<i>Candida albicans</i> and Zymosan A aggravate the inflammation in the chronic SCW model.

<p>On days 0, 7, 14, and 21, streptococcal cell wall (SCW) fragments were injected intra-articularly (i.a.) into the knee joint. Joint swelling (ratio of ^{99 m}Tc uptake in the treated right knee joint to that in the untreated left knee joint) were determined 1 day after every injection (A) and during the chronic phase of the model on day 28 (B; n=6 mice/group). In addition, the inflammatory cell influx (C) on histological slides was quantified on day 28. H&E stained frontal knee sections (original magnification 100×) (D) are shown (n=8 mice/group. Values are the mean ± SEM; ns=non significant, * P<0.05, ** P<0.01, *** P<0.001, by One-way ANOVA.</p

Exposure to <i>C. albicans</i> increases the cartilage destruction and bone erosion during chronic SCW arthritis.

<p>Analysis of destructive parameters after 4 relapsing flares of arthritis. On day 28, knee joints (n=8/group) were harvested for histological assessment. Knee joint sections were stained using Safranin O to determine the degree of proteoglycan (PG) depletion. H&E staining was used to score the degree of chondrocyte death, cartilage surface erosion and bone erosion (A). QPCR analysis of destructive related genes was performed on synovial biopsies (n=3/group) of day 22, one day after the last injection (B). Representative images of arthritic knee joints showing immunohistochemical staining for VDIPEN after the 4 repeated injections (day 28) (C). Besides, the quantitative measurement of VDIPEN expression (percentage of positively stained area) in the cartilage of the 3 groups of mice (n=8/group) was analyzed. Values are the mean ± SEM; ns=non significant * P<0.05, ** P<0.01, *** P<0.001, by One-way ANOVA.</p

Co-exposure to <i>C. albicans</i> induces Th17 cells in the joint.

<p>The mRNA expression of T-bet, GATA-3, RORγT and FOXP3 in synovial biopsies of the knee joints (n=3 mice/group) were measured by QPCR (A) The mRNA expression of IFNy, IL-17A, IL-17F, IL-21, IL-22 and IL-10 are shown (B). The knee joint synovium was dissected and prepared for enzymatic digestion. The cells were incubated with PMA/ionomycine and Golgiplug for 5 hours before flowcytometric analysis (n=6 mice/group) and stained for CD3, IL-17, and IFN-γ. Representative dot plots of the isolated cells gated on CD3+ are shown (C). The mean percentage of isolated IFN-γ and IL-17 producing T cells (CD3+ population) are shown (D). Results are mean ± SEM; ns=non significant *=P<0.05, **=P<0.01, by one-way ANOVA.</p

Effect of IL-17 on synoviolin expression in RA FLS.

<p>RA FLS were stimulated with IL-17A or IL-17F (50 ng/ml), IL-1 10 ng/ml, TNF 10 ng/ml or LPS 100 ng/ml for 24 h and synoviolin mRNA expression measured by real-time RT-PCR (<b>A, upper panel</b>). The results based on a ratio of synoviolin/β-actin mRNA amplification are presented as the fold induction in synoviolin mRNA expression relative to control samples, n = 3, mean ± SEM. * <i>P</i><0.05 compared to no treatment control. RA FLS were treated with IL-17A or IL-17F (50 ng/ml) for 24 h and synoviolin expression measured by Western Blotting (<b>A, lower panel</b>). The membrane was stripped and reprobed for β-actin and the result is representative of n = 3. RA FLS were treated with IL-17 5-200 ng/ml for 24 h (<b>B, left panel</b>) or IL-17 50 ng/ml over 24 h (<b>B, right panel</b>) and synoviolin expression measured by real-time RT-PCR, n = 3, mean ± SEM, * <i>P</i><0.05. Synoviolin mRNA expression in RA FLS, OA FLS and OA dermal fibroblasts that were untreated (–) or were treated with 50 ng/ml or 10 ng/ml of IL-1 for 24 h <b>(C).</b> * <i>P</i><0.05 versus <b>untreated FLS.</b> RA FLS were preincubated with PD98059 (50μM), SB203580 (5μM) or SP600125 (50μM) for 1 hour, then treated with IL-17 50 ng/ml for 1 hour. Synoviolin expression was measured by real-time PCR, mean ± SEM, n = 3 (<b>D</b>). * p<0.05 compared to IL-17 treatment.</p

Effect of IL-17 on RA FLS apoptosis.

