Sca-1+Lin−CD117− Mesenchymal Stem/Stromal Cells Induce the Generation of Novel IRF8-Controlled Regulatory Dendritic Cells through Notch −RBP-J Signaling This information is current as of September 29, 2021. Xingxia Liu, Shaoda Ren, Chaozhuo Ge, Kai Cheng, Martin Zenke, Armand Keating and Robert C. H. Zhao J Immunol published online 30 March 2015 http://www.jimmunol.org/content/early/2015/03/28/jimmun ol.1402641 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 30, 2015, doi:10.4049/jimmunol.1402641 The Journal of Immunology

Sca-1+Lin2CD1172 Mesenchymal Stem/Stromal Cells Induce the Generation of Novel IRF8-Controlled Regulatory Dendritic Cells through Notch–RBP-J Signaling

Xingxia Liu,*,1 Shaoda Ren,*,1 Chaozhuo Ge,* Kai Cheng,* Martin Zenke,† Armand Keating,‡,x and Robert C. H. Zhao*

Mesenchymal stem/stromal cells (MSCs) can influence the destiny of hematopoietic stem/progenitor cells (HSCs) and exert broadly immunomodulatory effects on immune cells. However, how MSCs regulate the differentiation of regulatory dendritic cells (regDCs) from HSCs remains incompletely understood. In this study, we show that mouse bone marrow–derived Sca-1+Lin2CD1172 MSCs can drive HSCs to differentiate into a novel IFN regulatory factor (IRF)8–controlled regDC population (Sca+ BM-MSC–driven

DC [sBM-DCs]) when cocultured without exogenous cytokines. The Notch pathway plays a critical role in the generation of the Downloaded from sBM-DCs by controlling IRF8 expression in an RBP-J–dependent way. We observed a high level of H3K27me3 methylation and a low level of H3K4me3 methylation at the Irf8 promoter during sBM-DC induction. Importantly, infusion of sBM-DCs could alleviate colitis in mice with inflammatory bowel disease by inhibiting lymphocyte proliferation and increasing the numbers of CD4+CD25+ regulatory T cells. Thus, these data infer a possible mechanism for the development of regDCs and further support the role of MSCs in treating immune disorders. The Journal of Immunology, 2015, 194: 000–000. http://www.jimmunol.org/ s an important component of the hematopoietic stem/ attractive because of their unique immunological characteristics, such progenitor cell (HSC) microenvironment, mesenchymal as low immunogenicity and immunoregulatory properties (3, 4). The A stromal/stem cells (MSCs) are capable of self-renewal and results from various studies have shown that MSCs are not able to multilineage differentiation (1, 2). However, MSCs are particularly stimulate T cell proliferation but can suppress T cell proliferation, and they also exert an inhibitory effect on the proliferation of B cells (5, 6). Additionally, MSCs might also act on dendritic cells (DCs) to *Center of Excellence in Tissue Engineering, Institute of Basic Medical Sciences, regulate immune responses (7); however, relatively little is known Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union about their effects on DC development and function.

