TLR2 signaling and cytokine production are induced by BMVs but not exosomes from Mtb-infected macrophages. (A) EVs released from Mtb-infected cells were purified by differential centrifugation and separated on sucrose density gradients. Fractions were pooled (#2-4, no vesicle control; #5-7, exosomes; #8-10 BMVs), concentrated, exchanged into DMEM culture medium, and analyzed for host and bacterial markers by Western blotting. Results are representative of two independent experiments. (B) HEK cells transfected with human TLR2 and CD14 were treated with the indicated sucrose gradient fractions for 24 h. TLR2 activation was assessed by ELISA for the production of IL-8. Results are the average of two replicates and are representative of two independent experiments. (C) Murine macrophages were incubated with the pooled sucrose gradient fractions for 24 h and TNF-α production was assessed by ELISA. Results are the average of two replicates and are representative of three independent experiments.