Cardiac endothelial cell-derived exosomes induce specific regulatory B cells

The mechanism of immune tolerance is to be further understood. The present study aims to investigate the role of the Cardiac endothelial cell (CEC)-derived exosomes in the induction of regulatory B cells. In this study, CECs were isolated from the mouse heart. Exosomes were purified from the culture supernatant of the primary endothelial cells. The suppressor functions of the regulatory B cells were determined by flow cytometry. The results showed that the CEC-derived exosomes carried integrin αvβ6. Exposure to lipopolysaccharide (LPS) induced B cells to express the latent transforming growth factor (TGF)-β, the latter was converted to the active form, TGF-β, by the exosome-derived αvβ6. The B cells released TGF-β in response to re-exposure to the exosomes in the culture, which suppressed the effector T cell proliferation. We conclude that CEC-derived exosomes have the capacity to induce B cells with immune suppressor functions.

B cells from the bone marrow and cultured in the presence of the exosomes or/and LPS for 7 days, and then the expression of the immune regulatory molecules of TGF-b and the latent associated proteins (LAP) by the B cells were assessed. The results showed that the exposure to LPS increased the levels of LAP ( Fig. 2A), but not TGF-b (Fig. 2B), in B cells. Exposure to exosomes alone also did not increase TGF-b (Fig. 2C); however, exposure to both LPS and exosomes markedly increased the levels of TGF-b in the B cells, which was abolished by the addition of TLR4 inhibitor to the culture (Fig. 2C) or exposure to exosomes produced by the b6-null CEC ( Fig. 2D-F).
Phenotypes of the TGF-b 1 B cells generated by the CEC-derived exosomes. Following the same procedures above, we treated naïve B cells with the CEC-derived exosomes and LPS in the culture for 7 days. The cells were analyzed by flow cytometry. About 64.4% B cells showed TGF-b 1 (Fig. 3A). Among the TGF-b 1 cells, high frequency of CD5 1 , CD38 1 , CD1d 1 , TIM1 1 , CD23 1 , CD27 1 cells were detected, and low frequency of IFN-c 1 and CD24 1 cells were also detected ( Fig. 3B-J).
CEC-derived exosomes induce TGF-b 1 B cells. We generated the exosome-specific TGF-b 1 B cells by exposing naïve B cells to the exosomes or/and LPS for 7 days. As shown by flow cytometry data, not much TGF-b1 B cells were induced when the cells were cultured in medium alone (Fig. 4A, 4E); exposure to the exosomes moderately increased the TGF-b 1 B cells (Fig. 4B, 4E), which was further increased by adding the exosomes and LPS to the culture (Fig. 4C, 4E). The presence of LPS in the culture alone did not induce TGF-b in the B cells (Fig. 4D, 4E). The results suggest that the CEC-derived exosomes are capable of inducing TGF-b expression in B cells, which can be promoted by the presence of LPS in the culture.
Exposure to CEC-derived exosomes induces TGF-b release from the TGF-b 1 B cells. We induced the TGF-b 1 B cells as indicated above; the cells were re-stimulated with exosomes or/and LPS. The supernatant was analyzed by ELISA. As shown by Fig. 5, the exposure to exosomes in the culture induced the release of TGF-b, which was further increased by the addition of LPS. Exposure to LPS alone did not induce the release of TGF-b. To elucidate if the avb6 carried by the exosomes played any roles in the TGF-b release from the B cells, we generated avb6-null exosomes. These avb6-null exosomes still induced the release of TGF-b, which was not further increased by the addition of LPS. The results suggest that re-exposure to the CECderived exosomes can induce the release of TGF-b from the B cells. Although avb6 is required in the generation of TGF-b 1 B cells, the avb6 in the exosomes does not play a critical role in the TGF-b release from the TGF-b 1 B cells. LPS can promote the release of TGF-b from the B cells in synergy with avb6.
Immune suppressor function of the CEC-derived exosomeinduced TGF-b 1 B cells. CD4 1 T cells play a critical role in the skewed immune response, such as the transplantation rejection 8 , which may be inhibited by the TGF-b 1 T cells 9 . To observe the immune suppressor function of the exosome-induced TGF-b 1 B cells, we generated the TGF-b 1 B cells using the above procedures and isolated CD4 1 CD25 2 T effector cells from the spleen. The T cells (labeled with CFSE) and B cells were cultured in the presence of anti-CD3/CD28 antibodies in the presence of exosomes or/and LPS for 3 days. The cells were then analyzed by flow cytometry. The results showed that after stimulating by anti-CD3/CD28, the T cells proliferated markedly ( Fig. 6A-C). The presence of the B cells did not suppress the T cell proliferation (Fig. 6D). Considering an activator might be needed for the B cell activation to release TGFb, we added exosomes to the culture, which partially suppressed the proliferation (Fig. 6E), and was significantly suppressed in the presence of both exosomes and LPS (Fig. 6F). To elucidate if the suppression was associated with the avb6 carried by the exosomes, we generated the avb6-null exosomes, which still showed the suppressor functions (Fig. 6G). The addition of a neutralizing anti-TGF-b antibody (Fig. 6H) or Etk inhibitor (I) to the culture efficiently inhibited the T cell proliferation. Exosomes alone did not show an inhibitory effect on the T cell proliferation (J). The summarized data of T cell proliferation are presented in Fig. 6K. The data indicate that the CEC-derived exosome-induced TGF-b 1 B cells can be activated by the exosomes to suppress effector T cell activities, which can be strengthened by LPS.

