Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

Abstract

IntroductionIntervertebral disc (IVD) degeneration is characterized by extracellular matrix breakdown and is considered to be a primary cause of discogenic back pain. Although increases in pro-inflammatory cytokine levels within degenerating discs are associated with discogenic back pain, the mechanisms leading to their overproduction have not yet been elucidated. As fragmentation of matrix components occurs during IVD degeneration, we assessed the potential involvement of hyaluronic acid fragments (fHAs) in the induction of inflammatory and catabolic mediators.MethodsHuman IVD cells isolated from patient biopsies were stimulated with fHAs (6 to 12 disaccharides) and their effect on cytokine and matrix degrading enzyme production was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The involvement of specific cell surface receptors and signal transduction pathways in mediating the effects of fHAs was tested using small interfering RNA (siRNA) approaches and kinase inhibition assays.ResultsTreatment of IVD cells with fHAs significantly increased mRNA expression levels of interleukin (IL)-1β, IL-6, IL-8, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1 and -13. The stimulatory effects of fHAs on IL-6 protein production were significantly impaired when added to IVD cells in combination with either Toll-like receptor (TLR)-2 siRNA or a TLR2 neutralizing antibody. Furthermore, the ability of fHAs to enhance IL-6 and MMP-3 protein production was found to be dependent on the mitogen-activated protein (MAP) kinase signaling pathway.ConclusionsThese findings suggest that fHAs may have the potential to mediate IVD degeneration and discogenic back pain through activation of the TLR2 signaling pathway in resident IVD cells.

Mechanisms of low back pain: a guide for diagnosis and therapy

“…A possible explanation could involve the growth of nerves capable of signaling pain deep into the annular structures 84 . Another hypothesis involves a class of molecules, called damage-associated molecular patterns (DAMPs), including hyaluronic acid and fibronectin fragments, able to stimulate sterile inflammation of the disc through the action of pro-inflammatory cytokines (IL-1beta, IL-6, and IL-8) and matrix degrading enzymes (MMP-1, MMP-3, and MMP-13) 83 .…”

“…Another hypothesis involves a class of molecules, called damage-associated molecular patterns (DAMPs), including hyaluronic acid and fibronectin fragments, able to stimulate sterile inflammation of the disc through the action of pro-inflammatory cytokines (IL-1beta, IL-6, and IL-8) and matrix degrading enzymes (MMP-1, MMP-3, and MMP-13) 83 . Also, subclinical anaerobic bacterial infection, encouraged by hypoxic conditions, could have a role in the development of discogenic pain 84 .…”

“…Clinical trials have demonstrated that some patients suffering from LBP have improvement following amoxicillin-clavulanate 84, 85 . Moreover, diabetes increases the risk of developing painful DD because advanced glycation end products (AGEs) induce catabolism and promote inflammation 86 .…”

Nerve Growth Factor Is Regulated by Toll-Like Receptor 2 in Human Intervertebral Discs

“…For example, a recent study found high mobility group B1, a TLR2 ligand, increases with the grade of degeneration in surgical specimens (35). Furthermore, TLR2 activation increases IL-1␤, IL-6, IL-8, matrix metalloproteinase-1 and -13, and COX-2 gene expression and IL-6 protein in a mixed population of NP and AF cells (11,36). These prior studies indicate that TLR2 could play an important role in the increase of inflammatory mediators and catabolic enzymes that contribute to disc degeneration.…”

“…TLR2 and TLR4 are thought to be the primary TLR subtypes that recognize ECM alarmins, where TLR4 functions as a homodimer and TLR2 functions as a homodimer or heterodimer with TLR1 or -6 (10). TLR activation increases the catabolic proteases matrix metalloproteinase-1 and -13 as well as IL-1␤, IL-6, and IL-8 in disc cells (11,36). Interestingly, in vivo injection of fibronectin fragments into rabbit discs induces degenerative changes (37), and exposure of NP cells to fibronectin fragments decreases proteoglycan synthesis and increases proteoglycan degradation (38).…”