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. 2015 Mar 10;6(2):e00133-15. doi: 10.1128/mBio.00133-15

FIG 5 .

FIG 5 

GAS SpyA activation of a caspase-1-dependent inflammasome response is required for bacterial clearance by cultured BMDMs and in vivo. (A) BMDMs were coincubated with GAS (AP) at an MOI of 20 plus 10 µM pan-caspase inhibitor zVAD-FMK (ZVAD) or 25 µM caspase-1 inhibitor Ac-YVAD-CHO (YVAD) for 2 h. Data shown are representative of the results of multiple experiments. (B) Relative levels of LDH release (percentages compared to levels in uninfected [UI] cells; left panel) and bacterial killing (right panel) by WT and Casp-1−/− BMDMs 2 h postinfection. (C) Relative levels of LDH release (left panel) and bacterial killing (right panel) 2 h postinfection (MOI = 5) by BMDMs isolated from wild-type (C57bl6), Nlrp3−/−, Asc−/−, and IL-1β−/− mice (n = 3). Data shown are representative of the results from two or more animals. (D) ELISA shows IL-1β secreted by BMDMs isolated from wild-type (C57bl6), Nlrp3−/−, Asc−/−, and IL-1β−/− mice after 2 h coincubation with GAS at an MOI of 20. Data shown are representative of the results from two animals. (E) CD1 mice were infected with 7 × 105 CFU of WT or ΔspyA GAS (AP) via tail-vein injection (n = 7 per group). Immediately after infection, anakinra (IL-1 receptor antagonist) was subcutaneously injected at 100 mg/kg over a 12-h interval until the end of survival curve. WT plus saline solution versus ΔspyA plus saline solution, *, P = 0.04; WT plus saline solution versus WT plus anakinra. **, P = 0.0036; WT plus anakinra versus ΔspyA plus anakinra, P = 0.06; ΔspyA plus saline solution versus ΔspyA plus anakinra, P = 0.07 (log rank test with 95% confidence interval); N.S., not significant (P > 0.05). (F) GAS-infected mice (n = 8 to 10) were sacrificed 2 days postinfection; heart, spleen, and liver were harvested for CFU enumeration; no statistically significant differences were observed between WT and ΔspyA-infected animals that received anakinra treatment or for ΔspyA-infected animals with saline solution treatment. Closed symbols = WT; open symbols = ΔspyA. Black = control mice, red = anakinra-treated mice. Error bars, SEM. Statistical analysis: *, P < 0.05; N.S., not significant (P > 0.05) (Student’s two-tailed unpaired t test). (G) BMDMs in RPMI plus 2% FBS were treated with 1 or 10 ng/ml of recombinant mouse IL-1β (PeproTech, Rocky Hill, NJ) at the time of infection. Cells were washed and CFU recovered 2 h postinfection to assess bacterial killing. Error bars, SEM. *, P < 0.05; **, P = 0.01; ***, P < 0.001; NS, not significant; n = 3 (Student’s unpaired two-tailed t test).