Chondroitin sulphate inhibits NF-κB activity induced by interaction of pathogenic and damage associated molecules  T.V. Stabler, Z. Huang, E. Montell, J. Vergés, V.B. Kraus  Osteoarthritis and Cartilage  Volume 25, Issue 1, Pages 166-174 (January 2017) DOI: 10.1016/j.joca.2016.08.012 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Correlation of serum LPS with radiographic knee OA severity. Significant positive correlation between serum LPS and radiographic knee joint space narrowing (JSN, sum of both knees) in a community OA cohort (n = 22). Osteoarthritis and Cartilage 2017 25, 166-174DOI: (10.1016/j.joca.2016.08.012) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Chondroitin sulfate (CS) reduces IL-1β release from macrophages produced by stimulated by LPS and HA fragments. THP-1 macrophages were activated with 200 nM phorbol ester (PMA) and primed with low concentrations (10 ng/ml) of LPS. Cells were untreated (control) or treated with HA fragments of varying mass or molecular weights (MW) ranging from small to large as follows: ultralow (ULMW, 7.5 kDa), low (LMW, 29 kDa), medium (MMW, 289 kDa) or full length, non-fragmented high (HMW, 1540 kDa). ULMW, LMW and MMW but not HMW induced a significant release of IL-1β from macrophages compared with control (LPS priming alone). A and B) addition of HA on an equal mass basis; C and D) addition of HA on an equimolar basis. Based on mass (A) or molar (C) concentration, A dose-dependent proinflammatory effect was apparent with the greatest effect produced by the smallest fragments and highest concentration of fragments. B) Dose response curves based on mass showed the estimated 50% effective proinflammatory concentration (EC50) was 0.8 μg/ml for ULMW, 1.8 μg/ml for LMW, and 2.5 μg/ml for MMW. C) The apparent size dependent gradient effect disappeared when comparing the different HA fragment preparations on the basis of numbers of fragments (equimolar concentrations); increasing numbers of fragments (independent of size) caused increasing IL-1β release with the effect plateauing at 1 pm. D) Dose response curves based on molarity showed the estimated EC50 was 297 fM for ULMW, 208 fM for LMW, and 233 fM for MMW. A control group without LPS priming and a 100 μg/ml ULMW HA group without LPS priming are also shown. Dot plots show experimental observations. Center lines with whiskers represent mean ± 95% CI. n = 4 for each treatment group. *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.0001 compared with control. Four parameter dose response curves show mean points. Osteoarthritis and Cartilage 2017 25, 166-174DOI: (10.1016/j.joca.2016.08.012) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Chondroitin sulphate (CS) reduces IL-1β release from macrophages treated with LPS and HA fragments. THP-1 cells were induced with 200 nM PMA and primed with low concentrations (10 ng/ml) LPS followed by stimulation with (10 μg/ml) of A) ULMW HA, B) LMW HA, or C) MMW HA fragments. CS in physiologically relevant doses (100–200 μg/ml) produced a dose-dependent reduction in IL-1β release from THP-1 macrophages treated HA fragments of the varying sizes. Dot plots show experimental observations. Center lines with whiskers represent mean ± 95% CI. CS alone had no effect on IL-1β release (dashed line). n = 4 for each treatment group. Osteoarthritis and Cartilage 2017 25, 166-174DOI: (10.1016/j.joca.2016.08.012) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 CS reduces NF-κB activity in macrophages treated with a TLR4 (LPS) or TLR2 (heat killed listeria monocytogenes, HKLM) stimulus. THP-1 cells were induced with 200 nM PMA and treated with LPS (1000 ng/ml, 100× greater than the priming dose) or HKLM (107 bacteria). Both stimuli significantly increased NF-κB activity (P < 0.0001) relative to the unstimulated control (dashed line) based on quantification in the THP1-Lucia NF-κB reporter cell line. C (50–200 μg/ml) produced a dose-dependent reduction in NF-κB activity in cells stimulated with either A) the TLR4 (LPS) stimulus, or B) the TLR2 (HKLM) stimulus. C) Based on dose response