C3f and V65 ELISA development and validation.

<p>(A) A typical standard curve for C3f sandwich ELISA. Graph represent the log of C3f peptide concentration (from 0–1 μg/ml) against reading of optical density (OD) values fitted with nonlinear regression curve (Four Parameters Logistic Regression(4PL)). (B) Recovery of C3f peptide from spiked normal human serum diluted 1:50 to 1:400. C3f peptide was added at 1 μg/ml into assay buffer as positive control (C3f control) and to human serum diluted at 1:50, 1:100, 1:200 and 1:400 to test recovery. Diluted serum without spiked C3f was tested as negative control. Recovery of spiked C3f in serum was calculated in comparison to C3f control and showed 65%, 86%, 88% and 96% recovery at 1:50, 1:100, 1:200 and 1:400 dilutions respectively. Data plotted as means ± SEM. (C) A typical standard curve for V65 competitive ELISA. Graph represent the log of V65 peptide concentration (from 0–4 μg/ml) fitted with nonlinear regression curve (4PL). (D) Graph represent the concentration of V65 peptide against OD values. Spiking experiment was carried out with 2-fold serial dilution starting at 1μg/ml of V65 peptide using normal human serum (at 1:100 or 1:400). Data plotted as means ± SEM.</p

Western blot of C3f and V65.

<p>(A) Reactivity of monoclonal and polyclonal antibodies anti-C3f (mAb-C3f and pAb-C3f respectively) to C3b (Complement C3b) and human vitronectin protein (V) on western blots. (B) Reactivity of polyclonal antibodies anti-V65 (pAb-V65) to C3b and vitronectin protein on western blots. (C) Reactivity of monoclonal antibody anti-C3f on Western blots of depleted and concentrated serum samples from OA and RA patients and synthetic C3f peptide. OA sample 1 and 2 were loaded at 21ng and 25ng per lane respectively. RA and C3f samples were loaded at 42ng and 7.5μg per lane respectively.</p

C3f in human serum samples.

<p>(A) Measurements of C3f in μg/ml in serum samples from OA patients (n = 13), RA patients (n = 13) and NC (n = 13). All samples were analysed at 1 in 50 dilution. Concentration of C3f was significantly increased in the RA group in comparison to the OA and NC group (***p>0.0001) and no increase was observed for the OA group. Data were analysed using Kruskal-Wallis with post hoc Dunn’s analysis and plotted as means ± SEM. (B) Schematic representation of human C3 complement cleavage to generated C3a, C3b, C3f and iC3b fragments. (C) C3f sandwich ELISA performed on filtered serum samples from the 3 study groups. RA patient’s sample (n = 5) and 1 NC sample were filtered using 3kDa and 10kDa cut-off filter and assessed by C3f sandwich ELISA. Unfiltered serum were tested as control. Data plotted as means ± SEM.</p

A-SAA levels in plasma and synovial fluid – A-SAA according to K&L grades in OA.

<p>(A) A-SAA profiles in blood and synovial fluid (SF) of OA and RA patients compared to those of matched healthy volunteers (HV). The boxes illustrate the 25th-75th percentiles, the horizontal lines the medians and the vertical lines the minimum and the maximum values. *p<0.05, **p<0.01 and ***p<0.001; non parametrical Mann-Whitney test. A-SAA expression level in (B) plasma and in (C) synovial fluid (SF) of OA patients classified according to their Kellgren and Lauwrence (K&L) grades. Correlation of A-SAA levels in plasma vs. A-SAA levels in SF for OA (D) and RA (E) patients.</p

Origine and roles of A-SAA in joints.

<p>A-SAA is probably mainly due to the passive diffusion process from blood into the joint cavity. However, A-SAA expression in joints may also arise from primary chondrocytes, FLS and preadipocytes under glucocorticoid treatment, from dedifferentiated chondrocytes under spontaneous release and from preadipocytes and adipose synovium under IL-1β treatment. Glucocorticoid-induced A-SAA expression is GR-dependent and can be downregulated in the presence of PPAR-γ agonists (genistein and rosiglitazone) or under the control of TGF-β, with ALK1 as a stimulatory pathway and ALK5 as an inhibitory pathway. Spontaneous secretion of A-SAA by dedifferentiated chondrocytes can also be inhibited by TGF-β. TLR4 can mediate A-SAA-induced cytokines and MMPs expression in both primary chondrocytes and FLS, and be selectively inhibited by TAK242. * Ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066769#pone.0066769-Vlasova1" target="_blank">[57]</a>.</p

Balance through either p-Smad2 or p-Smad1/5 signalling.