Medical College, Peking Union Medical College Hospital, Chinese Academy of by guest on September 29, 2021 Medical Sciences, Beijing 100005, People’s Republic of China; †Department of Cell DCs not only play a role in the initiation of immunity but also are Biology, Institute for Biomedical Engineering, Rhenish-Westphalian Technical indispensable for the preservation of tolerance. They work as pro- University, Aachen University Medical School, 52074 Aachen, Germany; and fessional APCs in promoting Ag-specific immune responses and are ‡Cell Therapy Program, Princess Margaret Hospital, Toronto, Ontario M5G 2M9, Canada; and xInstitute of Biomaterials and Biomedical Engineering, University of Tor- likewise implicated in tuning the balance between immunity and onto, Toronto, Ontario M5G 2M9, Canada tolerance induction (8, 9). DCs arise from HSCs and were initially 1X.L. and S.R. contributed equally to this work. identified by their potent activation of naive T cells (10). DCs can be Received for publication October 21, 2014. Accepted for publication February 27, divided into distinct subsets by anatomical location, and different 2015. subsets of classical DCs express a diversity of phenotype and function This work was supported by the National Key Scientific Program of China Grant and favor alternative modules of immunity (11–13). Recent findings 2011CB964901, Program for International Science and Technology Cooperation suggest that DC heterogeneity is developmentally determined, and it Projects of China Grant 2013DFG30680, National Natural Science Foundation of China Grants 81370879 and 81370466, National Science and Technology has been difficult to identify the relationships between these various Major Project of the Ministry of Science and Technology of China Grant cells based only on cell surface markers and functional responses. 2014ZX09101042, and by Key Program for Beijing Municipal Natural Science Foundation Grant 7141006. Consequently, understanding the molecular basis of DC development and diversification is important to better appreciate immune regula- Address correspondence and reprint requests to Prof. Robert C.H. Zhao, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic tion. Currently, the basis for DC development into the recognized Medicine, Peking Union Medical College, Peking Union Medical College Hospital, subsets/lineages is only partially understood, based on the require- Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences, 5 Dongdansantiao, Beijing 100005, People’s Republic of China. E-mail address: ments for several transcription factors, including PU.1, IFN regula- [email protected] tory factor (IRF)8, E2-2, IRF4, Batf3, Ikaros, GFi1, and ID2 (14-16). The online version of this article contains supplemental material. These transcription factors combine to form a transcriptional network Abbreviations used in this article: BM, bone marrow; BM-HSC, BM-derived hema- that gives rise to the phenotypically and functionally distinct subsets topoietic stem/progenitor cell; BM-MSC, BM-derived MSC; ChIP, chromatin immu- under steady-state conditions. It is now becoming evident that DC noprecipitation; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine development is guided by lineage-restricted transcription factors such t-butyl ester; DC, dendritic cell; H3K4me3, trimethylation at lysine 4 of histone H3; H3K27me3, trimethylation at lysine 27 of histone H3; HSC, hematopoietic as IRF8, E2-2, and Batf3 (17-19). However, little is known regarding stem/progenitor cell; IBD, inflammatory bowel disease; imDC, immature DC; IRF, how cytokines and lineage-restricted transcription factors operate at IFN regulatory factor; maDC, mature DC; MSC, mesenchymal stem/stromal cell; PRC, polycomb repressive complex; qRT-PCR, quantitative RT-PCR; regDC, regu- a molecular level to direct DC diversification and development. latory DC; sBM-DC, Sca+ BM-MSC–driven DC; siRNA, small interfering RNA; The Notch family provides an evolutionarily conserved signaling TCF, T cell–specific factor; TNBS, 2,4,6-trinitrobenzene sulfonic acid; TRAF, network that plays a key role in the development of a variety of TNFR-associated factor. immune cells. To date, four Notch receptor family members and Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 five Notch ligands have been identified in mammalian cells (20,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402641 2 INDUCED IRF8-CONTROLLED regDCs VIA NOTCH–RBP-J SIGNALING

21). Notch receptors are activated following binding of appro- acetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT; the cells in the priate ligands, which results in the nuclear translocation of the sBM-DCs plus DAPT group were treated with 20 mM DAPT that was Notch intracellular domain. The Notch intracellular domain dissolved in DMSO). DMSO or 20 mM DAPT (a Notch inhibitor, Tocris Bioscience) was added every 2 d; after coculture for 7 d, the remaining interacts with a number of cytoplasmic and nuclear proteins, loosely adherent cell clusters were collected for the following experiment. permitting signal transduction through several pathways that in- clude activation of the CBF-1/RBP-J transcription factor, which Flow cytometric analysis works as a negative regulator of lineage-specific gene expression Flow cytometric analysis was performed as previously described (39). The (22, 23). Several studies suggest possible involvement of Notch fluorescent Abs used in the study included FITC-conjugated anti-mouse d in myeloid cell differentiation (13, 24–26). It seems that there Sca-1, CD4, CD9, CD90, CD31, CD44, H-2K , Ia, CD11c, CD40, CD80, and CD86 and PE-conjugated anti-mouse CD25, CD45, CD73, and CD11b is reciprocal regulation of Notch in DC development, but the de- (BD Biosciences). For each Ab, IgG of the same isotype from the same tailed relationship and underlying mechanisms remain far from species was used as the isotype control (BD Biosciences). Analysis was being understood. performed on Accuri C6 flow cytometers with CFlow software (Accuri Recent data support the concept that specific gene expression Cytometers, Ann Arbor, MI). patterns are under the control of epigenetic alterations (27, 28). Endocytosis assay and MLCs Acetylation and methylation of specific lysine residues on N-ter- minal histone tails are fundamental for the formation of chromatin Endocytosis and MLCs were performed as previously described (39). domains. Two canonical modifications are trimethylation at lysine Cytokine analysis 27 of histone H3 (H3K27me3) and trimethylation at lysine 4 of The supernatant of the sBM-DCs was harvested in RPMI 1640 medium histone H3 (H3K4me3). It is known that H3K27me3 is a repressive without FBS for 4 h. The supernatant of the MLCs was harvested after 3 d. Downloaded from mark catalyzed by polycomb repressive complex (PRC)2 and is All supernatants and sera from mice were analyzed using ELISA kits (BD associated with promoters of inactive genes. Conversely, H3K4me3, Biosciences) according to the manufacturer’s instructions. catalyzed by the trithorax family of proteins, is a hallmark of tran- RNA isolation and quantitative RT-PCR analysis scriptional start sites and generally associated with transcriptionally active genes (29–32). A growing body of evidence has shown that Total RNA was extracted from cells with TRIzol reagent (Invitrogen) and then reverse transcribed using a Quantiscript RT kit (TaKaRa Bio). epigenetic alterations are involved in the regulation of various genes Quantitative RT-PCR (qRT-PCR) was performed on a StepOne system http://www.jimmunol.org/ expression (33–36), but little is known about epigenetic alterations (Applied Biosystems) with a SYBR Green real-time PCR kit (TaKaRa Bio). during the generation of DCs. Data were normalized to the reference gene b-actin. The primers used are Although several studies have demonstrated that MSCs can mod- listed in Supplemental Table I. ulate the development and function of DCs, the underlying mecha- Western blot analysis nisms remain to be determined. In this study, we report that Sca-1+ Lin2CD1172 bone marrow (BM)–derived MSCs (BM-MSCs) influ- The protein expression level was analyzed by Western blot as previously described (38). Abs were obtained from Cell Signaling Technology. enced the fate decision of BM-derived HSCs (BM-HSCs) and drove them to differentiate into distinct IRF8-controlled regulatory DCs Chromatin immunoprecipitation

