Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages

Abstract

Exosomes are small 30-100 nm membrane vesicles released from hematopoietic and nonhematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present in exosomes inducing these host responses were not defined. This study used LC-MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis-infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFPs) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP-treated J774 cells, the majority of which were also present in exosomes isolated from M. tuberculosis-infected cells. The exosomes from CFP-treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.

Figure 1

Characterization of mycobacterial proteins associated with exosomes from M.tb‐infected macrophages. Exosomes isolated from uninfected and 72 h post M.tb‐infected J774 cells were analyzed by Western blot for the indicated mycobacterial proteins and for the lysosomal‐associated membrane protein‐1 as a positive control for exosomes.

Figure 2

Venn diagram showing overlap between proteins identified in exosomes released by M.tb‐infected, irradiated M.tb‐infected and CFP‐treated J774 cells.

Figure 3

Exosomes from CFP‐treated macrophages induce TNF‐α and IL‐12p40 secretion in naïve macrophages and BMDC, respectively. (A) BMDCs were incubated for 24 h with exosomes from M.tb or uninfected (UI) macrophages and the culture supernatants were analyzed for IL‐12p40 by ELISA. (B) J774 cells were incubated for 24 h with exosomes from M.tb or uninfected (UI) macrophages and the culture supernatant was analyzed for TNF‐α by ELISA. Results are representative of at least two independent experiments.

Figure 4

Activation of splenocytes from CFP treated mice by exosomes from CFP‐pulsed macrophages. Splenocytes from CFP‐treated mice were cultured with CFP, exosomes from UT J774 cells, or exosomes from CFP‐treated cells. In total, 72 h post‐treatment, culture supernatants were analyzed for IFN‐γ (A) and TNF‐α (B) by ELISA. Results are representative of at least three independent experiments.

Figure 5

Exosomes from CFP‐treated macrophages induce antigen‐specific T‐cell activation. Lymphocytes from lungs, MLN and spleens were isolated from mice intranasally treated with PBS and exosomes and the isolated cells cultured in the presence or absence of CFP. Cells were stained with anti‐CD4‐PE, anti‐CD8‐PE and anti‐IFN‐γ‐FITC and analyzed by flow cytometry (A). Percentage of T cells stained for CD4 and intracellular IFN‐γ (B) and CD8 and intracellular IFN‐γ (C). Lymphocytes from lungs, MLN and spleens isolated from mice intranasally treated with PBS or exosomes were stained with CFSE (2.5 μM) and then cultured in the presence or absence of CFP for 72 h. Cells were then stained with anti‐CD4‐PE or anti‐CD8‐PE and analyzed for CFSE dilution by flow cytometry. Percentage of total CD4+ T cells which showed CFSE dilution (D) Percentage of total CD8+ T cells which showed CFSE dilution (E). Lymphocytes from lungs, MLN and spleens were isolated from mice intranasally treated with PBS or exosomes and cultured in the presence or absence of CFP. Cells were stained with anti‐CD4‐PE, anti‐CD8‐PE, antiCD44‐PECy5 and antiCD62L‐FITC and analyzed by flow cytometry. Expression of effector memory CD4+ T cells (CD44^hi and CD62L^low) (F) or effector memory CD8+ T cells (G). Shown is the percentage of total T cells which were CD44^hi and CD62L^low. Values are means±SD of two independent experiments. Total lymphocytes were gated using FSC/SSC. Cells were negative for staining with isotype control antibodies. Exo‐exosomes, UT, untreated.