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. 2013 Aug 22;15(4):R94.
doi: 10.1186/ar4274.

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

Lilian Quero, Marina Klawitter, Anja Schmaus, Melanie Rothley, Jonathan Sleeman, André N Tiaden, Juergen Klasen, Norbert Boos, Michael O Hottiger, Karin Wuertz, Peter J Richards

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

Lilian Quero et al. Arthritis Res Ther. .

Abstract

Introduction: Intervertebral 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.

Methods: Human 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.

Results: Treatment 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.

Conclusions: These 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.

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Figures

Figure 1
Figure 1
Preparation of fHAs. High molecular weight (HMW) hyaluronic acid (HA) was digested with bovine testis hyaluronidase. The resulting fragments were separated on a Bio Gel P10 column. For determination of fragment size fluorophore-assisted carbohydrate electrophoresis (FACE) analysis was performed. In this example, fractions 18, 23, 42, 48 and 64 derived from one HA-preparation were compared with a commercially-available HA standard (Sigma-Aldrich). M, HA standard marker; ds, disaccharide units.
Figure 2
Figure 2
Gene expression profile in IVD cells treated with fHAs. Human IVD cells were incubated for 18 hours with hyaluronic acid fragments (fHAs) at either 5 or 20 μg/ml (n = 4 to 6) and RNA harvested for analysis by qRT-PCR. Values were normalized to TATA-Box binding protein (TBP) mRNA and expressed as 2-ΔCT. Statistical analysis was performed using the Kruskal-Wallis one-way analysis of variance for multiple group comparisons followed by the Mann-Whitney U test for comparisons between two groups, * P <0.05, ** P <0.01 as compared to untreated IVD cells. IL, interleukin; TNF, tumor necrosis factor; MMP, matrix metalloproteinase; COX, cyclooxygenase; ADAMTS, A Disintegrin And Metalloproteinase with Thrombospondin Motifs.
Figure 3
Figure 3
Stimulation of IL-6 production in IVD cells. Human intervertebral disc (IVD) cells were incubated for 18 hours with either: hyaluronic acid fragments (fHAs) (20 μg/ml) (n = 4) (A); Pam3CSK4 (25 ng/ml) (n = 4) (B); lipopolysaccharide (LPS) (25 ng/ml) (n = 5) (C); or interleukin (IL)-1β (5 ng/ml) (n = 5) (D), and IL-6 protein levels determined in supernatants using a specific ELISA. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t-test, * P <0.05, ** P <0.01 as compared to untreated IVD cells.
Figure 4
Figure 4
The effect of gene silencing on fHA-mediated IL-6 production in IVD cells. A) siRNA-mediated knockdown of genes Toll like receptor (TLR)2, TLR4, CD44 and RHAMM was confirmed after 30 hours in intervertebral disc (IVD) cells by qRT-PCR (n = 4). In each case, gene expression was calculated as fold change as compared to untreated cells. The use of a non-specific scrambled siRNA (siRNA (S)), confirmed specificity of gene knockdown. B, C) Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) (n = 4) (B) and lipopolysaccharide (LPS)- (25 ng/ml) (n = 3). (C) stimulated IVD cells following gene knockdown of TLR2 or TLR4 respectively, as determined by IL-6 ELISA. (D) hyaluronic acid fragment (fHA)-treated (20 μg/ml) IVD cells following gene knockdown as determined by IL-6 ELISA (n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t-test, * P <0.01.
Figure 5
Figure 5
The effect of TLR2 inhibition on fHA-mediated IL-6 production in IVD cells. Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) (A) and hyaluronic acid fragment (fHA)-treated (20 μg/ml) (B) intervertebral disc (IVD) cells following antibody-mediated neutralization of Toll like receptor (TLR)2 activity (n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t-test, * P <0.05 as compared to cells treated with non-specific IgG control antibody.
Figure 6
Figure 6
The role of NF-κB in fHA-dependent IVD cell activation. (A) Immunofluorescence staining of intervertebral disc (IVD) cells for NF-κB (p65) (green) following treatment with either hyaluronic acid fragments (fHAs) (20 μg/ml) or interleukin (IL)-1β (5 ng/ml) at selected time points. Nuclear NF-κB (p65) is indicated by arrowheads. Untreated IVD cells served as a control. Scale bar = 50 μm. (B) Western blot analysis of nuclear extracts from untreated IVD cells or cells treated for 1 hour with either fHAs (20 μg/ml) or IL-1β (5 ng/ml). Levels of PARP1 protein served as a loading control. (C) NF-κB (p65) DNA binding activity in nuclear extracts from untreated IVD cells, or cells treated for one hour with IL-1β or fHAs (20 μg/ml) (n = 3). In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t-test, * P <0.01 as compared to untreated cells.
Figure 7
Figure 7
Role of MAP kinases in mediating the effects of fHAs in IVD cells. Cultured intervertebral disc (IVD) cells were treated for 15 minutes with hyaluronic acid fragments (fHAs) (20 μg/ml), interleukin (IL)-1β (5 ng/ml), lipopolysaccharide (LPS) (25 ng/ml) or left untreated and whole cell extracts harvested for Western blot analysis using antibodies specific to phosphorylated and non-phosphorylated SAPK/JNK (54/46) (A), ERK 1/2 (44/42) (B) and p38 (C). (D, E) IVD cells were treated with fHA (20 μg/ml) alone (Control) or in combination with MAP kinase inhibitors (10 μM) specific for p38 (SB203580), ERK 1/2 (PD98059) or SAPK/JNK (SP600125) and IL-6 (D) and matrix metalloproteinase (MMP)-3 (E) protein levels determined by specific ELISA (n = 4). In all cases, analyses were carried out in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using Students t-test, * P <0.05, ** P <0.01 as compared to cells treated with fHAs alone (Control).
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