Evaluation of the Inflammatory Response in Macrophages Stimulated with Exosomes Secreted by Mycobacterium avium-Infected Macrophages

Exosomes secreted from Mycobacterium avium-infected macrophages contain numerous antigens of both M. avium and the host cell and are involved in the induction and expression of the inflammatory responses in macrophages. The interaction between exosomes secreted from M. avium-infected macrophages and macrophage phagocytosis, cytokine secretion, immunostimulation, and apoptosis was analyzed. Upon stimulation with exosomes secreted from M. avium-infected macrophages, the phagocytosis of dextran by treated macrophages was increased. Furthermore, the expression of CD40, CD80, CD81, CD86, HLA-DR, and most notably CD195 was enhanced. Additionally, the secretion of IL-6, IL-8, IL-10, IFN-γ, and TNF-α was increased by stimulated macrophages. Exosome stimulation did not induce macrophage apoptosis when compared with macrophages infected with M. avium. Caspase expression, including that of caspases 3, 6, and 8, was also not altered in exosome stimulated macrophages. Thus exosomes trigger the inflammatory response in macrophages owing to the presence of bacterial antigens but have no effect on macrophage viability.


Introduction
Mycobacterium tuberculosis infection is a severe global health problem, and China is among the worst affected countries [1,2]. The immune response to M. tuberculosis infection requires the activation of alveolar macrophages and the development of a Th1-type CD4 + T cell response, leading to the formation of lung granulomas [3]. Alveolar macrophages, as the main resident immune cells in the lung, are activated to produce cytokines including interferon (IFN-), tumor necrosis factor (TNF-), interleukin 1 (IL-1), IL-10, and IL-12, which regulate the production of nitric oxide and reactive oxygen species to kill or inhibit mycobacteria [4,5]. Mycobacterium avium shares with M. tuberculosis a slow growth rate and an ability to generate granulomas and is itself pathogenic [6][7][8][9]. M. avium-and M. tuberculosis-containing phagosomes share important features including restricted fusing with endosomal/lysosomal compartments [10][11][12] and impaired acidification [13,14].
Exosomes are small membranous vesicles generated by inward budding of late endosomes, resulting in the formation of multivesicular bodies in the cell cytosol. Exosomes can be derived from B cells and antigen presenting cells such as macrophages, dendritic cells (DCs), and natural killer cells, which are enriched in proteins of the tetraspanin family including CD63 and CD81 [15] and molecules involved in antigen presentation to sensitized T cells (CD80, CD86, and MHC-II) [16]. Thus exosomes play pivotal roles in both physiological crosstalk between cells and disease pathogenesis.
In general, exosomes act as molecular carriers during immune cell-cell communication [17]. However, recent studies also show that exosomes carrying tumor antigens promoted antigen-specific T cell activation and tumor rejection in vivo [18]. Knowledge of the protein composition of exosomes suggests further functions for these extracellular vesicles, for instance, exosomes released from Mycobacteriuminfected macrophages carry mycobacterial antigens including 2 BioMed Research International lipoprotein and lipoarabinomannan [19]. Additionally, Hsp-70 in exosomes induces a proinflammatory response [20] and exosomes containing glycopeptidolipids of M. avium transfer them from infected to noninfected macrophages, resulting in a toll-like receptor-dependent proinflammatory response [21].
Exosomes can not only induce inflammatory responses, but could also modulate immune responses, including both immune stimulation and immune suppression [22]. Recent reports have also shown that exosomes containing microbial antigens were able to protect against microorganism infection. Studies by Colino and Snapper indicated that injecting mice with exosomes containing the capsular polysaccharide type 14 cross-reactive antigen of Streptococcus pneumoniae would induce a protective antibody response to resist S. pneumoniae [23]. Similarly, treating mice with exosomes derived from DCs pulsed with toxoplasma antigens was also shown to protect the mice against subsequent toxoplasma infection [24]. At present, there are approximately 29 kinds of M. tuberculosis proteins found 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 DC activation as well as activation of naïve T cells in vivo. This suggests that exosomes containing M. tuberculosis antigens may be alternative approach to developing a novel tuberculosis vaccine [25].
In this study, we analyzed the inflammatory response and apoptosis induced by exosomes secreted from M. aviuminfected macrophages.

