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Butyrate interferes with the differentiation and function of human monocyte-derived dendritic cells
Cellular Immunology 277 (2012) 66–73
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Butyrate interferes with the differentiation and function of human monocyte-derived dendritic cells Lu Liu a,1, Lin Li b,1, Jun Min c, Jie Wang c, Heng Wu a, Yujie Zeng a, Shuang Chen a, Zhonghua Chu a,⇑ a
Department of Gastrointestinal Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Department of Obstetrics and Gynecology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China c Department of Hepato-Biliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China b
a r t i c l e
i n f o
Article history: Received 18 August 2011 Accepted 18 May 2012 Available online 29 May 2012 Keywords: Dendritic cell Butyrate Immunosuppression
a b s t r a c t Dendritic cells (DCs) are specialized antigen-presenting cells that are uniquely capable of either inducing immune responses or maintaining a state of self-tolerance, depending on their stage of maturation. In the present study, we describe a way to interfere with DCs maturation. The compound butyrate can affect the differentiation of DCs generated from human monocytes and can inhibit T cell proliferation. We demonstrate that butyrate substantially down-regulates the expression of CD80, CD83, and MHC class II molecules; increases endocytic capability; reduces allostimulatory abilities; promote interleukin-10 (IL-10) production; and inhibits interleukin-12 (IL-12) and interferon-c (IFN-c) production. These results demonstrate a specific immune suppression property of butyrate and supports further investigation for butyrate as a new immunotherapeutic agent. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that play an important role in inducing primary T cell responses [1]. The differentiation process of DCs results in two main types of DCs: immature DCs (imDCs) and mature DCs (mDCs). The function of DCs depends on their stage of maturation. DCs are in the immature state in most tissues where they are capable of capturing antigens. ImDCs are poor stimulators of T cell proliferation, as they lack the requisite MHC molecules and costimulatory receptors. Upon encountering a powerful immunological stimulus, imDCs convert into mDCs, thus switching from an antigen-capturing mode to an antigen-presenting and T cell-stimulating mode [2]. MDCs are the most effective antigen presenting cells. They express high levels of costimulatory molecules, MHC molecules, and proinflammatory cytokines. While mDCs induce a state of immune activation, imDCs can induce a state of immune tolerance [3,4]. In fact,
Abbreviations: DCs, dendritic cells; APCs, antigen-presenting cells; imDCs, immature DCs; MDCs, mature DCs; HDAC, histone deacetylase; PBMCs, peripheral blood mononuclear cells; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; MLR, allogeneic mixed leukocyte reaction; FACS, fluorescence-activated cell sorter; MFI, fluorescence index. ⇑ Corresponding author. Address: Department of Gastrointestinal surgery, Sun Yat-sen memorial Hospital of Sun Yat-sen University, Yanjiang xilu, NO. 107, Guangzhou 510120, China. Fax: +86 20 34071091. E-mail address: [email protected] (Z. Chu). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.05.011
imDCs are the main target of medical research, including transplantation [5], autoimmune diseases [6], and immunotolerance to tumors [7]. However, there are difficulties in inducing DCs to form imDCs. Some research indicates that low doses of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF), transforming growth factor b1 (TGF-b1), or interleukin-10 (IL-10) can induce DCs to convert to the immature state, prolonging the survival time of grafts [8–10]. However, this effect is limited. When imDCs are stimulated by inflammatory factors, the imDCs differentiate into a mature state, mediating immunological rejection of the graft [11,12]. Histone deacetylase (HDAC) inhibitors can control the expression of genes by increasing histone acetylation as well as regulating chromatin structure and transcription. Butyrate, a kind of HDAC inhibitor, is a short-chain fatty acid derived from bacterial metabolism of dietary fiber in the colon. Butyrate has been studied not only as a differentiation stimulator of stem cells but also as an antineoplastic agent [13,14]. Recently, butyrate has been confirmed to inhibit T cell activation. In vitro and in vivo studies have also shown that butyrate can induce a state of T cell anergy [15,16]. Based on the observation that butyrate could alter the stimulatory function of APC and suppress subsequent T cell proliferation responses, it has been speculated that butyrate regulates T cell-mediated immune reactions through modulating APC function. In the present study, we investigated the influence of butyrate on the mature state of peripheral blood monocyte-derived DCs in vitro to explore the impact of an HDAC inhibitor on the process of DC maturity.
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2. Materials and methods
2.4. FACS analysis
2.1. Reagents and materials
To evaluate of surface-marker expression, 50 ll cells (5 105/ mL) were incubated with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mAb for 30 min at 4 °C. Nonbinding isotype-matched FITC- and PE-conjugated mouse IgG were used as control. Cells were analyzed on a FACSCalibur flow cytometer by CellQuest software. All mouse monoclonal antibodies were obtained from BD Bioscience (San Diego, CA, USA): anti-CD80 FITC, anti-CD86 FITC, anti-CD1a FITC, anti-CD83 PE, anti-HLA-DR PE.
RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), 100 lg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), and 10% fetal calf serum (FCS; Invitrogen, Carlsbad, CA, USA) was used as the culture medium. RhGM-CSF and recombinant human interleukin-4 (rhIL-4) were obtained from R&D Systems (Minneapolis, MN, USA). Mouse anti-human CD80, CD83, CD86, CD1a, and human leukocyte antigen-DR (HLA-DR) monoclonal antibodies (mAb) were obtained from BD Bioscience (San Diego, CA, USA). Lipopolysaccharide (LPS) (Escherichia coli 0111:B4), sodium butyrate, carboxyfluorescein diacetate succinimidyl ester (CFSE), and FITC-dextran (40000 MW) were obtained from Sigma– Aldrich (St. Louis, MO, USA). Human interleukin-12 (p40) (IL-12 p40) sandwich enzyme-linked immunosorbent assay (ELISA) kit was obtained from R&D Systems (Minneapolis, MN, USA). Human IL-10, interferon-c (IFN-c) ELISA kits were obtained from Uscn life Science & Technology (Wuhan, CHN). Ficoll lymphocyte isolation liquid (relative density 1.077 g/L) was obtained from Nycomed Pharma AS (Oslo, Norway).
2.5. Endocytosis assay To determine mannose receptor (MR)-mediated endocytosis, 5 105 cells/mL were incubated in medium with FITC-labeled dextran (molecular weight 40,000; Sigma–Aldrich, St. Louis, MO, USA) at a concentration of 1 mg/mL on day 7. After an incubation period of 2 h at 37 and 4 °C as a control, cells were recovered and washed extensively with cold phosphate-buffered saline (PBS) to stop endocytosis. Then cells were resuspended in PBS at a final concentration of 5 105 cells/mL and analyzed on a FACSCalibur. Fluidphase endocytosis was measured via cellular uptake of Lucifer yellow and was analyzed by flow cytometry.
