Effect of Lip-PLP on M1 and M2 marker expression within the synovium during ICA.

<p>A: Photomicrographs of frontal knee joint sections of mice with ICA at day 1 after treatment. B: Histological scoring of synovial infiltration mice with ICA at day 1 after treatment with saline or Lip-PLP. C+D: Expression of M1 (C) and M2 (D) markers in the synovium during ICA. RE = Relative Expression compared to values of GAPDH. Mice were treated at day 1 after induction of ICA and biopsies were obtained at day 1 after treatment. Data are expressed as mean +/− SD of eight animals. UD = undetectable. Statistical significance was determined by Student's t-test. * = P<0.05, ** = P<0.01 compared to saline treatment.</p

Liposomal targeting of PLP to the inflamed synovial lining strongly suppresses joint inflammation during AIA.

<p>A: Knee joint swelling as measured by ^99MTc-uptake is strongly suppressed after a single injection of Lip-PLP. B: Photomicrographs of frontal knee joint sections of mice with AIA at day 5 after treatment and naïve mice. Note that the inflammatory infiltrate is reduced in mice treated with Lip-PLP. Original magnification ×100, Asterisks points to synovial infiltrate, hash sign points to inflammatory exudates. C: Histological scoring of synovial infiltration at day 1 and day 5 after systemic treatment with Lip-PLP or saline. D: Silver staining of frontal knee joint sections of mice with AIA, treated by intravenous injection with gold-containing liposomes. Note that the silver staining of the gold particles is mostly observed within the synovial lining cells (arrows). Mice were treated at day 3 after induction of AIA. Values are the mean of 8 mice per group. Original magnification ×100; insert ×400. F = femur, JS = joint space. Statistical significance was determined by Student's t-test. * = P<0.05 compared to saline treatment.</p

Effect of Lip-PLP on M1 macrophages <i>in vitro</i>.

<p>Cells and supernatant of bone-marrow macrophages (BMM) and M1 macrophages (stimulated with LPS and IFN-γ for 24 hours) were obtained 24 hours after treatment with Lip-PLP or saline. A: Uptake of fluorescent liposomes by BMM and M1 macrophages as measured by flow cytometry. Note that uptake of liposomes is dependent on the amount of liposomes but not on PLP content. B: Protein levels of M1 cytokines TNF-α, IL-6 and IL-12 within the supernatant and surface expression of M1 marker CD86 as determined by flow cytometry. C: Gene expression of M2 markers. RE = Relative Expression compared to values of GAPDH. Data are expressed as mean +/− S.D. UD = undetectable. Three independent experiments were performed. Statistical significance was determined by Student's t-test. * = P<0.05, ** = P<0.01 compared to saline treatment.</p

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