Mycobacterium avium Culture. Mycobacterium avium sp.
Paratuberculosis (referred to as M. avium in this paper) was obtained from the Chinese Center for Disease Control and Prevention. Bacteria were grown on Middlebrooks 7H9 plates as previously described [26] for 4 weeks, at 37 ∘ C. Colonies were harvested by scraping, with 0.9% NaCl as vehicle, and Mycobacterium concentrations were calculated according to the McFarland Standards method. The concentration was then adjusted to 1.5 × 10 9 Mycobacterium/mL.

Mycobacterium avium Infection of Macrophages.
Macrophages were cultured at 1 × 10 6 cells per well (in 1 mL culture medium) in six-well plates and infected with M. avium at an MOI of 100 for 24 h, as previously described with modifications [26]. M. avium infection rate was determined by acid-fast staining test. Culture supernatants were collected and used for cytokine analysis and exosomes isolation. The macrophages were washed with phosphate-buffered saline (PBS) and apoptosis and the expression of cell surface molecules and caspase proteins analyzed.

Exosome Isolation.
Macrophage cell culture supernatants were centrifuged at 3,000 for 15 minutes to remove cells and cell debris; then supernatants were transferred to sterile tubes. The ExoQuick Exosome Precipitation Solution (System Biosciences (SBI), Mountain View, CA, USA) was added to cell culture supernatants, the tubes mixed by inverting, then refrigerated for 30 minutes. ExoQuick/biofluid mixture was centrifuged at 1,500 for 30 minutes; then the supernatant was aspirated. Spin-down residual ExoQuick solution was added and centrifuged at 1,500 for 5 minutes and all traces of fluid were removed by aspiration. Finally, exosome pellets were resuspended in 1/10 of the original volume using nuclease-free water. The exosomes pellets were mixed with 25 L of 9% sucrose containing protease inhibitors and stored at −80 ∘ C until use. All procedures were carried out at 4 ∘ C. The abbreviation (+)exosomes describes exosomes obtained from M. avium-infected macrophages, and the term (−)exosomes describes exosomes from noninfected macrophages. All exosomes samples were tested for endotoxin contamination using Limulus Amebocyte Lysate (LAL) test (Catalog number KT05, Houshiji, Co. Ltd.).

Apoptosis and Necrosis.
Macrophages cultured in sixwell plates were treated with LPS (50 ng/mL), (−)exosomes (50 g/mL), (+)exosomes (50 g/mL), or M. avium (MOI of 10) for 24 h. Apoptosis was quantitatively determined by flow cytometry using an annexin V-FITC/PI apoptosis detection kit (BD). Briefly, following treatment, cells were harvested by trypsinization, washed with PBS, and incubated with annexin V-FITC and PI at room temperature for 10 min in the dark. The stained cells were analyzed with a FACS Calibur flow cytometer and CellQuest analysis software (BD).
2.11. Statistical Analysis. All data are expressed as the mean ± SEM. Values were analyzed by SPSS version 16.0 software for Windows (IBM Corp., Armonk, NY, USA), and the statistical significance of differences between groups was evaluated by one-way analysis of variance. Values of < 0.05 were defined as statistically significant.

Analysis of Macrophage
Exosomes. TEM observation of phosphotungstic-stained, purified exosomes obtained from macrophages uninfected or infected with M. avium revealed a homogenous population of morphologically typical vesicles of 30 to 100 nm diameter (Figures 1(a) and 1(b)), similar in appearance and size to those in published reports [27,28]. Additionally, electrophoresis results showed that exosomes obtained from macrophages uninfected or infected with   M. avium were rich in proteins (Figure 1(c)), although there are obvious differences in components between (+)exosomes or (−)exosomes.

Characterization of Exosomes.
To confirm that exosomes secreted from M. avium-infected macrophages contained proteins from M. avium, exosomes were probed for a subset of M. avium proteins by western blotting. We found that exosomes released from M. avium-infected macrophages, but not from uninfected cells, contained ESAT-6, MPT63, SodA, MPT51, and antigen 85 complex (antigen 85-C) ( Figure 2). As expected, both exosome populations contained the host protein lysosomal associated membrane protein-1 (LAMP-1).