2.2. Cell separation The experiment was performed in the medical research center of Sun Yat-sen memorial hospital of Sun Yat-sen University. Five samples of heparinized blood were obtained from different healthy adult volunteers of Sun Yat-sen University. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over lymphoprep and resuspended in RPMI 1640 containing penicillin (100 U/mL), streptomycin (100 lg/mL), glutamine (2 mmol/L) and 10% FCS. Then, PBMCs were cultured for 2 h at 37 °C on plastic dishes. Subsequently, the suspended cells were collected as responder lymphocyte cell and used for mixed lymphocyte reactions, and the adherent cells were harvested to be used for the induction of DCs. 2.3. DC differentiation and maturation The adherent cells coming from CD14+ cells were cultured in RPMI 1640 containing 10% FCS medium in 25 cm2 plastic flasks at a cell density of 5 106 cells/mL at 37 °C in a humidified CO2containing atmosphere and divided into 4 groups based on different induction methods. r Group A (control): The adherent cells were cultured with 1000 U/mL rhGM-CSF and 500 U/mL rhIL-4 for 6 days. Then the DCs were cultivated in the medium without stimuli for 24 h. s Group B (LPS): The adherent cells were cultured with 1000 U/mL rhGM-CSF and 500 U/mL rhIL-4 for 6 days. Then the DCs were cultivated in the medium with 1 mg/L LPS as stimuli for 24 h. t Group C (Na-B): The adherent cells were cultured with 1000 U/mL rhGM-CSF and 500 U/mL rhIL-4 for 6 days. Then the DCs were cultivated in the medium with 1 mmol/L butyrate as stimuli for 24 h. We used 1 mmol/L butyrate because preliminary experiments showed 1.5 mmol/L butyrate had cytotoxicity on DCs in our culture condition. u Group D (Na-B + LPS): To study the influence of butyrate on the early stages of DC differentiation, adherent cells were cultured in the presence of butyrate (1 mmol/ L), GM-CSF (1000 U/mL) and rhIL-4 (500 U/mL) for 6 days. Then the DCs were cultivated in the medium with 1 mg/L LPS for 24 h after butyrate was depleted. DCs were harvested 24 h later to identify the cell phenotype using a fluorescence-activated cell sorter (FACS). The supernatant of cultures was collected and stored at 80 °C for detection of the secretion of IL-12 p40, IL-10, and IFN-c.
2.6. Allogeneic mixed leukocyte reaction (MLR) The stimulation of lymphocytes by DCs of different group was evaluated in the MLR assay. On day 7, each group of DCs were stimulated by adding mitomycin C at a final concentration of 25 mg/L; the cells were then washed extensively and incubated in the dark at 37 °C for 30 min. DCs were then washed with PBS three times and resuspended in RPMI 1640 culture medium. Next, the lymphocytes from different volunteer (responder cell) were washed twice with PBS and resuspended at 1 107/mL in PBS containing 0.25 lmol/L CFSE. The cell suspension was incubated at 37 °C for 15 min and immediately washed with RPMI-1640 culture medium to terminate the staining. Then, 2 104 stimulator DCs were added to 2 105 allogeneic CFSE-labeled lymphocytes in U-shaped 96well culture plates in RPMI 1640 medium supplemented with 10% FCS (total volume, 200 ll/well). At the same time, the CFSE-labeled lymphocytes without stimulated by DCs served as control. The cells were cultured in 5% CO2 atmosphere at 37 °C for 96 h. After collected and washed, the cells were analyzed on a FACSCalibur, and the data obtained were analyzed by Cellquest and Modfit software.
2.7. Measurement of cytokine IL-12 p40, IL-10, IFN-c production To assess IL-12p40, IL-10, and IFN-c secretion, cell-free supernatants were harvested 24 h after addition of the stimulus. Cytokines were measured by ELISA using matched-pair antibodies according to the manufacturer’s instructions. Wellscan MK3 enzyme-labeling instrument was used to detect the absorbance value following coloration at a wavelength of 450 nm. The concentrations of IL-12p40, IL-10, and IFN-c were determined from standard curves.
2.8. Statistical analysis Data in graphs were shown as mean ± SE. ANOVA followed by Student’s t-test was used for multiple comparisons. Statistical significance was set at P < 0.05.
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3. Results 3.1. Effect of butyrate on the growth and morphology of DCs After maturation was inducted with rhGM-CSF and rhIL-4, adherent cells gradually became suspended and increased in volume. During the process of maturation, DCs developed extensive cytoplasmic projections in many directions. A notable feature of activated DCs was the occurrence of large cell clusters several hours after the addition of LPS (Fig. 1B). Conversely, the morphology of butyrate-induced DCs on later stage was similar to imDCs; these cells were mostly suspended, but there was no change in cell volume. Only a few cells acquired multiple cytoplasmic projections. Compared with mDCs, little cell clustering occurred in butyrate-induced DCs on later stage (Fig. 1C). Compared with imDCs, butyrate-induced DCs on early stage grew slowly and became relatively small in the presence of butyrate for 6 days. Although butyrate was removed on day 6, DCs induced by LPS in sequence presented few small cytoplasmic projections from the cell, without changes to the volume or formation of cell clusters (Fig. 1D). The morphological differences of DCs demonstrated the significant influence of butyrate on the maturation process of cells.
marker CD83 was even lower than expression in imDCs. Furthermore, when the imDCs were first incubated with butyrate for 6 days and subsequently exposed to LPS for 24 h, interference in the DC maturation process was observed, as indicated by the inhibition of expression of CD83 and the reduction in CD80, CD1a, and MHC class II expression. But for the costimulatory molecule CD86, there were little difference between the four groups. 3.3. Butyrate increases the endocytic activity of DCs DCs are known to be capable of endocytosing fluorescence-labeled dextran via the cell surface mannose receptor; therefore, flow cytometry was applied and used to calculate the mean fluorescence index (MFI) of FITC in DC, indicating the endocytic activity and antigen intake of DCs. The endocytic capacity of imDCs gradually weakened as DCs matured. Fig. 3 shows that mDCs are capable of significantly lower endocytosis following the maturation process. The endocytic capacity in the presence or absence of butyrate on day 6 was nearly 2-fold higher than mDCs. Furthermore, endocytic activity of butyrate-induced DCs on early stage was upregulated when DCs were incubated with butyrate for 6 days, and addition of LPS could not inhibit this process. The endocytic capacity of butyrate-induced DCs on early stage was nearly 4-fold higher than mDCs.
