PGL I expression in live bacteria allows activation of a CD206/PPARγ cross-talk that may contribute to successful Mycobacterium leprae colonization of peripheral nerves
Fig 7
Crosstalk between PPARγ and CD206 mediates lipid droplet and PGE2 production in Schwann cells infected with PGL I-producing mycobacteria.
A. Competition assay suggesting cross-talk between PPARγ and CD206 in SCs. The addition of mannose at 100 μg/mL reduced BCG PGL I-induced PPARγ expression 48 h post-infection. PPARγ detection was performed using the specific rabbit polyclonal antibody (H-100) SC-7196 (Santa Cruz Inc., USA) followed by incubation with IgG anti-rabbit conjugated to Alexa Fluor 594 (Molecular Probes, USA) for immunofluorescence detection. Cells on coverslips were fixed with paraformaldehyde and stained with DAPI (blue) for nuclear localization. Representative fluorescence microscopy images showing the expression and localization of PPARγ after addition of mannose. Mean signal intensity per cell was quantified. Scale bar: 10μm (white line). B and C. Impact of the transcription factor PPARγ and the CD206 receptor on LD formation induced by M.leprae and BCG PGL I in SCs. Representative fluorescence microscopy images showing the effect of PPARγ antagonist GW9662 (5 μM) (B) and of mannose 100 μg/mL (C) on LD induction. The LD induction was estimated by microscopy after fluorescent Oil Red O staining and quantification of ORO-stained LDs was plotted as measurement of LD area/cell. The pretreatment with GW9662 or mannose respectively, reduced the LD formation induced by M.leprae or BCG PGL I 48 h post-infection. Scale bar: (A) 20μm (B) 10 μm and (C) 20 μm (white line). Data are shown as mean±SEM of three (7B) and five (7C) different experiments performed in triplicate. D and E. EIA was used to analyze the effect of the addition of GW9962 or mannose on the levels of PGE2 in the supernatants from the LD induction experiments. The results are the mean ± SEM from at least three independent experiments performed in triplicate. Statistical significance was calculated by ANOVA followed by Bonferroni’s multiple comparison test. *p < 0.05; ** p<0.01; ***p<0.001. F. M. leprae viability measured by qRT-PCR using the ratio of 16S rRNA/16S DNA 48 h after infection of ST8814 pre-treated or not with 100 μg/mL mannose. Data are shown as mean±SEM of six different experiments performed in triplicate. Statistical significance was calculated by Mann Whitney Test *p < 0.05.