Figure 8
From: Mito-xenophagic killing of bacteria is coordinated by a metabolic switch in dendritic cells
Silencing of aggrephagy-associated proteins increases the bacterial load and affects chlamydial antigen presentation in infected DCs. (A) DCs were siRNA-silenced for Parkin, HSP25/27 or HDAC6 and then infected with chlamydia. Cells were fixed and costained for LC3 (red), chlamydial LPS (green) and DNA (blue) (top panel). Enlarged photographs in the second panel from top (I, II, III and IV) show colocalisation between aggrephagy-associated proteins and chlamydial structures. The influence of siRNA silencing on the physical appearance of inclusions was visualised by DAPI (third panel from top). Enlarged photographs (lower panel, V, VI, VII and VIII) display the size and structure of inclusions. (B) The fluorescence intensity along a cellular cross section of interest (enlarged photographs I, II, III, and IV in (A) was measured. Obtained profiles were overlaid and coloured. (C) siRNA-silenced infected DCs (48 hpi) were analysed by flow cytometry using the IMAGEN kit. Summarised data from three independent experiments (D) show the number of chlamydia-positive cells, as well as the bacterial load (MFI) of infected cells. The MFI values obtained for infected cell cultures (control siRNA) were set to 1 arbitrary unit. (E) DCs were siRNA-silenced for Parkin, HSP25/27 or HDAC6 and infected with chlamydia. AllStars siRNA, non-infected cells and CD3/CD28 beads were used as controls. DCs were cocultured with chlamydia-sensitised CD8+ T cells for 48 h. IFN-γ secretion by CD8+ T cells was assayed by ELISA. Relative values of IFN-γ secretion are expressed in arbitrary units (maximum value was set to 10) and are the means ± SD of three independent experiments. Statistical analysis in (D and E) was performed as described in Methods (*p < 0.05; **p < 0.01; ***p < 0.001; n = 3).