Analysis of Phagocytosis.
We also investigated the phagocytic properties of macrophages preincubated with LPS, (−)exosomes, (+)exosomes, and M. avium compared with untreated macrophages. As shown in Figure 3, compared to medium-treated group, (−)exosomes treated macrophages  did not exhibit enhanced phagocytocity, while LPS, (+)exosomes, and M. avium-treated macrophages showed considerable elevation. It is noteworthy that (+)exosomes are significantly stronger stimuli than LPS and M. avium in enhancing macrophage phagocytic activity.

Discussion
Exosomes, 30-100 nm in diameter, are secreted vesicles derived from cell endosomal membrane system and have important implications in host biological functions. Exosomes secreted from M. avium-infected macrophages have been reported to contain many mycobacterial components including antigen 85-C, LpdC, PstS1, HspX, Mpt51, and Alanine [25], and they are proinflammatory [21]. Consistently, our results in the current study also demonstrated that (+)exosomes contained M. avium-derived proteins like ESAT-6, MPT63, SodA, MPT51, and antigen 85-C and these exosomes could induce macrophages to produce a panel of proinflammatory cytokines including IL-6, IL-8, IL-10, IFN-, and TNF-. More importantly, our study for the first time revealed that (+)exosomes could trigger comparable immune responses as M. avium infection in the means of enhancing signaling protein expression on cell surface and proinflammatory cytokine production. However, unlike M. avium infection, (+)exosomes had no apparent effect on cell apoptosis and necrosis. Despite being beyond the scope of the current study, it is warranted to investigate whether such results can be translated to animal models or even clinical trials.
In general, (+)exosomes could induce similar immune responses as M. avium infection; however, slight difference was observed. For instance, M. avium infection could enhance the expression of CD32 and CD163, but not CD195 on cell surface, while (+)exosomes enhanced CD195 expression but not the other two CD molecules. In addition, M. avium but not (+)exosomes induced TGF-1 level. CD32 negatively regulates IgG production by B cells [29]; CD163 is associated with a large range of inflammatory diseases including liver cirrhosis, type 2 diabetes, macrophage activation syndrome, Gaucher's disease, sepsis, HIV infection, rheumatoid arthritis, and Hodgkin lymphoma [30,31], while CD195 functions as a chemokine receptor and is involved in recruitment of immunocytes, especially T cells, to site of infection [32]. Given the functions of these three CD molecules, M. avium infection induced CD32 upregulation might be associated with one of the mechanisms in which M. avium escapes from host immune response, and CD163 upregulation might be involved in M. avium infection causing disease manifestations, while the (+)exosomes induced CD195 expression could be related to enhanced host immune responses, especially T cell related responses. However, to fully understand the mechanisms as well as biological significance of the subtle difference between M. avium and (+)exosomes induced immune responses, further in-depth investigation is required.
It is noteworthy that although (+)exosomes could induce immune responses comparable to M. avium infection, they caused neither cell apoptosis nor necrosis. These characteristics make (+)exosomes strong candidate as vaccine. Albeit the exact mechanism that (+)exosomes do not induce apoptosis or necrosis is yet to be determined, some clues can be obtained in the results of our current study. In the cytokine quantification assay, we observed that only M. avium infection, but not (+)exosomes treatment, induced high levels of TGF-1 expression. TGF-1, a polypeptide member of the transforming growth factor beta superfamily, performs a variety of cellular functions, including control of cell growth, proliferation, differentiation, and apoptosis [33]. Dysregulation of TGF-activation and signaling may result in apoptosis [34]. Consequently, TGF-1-related singling pathway, although other pathways may also be involved, is likely to be responsible for M. avium induced cell apoptosis.
Despite being beyond the scope of the current study, it is also interesting to determine which component(s) in M. avium can activate TGF-1 pathway.
In conclusion, (+)exosomes could induce inflammatory immune responses comparable to M. avium infection but do not cause cell apoptosis. This suggests that exosomes would make a good vehicle for vaccine delivery.