+ by guest on September 29, 2021 (regDCs, Sca BM-MSC–driven DCs [sBM-DCs]). The Notch sig- Chromatin immunoprecipitation (ChIP) was performed using an EZ-Magna naling pathway plays a critical role in the generation of the sBM-DCs ChIP kit (Millipore) according to the manufacturer’s instructions. ChIP- by controlling IRF8 expression in an RBP-J–dependent way. grade Abs specific for H3K4me3, H3K27me3, and EED were obtained from Millipore; anti-SIRT1 and anti-IRF8 Abs were obtained from Cell Signaling Technology; anti-WDR5 and anti-SUZ12 Abs were obtained Materials and Methods from Abcam; and anti-ASH2, anti-RbBP5, and anti-MLL1 Abs were Animals obtained from Bethyl Laboratories. The primers used are listed in Supplemental Table I. Five- to 6-wk-old BALB/C and C57BL/6 mice were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences Coimmunoprecipitation (Beijing, China). All mice were bred and maintained under specific pathogen-free conditions. Animal handling and experimental procedures Cells were harvested and lysed in RIPA buffer. The lysates were incubated were approved by the Animal Care and Use Committee of the Chinese with protein A/G agarose beads (Millipore); SIRT1 Ab (10 mg) was sub- Academy of Medical Sciences. sequently added, and the resulting mixture was incubated overnight at 4˚C. Beads conjugated with lysates and SIRT1 Abs were precipitated, washed Culture of mouse BM-MSCs and BM-DCs three times with RIPA buffer, and then analyzed by Western blot. MSCs were prepared and purified from mouse BM cells as previously Lentiviral vector preparation and infection and small described (37, 38). Mouse immature DCs (imDCs) and mature DCs interfering RNA knockdown assay (maDCs) were generated according to previously published protocols (39). Lentivirus production was carried out according to protocols from Gene- In brief, BM mononuclear cells were prepared from BALB/C mouse femur Pharma. The small interfering RNA (siRNA) sequences of RBP-J, IRF8, BM suspensions by depletion of red cells and then cultured at a density of and negative control were 59-GGTTACGCTGTGCTCTGAACA-39,59- 2 3 106 cells/ml in RPMI 1640 supplemented with 10% FBS, 10 ng/ml GCTGACTTGTGCATTGCTTCA-39, and 59-TTCTCCGAACGTGTCA- GM-CSF, and 5 ng/ml IL-4 (R&D Systems). For imDCs, nonadherent cells CGTTTC-39, respectively. GFP+ and red fluorescent protein+ cells were sorted were gently washed out on day 4, and the remaining loosely adherent cell by a BD FACSCalibur flow cytometer. clusters were collected. imDCs cultured for a further 4 d under the stim- ulation of 10 ng/ml bacterial LPS (Sigma-Aldrich) were used as maDCs. In vivo allogeneic delayed-type hypersensitivity assay Coculture experiment The allogeneic delayed-type hypersensitivity assay was performed as previously described (39). HSCs were enriched from BM cells using an EasySep mouse hematopoietic progenitor enrichment kit (StemCell Technologies). After HSCs were Preparation and treatment of inflammatory bowel disease + 2 2 purified, they were seeded onto Sca-1 CD117 Lin BM-MSC monolayers mouse model at a density of 1 3 105 cells in 2 ml per well in six-well plates, and MSC culture medium was replaced with RPMI 1640 supplemented with 10% sBM-DCs were i.p. injected (3 3 106 cells/mouse) into six BALB/C re- FBS. The ratio of MSCs/HSCs is 1:10. In some cases, the experiments cipient mice on days 26, 24, and 0. On day 0, the pretreated mice were were divided into two groups: sBM-DCs (the cells in the sBM-DC group injected by an intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid were treated with DMSO) and sBM-DCs plus N-[N-(3,5-difluorophen- (TNBS; Sigma-Aldrich) in 150 mg/kg 1:1 ethanol/PBS solution via a 4-cm The Journal of Immunology 3 catheter (TNBS plus sBM-DCs); six mice were injected with TNBS and Statistical analysis used as inflammatory bowel disease (IBD) model control (TNBS); six mice were injected with normal saline as normal controls. On day 5, the All statistical analysis was performed with SPSS software version 17.0. The , colon was photographed and stained with H&E. The levels of IL-12, IL-10, p values were calculated using a Student t test, and p values 0.05 were and TNF-a in serum were assessed by ELISA. The proportion of CD4+ considered to be statistically significant. CD25+ regulatory T lymphocytes in spleen was assessed using FACS analysis. CD11b+ splenocytes were isolated and used for detection IRF8 Results expression by Western blot analysis. Sca-1+CD1172Lin2 BM-MSCs induce the generation of Histology a novel DC population To investigate the influence of MSCs on the differentiation of HSCs, Colons of mice from different groups were harvested, fixed in 4% formalin + 2 2 for 48 h, and embedded in paraffin. Histological sections were cut and we isolated Sca-1 CD117 Lin MSCs from BM (Supplemental Fig. stained with H&E. 1A, 1B) and then seeded BM-HSCs on MSC monolayers at a ratio of Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Sca-1+CD1172Lin2 BM-MSCs induce the generation of regulatory sBM-DCs. (A) Morphology of sBM-DCs induced from BM-HSCs cocultured with Sca-1+CD1172Lin2 BM-MSCs for 7 d compared with imDCs and maDCs. Scale bars, 20 mm. (B) Expression of functional molecules on sBM-DCs, imDCs, and maDCs. Red lines represent cells stained with isotype-matched control Abs. (C) The expression of DC transcription factors of sBM- DCs, imDCs, and maDCs examined by Western blot. imDCs and maDCs were used as controls. Representative data from one of three independent experiments are shown. (D) Phagocytic ability of sBM-DCs and maDCs examined by flow cytometric analysis. The gray lines represent the controls (Ctrol). Representative data from one of three independent experiments are shown. (E) Different secretion of cytokines by sBM-DCs, imDCs, and maDCs. Cells (5 3 105) grown in RPMI 1640 without serum and the supernatants were collected after 4 h. The levels of IL-10, IL-12, and TNF-a were analyzed by ELISA. ★p , 0.05, ★★p , 0.01. 4 INDUCED IRF8-CONTROLLED regDCs VIA NOTCH–RBP-J SIGNALING

