Figure 1.
Activation of DCs infected with O. tsutsugamushi in vitro.
A. DCs were infected with O. tsutsugamushi for 24 h and stained with pooled scrub typhus patients' sera (green). The immunofluorescence image was merged with DIC image of the cells. B. DCs were stimulated with O. tsutsugamushi or LPS (0.5 µg/ml) for 20 h, stained with antibodies against the indicated surface molecules, and then analyzed by flow cytometer. Representative histograms of CD11c+-gated cells are presented. Gray filled: isotype control. C. The mean fluorescent intensity (MFI) of the surface markers from three separate experiments are presented. Error bar: mean+S.D., CNT: immature DCs, OT: DCs infected with O. tsutsugamushi, LPS: DCs stimulated LPS, OT2/LPS or OT6/LPS: DCs stimulated O. tsutsugamushi for 2 h (OT2) or 6 h (OT6) and then stimulated with LPS.
Figure 2.
Activation of DCs with increasing doses of live or inactivated O. tsutsugamushi.
DCs were stimulated with different doses (10 or 20 bacteria/cell) of live O. tsutsugamushi (OT), UV-inactive O. tsutsugamushi (UV-OT), heat-killed O. tsutsugamushi (H-OT), or LPS (0.5 µg/ml) for 20 h, stained with antibodies against the indicated surface markers, and analyzed by flow cytometric analysis. The mean fluorescent intensities (MFI) of the surface markers from three separate experiments are presented. Error bar: mean+S.D., CNT: immature DCs.
Figure 3.
Cytokine profiling of DCs infected with O. tsutsugamushi.
Secreted cytokines and chemokines from infected DCs were analyzed using a cytokine antibody array that simultaneously detects 62 cytokines and chemokines. Unstimulated immature DCs were used as a negative control (CNT). The membrane images were analyzed for the changes in signal intensities (top). Cytokines/chemokines with a normalized fold change greater than 1.2 are summarized (bottom). CNT: immature DC, OT: DCs infected with O. tsutsugamushi.
Figure 4.
Induction of autophagy in DCs infected with O. tsutsugamushi.
A and B. DCs were infected with O. tsutsugamushi for indicated time periods and subjected to immunoblot analysis for LC3 and β-actin as a loading control. LC3 II/LC3 I ratios were determined by densitometry of the immunoblot results from three independent experiments. Error bar: mean+S.D. C. DCs were infected with live O. tsutsugamushi (OT) or UV-inactivated bacteria (UV-OT) for 2 h and stained with scrub typhus patients' sera and anti-LC3 antibody. Colocalization of O. tsutsugamushi (green) with autophagosomes (red) was analyzed by confocal microscopy. CNT: immature DC, DIC: differential interference contrast.
Figure 5.
In vitro migration of DCs infected with O. tsutsugamushi in a 3D collagen matrix.
A. Single cell tracking was performed using Manual Tracking Plugin with Image J software. Thirty cells were randomly selected and tracked for 4 h. B. Speed, directionality, and Euclidean distance parameters were calculated by analyzing the acquired data from the Chemotaxis and Migration Tool Plugin software. The graphs represent velocity, directionality, and Euclidean distance, respectively. Red bars represent mean values. *: p<0.05, **: p<0.01, CNT: immature DCs, OT: DCs infected with O. tsutsugamushi, UV-OT: DCs infected with UV-inactivated O. tsutsugamushi, LPS: DCs stimulated with LPS, OT/LPS: DCs stimulated with O. tsutsugamushi and LPS.
Figure 6.
Ex vivo and in vivo migration of DCs infected with O. tsutsugamushi.
A. Ears of C57BL/6 mice were split into halves and CFSE-labeled DCs (green) were put on top of the dermis. Colocalization of DCs within LYVE-1+ lymphatic vessels (red) in dermis was analyzed by confocal microscopy after 2 h of incubation. B. The numbers of DCs within lymphatics of ear dermis were counted in different fields of confocal images from A. **: p<0.01. Red bars represent mean values. C. In vivo migration of DCs into lymph nodes was analyzed after subcutaneous injection of CFSE-labeled DCs into hind footpads of C57BL/6 mice. At 2 days after injection, CFSE-labeled CD11c+ cells in draining popliteal lymph nodes were detected by flow cytometer. Mean percentile of CFSE-labeled DCs in total lymph node cells from three separate experiments are presented. Error bar: mean+S.D., *: p<0.05. CNT: immature DCs, OT: DCs infected with O. tsutsugamushi, UV-OT: DCs stimulated with UV-inactivated O. tsutsugamushi, LPS: DCs stimulated with LPS.
Figure 7.
CCR7 expression on DCs infected with O. tsutsugamushi.
A. Representative histograms of CCR7 surface expression on DCs at 20 h after the indicated stimulation are presented. The numbers within the graphs indicate the percentage of CCR7+ DCs. Gray histogram: isotype control. B. Percentage of CCR7+ DCs after stimulation was examined in three separate experiments. Increasing amounts (10 or 20 bacteria/cell) of bacteria were also used to detect dose-dependent responses. Error bar: mean+S.D., CNT: immature DCs, OT: DCs infected with O. tsutsugamushi, UV-OT: DCs infected with UV-inactivated O. tsutsugamushi, LPS: LPS-stimulated DCs.
Figure 8.
Differential activation of MAP kinases in O. tsutsugamushi-infected DCs upon exposure to CCL19.
DCs were stimulated with O. tsutsugamushi (OT), or LPS for 18 h and then further incubated with CCL19 (200 ng/ml) for the indicated times. The activation of ERK and p38 MAP kinases was assessed by immunoblot using specific anti-phospho-ERK1/2 or phospho-p38 MAP kinases antibodies. ERK1/2, p38, and GAPDH were used as loading controls. CNT: immature DCs.