3.2. Effect of butyrate on the surface markers of DCs 3.4. Butyrate down-regulates the T cell stimulatory activity of DCs Because terminal DC maturation critically determines the outcome of immune responses, the surface markers of DCs were evaluated in each group to determine the maturation state of DCs. Flow cytometric analyses were performed to confirm phenotypic differences between each group. As shown in Fig. 2, the mDCs showed a phenotype characterized by up-regulation of CD1a, MHC class II molecules, the costimulatory molecule CD80, and the maturation marker CD83. However, butyrate-induced DCs on later stage showed a markedly different phenotype compared with mDCs. The expression of CD80, CD83, CD1a, and MHC class II molecules were reduced substantially, and expression of the maturation
Because of the expression of high levels of membrane MHC and co-stimulatory molecules, mDCs can activate T lymphocytes. As butyrate could down-modulate the expression of these surface molecules, butyrate’s effect on the ability of DCs to induce MLR was examined. The DCs/T cell ratio was 1/10. When the CFSE-labeled T lymphocyte split, CFSE fluorescence could be equally allocated into two daughter cells. By calculating the proportion of cells in each division peak and dividing by the expected progeny at those divisions, the number of cells that had entered division could be calculated. In Fig. 4A, discrete division cycles could be visualized
Fig. 1. The morphology of DCs in different groups on day 7. (A) Immature DCs (control group) (400). (B) Mature DCs induced by LPS (400). The suspension of mature DCs was shown as extensive dendrite formation and large cell clusters. (C) Immature DCs (butyrate-induced DCs on later stage) (400). The DCs were shown that only a few cells acquired multiple cytoplasmic projections and few cell clusters. (D) Immature DCs (butyrate-induced DCs on early stage) (400). The DCs were shown that few small cytoplasmic projections on the cells, without changes to the volume or formation of cell clusters.
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Fig. 2. Effect of butyrate on the phenotypic characterization of DCs. Monocytes were cultured for 6 days with GM-CSF (1000 U/mL) and IL-4 (500 U/mL). Subsequently, the immature DCs were activated with LPS (1 mg/L) or butyrate (1 mmol/L) for 24 h. In addition, to study the influence of butyrate on early stage of DC differentiation, monocytes were cultured in the presence of butyrate (1 mmol/L). Then the LPS (1 mg/L) was added into the culture after the butyrate was depleted on day 6. DCs were harvested 24 h later to detect the phenotypes of each group. Light curves were represented as control, and dark curve were represented as fluorescence intensity of DC’s surface markers (CD80, CD83, CD86, CD1a, and HLA-DR) in different induction groups. Numbers calculated by flow cytometry indicated the mean fluorescence intensity (MFI) of each sample. Control (group A); LPS (group B); Na-B (group C); Na-B + LPS (group D). Shown are the means ± SD (n = 5). ⁄P < 0.05; ⁄⁄P < 0.01.
by means of different CFSE signal peaks. The majority of the DCs induced by LPS underwent several cell divisions, visualized by the respective CFSE peaks, which had subsequently lost half of their CFSE signal with each division round. The results showed mDCs exhibited remarkable divisions of T lymphocyte, and there were little division of T lymphocyte in other groups. From histograms
on each plot (Fig. 4B), the division cycles could be visualized by using Proliferation kinetics model. The peak in dark grey represented non-divided CFSE-labeled responder lymphocyte (mother cell populations) whereas the different peaks in light grey indicated daughter cell populations. Next, the proliferation index was calculated by the proportions that progeny cells occupied progen-
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Fig. 3. Effect of butyrate on the phagocytic activity of DCs. Immature DCs were generated from monocytes cultured with GM-CSF (1000 U/mL) and IL-4 (500 U/mL), in the absence or presence of 1 mmol/L butyrate. On day 7, after using the different stimuli, the antigen capture capacity of DCs was evaluated using flow cytometry to assess single cell engulfment of FITC-labeled dextran. Light curves were represented as control, and dark curve were represented as fluorescence intensity of DC’s engulfment of FITClabeled dextran in different induction groups. Numbers calculated by flow cytometry indicated the mean fluorescence intensity (MFI) of each sample. Control (group A); LPS (group B); Na-B (group C); Na-B + LPS (group D). Shown are the means ± SD (n = 5). ⁄P < 0.05; ⁄⁄P < 0.01.
itor cells. The results (Fig. 4C) showed that butyrate-induced DCs on later stage for 24 h exhibited reduced allostimulatory capability, and the allogeneic proliferation index was 6-fold lower than that of mDCs. Furthermore, the proliferation index of butyrate-induced DCs on early stage was 12-fold lower than that of in the mDCs, and 2-fold lower than that of butyrate-induced DCs on later stage. These results indicate that the phenotypic changes induced by butyrate led to changes in lymphocyte proliferation. 3.5. Butyrate modulates IL-12, IL-10, and INF-c production of DCs IL-12 p40, IL-10, and IFN-c were assessed 24 h after the stimulation of each group to investigate the effects of butyrate on the regulation of the cytokine production of DCs. Compared with the mDCs, butyrate-induced DCs on later stage exhibited a nearly 3-fold decrease in IL-12 p40 secretion. Butyrate-induced DCs on early stage exhibited a similar IL-12 p40 secretion capacity to that of butyrate-induced DCs on later stage. Next, IFN-c was detected in each group. The results showed that IFN-c production of mDCs was nearly 5-fold higher than IFN-c production of butyrate-induced DCs. In contrast, IL-10 secretion by DCs of butyrate-induced DCs was nearly 11- and 7.5-fold higher than IL-10 secretion of mDCs (Fig. 5). 4. Discussion The short-chain fatty acid molecule butyrate, an HDAC inhibitor, has been widely applied induce stem cells differentiation and affect carcinoma cells invasion or metastasis [17,18]. However, very little research on the ability of butyrate to regulate DC function has been done. In this study, we provide morphological,
phenotypical, and functional evidence that butyrate suppresses the differentiation of monocyte-derived DCs in vitro. Our data showed that butyrate impaired DC function, which may represent a new approach in immunosuppression. The distinctive morphology of mDCs is dendrite formation, but we observed that DCs induced by butyrate exhibited a different morphology compared to LPS-induced DCs. Butyrate inhibited dendritic morphology formation, including the formation of clusters and increases in volume, causing the DCs to have a morphology similar to that of imDCs. Furthermore, the influence of butyrate was unaffected by the addition of LPS to early stage DCs. The inhibitory effects of butyrate in the early stages of DC maturation were more remarkable than the effects of adding butyrate on day 6. This suggests that butyrate could interfere with the differentiation and maturation process of DCs in early stages, keeping the cells in a stable, immature morphology. When stimulated with an inflammatory factor (LPS), the imDCs would convert into the mature phenotype, characterized by increased expression of MHC molecules, costimulatory molecules, and increased T-cell stimulating capacity [2,12]. We observed that butyrate down-regulated MHC molecules, costimulatory molecules (CD80), classical DC-associated molecules (CD1a), and DCspecific maturation molecules (CD83), which provide the cells with optimal T-cell stimulatory capacity. CD83 is a characteristic marker that is highly expressed on mDCs [19]; hence, the low expression of CD83 on butyrate-induced cells indicates a suppression of the DC maturation process. The marker CD80 has been shown to provide critical costimulatory signals through interaction with the receptor CD28 found on T cells [20]. Therefore, low CD80 expression may be an effective mechanism to down-regulate the T cell response. Furthermore our data show that butyrate does not remarkably down-regulate expression of the costimulatory
