Injectable hyaluronic acid down-regulates interferon signaling molecules, IGFBP3 and IFIT3 in the bovine intervertebral disc

Zepur Kazezian, Zhen Li, Mauro Alini, Sibylle Grad, Abhay Pandit

Low back pain which is a major cause of disability for people aged between 20 and 50 years imposes a serious socio-economic burden. The current focus of regenerative medicine is on identifying molecular markers to facilitate the design of targeted therapeutics. Previously, we have demonstrated that expression of the anti-proliferative interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and pro-apoptotic insulin-like growth factor-binding protein-3 (IGFBP3), are up-regulated as downstream targets of the inflammatory cytokine interferon α (IFNα) signaling pathway in the human annulus fibrosus (AF). Here, we hypothesized that injection of hyaluronic acid (HA) would have an anti-inflammatory and matrix modulatory effect on injured and IFNα2β inflamed bovine intervertebral discs (IVD). Discs with an AF defect and challenged with IFNα2β were used in a bovine IVD organ culture model to test the effect of HA on the IFNα2β pathway, as well as the matrix proteins aggrecan and collagen I. qRT-PCR was used to assess the gene expression of IFNα2β signaling molecules. Additionally, immunostaining was used to measure protein expression. Our results show that HA treatment significantly down-regulates IFNAR1, IFNAR2, STAT1/2, JAK1, IFIT3 and IGFBP3 mRNA expression in the inflamed groups. Protein analysis confirmed the PCR results. In the extracellular matrix, aggrecan and collagen I were up-regulated while ADAMTS4 was down-regulated upon treatment of the injured and inflamed discs with HA. Hence, HA demonstrates both an anti-inflammatory role, resulting in the down-regulation of IFIT3 and IGFBP3 in the AF, and a matrix modulatory effect by up-regulating aggrecan and collagen I expression. The pro-inflammatory environment of the degenerated IVD represents a challenge for regenerative therapies. The study demonstrates that hyaluronan acts as an anti-inflammatory molecule by down-regulating IFNAR1 and IFNAR2, the signalling molecules STAT1, STAT2, JAK1 and the downstream apoptotic targets IGFBP3 and IFIT3. We also demonstrated that hyaluronan modulates the disc matrix environment by increasing aggrecan and collagen I synthesis and down-regulating ADAMTS4 that degrades the matrix under inflammatory conditions. The significance of this work lies in the fact that hyaluronan acts as an anti-inflammatory molecule by shifting the disc environment towards a more anabolic state and by promoting native IVD matrix production.