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. 2022 Oct 13;90(11):e00417-22. doi: 10.1128/iai.00417-22

FIG 2.

FIG 2

The timing of RANKL exposure alters the relative impact of TLR2 on bacterial-induced osteoclastogenesis and cytokine responses. (A–C) BMDMs from WT, Tlr2−/−, Tlr9−/−, and Tlr2/9−/− mice were plated in CMG 14-12 supernatant + 35 ng/mL RANKL. After 1 day of culture, RANKL and CMG 14-12 supernatant stimulation was maintained, and cells were exposed to the indicated vol/vol percentage of S. aureus supernatant from the Δpsmα1-4 strain or the vehicle control. After 4 more days of culture, the cells were fixed and stained for TRAP, and TRAP+, multinucleated osteoclasts were quantified. (A) Representative images of the 5% treatment condition were taken at 10× magnification. (B) Counts of TRAP+ multinucleated cells were analyzed by a two-way ANOVA, and Dunnett’s multiple-comparisons test was used to compare between the treatment and the vehicle for each genotype. *, P < 0.05; **, P < 0.01; ***, P = 0.0001; ****, P < 0.0001. If not denoted with asterisks, the differences between treatments were not statistically significant. Error bars denote the SD. The results are representative of three biological replicates containing n = 3 technical replicates per group. (C) Changes in osteoclast number were calculated relative to the average vehicle count for the respective genotype. For each treatment, a one-way ANOVA was used to assess the effect of the genotype, and Dunnett’s multiple-comparisons test was used to compare each genotype to WT within each supernatant concentration. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. If not denoted with asterisks, the differences between genotypes were not statistically significant. Error bars denote the standard deviation (SD). The results are representative of three biological replicates containing n = 3 technical replicates per group. (D and E) BMDMs were seeded in media with or without RANKL. After 2 days of culture, cells incubated in media without RANKL were treated with 7% vol/vol Δpsmα1-4 Δspa supernatant (“No RANKL”) or RANKL + 7% vol/vol Δpsmα1-4 supernatant (“RANKL during stim.”). Cells incubated in media with RANKL were treated with 7% Δpsmα1-4 Δspa supernatant with RANKL maintained (“continuous RANKL”). After an additional 12 h of incubation, cytokines were measured from the cell culture media using Luminex technology. Cytokine concentrations were evaluated via two-way ANOVAs, and Dunnett’s multiple-comparisons test was used to compare each genotype to the WT. *, P < 0.05; **, P < 0.01; ***, P = 0.0001; ****, P < 0.0001. If not denoted with asterisks, the differences between genotypes were not statistically significant. Error bars denote the standard deviation (SD). The results represent one biological replicate with n = 3 technical replicates per group.