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Fig. 4. Effects of butyrate on the T cell allostimulatory capacity of DCs. Immature DCs were generated from monocytes cultured with GM-CSF (1000 U/mL) and IL-4 (500 U/ mL) in the absence or in the presence of 1 mmol/L butyrate. On day 6, immature DCs were further incubated with LPS or butyrate. After 24 h, the cells were harvested and used as stimulators of allogeneic T cell proliferation. The allogeneic lymphocytes (responder cells) were resuspended in PBS containing 0.25 lmol/L CFDA-SE and incubated at 37 °C for 15 min. Then the stimulator DCs were added to allogeneic CFSE-labeled lymphocytes at a ratio of 1:10. CFSE-labeled lymphocytes without stimulator DCs served as the control. The cells were cultured for 96 h and FACS detection was conducted. Flow histograms of CFSE fluorescence for allogeneic lymphocyte of different groups were shown in (A). Discrete division cycles could be visualized by means of different CFSE signal peaks, which had subsequently lost half of their CFSE signal with each division round. Proliferation kinetics model of lymphocytes were shown in (B). The peak in dark grey represented non-divided CFSE-labeled responder lymphocyte whereas the peak in light grey indicated daughter cell populations. The proliferation indexes of different groups were shown in (C). Control (group A); LPS (group B); Na-B (group C); Na-B + LPS (group D). Shown are the means ± SD (n = 5). ⁄⁄P < 0.01.
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Fig. 5. Effects of butyrate on DCs cytokine release. Immature DCs were generated from monocytes in the absence or presence of 1 mmol/L butyrate and stimulated with LPS (1 mg/L) or butyrate (1 mmol/L) for 24 h. Then IL-12, IL-10, and IFN-c were measured by ELISA using matched-pair antibodies. Wells from which IL-12 p40, IL-10, and IFN-c were collected contained 1 mL culture medium with 1 106 DCs. Control (group A); LPS (group B); Na-B (group C); Na-B + LPS (group D). Shown are the means ± SD (n = 5). ⁄ P < 0.05.
molecule CD86, compared with other surface markers of butyrateinduced DCs, especially CD80. It is different from previous reports [21,22]. There is still some controversy about role of CD80 and CD86 in triggering costimulation [23,24]. Both molecules are capable of binding CD28 on T cells, but some studies have shown that CD80 costimulation is superior to CD86 costimulation [25,26]. This would suggest that in our model, highly expression of CD86 alone was not sufficient to provide effective costimulation [25,27]. We observed a decrease in the expression of various surface molecules known to play important roles in the process of T cell activation by mediating intercellular contact and delivering essential costimulatory activity [28,29]. Our studies also showed that butyrate markedly reduced antigen-specific T cell proliferation. Therefore, the impaired ability of DCs to stimulate T cell proliferation function was most likely due to the down-regulation of MHC molecules and costimulatory molecules (CD80) on DCs [30,31]. Importantly, butyrate modulated cytokine production by DCs. The major Th1-skewing factors IL-12 and IFN-c were substantially suppressed by butyrate, which might critically influence the development of a subsequent T cell response. These results were consistent with previous reports [21,32,33]. Our study found that the anti-inflammatory cytokine IL-10 was increased in butyrate-induced DCs, similar to Millard’s research [34]. As a suppressive effect of the cytokine, IL-10 strongly inhibited proliferation of both CD4+ and CD8+ T cell, and shifted the T-helper cell (Th) response toward a predominant Th2 profile [35,36]. The alteration of DC-derived cytokine production by butyrate contributed to impairment of the DC immunostimulatory capacity, consistent with phenotypic changes and MLR results described above. Another notable characteristic of DCs induced with butyrate was that imDCs had an increased endocytic capacity. Moreover, the endocytic capacity of DCs could be further enhanced when butyrate was added in the early stage of differentiation. Furthermore, DC differentiation was inhibited by the addition of butyrate to DCs in early stages, keeping the cells in a stable morphology. Besides the morphological differences between DCs at different stages, the notable changes following butyrate addition to early stage DCs were the maturation of the phenotype and the continuous down-regulation of T-cell stimulatory activity. Importantly, the immature state of DCs could not be activated to a mature state by a stimulus such as LPS. This suggests that butyrate keeps the DCs in a stable, immature stage, which may be better than other inducing methods. The changes observed in cytokine production were similar with the addition of butyrate both in early stage and later stage, and the enhanced endocytic capacity of DCs would benefit the induction of Ag-specific immunological tolerance [37,38]. These data suggest that butyrate plays an important role in the early stage of DC differentiation by blocking the process of maturation from monocytic precursors into mDCs, and thus potentially impairing the normal function of DCs. Millard et al. [34] and Säemann et al. [22] combined butyrate and LPS on DC’s induction, and found butyrate could decrease the maturation markers of DCs and down-regulate their ability to prime
alloreactive naïve T cells. The effect of butyrate on the differentiation of DCs might be weakened when combined with stimulus such as LPS, which could not accurately reflect its suppression effects. Therefore, only butyrate was added to induce DCs final differentiation in our experiments and found butyrate had the suppressive effects on the later stage of DCs. Next, we also examined the effect of butyrate on early stage of DCs’ differentiation and found the suppressive effect persisted, even after butyrate was depleted and LPS was added on day 6 to induce final maturation. The results demonstrated that imDCs induced by butyrate on early stage of cultivation could resist extraneous stimulus, and maintain the immaturation stage of DCs. However, in Wang’s and Säemann’s studies, DCs were induced by GM-CSF and IL-4 for 5 or 7 days and then LPS for 2 days to explore effects of butyrate on early stage of DCs’ differentiation [21,22]. Butyrate was added throughout the whole culture period, which was different from our experiments. The depletion of butyrate could better explain the persistent suppressive effects on DCs. Our studiy found that butyrate could suppress the maturation procedure of DCs, which was similar to Säemann’s study. But Wang et al. [21] reported the opposite results. Wang reported that butyrate did not up-regulate maturation markers (CD83, CD86, MHC class I, and MHC class II molecules) of DCs, and there was no significant difference between butyrate-induced DCs and control DCs in MLR. It is probably due to the different concentration of butyrate in culture. The 0.5 mmol/L concentration of butyrate in Wang’s study might be too low to resist the maturation promoting effect of LPS, and our preliminary experiments showed that immunosuppression effect of 1 mmol/L butyrate were better than 0.5 mmol/L butyrate in our culture condition. Our results, demonstrating impairment of DC differentiation by butyrate, represent important evidence necessary to understand the mechanism of butyrate on immunological tolerance and support further interest in butyrate as an immunotherapeutic reagent. Because DCs are a crucial factor in the initiation of an effective immune response, the use of butyrate to interfere with DC maturation and differentiation may substantially modify the outcome of an immune response. Acknowledgments This study was supported by research funding from the National Natural Science Foundation of China (No. 81001306). References [1] W.J. Mayer, U.M. Irschick, P. Moser, M. Wurm, H.P. Huemer, N. Romani, E.U. Irschick, Characterization of antigen-presenting cells in fresh and cultured human corneas using novel dendritic cell markers, Invest. Ophthalmol. Vis. Sci. 48 (2007) 4459–4467. [2] M. Bros, F. Jährling, A. Renzing, N. Wiechmann, N.A. Dang, A. Sutter, R. Ross, J. Knop, S. Sudowe, A.B. Reske-Kunz, A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid, Blood 109 (2007) 3820–3829.