Discussion
The components of the endothelial cells may save as specific antigens to initiate a specific immune response to induce antigen specific immune reactions, such as to induce allograft rejection 10 or other immune responses. Thus, to create an alloantigen specific immune tolerance is expected to improve the survival of the allograft or ameliorate the alloantigen-induced immune response. The present data indicate that CEC-derived exosomes carry integrin avb6. After exposure to the exosomes, B cells differentiate into TGF-b 1 B cells. The TGF-b 1 B cells can be activated by re-exposure to the exosomes to release TGF-b into the culture supernatant and suppress effector T cell proliferation.
TGF-b is one of the major immune regulatory molecules 11 . TGFb-expressing T cells and B cells can be Tregs or Bregs. Thus, the TGF-b 1 B cells, we observed in the present study, can be Bregs. The TGF-b 1 B cells can be activated by exposure to exosomes, not by the bovine serum in the medium. The fact demonstrates that the TGF-b 1 B cells, generated by the CEC-derived exosomes, are a kind of ''exosome'' antigen-specific TGF-b 1 B cells. There are a number of  The data show that the CEC-derived exosomes carry avb6. This is in line with previous studies, such as Chen et al reports that intestinal epithelial cell-derived exosomes also carry avb6. avb6 is described by the early studies that can convert LTGFb 12 and followed by many others 13,14 . Thus, avb6 is an important molecule in the development of TGF-b 1 cells with a premise that the cells produce LTGFb. Although naïve B cells do not produce detectable LTGFb, after exposure to LPS, the expression of LTGFb is increased markedly as we observed in the present study. The data suggest that concurrent exposure to both avb6 and LPS may generate new immune regulatory cells. The inference is supported by our previous studies 6 and others 15,16 .
CD4 1 effector T cells are one of the major immune cells involving in a number of immune responses. In addition to the beneficial functions, CD4 1 effector T cells act as inflammatory cells in the induction of immune inflammation in the body. Thus, to suppress the activities of CD4 1 effector T cells to given extents has therapeutic effect on immune disorders. The regulatory cells, including Tregs and Bregs, can suppress immune inflammation associating with CD4 1 effector T cells 16,17 . It is suggested that the treatment with Tregs prevents chronic rejection of heart allografts 18 and inhibits intestinal inflammation 19 ; and B cells also play a critical role in the complex immunoregulatory network in organ transplantation 20  Different results about the production of TGF-b by B cells in response to LPS has been reported. Parekh et al indicate that LPS can induce TGF-b production by B cells in the culture 21 ; our data show that the exposure to LPS in the culture only induces the expression of LTGFb; the latter needs to be activated by the integrin avb6. Such a discordance may be because the LPS concentrations using in our experiments are different from that of Parekh et al; the highest concentration of LPS is 0.1 mg/ml in our study while Parekh's LPS concentration is 20 mg/ml. TGF-b is a latent form after synthesis, it requires being activated prior to obtaining its biological activities 22 ; the requirement might be met in the environment with relative high levels of LPS 21 . The inference is supported by published data that LPS can increase the expression of matrix metalloproteinase 23 ; the latter can activate TGF-b 24 , which still needs to be further investigated with B cells in the future studies.
In summary, the present data indicate that the CEC-derived exosomes carry avb6; the latter converts LTGFb to TGF-b in B cells. The generated TGF-b 1 B cells can be activated by re-exposure to the exosomes to release TGF-b, and suppress effector T cell proliferation. Mice. C57BL/6 mice were purchased from the Beijing Experimental Animal Center (Beijing, China). The mice were maintained in a pathogen free environment. The animal experimental procedures were approved by the Animal Ethic Committee at Experimental center of Beijing Fuwai Hospital in accordance to the guidelines.

Reagents
Isolation of CECs. CECs were isolated from C57BL/6 mice. The hearts were excised, rinsed with phosphate buffered saline (PBS) and cut into small pieces (2 3 2 3 2 mm). The tissue fragments were transferred to DMEM and incubated for 45-60 min at 37uC with mild shaking. The clumps of cells were dispersed by forcing through a sterile 18 G needle. The resulting tissue slurry was filtered through a 40-mm pore-size cell strainer and the single-cell suspension was washed in DMEM with centrifugation at 400 3 g for 5 min. The cell pellet was re-suspended in DMEM. The CECs were isolated with magnetic beads coated with an anti-CD31 antibody following the manufacturer's instructions. The isolated CECs were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 mM L-glutamine. The culture medium was changed in every 3 days. The CEC reached confluence about 9 days and used for further experiments.
