Background The combination of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), and endogenous danger-associated molecular patterns (DAMPs), such as hyaluronan (HA) fragments, are known to be able to induce an inflammatory response from macrophages characterised by the release of pro-inflammatory cytokines1. We have previously shown that pharmaceutical grade chondroitin sulphate (CS), commonly used in the symptomatic treatment of osteoarthritis (OA), can attenuate the inflammatory response.
Objectives To evaluate the synergistic effects of CS and glucosamine (GLU) in combination on LPS/HA mediated inflammatory responses of an immortalised human macrophage cell line (THP-1) and primary synovial fluid cells.
Methods THP-1 monocyte cells were grown and differentiated into mature macrophages by the addition of 200 nM of phorbol 12-myristate 13-acetate (PMA) as previously described1. Cells were cultured with a physiologically achievable range of concentrations of CS and GLU (0, 10, 50, 200 µg/ml of each, Bioibérica, S.A.U.) for 6 hours, then primed with physiologically relevant concentrations of LPS (10 ng/ml) (n=12/group). After 24 hours, cell culture media were replaced with serum free Opti-MEM supplemented with the previously mentioned concentrations of CS and GLU, LPS, and 10 µg/ml HA fragments (ultra-low molecular weight, Lifecore). After a further 24 hours, supernatants were harvested for NF-κB activity and pro-inflammatory cytokine (IL-1β, IL-6, IFN-γ, and TNF-α) assessment. Cell viability was measured using PrestoBlue reagent. The human knee primary synovial fluid cells were collected at the time of joint replacement and cultured with CS (200 µg/ml) and GLU (200 µg/ml), singly or in combination, with the addition of LPS and HA (n=2/group). After normalisation for cell viability, all results were expressed as fold change from the negative control (media only). One-way ANOVA with Dunnett’s post-hoc test was performed using GraphPad Prism.
Results CS and GLU in combination (200 µg/ml of each) significantly reduced NF-κB activity by 70% compared to the positive control group (LPS/HA only). Although CS (200 µg/ml) alone did not reduce NF-κB activity, the addition of the lower concentration of CS (10–50 µg/ml) to GLU (200 µg/ml) significantly reduced NF-κB activity compared with GLU (200 µg/ml) alone. Addition of lower concentrations of GLU (10–50 µg/ml) to CS (200 µg/ml) modestly reduced NF-κB activity (Fig 1). Similar trends were observed in secreted pro-inflammatory cytokines (IL-1β, IL-6, IFN-γ, and TNF-α); namely, CS and GLU in combination significantly attenuated the LPS/HA mediated pro-inflammatory responses (p<0.05) (Fig 1). Although, a diverse range of inflammatory responses to the LPS/HA activation was observed, constitutive pro-inflammatory cytokine production by primary synovial fluid cells was reduced by the combination of CS and GLU.
*p<0.05 for post-hoc test using placebo as control.
Conclusions Inflammatory reactions of THP-1 macrophages, induced by physiologically relevant concentrations of LPS and HA fragments, were suppressed synergistically by the combination of physiologically achievable concentrations of CS and GLU. A similar trend was observed in primary human synovial cells but further investigations are required. These data could explain, at least in part, the clinical efficacy of CS and GLU in combination observed in OA patients.
Reference  Stabler TV. Osteoarthritis Cartilage2017;25(1):166–174.
Disclosure of Interest M.-F. Hsueh Grant/research support from: Bioibérica, S.A.U., E. Montell Employee of: Bioibérica, S.A.U., V. Kraus Grant/research support from: Bioibérica, S.A.U.
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