L. Liu et al. / Cellular Immunology 277 (2012) 66–73 [3] S.M. Barratt-Boyes, A.W. Thomson, Dendritic cells: tools and targets for transplant tolerance, Am. J. Transplant. 5 (2005) 2807–2813. [4] L. Rober, F.N. Wan, M.S. Ralph, Dendritic cells in transplantation – friend or foe, Immunity 14 (2001) 357–368. [5] N. Tochiki, M. Narita, Z. Zheng, C. Lu, A. Saitoh, N. Watanabe, N. Satoh, T. Furukawa, K. Toba, I. Fuse, Y. Aizawa, M. Takahashi, Induction of recipient cellspecific donor T-cell anergy by UV-C-irradiated recipient immature monocytederived dendritic cells, Bone Marrow Transplant. 41 (2008) 1037–1045. [6] Y. Ikeda, F. Akbar, H. Matsui, M. Onji, Characterization of antigen-presenting dendritic cells in the peripheral blood and colonic mucosa of patients with ulcerative colitis, Eur. J. Gastroenterol. Hepatol. 13 (2001) 841–850. [7] P.T. Coates, F.J. Duncan, B.L. Colvin, Z. Wang, A.F. Zahorchak, W.J. Shufesky, A.E. Morelli, A.W. Thomson, In vivo-mobilized kidney dendritic cells are functionally immature, subvert alloreactive T-cell responses, and prolong organ allograft survival, Transplantation 77 (2004) 1080–1089. [8] F. Geissmann, P. Revy, A. Regnault, Y. Lepelletier, M. Dy, N. Brousse, S. Amigorena, O. Hermine, A. Durandy, TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells, J. Immunol. 162 (1999) 4567–4575. [9] M.B. Lutz, R.M. Suri, M. Niimi, A.L. Ogilvie, N.A. Kukutsch, S. Rössner, G. Schuler, J.M. Austyn, Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo, Eur. J. Immunol. 30 (2000) 1813–1822. [10] Z. Zheng, M. Narita, M. Takahashi, A. Liu, T. Furukawa, K. Toba, Y. Aizawa, Induction of T cell anergy by the treatment with IL-10-treated dendritic cells, Comp. Immunol. Microbiol. Infect. Dis. 27 (2004) 93–103. [11] R.M. Gorczynski, J. Bransom, M. Cattral, X. Huang, J. Lei, L. Xiaorong, W.P. Min, Y. Wan, J. Gauldie, Synergy in induction of increased renal allograft survival after portal vein infusion of dendritic cells transduced to express TGF-b and IL10, along with administration of CHO cells expressing the regulatory molecule OX2, Clin. Immunol. 95 (2000) 182–189. [12] A. Cignetti, A. Vallario, I. Roato, P. Circosta, B. Allione, L. Casorzo, P. Ghia, F. Caligaris-Cappio, Leukemia-derived immature dendritic cells differentiate into functionally competent mature dendritic cells that efficiently stimulate T cell responses, J. Immunol. 173 (2004) 2855–2865. [13] L. Wang, H.S. Luo, H. Xia, Sodium butyrate induces human colon carcinoma HT-29 cell apoptosis through a mitochondrial pathway, J. Int. Med. Res. 37 (2009) 803–811. [14] G. Liang, O. Taranova, K. Xia, Y. Zhang, Butyrate promotes induced pluripotent stem cell generation, J. Biol. Chem. 285 (2010) 25516–25521. [15] S.K. Jackson, A. DeLoose, K.M. Gilbert, The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors, Immunology 106 (2003) 486–495. [16] K.M. Gilbert, S.R. Boger, P. Price, E.K. Fifer, T cell tolerance induced by histone deacetylase inhibitor is mediated by P21cip1, Immunopharmacol. Immunotoxicol. 27 (2005) 545–564. [17] R. Joachimiak, A. Kaznica, T. Drewa, Influence of sodium butyrate on hepatocellular carcinoma (hepG2) and glioblastoma (C6) cell lines in vitro, Acta Pol. Pharm. 64 (2007) 561–563. [18] M. Zhou, P. Li, L. Tan, S. Qu, Q.L. Ying, H. Song, Differentiation of mouse embryonic stem cells into hepatocytes induced by a combination of cytokines and sodium butyrate, J. Cell. Biochem. 109 (2010) 606–614. [19] L.J. Zhou, T.F. Tedder, Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily, J. Immunol. 154 (1995) 3821– 3835. [20] A. Mondino, M.K. Jenkins, Surface proteins involved in T cell costimulation, J. Leukoc. Biol. 55 (1994) 805–815. [21] B. Wang, A. Morinobu, M. Horiuchi, J. Liu, S. Kumagai S, Butyrate inhibits functional differentiation of human monocyte-derived dendritic cells, Cell. Immunol. 253 (2008) 54–58. [22] M.D. Säemann, O. Parolini, G.A. Böhmig, P. Kelemen, P.M. Krieger, J. Neumuller, K. Knarr, W. Kammlander, W.H. Horl, C. Diakos, K. Stuhlmeier, G.J. Zlabinger,