10:1 (HSCs/MSCs). During the coculture, the nonadherent HSCs Fig. 1, sBM-DCs had a stable immature-like phenotype and secreted gradually extended small and short processes from different points of IL-10, an important inhibitory cytokine. We hypothesized that sBM- the cell body to become DC-like cells, which we termed sBM-DCs DCs may have immune regulatory functions. To confirm this hy- (Fig. 1A). Phenotype analysis (Fig. 1B) showed that sBM-DCs, pothesis, we added sBM-DCs into an allogeneic lymphocyte co- compared with maDCs, expressed a higher level of the myeloid culture system and showed that they significantly suppressed lineage marker CD11b but lower levels of functional markers Ia, lymphocyte proliferation (Fig. 2A). Meanwhile, we also observed CD80, CD86, and CD40, similar to imDCs. In contrast to imDCs, that IFN-g and IL-2 levels in culture supernatant were greatly re- addition of LPS to these cells could not increase the expression of the duced in the presence of sBM-DCs (Fig. 2B). Thus, we have shown above markers (Supplemental Fig. 2A). To understand the molecular that sBM-DCs are a novel DC population with low immunogenicity repertoires of sBM-DCs, based on the requirements for several and high immunoregulatory potential. transcription factors, we determined the expression of DC transcrip- Because sBM-DCs were potent inhibitors of the lymphocyte tion factors as reported by Steinman and Idoyaga (40) by Western proliferation in vitro, we wondered whether sBM-DCs could blot analysis (Fig. 1C). Interestingly, sBM-DCs expressed most of the also suppress allospecific immune reactions in vivo. An allogeneic aforementioned DC transcription factors such as IRF4, IRF8, PU.1, delayed-type hypersensitivity experiment was performed. As Ikaros, Batf3, Spib, and T cell–specific factor (TCF)4, but at lower shown in Fig. 2C, the footpad swelling of BALB/C mice receiving levels compared with imDCs and maDCs, indicating that sBM-DCs the alloantigen was suppressed significantly by infusion of sBM- represent a novel DC population with a similar phenotype but dif- DCs. Taken together, these results demonstrate that sBM-DCs ferent transcription factor pattern to imDCs. We also found that sBM- might be used as a negative regulator of immune responses and