Isolation of exosomes. The CECs were cultured with no-serum culture medium overnight. Exosomes were purified from the culture supernatant of CEC according to our established procedures that were published elsewhere 6 . Briefly, CEC culture supernatant was centrifuged at 1000 g and 8,000 g to eliminate cell debris, and  The TGF-b 1 B cells were exposed to the CEC-derived exosomes or and LPS in the culture overnight as denoted on the X axis. Exosome-b: The b6-null exosomes. The culture supernatant was analyzed by ELISA. The bars indicate the TGF-b levels in the culture supernatant (mean 6 SD; *, p , 0.01, compared to the medium group). Exosome (or exosomes-b) 5 5 mg/ ml. LPS 5 100 ng/ml. The data are a representative of 3 independent experiments.
www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 7583 | DOI: 10.1038/srep07583 subsequently centrifuged at 60,000 g. The pellet was then washed with PBS and pelleted again at 100,000 g. Isolated exosomes were resuspended in PBS and filtered twice through 0.22-mm filters. Sample exosomes were processed for electron microscopy imaging with our established procedures 6 .
Western blotting. Cells were lysed in RIPA buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing a protease-inhibitor cocktail on ice for 30 min. The cell lysates were centrifuged at 12,000 g for 15 min at 4uC. Proteins were fractioned by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST), followed by incubation with the primary antibodies (0.5-1 mg/ml) overnight at 4uC. After 3 washes in TBST, membranes were exposed to horseradish peroxidaseconjugated secondary antibody (151000) for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence (ECL) and exposed to X-ray films. b-actin was used as a loading control.
Flow cytometry. Cells were collected and analyzed by flow cytometry with our established procedures 25 . Briefly, in the surface staining, cells were blocked with 1% bovine serum albumin (BSA) for 30 min, incubated with fluorochrome-labeled antibodies (indicated in the figures) for 30 min on ice. In the case of together with the intracellular staining, after the surface staining, cells were fixed with 2% paraformaldehyde containing 0.1% Triton X-100 for 2 h; then stained with fluorochrome-labeled antibodies. Phorbol 12-myristate 13-acetate (PMA, 100 ng/ ml), ionomycin (500 ng/ml), and brefeldin A (10 mg/ml) were added to the medium for the last 4 hours of culture. After washing with PBS, the cells were analyzed with a flow cytometer (FACSCanto II, BD Bioscience). The data were analyzed with the software FlowJo.
Enzyme-linked immunosorbent assay (ELISA). The levels of TGF-b in culture supernatant were determined using a commercial reagent kit following the manufacturer's instructions.
Generation of TGF-b 1 B cells. CD19 1 IL-7 receptor 1 CD45 1 B cells were isolated from the mouse bone marrow by magnetic cell sorting (MACS). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine, 1 mg/ml anti-CD40 antibody, 100 ng/ml LPS and 5 mg/ml exosomes for 2-3 rounds (one round was 3 days). After 3-round cultures, the TGF-b 1 B cells were more than 80% as assessed by flow cytometry.
Assessment of TGF-b 1 B cell immune suppressor functions. CD4 1 CD25 2 T effector cells (Teff cells) were isolated from the mouse spleen with a commercial reagent kit (purity was greater than 98%) by MACS. The Teff cells were stained with Carboxyfluorescein succinimidyl ester (CFSE), cultured with the TGF-b 1 B cells at a ratio of 151 for 3 days in the presence of the exosomes (5 mg/ml) or/and LPS (100 ng/ ml). The cells were analyzed by flow cytometry.
Isolation of naïve B cells from the bone marrows. The femur bones were excised from C57BL/6 mice; the bone marrows were flushed with culture medium. The red blood cells were lysed with a lysis buffer. The bone marrow cells were incubated with magnetic bead-conjugated anti-CD19 antibody for 30 min on ice. The cell suspension was then allowed to run through a MACS column (Miltenyi Biotec) to allow for the retention of CD19 1 cells in the column. After elution, the CD19 1 cells were further incubated with magnetic bead-conjugated anti-CD45 and anti-CD127 (IL-7 receptor a chain) antibodies for 30 min on ice. The cell suspension was then allowed to run through a MACS column to allow for the retention of CD45 1 and CD127 1 cells in the column. The cells were eluted from the column and checked by flow cytometry; the purity of the cells was greater than 96% (checked by flow cytometry) and to be used in further experiments.
Preparation of the avb6-null exosomes. The isolated CECs were treated with a b6 shRNA reagent kit following the manufacturer's instructions. Briefly, in a 12-well plate, when the CEC reached 50% confluence, the cells were tranduced by lentivirus carrying b6 shRNA or control shRNA at a multiplicity of infection (MOI) of 50. Cells were harvested at 72 hours after infection and the knockdown efficiency of b6 was evaluated by quantitative real-time RT-PCR and western blot analysis. The b6-null CEC were cultured to generate exosomes in the same procedures as described above.
Statistics. The data are presented as means 6 SD. ANOVA was used to test differences between groups. A p , 0.05 was set as a significant criterion.