Immunosuppressive exosomes from TGF-β1 gene-modified dendritic cells attenuate Th17-mediated inflammatory autoimmune disease by inducing regulatory T cells

Cell Research, Dec 2011

Zhijian Cai, Wei Zhang, Fei Yang, Lei Yu, Zhou Yu, JihHung Pan, Lie Wang, Xuetao Cao, Jianli Wang

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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 Dear Editor 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 [1]. 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 [2]. 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 [3]. Despite the further understanding of the mechanisms of IBD, effective treatment is still lacking [4]. 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 [5]. It has been demonstrated that human tumor-derived exosomes can selectively impair lymphocyte responses to IL-2 and then impair their effector functions [6]. In addition, exosomes from immune inhibitory gene-modified bone marrow-derived dendritic cell (BMDC) have been found to suppress inflammation and collagen-induced arthritis [7]. 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 [ 8 ]. 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-? [ 9 ]. 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 [ 10 ]. 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 [ 11 ]. 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 | Cell Research Zhijian Cai et al. npg 609 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. Acknowledgments 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 1 Kaser A , Zeissig S , Blumberg RS . Inflammatory bowel disease . Annu Rev Immunol 2010 ; 28 : 573 - 621 . 2 Sarra M , Pallone F , Macdonald TT , Monteleone G . IL -23/IL17 axis in IBD . Inflamm Bowel Dis 2010 ; 16 : 1808 - 1813 . 3 Barnes MJ , Powrie F . Regulatory T cells reinforce intestinal homeostasis . Immunity 2009 ; 31 : 401 - 411 . 4 Sands BE . Inflammatory bowel disease: past, present, and future . J Gastroenterol 2007 ; 42 : 16 - 25 . 5 Mincheva-Nilsson L , Baranov V . The role of placental exosomes in reproduction . Am J Reprod Immunol 2010 ; 63 : 520 - 533 . 6 Clayton A , Mitchell JP , Court J , Mason MD , Tabi Z. Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2 . Cancer Res 2007 ; 67 : 7458 - 7466 . 7 Kim SH , Bianco NR , Shufesky WJ , Morelli AE , Robbins PD . Effective treatment of inflammatory disease models with exosomes derived from dendritic cells genetically modified to express IL-4 . J Immunol 2007 ; 179 : 2242 - 2249 . 8 Morelli AE , Larregina AT , Shufesky WJ , et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells . Blood 2004 ; 104 : 3257 - 3266 . 9 Ogino H , Nakamura K , Ihara E , Akiho H , Takayanagi R. CD4 +CD25+ regulatory T cells suppress Th17-responses in an experimental colitis model . Dig Dis Sci 2011 ; 56 : 376 - 386 . 10 Chen W , Jin W , Hardegen N , et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3 . J Exp Med 2003 ; 198 : 1875 - 1886 . 11 Ziegler SF , Buckner JH . FOXP3 and the regulation of Treg/ Th17 differentiation . Microbes Infect 2009 ; 11 : 594 - 598 .

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Zhijian Cai, Wei Zhang, Fei Yang, Lei Yu, Zhou Yu, JihHung Pan, Lie Wang, Xuetao Cao, Jianli Wang. Immunosuppressive exosomes from TGF-β1 gene-modified dendritic cells attenuate Th17-mediated inflammatory autoimmune disease by inducing regulatory T cells, Cell Research, 2011, 607-610, DOI: 10.1038/cr.2011.196