DCs had a greater phagocytic capacity compared with maDCs (Fig. have the potential to treat immune dysregulation diseases. Downloaded from 1D). To further characterize sBM-DCs, we determined their cytokine Infusion of sBM-DCs alleviates colitis in mice with IBD expression patterns by ELISA (Fig.1E).IncontrasttomaDCs,sBM- DCs secreted more IL-10 but less IL-12 and TNF-a, and this profile Because sBM-DCs were potent in inhibiting lymphocyte prolif- was not altered after LPS stimulation (Supplemental Fig. 2B), sug- eration both in vitro and in vivo, we next tested whether sBM-DCs gesting that sBM-DCs maybe be involved in immune regulation. were able to suppress T cell–mediated inflammation in vivo. IBD + 2 2 These results demonstrate that mouse BM-derived Sca-1 CD117 Lin is a well-known preclinical inflammatory disease model and is http://www.jimmunol.org/ MSCs drive HSCs to differentiate into a unique population of DCs. often used to investigate pathophysiology and test emerging therapeutic strategies. Thus, we studied the therapeutic effect of The low immunogenicity and immunoregulatory properties of sBM-DCs in an IBD mouse model (41). Macroscopic findings sBM-DCs both in vitro and in vivo showed that mice in the TNBS group developed clinical signs of To investigate whether the ability of sBM-DCs to stimulate colitis with severe edema of the colon and fecal blood. Amelio- lymphocyte proliferation was different from that of maDCs, MLC ration of the signs was observed after treatment with sBM-DCs was performed. The results demonstrate that sBM-DCs could only (Fig. 3A). Furthermore, we observed severe damage of the epi- slightly stimulate lymphocyte proliferation (Fig. 2A). As shown in thelium, infiltration of inflammatory cells, and disruption of the by guest on September 29, 2021

FIGURE 2. The low immunogenicity and immunoregulatory properties of sBM-DCs both in vitro and in vivo. (A) Lymphocytes from C57BL/6 mice (C57-Lym) were stained with CFSE and cocultured alone or with sBM-DCs or lymphocytes from BALB/C mice (B/C-Lym) in 10:1 for 3 d. Black lines represent lymphocytes from C57L/6 alone as control. The influence of sBM-DCs on lymphocyte proliferation was assessed by FACS. A FACS analysis representa- tive of three independent experiments with similar results is shown. (B)IFN-g and IL-2 secretion by lymphocytes treated as described in (A) was an- alyzed by ELISA performed on supernatants. All data represent means 6 SD. ★p , 0.05, ★★p , 0.01. (C) Footpad swelling of BALB/c mice re- ceiving C57BL/6 splenocytes was measured after i.p. infusion of sBM-DCs, maDCs, or normal saline (NS) (n = 5). All data represent means 6 SD. ★p , 0.05. NS, normal saline. The Journal of Immunology 5

FIGURE 3. sBM-DCs alleviate im- mune pathological changes of IBD. (A) Macroscopic findings of the colon from different groups. Scale bars, 2 mm. (B) Histological observations of the colon stained with H&E. Scale bars, 100 mm. (C) The serum was Downloaded from collected and levels of IL-12, IL-10, and TNF-a were assessed by ELISA. Data represent means 6 SD. ★★p , 0.01, ★★★p , 0.001. (D) The pro- portion of CD4+CD25+ regulatory T lymphocytes in spleen was assessed

using CD4 and CD25 Ab and FACS http://www.jimmunol.org/ analysis. (E)CD11b+ splenocytes were isolated and used for detection of IRF8 expression by Western blot. Repre- sentative data from one of three inde- pendent experiments are shown. NC, normal control. by guest on September 29, 2021

