1. 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 (January 2017)
DOI: /j.joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

2. 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  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

3. 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 <  compared with control. Four parameter dose response curves show mean points.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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4. 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  , DOI: ( /j.joca )
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5. 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  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

6. 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  , DOI: ( /j.joca )
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7. 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  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

8. 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  , DOI: ( /j.joca )
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9. 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  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions