study further tested the hypothesis that HMGB1 promotes inflammatory signaling driving DD in human nucleus pulposus (NP) cells and tissue. Immunofluorescence and western blot analysis confirmed the expression of HMGB1 and its extracellular release by NP cells under cell stress. Gene expression and protein quantification indicate that HMGB1 stimulates the expression IL‐6 and MMP‐1 in a dose‐dependent manner. The contributions of toll‐like receptor (TLR) −2, −4 and receptor for advanced glycation end products (RAGE) as receptors mediating HMGB1 signaling was examined using small molecule inhibitors. Inhibition of TLR‐4 signaling, with TAK‐242, completely abrogated HMGB1 induced IL‐6 and MMP‐1 expression, whereas inhibition of TLR‐2, with O‐vanillin, or RAGE, with FPS‐ZM1, had mild inhibitory effects. HMGB1 stimulation activated NF‐ĸB signaling while TAK‐242 co‐treatment abrogated it. Lastly, effects of HMGB1 on matrix deposition was evaluated in a 3D culture system of human NP cells. These results implicate HMGB1 as a potent DAMP that promotes inflammation in NP cells and degradation of NP tissues. TLR4‐HMGB1 axis is a potential major pathway to alleviate disc inflammation and mitigate DD. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Multi‐joint disease pathologies in the lumbar spine, including ligamentum flavum (LF) hypertrophy and intervertebral disc (IVD) bulging or herniation contribute to lumbar spinal stenosis (LSS), a highly prevalent condition characterized by symptomatic narrowing of the spinal canal. Clinical hypertrophic LF is characterized by a loss of elastic fibers and increase in collagen fibers, resulting in fibrotic thickening and scar formation. In this study, we created an injury model to test the hypothesis that LF needle scrape injury in the rat will result in hypertrophy of the LF characterized by altered tissue geometry, matrix organization, composition and inflammation. An initial pilot study was conducted to evaluate effect of needle size. Results indicate that LF needle scrape injury using a 22G needle produced upregulation of the pro‐inflammatory cytokine Il6 at 1 week post injury, and increased expression of Ctgf and Tgfb1 at 8 weeks post injury, along with persistent presence of infiltrating macrophages at 1, 3, and 8 weeks post injury. LF integrity was also altered, evidenced by increases in LF tissue thickness and loss of elastic tissue by 8 weeks post injury. Persistent LF injury also produced multi‐joint effects in the lumbar IVD, including disc height loss at the injury and adjacent to injury level, with degenerative IVD changes observed in the adjacent level. These results demonstrate that LF scrape injury in the rat produces structural and molecular features of LF hypertrophy and IVD height and histological changes, dependent on level. This model may be useful for testing of therapeutic interventions for treatment of LSS and IVD degeneration associated with LF hypertrophy.

Objective: Low back pain (LBP) is the leading cause of global disability and is thought to be driven primarily by intervertebral disc (IVD) degeneration (DD). Persistent upregulation of catabolic enzymes and inflammatory mediators have been associated with severe cases of DD. Nuclear factor kappa B (NF-κB) is a master transcription regulator of immune responses and is over expressed during inflammatory-driven musculoskeletal diseases, including DD. However, its role in triggering DD is unknown. Therefore, this study investigated the effect of NF-κB pathway over-activation on IVD integrity and DD pathology. Methods: Using skeletally mature mouse model, we genetically targeted IVD cells for canonical NF-κB pathway activation via expression of a constitutively active form of inhibitor of κB kinase B (IKKβ), and assessed changes in IVD cellularity, structural integrity including histology, disc height, and extracellular matrix (ECM) biochemistry, biomechanics, expression of inflammatory, catabolic, and neurotropic mediators, and changes in macrophage subsets, longitudinally up to 6-months post activation. Results: Prolonged NF-κB activation led to severe structural degeneration, with a loss of glycosaminoglycan (GAG) content and complete loss of nucleus pulposus (NP) cellularity. Structural and compositional changes decreased IVD height and compressive mechanical properties with prolonged NF-κB activation. These alterations were accompanied by increases in gene expression of inflammatory molecules (Il1b, Il6, Nos2), chemokines (Mcp1, Mif), catabolic enzymes (Mmp3, Mmp9, Adamts4), and neurotrophic factors (Bdnf, Ngf) within IVD tissue. Increased recruitment of activated F4/80+ macrophages exhibited a greater abundance of pro-inflammatory (CD38+) over inflammatory-resolving (CD206+) macrophage subsets in the IVD, with temporal changes in the relative abundance of macrophage subsets over time, providing evidence for temporal regulation of macrophage polarization in DD in vivo, where macrophages participate in resolving the inflammatory cascade but promote fibrotic transformation of the IVD matrix. We further show that NF-κB driven secretory factors from IVD cells increase macrophage migration and inflammatory activation, and that the secretome of inflammatory-resolving macrophages mitigates effects of NF-κB overactivation. Conclusion: Overall the observed results suggest prolonged NF-κB activation can induce severe DD, acting through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and inflammatory activation of a macrophage cell populations, that can be mitigated with inflammatory-resolving macrophage secretome.

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