T V Stabler1, Z Huang2, E Montell3, J Vergés4, V B Kraus5. 1. Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, USA. Electronic address: stablert@hotmail.com. 2. Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, USA; Department of Orthopedic Surgery, West China Hospital, West China Medical School, SiChuan University, ChengDu, SiChuan Province, People's Republic of China. Electronic address: Zey.huang@gmail.com. 3. Pre-Clinical R&D Area, Bioibérica, S. A., Barcelona, Spain. Electronic address: lmontell@bioiberica.com. 4. Pre-Clinical R&D Area, Bioibérica, S. A., Barcelona, Spain. Electronic address: jverges@bioiberica.com. 5. Duke Molecular Physiology Institute, Duke University School of Medicine, Durham, USA; Division of Rheumatology, Duke University School of Medicine, Durham, USA. Electronic address: kraus004@duke.edu.
Abstract
OBJECTIVE: To evaluate the anti-inflammatory mechanism of action of Chondroitin Sulphate (CS). DESIGN: THP-1 macrophages were cultured with a range of sizes and concentrations of HA fragments with TLR4 (LPS in a physiologically relevant concentration determined by analyses of sera of a community clinic ascertained knee osteoarthritis (OA) cohort) or TLR2 (heat killed listeria bacteria) agonists and varying concentrations of CS in a physiologically relevant range (10-200 μg/ml). We measured IL-1β release, intracellular IL-1β, proIL-1β, caspase-1 and NF-κB activity and DNA binding activity of NF-κB transcription factors from nuclear and cytoplasmic extracts. RESULTS: Serum LPS was significantly associated with radiographic knee joint space narrowing (JSN) (P = 0.02) in the OA cohort (n = 40). The priming dose of LPS used for these experiments (10 ng/ml) was below the lowest serum concentration of the OA cohort (median 47.09, range 14.43-81.36 ng/ml). Priming doses of LPS and HA fragments alone did not elicit an inflammatory response. However, primed with LPS, HA fragments produced large dose-dependent increases in IL-1β that were inhibitable by CS. CS did not inhibit caspase-1 activity but in physiologically achievable concentrations, attenuated NF-κB activity induced by either the TLR4 (LPS 1000 ng/ml) or TLR2 agonists alone or in combination with HA fragments. LPS induced and CS significantly reduced activity of canonical NF-κB transcription factors, p65, p50, c-Rel and RelB. CONCLUSIONS: Subinflammatory concentrations of pathogenic (LPS, listeria) and damage associated (HA) molecules interact to induce macrophage-related inflammation. CS works upstream of the inflammasome by inhibiting activation of NF-κB transcription factors.
OBJECTIVE: To evaluate the anti-inflammatory mechanism of action of Chondroitin Sulphate (CS). DESIGN:THP-1 macrophages were cultured with a range of sizes and concentrations of HA fragments with TLR4 (LPS in a physiologically relevant concentration determined by analyses of sera of a community clinic ascertained knee osteoarthritis (OA) cohort) or TLR2 (heat killed listeria bacteria) agonists and varying concentrations of CS in a physiologically relevant range (10-200 μg/ml). We measured IL-1β release, intracellular IL-1β, proIL-1β, caspase-1 and NF-κB activity and DNA binding activity of NF-κB transcription factors from nuclear and cytoplasmic extracts. RESULTS: Serum LPS was significantly associated with radiographic knee joint space narrowing (JSN) (P = 0.02) in the OA cohort (n = 40). The priming dose of LPS used for these experiments (10 ng/ml) was below the lowest serum concentration of the OA cohort (median 47.09, range 14.43-81.36 ng/ml). Priming doses of LPS and HA fragments alone did not elicit an inflammatory response. However, primed with LPS, HA fragments produced large dose-dependent increases in IL-1β that were inhibitable by CS. CS did not inhibit caspase-1 activity but in physiologically achievable concentrations, attenuated NF-κB activity induced by either the TLR4 (LPS 1000 ng/ml) or TLR2 agonists alone or in combination with HA fragments. LPS induced and CS significantly reduced activity of canonical NF-κB transcription factors, p65, p50, c-Rel and RelB. CONCLUSIONS: Subinflammatory concentrations of pathogenic (LPS, listeria) and damage associated (HA) molecules interact to induce macrophage-related inflammation. CS works upstream of the inflammasome by inhibiting activation of NF-κB transcription factors.