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
73
Bacterial metabolite interference with maturation of human monocytederived dendritic cells, J. Leukoc. Biol. 71 (2002) 238–246. K.S. Hathcock, G. Laszlo, C. Pucillo, P. Linsley, R.J. Hodes, Comparative analysis of B7–1 and B7–2 costimulatory ligands: expression and function, J. Exp. Med. 180 (1994) 631–640. S. Salek-Ardakani, R. Arens, R. Flynn, A. Sette, S.P. Schoenberger, M. Croft, Preferential use of B7.2 and not B7.1 in priming of vaccinia virus-specific CD8 T cells, J. Immunol. 182 (2009) 2909–2918. U. Matulonis, C. Dosiou, G. Freeman, C. Lamont, P. Mauch, L.M. Nadler, J.D. Griffin, B7–1 is superior to B7–2 costimulation in the induction and maintenance of T cell-mediated antileukemia immunity. Further evidence that B7–1 and B7–2 are functionally distinct, J. Immunol. 156 (1996) 1126– 1131. P. Rajavelu, S.D. Das, Expression of co-stimulatory molecules B7.1 & B7.2 on macrophages infected with various strains of Mycobacterium tuberculosis & its influence on T-cell apoptosis Indian, J. Med. Res. 127 (2008) 388–394. G.A. Bohmig, P.M. Krieger, M.D. Saemann, C. Wenhardt, E. Pohanka, G.J. Zlabinger, N-butyrate downregulates the stimulatory function of peripheral blood-derived antigen-presenting cells: a potential mechanism for modulating T-cell responses by short-chain fatty acids, Immunology 92 (1997) 234–243. A. Gruber, T. Brocker, MHC class I-positive dendritic cells (DC) control CD8 T cell homeostasis in vivo: T cell lymphopenia as a prerequisite for DC-mediated homeostatic proliferation of naive CD8 T cells, J. Immunol. 175 (2005) 201– 206. S.Y. Tseng, M. Otsuji, K. Gorski, X. Huang, J.E. Slansky, S.I. Pai, A. Shalabi, T. Shin, D.M. Pardoll, H. Tsuchiya, B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells, J. Exp. Med. 193 (2001) 839–846. F. Fu, Y. Li, S. Qian, L. Lu, F. Chambers, T.E. Starzl, J.J. Fung, A.W. Thomson, Costimulatory molecule-deficient dendritic cell progenitors (MHC class II+, CD80dim, CD86-) prolong cardiac allograft survival in nonimmunosuppressed recipients, Transplantation 62 (1996) 659–665. L. Lu, D. McCaslin, T.E. Starzl, A.W. Thomson, Bone marrow-derived dendritic cell progenitors (NLDC 145+, MHC class II+, B7–1dim, B7–2-) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes, Transplantation 60 (1995) 1539–1545. F. Miro, C. Nobile, N. Blanchard, M. Lind, O. Filipe-Santos, C. Fieschi, A. Chapgier, G. Vogt, L. de Beaucoudrey, D.S. Kumararatne, F. Le Deist, J.A. Casanova, S. Amigorena, C. Hivroz, T cell-dependent activation of dendritic cells requires IL-12 and IFN-gamma signaling in T cells, J. Immunol. 177 (2006) 3625–3634. D. Messmer, I. Hatsukari, N. Hitosugi, I.G. Schmidt-Wolf, P.C. Singhal, Morphine reciprocally regulates IL-10 and IL-12 production by monocytederived human dendritic cells and enhances T cell activation, Mol. Med. 12 (2006) 284–290. A.L. Millard, P.M. Mertes, D. Ittelet, F. Villard, P. Jeannesson, J. Bernard, Butyrate affects differentiation, maturation and function of human monocyte-derived dendritic cells and macrophages, Clin. Exp. Immunol. 130 (2002) 245–255. J. Peguet-Navarro, C. Moulon, C. Caux, C. Dalbiez-Gauthier, J. Banchereau, D. Schmitt, Interleukin-10 inhibits the primary allogeneic T cell response to human epidermal Langerhans cells, Eur. J. Immunol. 24 (1994) 884–891. A.M. Stax, C. Crul, S.W. Kamerling, N. Schlagwein, R.N. van der Geest, A.M. Woltman, C. van Kooten, CD40L stimulation of rat dendritic cells specifically favors the IL-12/IL-10 ratio resulting in a strong T cell stimulatory capacity, Mol. Immunol. 45 (2008) 2641–2650. H. Toyokawa, A. Nakao, R.J. Bailey, M.A. Nalesnik, T. Kaizu, J.L. Lemoine, A. Ikeda, K. Tomiyama, G.D. Papworth, L. Huang, A.J. Demetris, T.E. Starzl, N. Murase, Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation, Liver Transpl. 14 (2008) 346–357. M. Nouri-Shirazi, E. Guinet, Direct and indirect cross-tolerance of alloreactive T cells by dendritic cells retained in the immature stage, Transplantation 74 (2002) 1035–1044.
