Fig 2: Attenuation of arthritis via regulation of the phenotype of immune cells in p40-EBI3 transgenic C57BL/6 mice achieved by microinjection of the transgene. Collagen-induced arthritis (CIA) was induced in WT or p40-EBI3 transgenic C57BL/6 mice as described in the Methods section. 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 The concentrations of total IgG in sera from mice in each group at 12 weeks after CII immunization were determined by ELISA. C Spleens were obtained from mice in each group at 12 weeks after CII immunization and analyzed by flow cytometry for B220+GL-7+ germinal center B cells and B220-CD138+ plasma cells. Representative flow cytometry plots from one of three independent experiments are shown (left); the results are depicted as the mean ± SD of three mice per group (right). D Mice were sacrificed at 12 weeks after CIA induction; the populations of IFN-?+, IL-4+, IL-17+, and CD25+Foxp3+ cells among splenic CD4+ T cells were analyzed by flow cytometry. Representative flow cytometry plots from one of three independent experiments are shown (left); the results are depicted as the mean ± SD of three mice per group (right) (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: 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).

Fig 4: 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 5: Identification of the p40-EBI3 protein in mice. A Splenocytes isolated from normal BALB/c mice were stimulated with LPS, zymosan, or Con A or were untreated (Nil). The expression levels of p40-EBI3 in culture supernatants were detected using ELISA and converted into OD (405 nm) and pg/mL values. B Detection of p40-EBI3 protein expression in splenic non-T cells by immunoprecipitation with western blot analysis. Splenic non-T cells isolated from WT BALB/c mice were stimulated under different conditions for 3 days. Prepared cell lysates were immunoprecipitated with an anti-EBI3 antibody and subjected to western blotting with an anti-p40 antibody. C CD11c+ splenic dendritic cells isolated from normal BALB/c mice were stimulated under different conditions. The expression levels of the p40-EBI3 protein in culture supernatants were determined by ELISA. D Detection of p40-EBI3 protein expression in splenic non-T cells by EBI3 immunoprecipitation with p40 western blot analysis. Splenic non-T cells isolated from WT BALB/c mice were stimulated with 5 µg/mL ConA for 3 days. The p40-EBI3 protein was detected in the cell lysates and culture supernatant. E Schematic map of the p40-EBI3 plasmid vector (details described in the Methods section). F HEK293 cells were transfected with the p40-EBI3 plasmid vector. The expression levels of p40, EBI3, and FLAG in transfected cells were determined by western blotting. G The expression levels of p40-EBI3 in the culture supernatants in E were determined by ELISA (*p < 0.05, **p < 0.01, ***p < 0.001).