Figure 3. Proinflammatory cytokine production induced by Brucella is partially dependent on STING pathway.

BMDM derived from C57BL/6, STING^−/− and cGAS^−/− mice were transfected with DNA purified from B. abortus (1 μg/well) encapsulated with lipofectamine or infected with B. abortus (MOI 100:1). Total RNA was extracted and qPCR was performed to measure IFN-β (A) expression. Culture supernatants were harvested 17 hrs after treatment to measure CXCL10 (B), TNF-α (C), IL-6 (D) and IL-1β (E) by ELISA assay. Where indicated, cells were treated with 100 U/ml IFN-β 18 hrs prior the course of infection or were untreated. (F) The same culture supernatants or cell lysates were harvested 17 hrs after infection and pro-IL-1β (cell lysates), IL-1β (supernatant) and caspase-1 processing were determined by Western blot. Equal loading was controlled by measuring β-actin in the corresponding cell lysates. (G) NOS2 and (H) Arg1 gene expression was determined in macrophages infected with Brucella abortus (MOI 100:1) for 24 hours by qPCR. (I) Macrophages from C57BL/6 and MAVS^−/− mice were stimulated with B. abortus S2308 (MOI 100:1) for 24 hrs and qPCR analysis was performed for IFN-β expression. Data are representative of at least three independent experiments and three replicates in each experimental group. Significant differences comparing WT versus STING are denoted by an asterisk, WT versus cGAS are denoted by # and STING versus cGAS are denoted by & (two-way ANOVA, p< 0.05). *** P< 0.0001 for statistically significant differences from MAVS^−/− compared to C57BL/6.