Highly reduced Th17 differentiation in the absence of both IL-6 and IL-21 signaling.

<p>WT and IL-21R^-/- naïve T cells were stimulated for four days with a differentiation cocktail either with or without IL-6 as described in <i>Methods</i>. Representative flow cytometry dot plots reflecting the IL-17+ fraction of the CD4+ population. Gates were set at a maximum of 0.3% IL-17-positive cells in the ‘fluorescence minus one’ control per condition, without addition of IL-17A antibodies (A). Summary of the relative proportion of IL-17+ cells within the CD4+ population (B). Culture supernatant levels of IL-17 (C), IL-21 (D), and IL-22 (E). 6–10 mice/group; *p<0.05, **p<0.01, ***p<0.001 versus WT + IL-6; ^###p<0.001 versus WT—IL-6; ^p<0.05, ^^^p<0.001 versus IL-21R^-/- + IL-6; A—Kruskal-Wallis, B-D—One-way ANOVA.</p

IL-6 and IL-21 play an important role during in vivo Th17 differentiation and antibody production.

<p>AIA was induced in WT, IL-6^-/-, IL-21R^-/-, and IL-6^-/- x IL-21R^-/- mice. Draining lymph node CD4+IL-17+ cell fraction two days after arthritis induction as measured using flow cytometry (A; n = 10/group). Serum IgG1, IgG2b, and total IgG levels as measured by ELISA (B; n = 5/group). *p<0.05, **p<0.01, ***p<0.001 versus WT; ^##p<0.01 versus IL-6^-/-; ^^^p<0.001 versus IL-21R^-/-; A—One-way ANOVA, B—Kruskal-Wallis.</p

Arthritis incidence and severity of mice receiving anti-IL-6 and/or anti-IL-21 treatment late in disease development.

<p>Collagen induced arthritis was initiated in DBA-1 mice, subsequently treated with anti-IL-6R antibodies and/or sIL-21R.Fc from the day of booster injection (d = 21; n = 5/group). Arthritis incidence (A) and severity (B) based on macroscopic scoring. Bone damage as measured using the Faxitron depicted as radiological damage (C). Histologically scored inflammation, bone erosion (both H&E staining, scale bar 500 μM), and cartilage proteoglycan depletion (SafO staining, scale bar 200 μM) (D). *p<0.05 versus Rat IgG1; One-way ANOVA.</p

Effect of anti-IL-6 and/or anti-IL-21 treatment on Th17, Th1, and antibody development in CIA mice.

<p>Th17 (A) and Th1 (B) levels in draining lymph nodes as measured using flow cytometry, and anti-CII antibody level in serum (C) as measured by ELISA of mice with CIA receiving anti-IL-6 and/or anti-IL-21 treatment. n = 5/group; **p<0.01 versus Rat IgG1; A+C—Kruskal-Wallis, B—One-way ANOVA.</p

Antigen-induced arthritis severity is potently reduced by combinatorial blockade of IL-6 and IL-21 signaling pathways.

<p>Joint swelling of WT, IL-6^-/-, IL-21R^-/-, and IL-6^-/- x IL-21R^-/- mice at day 1 (A), day 2 (B), and day 4 (C) after arthritis induction, depicted as ratio between right and left knee joint, measured by ^99mTechnetium pertechnetate uptake in the joint (n = 6/group). Histologically scored inflammation, bone erosion (both H&E staining, scale bar 500 μM), and cartilage proteoglycan depletion (SafO staining, scale bar 200 μM) (D; n = 10/group). *p<0.05, **p<0.01, ***p<0.001 versus WT; ^#p<0.05, ^###p<0.001 versus IL-6^-/-; ^p<0.05 versus IL-21R^-/-; A+B One-way ANOVA, C+D Kruskal-Wallis.</p

Additional file 4: of Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis

NIMPR14- and F4/80-positive cells in the infiltrate and exudate in the joints of FcγRI,II,III−/− mice and their WT controls. Representative photomicrographs of (a) NIMPR14 and (b) F4/80 staining showing neutrophils and macrophages in the infiltrate and exudate of the knee joints of FcγRI,II,III−/− mice and their WT controls at day 7 after induction of antigen-induced arthritis. Original magnification ×400 for infiltrate and ×200 and ×400 for exudate. (PDF 401 kb

Additional file 2: of FcÎł receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis

Gating strategy for flow cytometric analysis. Gating strategy for flow cytometric analysis used to identify CD11bposLy6Chigh and CD11blow/negLy6Chigh osteoclast precursor populations. First, single cells were selected. For identification of CD11bposLy6Chigh monocytes, cells negative for CD90.2, CD45R/B220, CD49b, NK1.1, and Ly6G and positive for CD11b were selected (gate A). Subsequently, cells were back-gated for side scatter and forward scatter to exclude cells with high granulosity (gate B), and finally Ly6Chigh cells were selected (gate C). For identification of CD11blow/negLy6Chigh, after exclusion of CD90.2-, CD45R/B220-, CD49b-, NK1.1-, Ly6G-positive cells (gate D), cells were gated for their expression of CD11b and Ly6C (CD11Blow/negLy6Chigh) (gate E). (PDF 299 kb

Additional file 3: of Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis

NIMPR14- and F4/80-positive cells in the infiltrate and in the exudate in the joints of FcγRI,II,III,IV−/− mice and their WT controls. Representative photomicrographs of (a) NIMPR14 and (b) F4/80 staining showing neutrophils and macrophages in the infiltrate and exudate of the knee joints of FcγRI,II,III,IV−/− mice and their WT controls at day 7 after induction of antigen-induced arthritis. Original magnification ×400 for infiltrate and ×200 and ×400 for exudate. (PDF 422 kb

Additional file 1: of Fcγ receptor-mediated influx of S100A8/A9-producing neutrophils as inducer of bone erosion during antigen-induced arthritis

Graphical representation of bone erosion scoring method and quantification of noncartilage collagenous tissue and proteoglycan (PG) depletion. a Graphical representation of the 13 locations along the patella, femur, tibia, and cruciate ligament where bone erosion was scored. b Quantification of the noncartilage collagenous tissue (blue staining in Safranin O/Fast Green staining) in femur and tibia showed no differences between naive FcγRI,II,III,IV−/− and wild-type (WT) mice. ns Not significant. c Quantification of PG depletion showed a significant decrease at the tibiofemoral area in the joints of FcγRI,II,III,IV−/− mice as compared with their WT controls (n = 10 and 14 joints per group, respectively) at both 7 and 21 days after AIA induction. Scatterplots are shown, with horizontal and vertical lines showing mean ± SEM values. ns Not significant. * P < 0.05, ** P < 0.01 versus WT controls. (PDF 437 kb