Exosomes isolated from mycobacteria-infected THP-1 cells or from M bovis BCG–infected mice induce a proinflammatory response in vitro and in vivo. IL-12 p40 (A) and TNF-α (B) protein levels in lysates of mouse lungs 1 day after intranasal injection with 20 μg exosomes derived from uninfected, M bovis BCG–infected, or M tuberculosis H37Rv–infected THP-1 cells as quantified by ELISA. Background cytokine levels (ie, PBS injection) are subtracted from all values. Shown are the means plus or minus standard deviations for 3 mice and are representative of 2 separate experiments. (C) Neutrophil infiltration in the BALF of mice injected with the various exosome preparations or PBS control at 1 day after injection. Data are representative of 2 experiments. (D) Exosomes (3 μg) isolated from the BALF of uninfected (UI) or M bovis BCG–infected C57BL/6 mice were analyzed by Western blot using specific antibodies for the host protein hsp70 and for the mycobacteria 19-kDa lipoprotein and LAM. (E) C57BL/6 BMMs were treated with 200 ng of exosomes isolated from the BALF of uninfected or M bovis BCG–infected mice. Cell-conditioned media were assayed for TNF-α by ELISA 24 hours after exosome treatment. Data are representative of 2 experiments and are expressed as means plus or minus standard deviation for duplicate wells.