curves, the estimated 50% IC50 was 146 μg/ml CS for LPS stimulated cells and 64 μg/ml for HKLM stimulated cells. Dot plots show experimental observations. Center lines with whiskers represent mean ± 95% CI. n = 4 for each treatment group. Four parameter logistic dose response curve shows mean points. Osteoarthritis and Cartilage 2017 25, 166-174DOI: (10.1016/j.joca.2016.08.012) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 CS inhibits the activation of NF-κB by interaction between HA fragments and TLR4 or TLR2 stimuli. THP-1 cells were induced with 200 nM PMA then treated with ULMW HA (10 μg/ml), or LPS (1000 ng/ml) or HKLM (107 bacteria), singly and in combination with measurement of NF-κB activity in the THP1-Lucia NF-κB reporter cell line. A) HA fragments alone produced no increase in NF-κB activity but synergistically induced NF-κB activity through both the TLR4 (HA+LPS) or TLR2 (HA+HKLM) pathways. CS (200 μg/ml) reduced NF-κB activity induced through either the TLR4 (HA+LPS) or TLR2 (HA+HKLM) pathways. B) Inhibition of caspase-1 activity with a cell-permeable inhibitor (sequence Ac-AAVALLPAVLLALLAPYVAD-CHO, 10 μM) completely blocked the synergistic effect of HA fragments on the TLR4 and TLR2 pathways consistent with inflammasome (caspase-1) activation by HA fragments. Dot plots show experimental observations. Center lines with whiskers represent mean ± 95% CI. n = 4 for each treatment group. Osteoarthritis and Cartilage 2017 25, 166-174DOI: (10.1016/j.joca.2016.08.012) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 CS inhibits activation of canonical NF-kB transcription factors in the nucleus and cytoplasm of THP-1 macrophages. THP-1 cells were treated with CS (200 μg/ml) for 24 h followed by addition of LPS (1000 ng/ml) for 24 h. Nuclear and cytoplasmic extracts were assessed for DNA binding activity of NF-κB transcription factors p65, p50, C-Rel, and RelB and p52 in the presence and absence of CS. LPS increased the activity of NF-κB transcription factors p65, p50, and RelB in the A) nucleus and the B) cytoplasm as well as C-Rel in the nucleus only; the activity of each of these factors was reduced by CS. LPS had no appreciable effect on the non-canonical NF-κB associated p-52 factor and no effect of CS on this factor could therefore be discerned. Dot plots show experimental observations. Center lines with whiskers represent mean ± 95% CI. n = 3 for each treatment group. Osteoarthritis and Cartilage 2017 25, 166-174DOI: (10.1016/j.joca.2016.08.012) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Supplemental Fig. 1 Chondroitin sulphate (CS) does not inhibit intracellular caspase-1 activity. THP-1 cells were induced with 200 nM PMA, without (A) and with (B) priming with low concentrations (10 ng/ml) LPS followed by stimulation with ULMW HA fragments (10 μg/ml) with and without CS (200 μg/ml). A) ULMW HA fragments significantly increased (P < 0.0001) intracellular caspase-1 activity, CS had no effect on this activity. n = 3 for each treatment group. B) CS significantly reduced (P < 0.0001) intracellular IL-1β and proIL-1β in cells treated with LPS plus ULMW HA but did not alter the IL-1β to proIL-1β ratio consistent with lack of caspase-1 inhibition by CS. n = 6 for each treatment group. Dot plots show experimental observations. Center lines with whiskers represent mean ± 95% CI. Osteoarthritis and Cartilage 2017 25, 166-174DOI: (10.1016/j.joca.2016.08.012) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Supplemental Fig. 2 Chondroitin sulphate (CS) does not directly inhibit NF-κB DNA binding activity. Cytoplasmic extracts from THP-1 cells treated with LPS (1000 ng/ml) were tested with and without CS (200 μg/ml) added directly to the binding buffer of the transcription factor assay to act as a competitor to block subunit binding. No inhibition was seen. n = 3 for each treatment group. Dot plot shows experimental observations. Center line with whiskers represents mean ± 95% CI. Osteoarthritis and Cartilage 2017 25, 166-174DOI: (10.1016/j.joca.2016.08.012) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions