Immunosuppressive exosomes from TGF-β1 gene-modified dendritic cells attenuate Th17-mediated inflammatory autoimmune disease by inducing regulatory T cells
Cell Research |
Immunosuppressive exosomes from TGF-?1 gene-modified dendritic cells attenuate Th17-mediated inflammatory autoimmune disease by inducing regulatory T cells
0 Institute of Immunology, Zhejiang University School of Medicine , Hangzhou, Zhejiang 310058 , China
1 Hangzhou Wahaha group Co. Ltd. , Hangzhou, Zhejiang 310018 , China
Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is generally believed to originate from the complicated interactions between genetic factors and commensal flora. This leads to the breakdown of innate immunity and the aberrant activation of the immune system, which is largely responsible for tissue damages in patients and animal models . A new CD4+ T cell lineage, Th17, which predominantly produces IL-17, has been revealed recently to play a key role in the development of IBD. Studies in IL-17 receptor A (IL-17RA)-knockout mice have demonstrated that IL-17 is necessary for the development of IBD. Blockade of IL-17 signaling by an IL-17RA-IgG1 fusion protein significantly attenuated IBD . It has also been shown that the CD4+ T cells that express Foxp3 (Tregs) are the major regulatory T cells that help maintain intestinal homeostasis . Despite the further understanding of the mechanisms of IBD, effective treatment is still lacking . Therefore, it is necessary to explore new clues and targets for therapeutic interventions. Exosomes are small vesicles released by various live cells with a structure of lipid bilayer and diameter ranging from 50 to 100 nm. They are formed by the fusion of multivesicular bodies with the plasma membrane followed by exocytosis . It has been demonstrated that human tumor-derived exosomes can selectively impair lymphocyte responses to IL-2 and then impair their effector functions . In addition, exosomes from immune inhibitory gene-modified bone marrow-derived dendritic cell (BMDC) have been found to suppress inflammation and collagen-induced arthritis . Therefore, it is conceivable that exosomes may be used as vehicles for therapeutic intervention of autoimmune diseases. In the present study, we were interested in whether exosomes derived from the TGF-?1 gene-modified BMDC (TGF-?1-EXO) have the immunosuppressive function and play a protective role during IBD develop-
ment. First, we identified the profile of exosomes that we
prepared. Under the electron microscope, the exosomes
displayed typical characteristic of round morphology
with a diameter of 50-100 nm. Immunoblotting results
demonstrated that all types of exosomes contained the
exosome-associated proteins Hsp70, Tsg101 and CD9,
whereas ER-residing protein Grp94 was absent in
exosomes (Supplementary information, Figure S1). We also
examined the level of TGF-?1 in exosomes. The total
amount of TGF-?1 in TGF-?1-EXO was about 15 pg/?g
of exosomes and no TGF-?1 was expressed in exosomes
derived from non-gene-modified BMDC (Control-EXO)
or LacZ gene-modified BMDC (LacZ-EXO) (data not
shown). When examined in mixed lymphocyte
reaction system, Control-EXO and LacZ-EXO inhibited T
cell proliferation at low-dose level. It was proposed that
the inhibition was due to their low-level expression of
costimulatory molecules. The T cell proliferation was
promoted when dose was increased. Exosomes contain
a large amount of allo-MHC molecules, which can be
taken up by dendritic cells (DCs) and presented to T cells
for allostimulation. This might be the reason why
Control-EXO or LacZ-EXO did not inhibit, but promoted
T-cell proliferation at a higher dose. On the other hand,
TGF-?1-EXO showed a stronger inhibitory effect on T
cell proliferation than on Control-EXO or LacZ-EXO
via TGF-?1 and the effect was dose dependent
(Supplementary information, Figure S2). We also analyzed the
phenotype of exosomes by FACS (Supplementary
information, Figure S3). In our results, there were no obvious
difference in the expression level of costimulatory and
adhesion molecules among Control-EXO, LacZ-EXO
and except that TGF-?1-EXO express lower MHC class
II molecules, which could explain that TGF-?1-EXO still
showed stronger inhibitory effect than Control-EXO and
LacZ-EXO after TGF-?1 was bloked. Moreover,
TGF?1-EXO plus anti-CD9 showed similar inhibitory effect
to TGF-?1-EXO alone, excluding the possibility that
anti-TGF-?1 can affect T cell modulatory properties of
TGF-?1-EXO through enhancing exosome capture by
BMDC after antibody binding. These results further
confirm that the inhibitory effect of TGF-?1-EXO is TGF-?1
dependent (Supplementary information, Figure S2).
Then we explored the effect of TGF-?1-EXO during
IBD development. We found that in vivo administration
of Control-EXO or LacZ-EXO did not prevent
DSSinduced weight loss, whereas administration of
TGF-?1EXO significantly prevented the weight loss (Figure 1A).
The disease activity index (DAI) scores and intestinal
bleeding were also significantly reduced by the
administration of TGF-?1-EXO, but not Control-EXO or
LacZEXO (Figure 1B and 1C). Histological assessment of
colonic damage revealed that the mice which were
untreated or received Control-EXO or LacZ-EXO suffered
severe diffuse inflammation involving the mucosa,
submucosa, and in some cases extending through all
intestinal layers (transmural inflammation). There was also
pronounced disruption of the normal architecture and crypt
loss. In contrast, DSS-mice treated with TGF-?1-EXO
had much less damage in colon tissues, showing more
conserved glandular structure and limited leukocyte
infiltrations in mucosa and submucosa (Figure 1D). These
results indicated that in vivo administration of
TGF-?1EXO could effectively inhibit the development of
DSSinduced murine IBD. To further reveal the protective
advantage of TGF-?1-EXO during the development of
IBD, we compared the protective effect of TGF-?1-EXO
and TGF-?1 cytokine. We found that 450 pg TGF-?1,
the total amount in TGF-?1-EXO that we injected,
showed no protective effect to IBD, and even if a higher
dose of 4 500 pg TGF-?1 was injected, it showed only
slight protective effect. The protective effect of
TGF-?1EXO was much better than TGF-?1 alone (Figure 1E).
Although TGF-?1 is proved to play an important role in
the protection of IBD, we could not detect the increase
of TGF-?1 secretion at the inflammatory site (data not
shown). The total TGF-?1 in exosomes that we injected
into mice was about 450 pg; after diffusion in vivo, it
was far from the detectable level. We supposed that the
stability of TGF-?1 in TGF-?1-EXO would be favorable
for the protective effect of TGF-?1-EXO, because we
found that TGF-?1 presented in the form of exosomes
showed stronger stability than TGF-?1 cytokine when
digested by trypsin (Supplementary information,
Figure S4). Moreover, the stability was related to the intact
membrane structure of exosomes because disruption of
the membrane structure of exosomes could decrease the
ability of TGF-?1 and TSG101, another exosomal
protein, in TGF-?1-EXO to resist the digestion of trypsin
(Figure 1F). Inflammatory cells secret large amounts
of proinflammatory cytokines such as TNF-? and IL-1,
which increase the endotheliocytes to express ligands of
adhesion molecule. Exosomes from BMDC are reported
to express many kinds of adhesion molecules [
]. It is
possible that exosomes will be recruited to an
inflammatory site and cause the enriched effect of TGF-?1, which
may be another factor that TGF-?1-EXO possess better
protective effect than TGF-?1 alone.
Tregs were capable of suppressing colonic
inflammation in IBD mice by downregulating Th17 responses
via TGF-? [
]. We speculated that TGF-?1-EXO might
induce the Tregs to exert protective effect. As expected,
we found that TGF-?1-EXO could effectively induce
CD4+Foxp3+ Tregs via TGF-?1 in vitro (Supplementary
information, Figure S5), and we also found that
TGF-?1EXO induced CD4+Foxp3+ Tregs in lymphocytes from
mesentery lymph nodes (mLNs) of inflammatory site
(Figure 1G), but not in splenocytes (data not shown).
It is known that the TCR signaling is required for the
generation of Tregs, but not for TGF-?1 signaling [
Compared with lymphocytes of the inflammatory site, the
possibility of splenocytes to contact intestinal antigen,
which could provide the TCR signaling, was small. In
addition, Tregs might transfer to spleen from mLNs and
increase the Treg level in spleen. However, the increase
of the transferred Tregs was too small to be detected in
total CD4+ T cells in the spleen. Therefore,
TGF-?1-EXOinduced CD4+Foxp3+ Tregs were mainly from mLNs of
the inflammatory site, but not in the remote splenocytes.
There is a delicate balance between Th17 and Treg.
A signature transcription factor, ROR?T for Th17 is also
induced by TGF-?1. In the absence of IL-6, Foxp3 can
inhibit ROR?T function and drive Treg differentiation.
However, when the cells also receive a signal from IL-6,
Foxp3 function is inhibited and the Th17 differentiation
pathway is induced [
]. Similar to CD4+Foxp3+ Tregs,
we found that TGF-?1-EXO could decrease Th17 in
lymphocytes from mLNs (Figure 1H), but not in
splenocytes (data not shown). Furthermore, the levels of IL-17
and IL-6 in colon tissues were also inhibited by
TGF-?1EXO and results even showed that the serum level of
IL17 was decreased in TGF-?1-EXO-treated mice
(Supplementary information, Figure S6), suggesting that
TGF?1-EXO could affect Th17 cells systemically.
To further confirm the protective effect of CD4+Foxp3+
Tregs, they were depleted by the pretreatment of mice
with anti-CD25 monoclonal antibody (mAb). Then we
found that the protective effect of TGF-?1-EXO against
DSS-induced IBD was totally abrogated (Figure 1I) and
the inhibitory effect of TGF-?1-EXO on IL-17 in colon
tissues and serum also could not be observed (Figure 1J).
These results further support the notion that CD4+Foxp3+
Tregs play a role in the prevention of DSS-induced IBD
www.cell-research.com | Cell Research
Zhijian Cai et al. npg
npg Treatment of IBD by immunosuppressive exosomes
by treatment with TGF-?1-EXO.
This is the first report about utilizing exosomes that
were derived from TGF-?1 gene-modified BMDCs
to delay DSS-induced IBD. TGF-?1-EXO induced
CD4+Foxp3+ Tregs and decreased the proportion of Th17
in lymphocytes from mLNs of the inflammatory site. As
acellular vesicles, exosomes can be preserved for a long
time. Unlike DC, exosomes are derived from the DC and
presumably reflect the function of the DC at the time of
isolation. Moreover, the protective effect of
TGF-?1EXO in IBD was much better than TGF-?1 cytokine. All
these specific characters make exosomes to be one of the
attracting candidates for clinical application of
autoimmune diseases. Experimental materials and methods
aredescribed in the Supplementary information, Data S1.
We thank Ms Diya Yang for technical assistance. This work
was supported by the National Key Basic Research Program of
China (2007CB512403), the National Natural Science
Foundation of China (30972725 and 31070795), the National Technology
Key Projects of China (008ZX1002-008) and the Natural Science
Foundation of Zhejiang Province (Z2090042).
*These three authors contributed equally to this work.
Correspondence: Jianli Wang
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