<p>RA FLS were treated with SNP 0.01-1 µM for 24 h and apoptosis measured by SS DNA apoptosis kit (<b>A</b>). The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * <i>P</i><0.05 compared to no treatment control. RA FLS were treated with SNP 0.01–1 µM for 24 h (<b>B, left panel</b>) or 0.1 µM over 24 h (<b>B, right panel</b>) and synoviolin expression measured by real-time RT-PCR. The results are expressed as the ratio of synoviolin/β-actin mRNA amplification, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * <i>P</i><0.05 compared to no treatment control. RA FLS were pretreated with IL-17 100 ng/ml for 2 h then cotreated with SNP 0.1-1 µM for 24 h and apoptosis measured by SS DNA apoptosis kit (<b>C</b>). The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * <i>P</i><0.05 compared to no treatment control. RA FLS were treated as above and annexin V analysed. Representative serial fluorescent microscopic pictures of Annexin V positive cells (green), double labelled with Propidium iodide (PI, red) at magnification x 200 (<b>D</b>).</p

<i>C. albicans</i> skews T-cell cytokine profile.

<p>Serum levels of anti-SCW-specific total IgG, IgG1, and IgG3 antibodies (A). Draining lymph node cells (1*10⁵/well) were stimulated with anti-CD3/anti-CD28 antibodies for 72 hours (n=6/group). Levels of IFN-γ, IL-17 and IL-10 were determined by Luminex in the supernatants of the stimulated lymph node cells (B) and washouts of synovial biopsies of day 22 (C). Results are the mean ± SEM; ns=non significant *=P<0.05; **=P<0.01, ***=P<0.001, by One-Way ANOVA.</p

Effect of IL-17 and TNF on RA FLS synoviolin expression and apoptosis.

<p>RA FLS were stimulated with IL-17A (50 or 100 ng/ml) and/or TNF (1 or 10 ng/ml) for 24 h and synoviolin mRNA expression measured by real-time RT-PCR (<b>A</b>). The results based on a ratio of synoviolin/β-actin mRNA amplification are presented as the fold induction in synoviolin mRNA expression relative to control samples, n = 3, mean ± SEM. * <i>P</i><0.05 compared to no treatment control. † <i>P</i><0.05 compared to TNF treated samples. RA FLS were pretreated with IL-17 100 ng/ml and TNF 10 ng/ml for 2 h then cotreated with SNP 0.5 µM (<b>B, left panel</b>) or 0.1 µM (<b>B, right panel</b>) for 24 h and apoptosis measured by SS DNA apoptosis kit. The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * <i>P</i><0.05 compared to SNP treatment in S-08 nucleofected RA FLS.</p

Severity of chronic streptococcal cell wall-induced arthritis in wild-type (WT) or IL-17R ^−/− mice A).

<p>(A representative image of haematoxylin and eosin-stained synovial knee joint sections in WT or IL-17R <b>⁻</b>^/<b>−</b> mice at day 42 after 5 repeated injections of streptococcal cell wall (SCW) fragments (magnification ×40). Arthritis severity and inflammatory infiltrate were scored histologically in injected joints of WT or IL-17R <b>⁻</b>^/<b>−</b> mice. Arthritis severity was scored on a scale of 0–2 and synovial infiltrate on a scale of 0–3. Results are expressed as mean ± SEM of 2 separate experiments, n = 10 mice per group per experiment, * <i>P</i><0.05 compared to WT mice. <b>Synoviolin expression is reduced in IL-17R ^−/− mice with chronic SCW-induced arthritis</b> (<b>B</b>). Synoviolin expression in sections of arthritic knee joints from, a wild-type (WT) or IL-17R <b>⁻</b>^/<b>−</b> mouse after 5 repeated injections of SCW fragments (magnification x 40, day 42, n = 10 mice in WT or IL-17R <b>⁻</b>^/<b>−</b> groups from 2 separate experiments). Synoviolin expression was quantified in 10 high power fields, averaged and expressed as the number of synoviolin positive cells/mm², * <i>P</i><0.05 compared to WT mice. <b>Synovial apoptosis in WT or IL-17R ^−/− mice with chronic streptococcal cell wall-induced arthritis</b> (<b>C</b>). A representative image of increased TUNEL staining in IL-17R <b>⁻</b>^/<b>−</b> synovium (magnification ×200.). The number of TUNEL-positive cells was quantified in 10 high power fields, averaged and expressed as the number of TUNEL positive cells/mm², mean ± SEM, * <i>P</i><0.05 compared to WT mice. <b>Synovial PCNA staining in knee joint sections from WT or IL-17R ^−/− mice with chronic streptococcal cell wall-induced arthritis</b> (<b>D</b>). A representative image of PCNA staining in synovial sections (magnification ×200). The number of PCNA-positive cells was quantified in 10 high power fields, averaged and expressed as the number of PCNA positive cells/mm², mean ± SEM, * <i>P</i><0.05 compared to WT mice.</p