Contents lists available at SciVerse ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Butyrate interferes with the differentiation and function of human monocyte-derived dendritic cells Lu Liu a,1, Lin Li b,1, Jun Min c, Jie Wang c, Heng Wu a, Yujie Zeng a, Shuang Chen a, Zhonghua Chu a,⇑ a
Department of Gastrointestinal Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Department of Obstetrics and Gynecology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China c Department of Hepato-Biliary Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China b
a r t i c l e
i n f o
Article history: Received 18 August 2011 Accepted 18 May 2012 Available online 29 May 2012 Keywords: Dendritic cell Butyrate Immunosuppression
a b s t r a c t Dendritic cells (DCs) are specialized antigen-presenting cells that are uniquely capable of either inducing immune responses or maintaining a state of self-tolerance, depending on their stage of maturation. In the present study, we describe a way to interfere with DCs maturation. The compound butyrate can affect the differentiation of DCs generated from human monocytes and can inhibit T cell proliferation. We demonstrate that butyrate substantially down-regulates the expression of CD80, CD83, and MHC class II molecules; increases endocytic capability; reduces allostimulatory abilities; promote interleukin-10 (IL-10) production; and inhibits interleukin-12 (IL-12) and interferon-c (IFN-c) production. These results demonstrate a specific immune suppression property of butyrate and supports further investigation for butyrate as a new immunotherapeutic agent. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that play an important role in inducing primary T cell responses [1]. The differentiation process of DCs results in two main types of DCs: immature DCs (imDCs) and mature DCs (mDCs). The function of DCs depends on their stage of maturation. DCs are in the immature state in most tissues where they are capable of capturing antigens. ImDCs are poor stimulators of T cell proliferation, as they lack the requisite MHC molecules and costimulatory receptors. Upon encountering a powerful immunological stimulus, imDCs convert into mDCs, thus switching from an antigen-capturing mode to an antigen-presenting and T cell-stimulating mode [2]. MDCs are the most effective antigen presenting cells. They express high levels of costimulatory molecules, MHC molecules, and proinflammatory cytokines. While mDCs induce a state of immune activation, imDCs can induce a state of immune tolerance [3,4]. In fact,
Abbreviations: DCs, dendritic cells; APCs, antigen-presenting cells; imDCs, immature DCs; MDCs, mature DCs; HDAC, histone deacetylase; PBMCs, peripheral blood mononuclear cells; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; MLR, allogeneic mixed leukocyte reaction; FACS, fluorescence-activated cell sorter; MFI, fluorescence index. ⇑ Corresponding author. Address: Department of Gastrointestinal surgery, Sun Yat-sen memorial Hospital of Sun Yat-sen University, Yanjiang xilu, NO. 107, Guangzhou 510120, China. Fax: +86 20 34071091. E-mail address: [email protected] (Z. Chu). 1 These authors contributed equally to this work. 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.05.011
imDCs are the main target of medical research, including transplantation [5], autoimmune diseases [6], and immunotolerance to tumors [7]. However, there are difficulties in inducing DCs to form imDCs. Some research indicates that low doses of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF), transforming growth factor b1 (TGF-b1), or interleukin-10 (IL-10) can induce DCs to convert to the immature state, prolonging the survival time of grafts [8–10]. However, this effect is limited. When imDCs are stimulated by inflammatory factors, the imDCs differentiate into a mature state, mediating immunological rejection of the graft [11,12]. Histone deacetylase (HDAC) inhibitors can control the expression of genes by increasing histone acetylation as well as regulating chromatin structure and transcription. Butyrate, a kind of HDAC inhibitor, is a short-chain fatty acid derived from bacterial metabolism of dietary fiber in the colon. Butyrate has been studied not only as a differentiation stimulator of stem cells but also as an antineoplastic agent [13,14]. Recently, butyrate has been confirmed to inhibit T cell activation. In vitro and in vivo studies have also shown that butyrate can induce a state of T cell anergy [15,16]. Based on the observation that butyrate could alter the stimulatory function of APC and suppress subsequent T cell proliferation responses, it has been speculated that butyrate regulates T cell-mediated immune reactions through modulating APC function. In the present study, we investigated the influence of butyrate on the mature state of peripheral blood monocyte-derived DCs in vitro to explore the impact of an HDAC inhibitor on the process of DC maturity.
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2. Materials and methods
2.4. FACS analysis
2.1. Reagents and materials
To evaluate of surface-marker expression, 50 ll cells (5 105/ mL) were incubated with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mAb for 30 min at 4 °C. Nonbinding isotype-matched FITC- and PE-conjugated mouse IgG were used as control. Cells were analyzed on a FACSCalibur flow cytometer by CellQuest software. All mouse monoclonal antibodies were obtained from BD Bioscience (San Diego, CA, USA): anti-CD80 FITC, anti-CD86 FITC, anti-CD1a FITC, anti-CD83 PE, anti-HLA-DR PE.
RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), 100 lg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA), and 10% fetal calf serum (FCS; Invitrogen, Carlsbad, CA, USA) was used as the culture medium. RhGM-CSF and recombinant human interleukin-4 (rhIL-4) were obtained from R&D Systems (Minneapolis, MN, USA). Mouse anti-human CD80, CD83, CD86, CD1a, and human leukocyte antigen-DR (HLA-DR) monoclonal antibodies (mAb) were obtained from BD Bioscience (San Diego, CA, USA). Lipopolysaccharide (LPS) (Escherichia coli 0111:B4), sodium butyrate, carboxyfluorescein diacetate succinimidyl ester (CFSE), and FITC-dextran (40000 MW) were obtained from Sigma– Aldrich (St. Louis, MO, USA). Human interleukin-12 (p40) (IL-12 p40) sandwich enzyme-linked immunosorbent assay (ELISA) kit was obtained from R&D Systems (Minneapolis, MN, USA). Human IL-10, interferon-c (IFN-c) ELISA kits were obtained from Uscn life Science & Technology (Wuhan, CHN). Ficoll lymphocyte isolation liquid (relative density 1.077 g/L) was obtained from Nycomed Pharma AS (Oslo, Norway).
2.5. Endocytosis assay To determine mannose receptor (MR)-mediated endocytosis, 5 105 cells/mL were incubated in medium with FITC-labeled dextran (molecular weight 40,000; Sigma–Aldrich, St. Louis, MO, USA) at a concentration of 1 mg/mL on day 7. After an incubation period of 2 h at 37 and 4 °C as a control, cells were recovered and washed extensively with cold phosphate-buffered saline (PBS) to stop endocytosis. Then cells were resuspended in PBS at a final concentration of 5 105 cells/mL and analyzed on a FACSCalibur. Fluidphase endocytosis was measured via cellular uptake of Lucifer yellow and was analyzed by flow cytometry.
