Additional file 2: of Are online symptoms checkers useful for patients with inflammatory arthritis?

Dataset used for this study. (XLSX 70 kb

Th17 polarising cytokines reduce CTLA-4 expression.

<p>Cell trace-labelled CD4+CD25- T cells were stimulated for four days with antiCD3CD28 beads under no cytokine supplement (Th0), with TGFβ alone or with the pro-Th17 cocktail (TGFβ with IL-1β, IL-6 and IL-23) as indicated and expression of total CTLA-4 and Foxp3 assessed by flow cytometry. <b>A)</b> Representative FACS plots showing CTLA-4 against Foxp3 expression and cell division, indicated by cell-trace dilution. <b>B)</b> Summary of CTLA-4 expression for 12 donor donors. Bars indicate mean values and error bars show standard deviation. Significance was tested by repeated measures, single factor within subject analysis (* = P<0.05, *** = P<0.001).</p

1,25(OH)₂D₃ promotes CTLA-4-mediated B7 depletion from dendritic cells and suppression of T cell proliferation.

<p>CTLA-4 expressing ‘suppressor cells’ were prepared by stimulating CD4+CD25- T cells under Th0 or Th17 conditions in the presence or absence of 1,25(OH)₂D₃. <b>A)</b> Suppressor cells were cultured with autologous DCs and antiCD3 for 24 hours with or without CTLA-4 blocking antibody. Expression of CD80, CD86, CD11c and CD40 by DCs was measured by flow cytometry. Dot plots show the ratio of marker expression in control versus anti-CTLA-4-treated cultures for four donors. <b>B)</b> Suppressor cells were CFDA-SE labeled and added to autologous DC plus antiCD3 stimulations of allogeneic cell trace violet labeled CD4+CD25- T cells (responders) with or without anti-CTLA-4. Parallel stimulations were also prepared in which CFDA-SE-labeled CD4+CD25- were added in place of suppressor T cells as a control for cell number. At five days, proliferation of responder T cells was assessed by flow cytometry. Data are from one donor but representative of four. Shaded histograms show proliferation in the absence of suppressors. Dotted and solid lines indicate proliferation in the presence versus the absence of anti-CTLA-4 respectively.</p

1,25(OH)₂D₃ maintains a regulatory T cell phenotype even under inflammatory, Th17 polarising conditions.

<p>CD4+CD25- T cells were stimulated in the presence of recombinant cytokines IL-1β, IL-6, IL-23 and TGFβ as indicated with or without 1,25(OH)₂D₃ and expression of regulatory-associated markers CTLA-4, Foxp3 and CD25 assessed at four days and cytokines IL-2, IL-17, IFNγ, IL-21 and IL-10 measured at five days by flow cytometry. Data are summarised for n≥5 donors. Bars indicate mean values and error bars show the standard deviation. Repeated measures, two factor within subject analysis was used to test interaction between 1,25(OH)₂D₃ and cytokine treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131539#pone.0131539.s002" target="_blank">S1 Table</a>). For markers that did not show interaction the two factor analysis was re-run in the absence of interaction and P values for each factor are shown (1,25(OH)₂D₃ = <i>P</i>_<i>D3</i> and cytokine treatment = <i>P</i>_<i>Cyt</i>. Where interaction was detected, single factor analysis was performed. P values are shown for the effect of cytokine treatment under control (<i>P</i>_{<i>cyt—D3</i>}) and 1,25(OH)₂D₃ (<i>P</i>_{<i>cyt + D3</i>}) conditions separately. Significant contrasts between cytokine treatments are indicated by stars (* = P<0.05, ** = P<0.01, *** = P<0.001).</p

Transendocytic function of CTLA-4 is promoted by 1,25(OH)₂D₃ and maintained under inflammatory conditions.

<p>CTLA-4 transendocytic function was tested as described in the methods. <b>A)</b> Gating strategy to ensure exclusion of CD86-GFP donor cells from the measurement of T cell GFP acquisition. <b>B)</b> Representative FACS plots of CD86-GFP acquisition versus trafficking CTLA-4. <b>C)</b> Total CTLA-4 mediated CD86 acquisition by T cells summarized for n = 5 donors. Bars indicate mean values and error bars show the standard deviation. P values for the separate effects of Th17 cytokines (P_cyt) and 1,25(OH)₂D₃ (P_D3) are shown as determined by the 2 factor without interaction model since no interaction was detected (P = 0.146).</p

Suppression of CTLA-4 by Th17 polarising cytokines is not specific to IL-17+ T cells.

<p>CD4+CD25- T cells were stimulated in the presence of Th17 polarising cytokines for four days and assessed for IL-17, IFNγ, IL-21, TNFα, IL-2 or Foxp3 in combination with CTLA-4 by flow cytometry. <b>A)</b> Frequency of total CTLA-4+ cells. <b>B)</b> Representative bivariate FACS plot of CTLA-4 versus IL-17 for cells cultured under Th17 polarising conditions. <b>C)</b> CTLA-4 expression in CTLA-4+ T cells gated according to IL-17 expression. <b>D)</b> CTLA-4 expression by CTLA-4+ T cells that expressed IL-17, IFNγ, IL-21, TNFα or IL-2. <b>E)</b> CTLA-4 expression in CTLA-4+ T cells defined by FoxP3 expression. In C, D and E expression under Th17 conditions is expressed relative to the level under Th0 conditions. Data are summarised for n≥7 donors. Bars indicate median values and error bars show the semi interquartile range. Significance with respect to cells expressing the marker under Th0 conditions was tested by Wilcoxon matched paired tests. (* = P<0.05, ** = P<0.01, *** = P<0.001).</p

Serum response in arthritis derived fibroblasts within anatomical sites.

<p>The figure describes the relationships between RA and OA derived fibroblasts in low and high serum conditions using PCA. Bone marrow (A), skin (B) and synovium (C) derived fibroblasts are represented separately.</p

Ingenuity Network analyses.

<p>The three major networks identified were: (A) Actin cytoskeleton remodelling by extracellular insulin-like growth factor binding proteins through gonadotropic hormones. (B) ITGB3 signalling connected to differentiation and angiogenesis. (C) Regulation of apoptosis through CD44. Yellow nodes represent genes resulting from input whereas empty nodes represent added genes by Ingenuity. Dashed and solid lines represent indirect and direct relationships respectively.</p

Differential gene expression analyses at tissue, serum and disease level.

<p>The figure shows the differential gene expression analysis performed within each level of the study. For each level of organisation we quantified the number of differentially expressed genes at an FDR < 5% followed by a 2-fold filter. This allowed us to devise a hierarchy of organisation that follows Anatomical Location then serum response and finally disease status (A). A more detailed tissue-by-tissue analysis to identify serum response genes in RA and OA fibroblasts (B) and to identify disease genes within high and low serum (C) was then performed. The arrows in panel A represent the direction of the hierarchy defined by the number of genes differentially expressed. BoM, Bone marrow.</p

Identification of a hierarchy of molecular signatures in arthritis derived fibroblasts.

<p>The relative similarity of the different groups of fibroblast in a principal component (PC) plot is shown. Groups defined by disease state and anatomical location are indicated by coloured symbols whereas individual samples response to serum (Hi, High serum; Lo, low serum) is shown by a solid grey line. The PCA clearly separates synovium (Syn, red and orange) from bone marrow (BoM, blue and grey) and skin (Skn, green and light blue) on the first PC. Skin and bone marrow samples are only separated on the second PC. Additionally it can be observed that all samples are separated by their serum response mainly on the first PC.</p