cryptal glands in colon histological sections from TNBS mice. infusion of sBM-DCs induced the increase of CD4+/CD25+ Treg However, in the sBM-DC–treated group, the colitis was alleviated cells is dependent on MHC class II expression by sBM-DCs is not (Fig. 3B). Furthermore, IL-2 and TNF-a levels in the sBM-DC– mentioned in the present work and needs further study. However, treated group were significantly lower compared with the TNBS these data provide further evidence that sBM-DCs have the po- group, and the level of IL-10, a type of anti-inflammatory cyto- tential to treat immune disorders. kine, was increased significantly (Fig. 3C). As CD4+ T cells have been shown to play a central role in The Notch signaling pathway plays an essential role in the TNBS colitis, we were interested in knowing whether sBM-DCs generation of sBM-DCs can modulate CD4+ T cell proliferation in vivo. To investigate this A previous study using a Transwell culturing system showed that question, the ratios of CD4+ and CD25+ regulatory T cells were direct cell–cell contact was important in generating sBM-DCs analyzed in splenocytes isolated from different groups. Compared (data not shown). We were interested in determining whether with the TNBS group, the proportion of CD4+ T cells in the sBM- the Notch signaling pathway influences sBM-DCs, given that it is DC–treated group was decreased, and the percentage of CD4+ evolutionarily conserved and crucial for stem cell development CD25+ regulatory T cells was increased (Fig. 3D). We also found and differentiation. To test the effect of Notch signaling on sBM- that CD11b+ splenocytes from sBM-DC–treated mice expressed DC production, we added DAPT (a kind of Notch inhibitor) to the lower levels of IRF8 (Fig. 3E), indicating that sBM-DCs might coculture system. Interestingly, addition of DAPT changed the exert their inhibitory effects through more than one mechanism. phenotypes of sBM-DCs by increasing expression of Ia, CD80, Darrasse-Je`ze (42) reported that the DC-dependent feedback loop CD86, and CD40 and decreasing expression of CD11b (Fig. 4A), that controls regulatory T cell numbers and homeostatic division is and it also altered the expression of most DC-specific transcription dependent on MHC class II expression by DCs in vivo. Whether factors. As shown in Fig. 4B, the expression of IRF8, TNFR- 6 INDUCED IRF8-CONTROLLED regDCs VIA NOTCH–RBP-J SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. The Notch signaling pathway plays an essential role in the generation of sBM-DCs. After HSCs were cocultured with Sca-1+CD1172Lin2 BM-MSCs treated with DMSO or DAPT for 7 d, the induced cells were collected and purified. (A) The cells were analyzed for the expression of functional molecules (black lines) by FACS. Red lines represent cells stained with isotype-matched control Abs. Representative data from one of three independent experiments are shown. (B) The Notch signaling pathway alters the expression of sBM-DC transcription factors detected by Western blot. The results are representative of three independent experiments. (C) The Notch signaling pathway suppresses proinflammatory cytokine production in sBM-DCs measured by ELISA. All data represent means 6 SD. ★★p , 0.01, ★★★p , 0.001. (D) The Notch signaling pathway affects the immune regulatory function of sBM- DCs. CFSE-labeled lymphocytes from C57BL/6 mice (C57-Lym) were cultured alone or cocultured with sBM-DCs or DAPT-treated sBM-DCs for 3 d in the absence or presence of lymphocytes from BALB/C mice (B/C-Lym), then assessed by FACS. A FACS analysis representative of three independent experiments with similar results is shown. associated factor (TRAF)6, IRF4, PU.1, Batf3, and TCF4 in- expression of IRF8 and IL-12 in sBM-DCs. We then wanted to creased in DAPT-treated sBM-DCs and the expression of Ikaros know whether IRF8 has any relationship with the secretion of and SpiB decreased significantly compared with that of sBM-DCs. inflammatory cytokine by sBM-DCs. We used the RNA silencing Moreover, the results from ELISA experiments showed that sBM- technique to knock down IRF8 expression and found that the RNA DCs treated with DAPT secreted higher levels of IL-12 and TNF-a and protein levels of IL-12 were markedly reduced (Fig. 5A, 5B). (Fig. 4C), and an MLR assay showed that DAPT-treated sBM-DCs We also used a ChIP assay to explore the potential direct controls are less capable of suppressing lymphocyte proliferation than are of IL-12 expression in sBM-DCs by IRF8. The results of the ChIP sBM-DCs (Fig. 4D). These results demonstrate that the generation assay showed that IRF8 directly controlled the expression of IL-12 and function of sBM-DCs is Notch-dependent. by binding to the promoter region of IL-12 (Fig. 5C). Our data show that Notch inhibits the expression of IRF8 and that IRF8 The suppression of IRF8 expression by Notch signaling determines the secretion of IL-12 in sBM-DCs, which implies that determines inflammatory cytokine secretion by sBM-DCs IRF8 has a central role in the generation of sBM-DCs. IRF8 was recently found to promote DC lineage commitment, but the molecular mechanisms regulated by IRF8 that drive the gen- Notch-controlled IRF8 expression is RBP-J–dependent eration and control of DC functions remain largely unknown. From RBP-J plays a critical role in signal transduction via the canonical results shown in Fig. 4B and 4C, we know that Notch inhibits the Notch pathway. However, Notch-independent RBP-J activities The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Knockdown of IRF8 reduces IL-12 secretion of sBM-DCs and Notch-controlled IRF8 expression is RBP-J–dependent. HSCs were trans- fected without any siRNA (NO) or