FIG 2.

Myd88 and Trif are required for cytokine production and the restriction of intracellular Coxiella burnetii Nine Mile II replication in C57BL/6 macrophages. (A and B) C57BL/6, Trif^−/−, Myd88^−/−, and Myd88^−/− Trif^−/− BMDMs were infected with WT C. burnetii NMII at MOIs of 5 and 50 for 24 h. Levels of TNF and IL-6 in the supernatants were measured by ELISA. Graphs show the means ± SEM from triplicate wells. Results are representative of two independent experiments. (C) C57BL/6, Trif^−/−, Myd88^−/−, and Myd88^−/− Trif^−/− BMDMs were infected with WT C. burnetii NMII at an MOI of 100. At days 1 and 7 postinfection, bacterial uptake and replication were measured as genomic equivalents (GEs) by qPCR. Graphs show the fold change in GEs relative to the GEs measured on day 1 ± SEM from triplicate wells. (D) Seven days postinfection, BMDMs of the indicated genotypes infected with mCherry-expressing WT C. burnetii NMII at an MOI of 100 were fixed, stained with DAPI, and examined by fluorescence microscopy. The number of large NMII-containing vacuoles was determined and calculated as a percentage of the total cell number on day 7 postinfection. Graphs show the mean percentage of cells containing C. burnetii vacuoles ± SEM from triplicate coverslips. At least 300 cells were counted per coverslip. Results are representative of two independent experiments. (E) Representative fluorescence micrographs of BMDMs of the indicated genotypes infected with mCherry-expressing C. burnetii (Cb) NMII at an MOI of 100 and fixed and stained with DAPI on day 7 postinfection. Images were taken at ×40 magnification. Scale bars represent 25 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance.