Fig 2: In vivo antiosteoclastogenic and anti-inflammatory properties of the p40-EBI3-Fc protein with respect to the development of collagen-induced arthritis (CIA). Mice with CIA received the p40-EBI3-Fc protein (2.5 mg/kg) or vehicle intraperitoneally daily after induction of CIA for 9 weeks. A Representative results from one of three independent experiments are shown for the clinical scores (left) and incidences of arthritis (right) for each treatment group over time. B At 63 days after initial CII immunization, tissues were obtained from the ankle joints of mice with CIA and stained with hematoxylin and eosin (H&E; original magnification, ×40), safranin O (original magnification, ×200), toluidine blue (original magnification, ×200), or tartrate-resistant acid phosphatase (TRAP; original magnification, ×200) to examine arthritis severity. C Isolated splenic CD4+ T cells from each group of mice were cultured for 3 days. T cell proliferation was determined by a [3H] thymidine incorporation assay. D The concentrations of CII-specific IgG, IgG1, and IgG2a in sera from mice in each group were determined by ELISA. Bars show the mean ± SD of four mice per group. E Tissue sections generated from the ankle joints of mice with CIA treated with or without p40-EBI3-Fc were stained with antibodies against TNFa, IL-1ß, IL-6, RANKL, VEGF, and HIF-1a or with an isotype control. Cells stained with each antibody are shown in brown (upper). The numbers of positive cells were counted in high-power fields (magnification, ×400) with the aid of Adobe Photoshop software and averaged for three randomly selected fields per tissue section. Bars show the mean ± SD of three mice per group (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: W37 of murine p40 is important for binding of HIL-23 to murine IL-12Rß1.A, alignment of murine and human p40 N-terminal amino acids Y31 to P39. Single, double, and triple substitutions within mp40 are highlighted in red. B, Western blot analysis of secreted murine HIL-23 variants from transfected CHO-K1 cells. C, cellular proliferation of Ba/F3-gp130-mIL-12Rß1-mIL-23R cells. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells). Parental Ba/F3-gp130 cells were used as controls. The results of one representative experiment of three are shown. Error bars represent S.D. for technical replicates. Statistical analysis used a one-way ANOVA, followed by Bonferroni correction (n = 3), ***p = 0.001, ns not significant. D, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rß1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of three. E, co-IP of FLAG-tagged murine HIL-23 variants (wild-type, D36K, W37K and T38K) and full-length mIL-12Rß1. The position of mIL-12Rß1 and HIL-23 variants is indicated by arrows. One of two independent experiments is shown. F, co-IP of FLAG-tagged murine HIL-23 variants (wild-type, D36K/W37K/T38K, D36K/W37K, W37K/T38K) and full-length mIL-12Rß1. The position of mIL-12Rß1 and HIL-23 variants is indicated by arrows. One of two independent experiments is shown.

Fig 4: Alanine substitutions of p40 D36, W37 and T38 impaired HIL-23 activity.A, alignment of murine and human p40 N-terminal amino acids Y31 to P39. Single and triple substitutions within mp40 are highlighted in red. B, Western blot analysis of secreted murine HIL-23 variants from transfected CHO-K1 cells. Cytokine variants were transiently expressed with comparable efficiency. C, analysis of STAT3 and ERK1/2 activation. Ba/F3-gp130-mIL-12Rß1-mIL-23R cells were washed, starved, and stimulated with the indicated cytokines (10% conditioned cell culture supernatant of transfected CHO-K1 cells) for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 µg/lane) were loaded on SDS-PAA gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-ERK1/2, and ERK1/2. Western blotting data show results of one representative experiment of three. D, cellular proliferation of Ba/F3-gp130-mIL-12Rß1-mIL-23R cells. The cells were cultured for 3 days in the presence of the indicated cytokines (0.04–20% conditioned cell culture supernatant of transfected CHO-K1 cells). The results of one representative experiment of four are shown. Error bars represent S.D. for technical replicates.

Fig 5: The p40-EBI3-Fc protein reduced proinflammatory cytokine levels and regulated the Th17/Treg cell balance. A Schematic map of the p40-EBI3-Fc expression vector used in CHO cells. B Western blotting for IgG Fc and IL-12p40 under treatment with different concentrations of p40-EBI3-Fc. C p40-EBI3-Fc protein expression was determined by ELISA. D Various proteins (p40-EBI3-Fc, recombinant IL-12p40, p(40)2, IL-12, IL-23, and IL-27) were immunoprecipitated with an anti-EBI3 antibody and subjected to western blotting with an anti-p40 antibody. E The expression levels of p40-EBI3-Fc and each recombinant protein were determined by p40-EBI3 ELISA. F The concentrations of TNF-a, IL-6, and IL-17 in the culture supernatants of cytokine-stimulated murine splenocytes were detected by ELISA. G The concentrations of TNF-a, IL-6, and IL-17 in the culture supernatants of LPS-stimulated murine splenocytes treated with or without the synthesized p40-EBI3-Fc protein at various concentrations (0.01-1 µg/mL). H Splenic CD4+ T cells from C57BL/6 mice were cultured under Th17 cell-inducing conditions in the presence or absence of the p40-EBI3-Fc protein (1 µg/mL). Three days later, the cells were stained with antibodies against CD4, IL-17, CD24, and Foxp3. Plot from a representative experiment showing the frequencies of IL-17+ and CD25+Foxp3+ cells among CD4+ T cells. Data are representative of four independent experiments with similar results. I IL-17 and Foxp3 mRNA expression levels were determined by real-time polymerase chain reaction. The values shown represent the mean ± SD. J Plot from a representative experiment showing the frequencies of CD25+Foxp3+ cells among CD4+ T cells under Th0, Th1, Th2, Th17, or Treg cell-inducing conditions. Data are representative of four independent experiments with similar results (*p < 0.05, **p < 0.01, ***p < 0.001).