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
A. Competition assay suggesting the Mannose Receptor (CD206) as a receptor candidate to mediate the internalization of BCG WT in SC. After pre-infection with M. leprae or BCG PGL I, the addition of mannose at 100 or 1000 μg/mL reduced the BCG WT internalization rate 48 h post-infection. In all experiments, the degree of internalization of PKH67-labeled BCG WT was determined after Trypan Blue quenching by flow cytometry. B. Pre-infection with PGLI-expressing bacteria MOI 10:1 favors the internalization of ManLAM-covered latex beads, which were incubated with SC for 48 h at 33°C and a 50:1 proportion. MFI was determined using the flow cytometry FL1-A channel. Green fluorescent latex beads covered with ManLAM showed a higher degree of internalization in comparison to the control green fluorescent latex beads. C. Normalized relative expression of mrc1 (delta delta Ct) in SC infected for 4 h, 24 h or 48 h with either BCG PGL I, M. leprae, or BCG WT at MOI 50:1. Results are presented in terms of fold changes after normalization with rpl13 mRNA. D. CD206 expression in SC after 24 h of infection was measured by flow cytometry. Immunofluorescent labeling of CD206 was carried out using the FITC conjugated anti-CD206 antibody. The conditions were: uninfected (NI), isotype control, and treatment with BCG WT, BCG PGL I, or M. leprae. A representative histogram plot shows fluorescence at the FL1-A channel. E. Representative images of fluorescence microscopy showing CD206 expression in uninfected SC and M. leprae or BCG PGL I-infected SC after 24 h of incubation. Cells on coverslips were fixed with paraformaldehyde and stained with DAPI (blue) for nuclear localization. Cells were immunolabeled with FITC conjugated anti-CD206 antibody or a FITC conjugated isotype and then examined by fluorescence microscopy. The mean CD206 signal intensity per cell was quantified. Scale bar: 10μm (white line) A-E. Results are represented as mean ± SEM of three or more independent biological replicates. Statistical significance was calculated by ANOVA followed by Bonferroni’s multiple comparison test. *p < 0.05; ** p<0.01; *** p<0.001.