Targeting the inflammasome and adenosine type-3 receptors improves outcome of antibiotic therapy in murine anthrax

Figure 1 Effects of IB-MECA on phosphorylation of AKT and cAMP level in human small airway lung epithelial cells. A: IB-MECA upregulates AKT phosphorylation in cultured human small airway lung epithelial cells (HSAECs) and prevents downregulation of AKT phosphorylation caused by exposure of the cells to B. anthracis Sterne spores (MOI of 10) when added to the culture medium for 5 min at the indicated times (B); C: IB-MECA treatment (2.5 nmol/L) downregulated EdTx-mediated cAMP production in HSAECs. Cells were starved in serum-free medium for 2 h and treated with EdTx (1 μg/mL edema factor and 5 μg/mL protective antigen) in complete culture medium containing 250 nmol/L IBMX, in the presence or absence of IB-MECA for 2 h. Cell lysates were analyzed with western blotting using antibodies specific for phospho-AKT (pAKT, S473) and total AKT (tAKT) (A and B), and competitive cAMP ELISA (C). Asterisks indicate the corresponding pairs of measurements (mean ± SD, n = 3) used for statistical comparison by the two-tailed t test. ^aP = 0.05, ^bP = 0.01.

Figure 2 Survival of B. anthracis Sterne-challenged DBA/2 mice after treatment. Mice challenged with B. anthracis Sterne spores (i.p.) were treated (s.c.) with indicated daily bolus doses. A: A3R agonist Cl-IB-MECA (0.05, 0.15 and 0.3 mg/kg); B: combination of Cl-IB-MECA (0.05, 0.15 and 0.3 mg/kg) with ciprofloxacin (50 mg/kg); C, D: Caspase-1 inhibitor YVAD (C: 2.5 mg/kg; D: 12.5 mg/kg) in combination with ciprofloxacin (50 mg/kg); E: Triple combination of caspase-1 inhibitor YVAD (2.5 mg/kg), A3AR agonist Cl-IB-MECA (0.15 mg/kg) and ciprofloxacin (50 mg/kg); F: Triple combination of caspase-1 inhibitor YVAD (12.5 mg/kg), A3AR agonist Cl-IB-MECA (0.3 mg/kg) and ciprofloxacin (50 mg/kg). Kaplan–Meier statistical log-rank analysis was used for survival data.