with negative control of siRNA (NC) or with siRNA-IRF8 (IRF8-I) or siRNA–RBP-J (RBP-J-I) and then cocultured with Sca-1+CD1172Lin2 BM-MSCs for 7 d. (A) The expression of IRF8 protein in the cells was then assayed by Western blot. To determine IL-12 secretion, supernatants were collected from 5 3 105 cells grown in RPMI 1640 without serum for 4 h and then assayed by ELISA for the IL-12 level. (B) The expression of Irf8 and Il12 RNA in the cells was assessed by qRT-PCR. (C) The recruitment of IRF8 to the promoter region of Il12 in sBM-DCs was tested by ChIP assay. All data represent means 6 SD. (D) The influence of RBP-J on IRF8 expression in sBM-DCs was analyzed by Western blot and qRT-PCR. (E) The influence of RBP-J on IL-12 expressed in sBM-DCs. The expression of Il12 RNA was assayed by qRT-PCR. To determine IL-12 secretion, supernatants were collected from 5 3 105 cells grown in RPMI 1640 without serum for 4 h and then assayed by ELISA for IL-12 level. (F) Immuno- precipitation (IP)/Western blot (WB) analysis was performed to investigate the influence of the Notch pathway on the direct binding between SIRT1 and RBP-J. SIRT1 was immunoprecipitated using SIRT1 Ab. A Western blot using RBP-J Ab was then used to confirm the level of SIRT1 bound to RBP-J. One representative experiment of three is shown. (G) The impacts of Notch signaling on IRF8 expression and IL-12 secretion in RBP-J–silenced cells. The expression of IRF8 was analyzed by Western blot, and the secretion level of IL-12was determined by ELISA. ★p , 0.05, ★★p , 0.01, ★★★p , 0.001. have also been reported (43). We then focused on whether Notch- IRF8 though RBP-J, we added DAPT in RBP-J–silenced cells and controlled IRF8 expression is RBP-J–dependent. We knocked measured its impacts on IRF8 expression and IL-12 secretion. The down RBP-J expression in HSCs by siRNA and then cocultured results revealed that Notch and RBP-J act in the same linear the cells with Sca-1+CD1172Lin2 BM-MSCs. The expression of pathway, and DAPT is unable to increase the expression of these IRF8 from HSCs transfected with siRNAs against RBP-J was messengers in RBP-J–silenced cells (Fig. 5G). significantly reduced compared with the control group, suggesting that IRF8 is a target of RBP-J–mediated regulation (Fig. 5D). We The Notch signaling pathway regulates the expression of IRF8 also observed that changes in IL-12 secretion are in accordance through histone modification with those of IRF8 (Fig. 5E). These data suggest that Notch Epigenetic alterations are often involved in the regulation of gene regulates the expression of IRF8 via RBP-J. To further explore expression. H3K4me3 and H3K27me3 are mediated by the his- underlying mechanisms, we assessed the acetylation state of RBP- tone methyltransferase complexes MLL (comprising MLL1, WDR5, J both in sBM-DCs and DAPT-treated sBM-DCs using a coim- RbBP5, and ASH2L) and PRC2 (comprising SUZ12 and EED), munoprecipitation assay and found that RBP-J bound more to respectively. To test the hypothesis that Notch signaling also SIRT1 (a deacetylase) in sBM-DCs compared with DAPT-treated regulates the expression of IRF8 via epigenetic mechanisms, we sBM-DCs in which Notch signaling was blocked, indicating that assessed the levels of H3K4me3 and H3K27me3 in the Irf8 Notch controlled IRF8 expression via the acetylation changes of promoter regions by ChIP. The results showed that levels of RBP-J (Fig. 5F). To strengthen our indication that Notch controls H3K27me3 at the Irf8 promoter increased in sBM-DCs, whereas 8 INDUCED IRF8-CONTROLLED regDCs VIA NOTCH–RBP-J SIGNALING levels of H3K4me3 at the Irf8 promoter increased in DAPT- we found that Sca-1+CD1172Lin2 BM-MSCs can directly drive treated sBM-DCs (Fig. 6A). These results indicate that the chro- BM-HSCs to differentiate into regulatory sBM-DCs. SBM-DCs matin structure of the Irf8 gene is maintained in a repressive state differ from sDCs (38), which are driven by MSCs isolated from in sBM-DCs, but it may be changed to an active state in the mouse embryonic fibroblasts. SBM-DCs expressing CD11c and Ia presence of DAPT. Our data show that WDR5 of the MLL com- at low levels are phenotypically distinguishable from the CD11chi plex and SUZ12 of the PRC2 complex are mainly responsible for Iahi regDCs induced by mouse GM-CSF, mouse IL-10, and human the H3K4me3 and H3K27me3 modifications, respectively, of Irf8 TGF-b (47). Because CD80 and CD86 are expressed at low levels in sBM-DCs (Fig. 6B). Taken together, the results suggest that the by sBM-DCs, they are also different from the CD80hiCD86hi Notch signaling pathway strongly downregulates the expression CD40+Iaint DCs reported by Akbari et al. (48). These results show of IRF8 in sBM-DCs through the Notch–RBP-J signaling and that sBM-DCs are a phenotypically novel regDC population. Cell changes in histone methylation (Fig. 7). fate decisions in the hematopoietic system involve the actions of a small number of regulatory transcription factors, which establish Discussion specification in pluripotent progenitors and induce commitment MSCs serve as an important supporting population for HSC de- and differentiation to the blood/immune cell lineages (49). velopment. Identifying the molecular pathways regulating HSC Experiments with transcriptional arrays have recently been per- specification, self-renewal, and expansion remains a fundamental formed on DC subsets and have revealed discrete programs for the goal of both basic and translational research (44, 45). In this study, DC subsets. PU.1 has been identified as a key selector gene that we show that Sca-1+CD1172Lin2 BM-MSCs could drive BM- functions during the macrophage versus DC fate decision. High

