Fig 4. S. aureus stimulates RANKL in isolated mouse calvarial osteoblasts independent on cytokine but dependent on prostaglandin productions and TLR2.

(A) S. aureus time-dependently stimulated Tnfsf11 mRNA without affecting Tnfrsf11b mRNA in mouse osteoblasts. (B) Concentration-dependent stimulation of Tnfsf11 mRNA, with no effect on Tnfrsf11b mRNA, by S. aureus (3x10⁶ CFU/ml). (C) S. aureus (3x10⁶ CFU/ml) upregulated the mRNA expression of Il1b, Il11, Il6, Lif, Osm and Tnfsf2 in osteoblasts. (D) The stimulatory effect by S. aureus (3x10⁶ CFU/ml) on Tnfsf11 mRNA expression in osteoblasts was unaffected by adding a mixture of antibodies neutralizing IL-1β, IL-6, IL-11, LIF, OSM and TNF-α. (E) S. aureus (3x10⁶ CFU/ml) stimulated Ptgs2 mRNA in mouse osteoblasts. (F) Indomethacin (1 μmol/l) abolished Tnfsf11 mRNA in osteoblasts induced by S. aureus (3x10⁶ CFU/ml). (G, H) The stimulatory effect by S. aureus (3x10⁶ CFU/ml) on Tnfsf11 and Ptgs2 mRNA was observed in osteoblasts from wild type mice but not from Tlr2 deficient mice. Data are means of 5 observations and SEM is given as vertical bars when larger than the radius of the symbol. In Fig 4A, effects on Tnfsf11 mRNA were statistically significant at 1–48 h (P<0.001). In Fig 4B, effects on Tnfsf11 mRNA were statistically significant at 10⁶ and 3x10⁶ (P<0.01) and at 10⁷ and 3x10⁷ (P<0.001) CFU/ml. ***P<0.001 compared to unstimulated control (D-G) or to S. aureus stimulated osteoblasts (G).