FIG. 5.

Internalization is a critical event for stimulation of cytokine production by live spirochetes. A. B. burgdorferi-GFP was incubated with PBMCs in the presence of LysoTracker Red for 4 h at an MOI of 10:1 prior to fluorescence microscopy. The merged image (yellow) shows colocalization of GFP (green) with endosomal compartments (red). B. PBMCs were incubated with T. pallidum at an MOI of 10:1 in the presence of LysoTracker Red for 4 h prior to IFA and fluorescence microscopy. C. Effect of CytoD on the TNF-α response of PBMCs to live and lysed B. burgdorferi (Bb). Shown are results from one of two experiments; error bars represent the standard deviations for duplicate samples. D. CytoD inhibits uptake of live B. burgdorferi by monocytes. PBMCs were preincubated with cytochalasin D (10 μg/ml) for 1 h prior to the addition of B. burgdorferi-GFP at various MOIs for 8 hours. The cells then were stained with CD14 and analyzed by flow cytometry to determine the percentage of monocytes that had internalized spirochetes (GFP-positive cells) and the number of spirochetes taken up per monocyte (represented by the mean fluorescence intensity [MFI] of GFP-positive cells). Shown are the means ± standard deviations from four independent experiments. Asterisks indicate P values of ≤0.05 for CytoD-treated and untreated samples. UN, unstimulated.