Figure. 1. AEBS suppresses inflammatory arthritis in CIA mice. Both the arthritis score and the incidence of arthritis were reduced in CIA mice given AEBS. Mice were immunized with CII. Seven days later, mice were given oral AEBS (60 mg/kg) or vehicle, daily, for 8 weeks. (A) The mean arthritis scores ±± SDs (left panel) and arthritis incidence scores (right panel). (B) Histological data on joints from CIA mice given AEBS or vehicle. Mice were sacrificed on day 49 after immunization. Joint tissue sections were stained with H&E, toluidine blue, and safranin O. Joints from mice given AEBS exhibited mild erosive arthritis, whereas those from vehicle-treated mice exhibited markedly erosive destructive arthritis. Representative photographs are shown. Original magnifications ×40 or ×200, as indicated. The histological scores (measuring inflammation and the extent of cartilage damage) in mice given AEBS (n = 5) or vehicle (n = 5) are shown in the right graph. Data are expressed as means ± ± SDs. ***P < 0.01 compared to the vehicle-treated control group. (C) Tissue sections from joints of CIA mice given AEBS or vehicle were stained with anti-nitrotyrosine and anti-iNOS antibodies. Cells stained with either antibody are brown in color. Original magnification ×400. The cells showing positive nitrotyrosine, and iNOS were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented (cells/field). (D) Levels of total circulating IgG, IgG1, and IgG2a in CIA mice given AEBS (n = 8) or vehicle (n = 8). Total IgG, IgG1, and IgG2a levels were determined in sera of individual mice via ELISA. Data are expressed as means ±± SDs. * P < 0.05, *** P < 0.001 compared to the vehicle-treated group. Each experiment was performed 3 times. Figure. 2. AEBS reduces Th17 cell numbers in CIA mice. Tissues were obtained and stained when the mice were sacrificed (8weeks from immunization). (A) AEBS reduced the expression levels of IL-17, IL-6, TNF-α, and IL-1β in synovial tissues. Tissue sections from joints of CIA mice given AEBS (n = 5) or vehicle (n = 5) were stained with anti-IL-17, anti-IL-6, anti-TNF-α, and anti-IL-1β antibodies; or anti-isotype control antibodies. Stained cells are brown in color. Original magnification ×400. The cells showing positive IL-17, IL-6, TNF- α, and IL-1β were enumerated visually at higher magnification (projected on a screen) by four individuals, and the mean values are presented (cells/field). (B) Pooled splenocytes from CIA mice given AEBS were cultured with PMA (25 ng/ml) and ionomycin (250 ng/ml) for 30 min and p-IκB levels determined via Western blotting. Data are expressed as means ±± SDs. *P < 0.05 compared to the vehicle-treated group. Each experiment was performed 3 times. Figure. 3. AEBS decreases Th17 cell numbers in CIA mice. (A) Spleens from each mouse were stained for CD4+p-STAT3(Y705)+, CD4+p-STAT3(S727)+, CD4+STAT3+, and CD4+IL-17+ T cells using antibodies specific for CD4, STAT3Y705, STAT3S727, and IL-17. The cell populations were analyzed via laser confocal microscopy (original magnification ×400). (B) The numbers of T cells positive for CD4+STAT3Y705+, CD4+STAT3S727+, CD4+STAT3+, and CD4+IL-17+ in each mouse were visually counted at a higher magnification (after projection of fields onto a screen) and mean values are shown. (C) Spleens from each mouse were stained for CD4+p-STAT5(Y694)+, CD4+STAT5+, and CD4+CD25+Foxp3+. (D) The numbers of T cells positive for CD4+p-STAT5(Y694)+, CD4+STAT5+, and CD4+CD25+Foxp3+ in each mouse were visually counted at a higher magnification (after projection of fields onto a screen) and mean values are shown. (Number of AEBS treated mice = 5, Number of vehicle treated mice = 5) Data are expressed as means ±± SDs. *P < 0.05, **P < 0.01. Each experiment was performed 3 times. Figure. 4. AEBS represses IL-17 synthesis by mouse CD4+ T cells. (A) CD4+ T cells isolated from the spleens of naïve DBA/1J mice were cultured under Th17-polarizing conditions in the presence or absence of AEBS (50–200μg/ml). Three days later, the cells were stained with antibodies against CD4 and IL-17, as described in Materials and Methods. (B) The IL-17 levels in the culture supernatants described in (A) above were measured via ELISA. (C) The levels of IL-17 and Ahr mRNAs were determined by real-time PCR. (Number of AEBS treated mice = 3, Number of vehicle treated mice = 3) Each experiment was performed 3 times. Figure. 5. AEBS represses IL-17 synthesis by human PBMCs. (A) CD4+ T cells isolated from PBMCs of normal healthy volunteers were cultured under Th17-polarizing conditions in the presence or absence of AEBS (50–200μg/ml). Three days later, the cells were stained with antibodies against CD4 and IL-17, as described in the Materials and Methods section. The proportion of Th17 cells were measured by Side-scattered light (SSC) method. (B) The levels of IL-17 in the culture supernatants described in (A) above were measured using sandwich ELISA (R&D Systems, Minneapolis, MN). (C) The levels of IL-17 and RORc mRNAs were determined using real-time PCR. (Number of AEBS group = 3, Number of vehicle group = 3) Each experiment was performed 3 times. Figure. 6. AEBS inhibits osteoclast formation in DBA/1J mice. (A) In vitro, AEBS inhibited osteoclast formation in a dose-dependent manner. BMM cells from naïve DBA/1J mice treated with vehicle were cultured in the presence of M-CSF (10 ng/ml), and/or RANKL (50 ng/ml), and/or AEBS (50–150 μg/ml). The medium was changed every 2 days. After 7 days, cells were stained to detect TRAP activity. Original magnification ×100. ***P < 0.001. (B) The levels of mRNAs encoding the osteoclastogenic markers MMP-9, the calcitonin receptor, TRAP, OSCAR, and NFATc1 were measured via real-time PCR under the experimental conditions described in (A) above. **P < 0.01, ***P < 0.001. (Number of AEBS treated mice = 3, Number of vehicle treated mice = 3) Each experiment was performed 3 times. Figure. 7. AEBS inhibits the differentiation of human monocytes into osteoclasts. (A) AEBS inhibited osteoclast formation, in a dose-dependent manner, in the presence of M-CSF and RANKL. Human monocytes from healthy volunteers (n = 3) were cultured in the presence of M-CSF (25 ng/ml), and/or RANKL (30 ng/ml), and/or AEBS (50–150 μg/ml). After 9 days, cells were stained to detect TRAP activity. Representative photographs from each group are shown in the left panels. The numbers of multinucleated TRAP+ cells are shown in the right panel. Original magnification ×100. ***P < 0.001. (B) The levels of mRNAs encoding TRAP, MMP-9, and cathepsin K were quantified via real-time PCR. **P < 0.01, ***P < 0.001, compared to treatment with M-CSF and RANKL. (Number of AEBS group = 3, Number of vehicle group = 3) Each experiment was performed 3 times.