Figure 1.
C. burnetii persistence in AT from mice.
Abdominal, inguinal, and dorsal (25 mg) AT from mice inoculated with C. burnetii were collected after different periods of time, DNA was extracted in a 100 µl volume and the presence of C. burnetii DNA was determined by qPCR using a 5 µl DNA extract. AT from BALB/c mice inoculated with C. burnetii via IP route (A), from BALB/c mice infected via the IT (B) route and from C57BL/6 mice infected via the IP route (C) were sampled. Results represented the number of DNA copies for total sample (25 mg) and were expressed as mean ±SEM.
Figure 2.
Cell culture and immunostaining of C. burnetii within adipose tissue.
A, C. burnetii was detected in dilacerated inguinal AT at day 60 p.i. incubated with L929 cells after 40 days of culture. Bacteria were detected using specific antibodies conjugated with Alexa 488 (in green). Original magnification: X100. B, The presence of C. burnetii was determined in sections of paraffin-embedded abdominal AT at day 30 p.i. using a rabbit anti-C. burnetii polyclonal antibodies. Bacteria were revealed using biotin-conjugated antibodies and peroxidase-labeled streptavidin. Labeled bacteria appear in red in the inflammatory infiltrate (circle) and in adipocytes (arrows). Original magnification: X400. C, Immunodetection of C. burnetii in sections of paraffin-embedded abdominal AT at day 30 p.i. Bacteria were revealed using rabbit anti-C. burnetii polyclonal antibodies and Alexa 555-conjugted anti-rabbit IgG (red). Labeled bacteria in red (arrows) were observed in AT (green, artificially attributed color) using confocal microscopy. D, Sections of paraffin-embedded abdominal AT from C57BL/6 transgenic mice with GFP-labeled immune cells inoculated with C. burnetii via the IP route at day 10 p.i. were incubated with rabbit anti-C. burnetii polyclonal antibodies. Bacteria were revealed using Alexa 555-conjugated F(ab') anti-rabbit IgG. Bacteria appear in red in the macrophages and in adipocytes. Original magnification: X100.
Table 1.
Mean DNA copies of C. burnetii in the organs of nude mice transplanted with BALB/c-infected abdominal adipose tissue.
Figure 3.
Intracellular fate of C. burnetii in adipocytes.
A-C, Adipocytes differentiated from the fibroblast cell line 3T3-L1 were incubated with increasing doses of C. burnetii (A). Adipocytes differentiated from the fibroblast cell line 3T3-L1 (B) or stromal vascular cells (C) were incubated with 10 bacteria per cell for 4 hours. After washing (defined as day 0), adipocytes were incubated at 37°C for different periods of time. Bacteria were detected using qPCR and the results are represented as the mean values ±SEM (n = 8). D, Bacteria and lipid droplets were labeled with human anti-C. burnetii antibodies and bodipy 493/503, respectively. A representative micrographs obtained by confocal microscopy is shown. Bacteria are displayed as red, and lipid droplets are displayed as green. E, 3T3-L1-differentiated adipocytes were incubated with 10 C. burnetii per cell for 4 hours, washed to remove unbounded bacteria, and cultured for 8 days. Infected cells were labeled with anti-C. burnetii (Alexa 555), anti-Lamp-1 (Alexa 488), anti-cathepsin D (CatD) (Alexa 647) antibodies and bodipy phallacidin (to label filamentous actin) and were analyzed by laser scanning confocal microscopy. The co-localization of bacteria (red) with Lamp-1 (green) and cathepsin D (blue) is shown by merging the respective fluorescent images. C. burnetii co-localized only with Lamp-1 marker (yellow).
Figure 4.
Microarray analysis of adipocytes stimulated by C. burnetii.
Adipocytes differentiated from the fibroblast cell line 3T3-L1 were incubated with 50 bacteria per cell for 8 hours. A, Heatmap representation of modulated genes selected by supervised analysis in stimulated and unstimulated adipocytes. Genes (in rows) and samples (in columns) were organized by hierarchical clustering (Pearson correlation distance, average linkage). Gene expression levels are color coded from blue (down-modulated) to red (up-modulated). B-C, Functional annotation of modulated genes and qRT-PCR results. The modulated genes in the most enriched GO terms are shown (B). qRT-PCR confirmation of some genes of the most enriched GO terms identified by microarray analysis (C). qRT-PCR ratios are presented as bleu bars and microarray ratios are presented as red bars. Both methods gave similar results (Spearman correlation coefficient = 0.85, P value = 0.006).
Figure 5.
Networks induced in C. burnetii-stimulated adipocytes.
The pathways induced in cultured adipocytes by C. burnetii stimulation were generated using the PathwayStudioTM software. Note that networks were organized around IL6, TNF, CCL2, CCL5, NF-kB and TLR2 genes.