HSCs to differentiate into a distinct IRF8-controlled regulatory expression of PU.1 is required to induce a DC fate in monocytes Downloaded from sBM-DC subset in the absence of exogenous cytokines. The Notch and to antagonize the macrophage-inducing transcription factors pathway is responsible for sBM-DC generation by inhibiting IRF8 c-Maf and MafB (50, 51). In accordance with these earlier results, expression through an RBP-J–dependent means and by regulat- we found a marked increase in the expression of PU.1 and no ing histone H3 modification. Importantly, sBM-DCs can alleviate TRAF6 and c-Maf expression upon differentiation of HSCs into colitis in IBD mice by increasing the number of CD4+CD25+ sBM-DCs in cocultures. + regulatory T cells and by suppressing CD4 T cell proliferation Genetic deletion studies reveal key roles of several transcription http://www.jimmunol.org/ in vivo. factors in DC development. Transcription factors required by Our previous studies have shown that MSCs can regulate the specific DC subsets have recently been identified, such as E2-2 for differentiation of monocyte-derived DCs (46). In the present study, the development of plasmacytoid DCs (17), Batf3 for CD8+ DCs by guest on September 29, 2021

FIGURE 6. The Notch pathway regulates the expression of IRF8 through histone modifications. (A) ChIP was performed to determine the histone H3 methylation sta- tus in the promoter regions of Irf8 in sBM- DCs in the presence or absence of DAPT on day7. The values were normalized to the inputs for each sample. (B) ChIP was performed on sBM-DCs or DAPT-treated sBM-DCs on day 7 using Abs specific for the core components of the MLL and PRC2 complexes to examine the recruitment of these components to the promoter regions of Irf8. The values were normalized to the input for each sample. All data represent means 6 SD. ★p , 0.05, ★★p , 0.01. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. A proposed schema of the mechanism of action of the Sca-1+CD1172Lin2 BM-MSC–mediated immune regulation of sBM-DCs. Sca-1+ CD1172Lin2 BM-MSCs drive HSCs to differentiate into a novel IRF8-controlled regDC population (sBM-DCs) when cocultured without exogenous cytokines. The Notch pathway regulates the generation of the sBM-DCs by controlling IRF8 expression in an RBP-J–dependent way; furthermore, IRF8 determines the secretion of IL-12 directly. Moreover, IRF8 expression is also regulated by the stable reciprocal changes in the levels of H3K4me3 and H3K27me3 mediated by the MLL and PRC2 complexes, respectively. WDR5 (red) of the MLL complex and SUZ12 (red) of the PRC2 complex are mainly responsible for the histone H3 modifications of the Irf8 promoter in sBM-DCs.

(19), and IRF4 for CD11b+ DCs (52). The interplay of these IL-12 secretion by sBM-DCs, and we showed that IRF8 regulated different transcription factors is essential to shape the rich het- the transcription of IL-12 directly, and knockdown of IRF8 ef- erogeneity of the DC family. Thus, we identified the pattern of fectively reduced IL-12 secretion of sBM-DCs. These results transcription factors for driving the differentiation programs of suggest that IRF8 is an essential component of sBM-DC function sBM-DCs (Fig. 1C) according to previously published reports by and sBM-DC–mediated innate immune response. These results Steinman and Idoyaga (40), and we established how they instruct confirm that, functionally, sBM-DCs are an IRF8-controlled sBM-DC development and function. Interestingly, sBM-DCs, regDC population. Strikingly, IRF8 was also reported to play a criti- expressing lower levels of PU.1, IRF4, IRF8, SpiB, Batf3, cal role in the regulation of several proinflammatory genes (including TCF4, and Ikaros, as well as dim levels of TRAF6, Relb, and c- IL-12) that function during macrophage activation (53–55). Whether Maf, are quite different from imDCs and maDCs. These results there is any relationship in IRF8 function between sBM-DC gener- show that sBM-DCs are a novel regDC population with respect ation and macrophage activation deserves further study. to gene expression profile. Among the transcription factors, the The fate decision of hematopoietic cells occurs throughout present study focused more on IRF8. IRF8, also known as IFN embryogenesis and adulthood, much of which is controlled by the consensus sequence–binding protein, is a component of the IRF Notch signaling pathway (56). However, the molecular mecha- transcription factor family. IRF8 is differentially expressed in DCs nisms regulated by Notch that drive the generation and control and is particularly highly expressed in common DC progenitors, the functions of DCs remain largely unknown. In this study, we plasmacytoid DCs, CD11b+ conventional DCs, and CD8a+ DCs show that Sca-1+CD1172Lin2 BM-MSCs express high levels of (18). In this study, we detected the expression of IRF8 in imDCs, Jagged-1, a type of Notch ligand (Supplemental Fig. 1C, 1D) maDCs, and sBM-DCs and found that sBM-DCs express lower through which the Notch signaling pathway is activated to regu- levels of IRF8 compared with imDCs and maDCs. Furthermore, late the generation of sBM-DCs. When DAPT was added, sig- inhibiting secretion of inflammatory cytokine is the hallmark of nificant changes in sBM-DCs were observed. Further microRNA regDCs, but the related molecular mechanisms are not well un- interference assays demonstrated that secretion of IL-12 is IRF8- derstood. In the present study, we observed a decreased level of dependent and that IRF8 controls the secretion of IL-12 in an 10 INDUCED IRF8-CONTROLLED regDCs VIA NOTCH–RBP-J SIGNALING

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