Fig. 5
TLR-2 stimulating CFP-10-derived C5 peptide enhances autophagy in MΦs through LC3 binding. a, b RAW.A4 MΦs transfected with gfpLC3 were activated or not with TLR-2 stimulating peptides (TSPs) (1 µg/mL for 4 h), and infected with rfp-BCG and washed. Likewise, primary MΦs from either wt-C57Bl6 mice or TLR-2-KO mice were infected with rfp-BCG with or without TSPs, washed, fixed, and stained using antibodies to LC3 and imaged using confocal microscopy. Percent autophagosomes colocalizing with BCG were calculated from three separate experiments and plotted (*p < 0.009). rfp-BCG did not colocalize with LC3 in TLR-2 KO MΦs. C5 peptide alone had no effect on the numbers of autophagic puncta (not shown). c wt-C57Bl/6 MΦs were infected as above and stained for lysosome markers LAMP1 and CD68 followed by quantitation of labeling. d MΦs were either treated or not with siRNA vs. beclin1 to selectively block autophagy and scrambled siRNA as control, followed by infection with wt-BCG, or its combination with C5 peptide at a dose (1 µg/mL; 0.5 µM) known to enhance antigen presentation. BB7 CD4 T cells were overlaid on the MΦs, and IL-2 determined using sandwich ELISA. When autophagy was not blocked (blue fill), TSPs combined with BCG increased antigen presentation. When autophagy was inhibited using siRNA beclin1 (purple fill) antigen presentation was decreased (*p < 0.01 vs. scrambled siRNA control; two experiments, ± SD). P-values were determined using one-way ANOVA with Dunnett’s post test. e Lysates of MΦs from one of the ELISA experiments were analyzed using an antibody to LC3; they show reduced lipidation of LC3 bands after siRNA vs. beclin1 treatment
