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 5
Mannose receptor mrc1 knockdown diminishes BCG WT and M. leprae entry into Schwann cells.
A. Verification of mrc1 gene knockdown. SC were transfected with control siRNA or two siRNA targeting mrc1 for 24 h before infection. Flow cytometry result showing that knockdown with mrc1siRNA reduces CD206 expression in M. leprae-infected SC. Representative histogram plots show fluorescence at the FL1-A channel. SCs were submitted to immunofluorescent labeling of CD206 with the FITC conjugated anti-CD206 antibody (clone15-2, Mouse IgG1, κ1, Biolegend). SC expression of CD206 after 24 h of infection at MOI 50:1, 33°C was measured using the flow cytometry FL1-A (green) channel to determine MFI. B. Flow cytometry result showing the effect of mrc1 knockdown on the degree of association of PKH26-(red) labeled M. leprae or BCG WT. For BCG WT, the previously described co-infection assay was applied. MFI was determined at the FL2-A channel. C. SC on coverslips were stimulated with M. leprae or BCG WT for 48 h. The cells were fixed with paraformaldehyde, stained with DAPI (blue) for nuclear localization, and examined by fluorescence microscopy. The upper panel shows (a-b) BCG WT association with and without control siRNA, (c) co-infected SC with M. leprae and PKH26-labeled BCG WT. The middle panel shows (d) co-infection in the presence of control siRNA, (e-f) co-infection in the presence of two types of mrc1 siRNA. The lower panel shows PKH26-labeled M. leprae associated to SC in the presence of (g) control siRNA, (h- i) tow types of mrc1 siRNA. Scale (white line) represents 10 μm. Results are represented as mean ± SEM of three 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.