Functional Tissue Analysis Reveals Successful Cryopreservation of Human Osteoarthritic Synovium

<div><p>Osteoarthritis (OA) is a degenerative joint disease affecting cartilage and is the most common form of arthritis worldwide. One third of OA patients have severe synovitis and less than 10% have no evidence of synovitis. Moreover, synovitis is predictive for more severe disease progression. This offers a target for therapy but more research on the pathophysiological processes in the synovial tissue of these patients is needed. Functional studies performed with synovial tissue will be more approachable when this material, that becomes available by joint replacement surgery, can be stored for later use. We set out to determine the consequences of slow-freezing of human OA synovial tissue. Therefore, we validated a method that can be applied in every routine laboratory and performed a comparative study of five cryoprotective agent (CPA) solutions. To determine possible deleterious cryopreservation-thaw effects on viability, the synovial tissue architecture, metabolic activity, RNA quality, expression of cryopreservation associated stress genes, and expression of OA characteristic disease genes was studied. Furthermore, the biological activity of the cryopreserved tissue was determined by measuring cytokine secretion induced by the TLR ligands lipopolysaccharides and Pam3Cys. Compared to non frozen synovium, no difference in cell and tissue morphology could be identified in the conditions using the CS10, standard and CryoSFM CPA solution for cryopreservation. However, we observed significantly lower preservation of tissue morphology with the Biofreeze and CS2 media. The other viability assays showed trends in the same direction but were not sensitive enough to detect significant differences between conditions. In all assays tested a clearly lower viability was detected in the condition in which synovium was frozen without CPA solution. This detailed analysis showed that OA synovial tissue explants can be cryopreserved while maintaining the morphology, viability and phenotypical response after thawing, offering enhanced opportunities for human <i>in vitro</i> studies.</p></div

Effect of synoviolin knockdown on apoptosis in RA FLS.

<p>RA FLS were nucleofected (amaxa) for 24 h with 0.5 µg of 4 synoviolin siRNA duplexes (S-01 to S-04), synoviolin smartpool siRNA duplex or siCONTROL siRNA (sictl) and synoviolin expression analysed by real-time RT-PCR <b>(A, left panel)</b> or Western Blot <b>(A, right panel).</b> Synoviolin mRNA was normalized to GAPDH and results expressed as the percentage fold reduction compared to sictl treated samples, n = 5, mean ± SEM of duplicate experiments. * <i>P</i><0.05, compared to sictl nucleofected RA FLS. (<b>B</b>), RA FLS were nucleofected as above then treated with SNP 0.1–1 µM overnight and apoptosis measured by the SS DNA apoptosis assay. Results are expressed as fold induction in apoptosis, n = 3 from 3 separate RA donors, mean ± SEM of duplicate experiments. * <i>P</i><0.05, compared to sictl nucleofected RA FLS. (<b>C</b>), RA FLS were nucleofected as above, serum starved overnight then pretreated with 100 ng/ml IL-17A for 2 h then cotreated with SNP 0.1–1 µM overnight and apoptosis measured by SS DNA apoptosis assays expressed as fold induction of apoptosis, n = 3 from 3 separate RA donors, mean ± SEM of duplicate experiments. * <i>P</i><0.05, compared to sictl nucleofected RA FLS. † <i>P</i><0.05 compared to SNP treatment in S-08 nucleofected RA FLS.</p