<p>(A) Human chondrocytes were stimulated at day 7 for 48h in the presence or absence of prednisolone (1 µM), dexamethasone (1 µM), compound A (CpdA) (1 µM) and/or TGF-β (10 ng/mL). A-SAA was determined by ELISA in the culture supernatants. p-Smad2, Smad2, p-Smad1/5 and Smad1 expression were detected by western blotting in cell extracts. Quantification was performed using ImageQuant LAS 4000 software. Ratio [p-Smad1/5]/[p-Smad2] is also indicated. (B) Primary human chondrocytes were stimulated at day 1 during 72h in the presence or absence of prednisolone (1 µM), TGF-β (10 ng/mL) and SB431542 (1.5 µM). A-SAA (ELISA) and Smad1 expression (Western blotting) in dedifferentiated chondrocytes (C) and FLS (D) transduced with lentiviral particles for shRNA control or three different shRNA of Smad1. *a, statistically different from control. *b, statistically different from prednisolone-treated cells. *c, statistically different from prednisolone and TGF-β treated cells.</p

A-SAA secretion by several cell types and tissues originated from OA patients.

<p>Detection of A-SAA secreted by (A) chondrocytes along dedifferentiation process, (B) primary human chondrocytes after 48h of treatment, (C) fibroblast-like synoviocytes after 1 week of treatment, (D) preadipocytes after 1 week of treatment with TNF-α (10 ng/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL), LPS (10 ng/mL), rosiglitazone (10 µM), leptin (100 nM), insulin (100 nM) and prednisolone (1 µM), (E) Secretion of A-SAA by cartilage shavings (0.2 g) after 1 week of treatment with prednisolone (1 µM). (F) Spontaneous secretion of A-SAA by fat synovial membrane after 48h in culture cell medium. A-SAA was determined in the culture supernatants (ELISA) and in cell extracts (Western blotting).</p

Glucocorticoid-induced A-SAA expression is GR-dependent and downregulated in the presence of PPAR-γ agonists.

<p>(A) OA-fibroblast-like synoviocytes (FLS) were stimulated during 1 week with prednisolone (1 µM), mifepristone (5 µM), eplerenone (5 µM), spironolactone (5 µM) and aldosterone (1 µM). Prednisolone-induced A-SAA expression was inhibited by the GR inhibitor mifepristone but not by MR inhibitors (eplerenone and spironolactone). A-SAA expression was barely detectable under aldosterone treatment, a MR-dependent glucocorticoid. *a, statistically different from control. *b, statistically different from prednisolone-treated cells. A-SAA expression level by (B) FLS after 1 week and (C) primary human chondrocytes after 72h (A) of treatment with prednisolone (1 µM), genistein (50 µM), rosiglitazone (10 µM) and/or insulin (100 nM). *a, statistically different from prednisolone-treated cells; *b statistically different from prednisolone- and insulin-treated cells.</p

TLR4 mediates A-SAA-induced cytokines and MMPs.

<p>Blockade of rhSAA (2.5 µg/mL)-induced cytokines and MMPs by specific inhibitors: lipoxin A4 (LX4; 1 µM), anti-SR-B1 antibody (5 µg/mL) and its isotype IgG1 control antibody (5 µg/mL), anti-RAGE (10 µg/mL) and its isotype IgG control antibody (10 µg/mL) and TAK242 (1 µM) for FPRL1, SR-B1, RAGE and TLR4 recepetors, respectively, on A) OA primary chondrocytes and B) OA FLS after 24h of stimulation. Cytokines and MMPs levels were quantified by ELISA in the culture supernatants. *a, statistically different from control. *b, statistically different from rhSAA-treated cells.</p

Endotoxin contamination - Polymyxin B and Proteinase K digestion treatment.

<p>OA fibroblast-like synoviocytes (A–C) and human primary chondrocytes at day 1 (B–D) were stimulated during 24h with rhSAA (5 µg/mL) and LPS (2 ng/mL) in the presence or not of A–B) polymyxin B (0.2 µg/mL) or C–D) Proteinase K (10 µg/mL) and PMSF (10 µM). IL-6 level was quantified by ELISA in the culture supernatants. *a, statistically different from control. *b, statistically different from LPS-induced cells. c*, statistically different from SAA-induced cells.</p