2.2. Cell separation The experiment was performed in the medical research center of Sun Yat-sen memorial hospital of Sun Yat-sen University. Five samples of heparinized blood were obtained from different healthy adult volunteers of Sun Yat-sen University. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over lymphoprep and resuspended in RPMI 1640 containing penicillin (100 U/mL), streptomycin (100 lg/mL), glutamine (2 mmol/L) and 10% FCS. Then, PBMCs were cultured for 2 h at 37 °C on plastic dishes. Subsequently, the suspended cells were collected as responder lymphocyte cell and used for mixed lymphocyte reactions, and the adherent cells were harvested to be used for the induction of DCs. 2.3. DC differentiation and maturation The adherent cells coming from CD14+ cells were cultured in RPMI 1640 containing 10% FCS medium in 25 cm2 plastic flasks at a cell density of 5 106 cells/mL at 37 °C in a humidified CO2containing atmosphere and divided into 4 groups based on different induction methods. r Group A (control): The adherent cells were cultured with 1000 U/mL rhGM-CSF and 500 U/mL rhIL-4 for 6 days. Then the DCs were cultivated in the medium without stimuli for 24 h. s Group B (LPS): The adherent cells were cultured with 1000 U/mL rhGM-CSF and 500 U/mL rhIL-4 for 6 days. Then the DCs were cultivated in the medium with 1 mg/L LPS as stimuli for 24 h. t Group C (Na-B): The adherent cells were cultured with 1000 U/mL rhGM-CSF and 500 U/mL rhIL-4 for 6 days. Then the DCs were cultivated in the medium with 1 mmol/L butyrate as stimuli for 24 h. We used 1 mmol/L butyrate because preliminary experiments showed 1.5 mmol/L butyrate had cytotoxicity on DCs in our culture condition. u Group D (Na-B + LPS): To study the influence of butyrate on the early stages of DC differentiation, adherent cells were cultured in the presence of butyrate (1 mmol/ L), GM-CSF (1000 U/mL) and rhIL-4 (500 U/mL) for 6 days. Then the DCs were cultivated in the medium with 1 mg/L LPS for 24 h after butyrate was depleted. DCs were harvested 24 h later to identify the cell phenotype using a fluorescence-activated cell sorter (FACS). The supernatant of cultures was collected and stored at 80 °C for detection of the secretion of IL-12 p40, IL-10, and IFN-c.
2.6. Allogeneic mixed leukocyte reaction (MLR) The stimulation of lymphocytes by DCs of different group was evaluated in the MLR assay. On day 7, each group of DCs were stimulated by adding mitomycin C at a final concentration of 25 mg/L; the cells were then washed extensively and incubated in the dark at 37 °C for 30 min. DCs were then washed with PBS three times and resuspended in RPMI 1640 culture medium. Next, the lymphocytes from different volunteer (responder cell) were washed twice with PBS and resuspended at 1 107/mL in PBS containing 0.25 lmol/L CFSE. The cell suspension was incubated at 37 °C for 15 min and immediately washed with RPMI-1640 culture medium to terminate the staining. Then, 2 104 stimulator DCs were added to 2 105 allogeneic CFSE-labeled lymphocytes in U-shaped 96well culture plates in RPMI 1640 medium supplemented with 10% FCS (total volume, 200 ll/well). At the same time, the CFSE-labeled lymphocytes without stimulated by DCs served as control. The cells were cultured in 5% CO2 atmosphere at 37 °C for 96 h. After collected and washed, the cells were analyzed on a FACSCalibur, and the data obtained were analyzed by Cellquest and Modfit software.
2.7. Measurement of cytokine IL-12 p40, IL-10, IFN-c production To assess IL-12p40, IL-10, and IFN-c secretion, cell-free supernatants were harvested 24 h after addition of the stimulus. Cytokines were measured by ELISA using matched-pair antibodies according to the manufacturer’s instructions. Wellscan MK3 enzyme-labeling instrument was used to detect the absorbance value following coloration at a wavelength of 450 nm. The concentrations of IL-12p40, IL-10, and IFN-c were determined from standard curves.
2.8. Statistical analysis Data in graphs were shown as mean ± SE. ANOVA followed by Student’s t-test was used for multiple comparisons. Statistical significance was set at P < 0.05.
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3. Results 3.1. Effect of butyrate on the growth and morphology of DCs After maturation was inducted with rhGM-CSF and rhIL-4, adherent cells gradually became suspended and increased in volume. During the process of maturation, DCs developed extensive cytoplasmic projections in many directions. A notable feature of activated DCs was the occurrence of large cell clusters several hours after the addition of LPS (Fig. 1B). Conversely, the morphology of butyrate-induced DCs on later stage was similar to imDCs; these cells were mostly suspended, but there was no change in cell volume. Only a few cells acquired multiple cytoplasmic projections. Compared with mDCs, little cell clustering occurred in butyrate-induced DCs on later stage (Fig. 1C). Compared with imDCs, butyrate-induced DCs on early stage grew slowly and became relatively small in the presence of butyrate for 6 days. Although butyrate was removed on day 6, DCs induced by LPS in sequence presented few small cytoplasmic projections from the cell, without changes to the volume or formation of cell clusters (Fig. 1D). The morphological differences of DCs demonstrated the significant influence of butyrate on the maturation process of cells.
marker CD83 was even lower than expression in imDCs. Furthermore, when the imDCs were first incubated with butyrate for 6 days and subsequently exposed to LPS for 24 h, interference in the DC maturation process was observed, as indicated by the inhibition of expression of CD83 and the reduction in CD80, CD1a, and MHC class II expression. But for the costimulatory molecule CD86, there were little difference between the four groups. 3.3. Butyrate increases the endocytic activity of DCs DCs are known to be capable of endocytosing fluorescence-labeled dextran via the cell surface mannose receptor; therefore, flow cytometry was applied and used to calculate the mean fluorescence index (MFI) of FITC in DC, indicating the endocytic activity and antigen intake of DCs. The endocytic capacity of imDCs gradually weakened as DCs matured. Fig. 3 shows that mDCs are capable of significantly lower endocytosis following the maturation process. The endocytic capacity in the presence or absence of butyrate on day 6 was nearly 2-fold higher than mDCs. Furthermore, endocytic activity of butyrate-induced DCs on early stage was upregulated when DCs were incubated with butyrate for 6 days, and addition of LPS could not inhibit this process. The endocytic capacity of butyrate-induced DCs on early stage was nearly 4-fold higher than mDCs.
3.2. Effect of butyrate on the surface markers of DCs 3.4. Butyrate down-regulates the T cell stimulatory activity of DCs Because terminal DC maturation critically determines the outcome of immune responses, the surface markers of DCs were evaluated in each group to determine the maturation state of DCs. Flow cytometric analyses were performed to confirm phenotypic differences between each group. As shown in Fig. 2, the mDCs showed a phenotype characterized by up-regulation of CD1a, MHC class II molecules, the costimulatory molecule CD80, and the maturation marker CD83. However, butyrate-induced DCs on later stage showed a markedly different phenotype compared with mDCs. The expression of CD80, CD83, CD1a, and MHC class II molecules were reduced substantially, and expression of the maturation
Because of the expression of high levels of membrane MHC and co-stimulatory molecules, mDCs can activate T lymphocytes. As butyrate could down-modulate the expression of these surface molecules, butyrate’s effect on the ability of DCs to induce MLR was examined. The DCs/T cell ratio was 1/10. When the CFSE-labeled T lymphocyte split, CFSE fluorescence could be equally allocated into two daughter cells. By calculating the proportion of cells in each division peak and dividing by the expected progeny at those divisions, the number of cells that had entered division could be calculated. In Fig. 4A, discrete division cycles could be visualized
Fig. 1. The morphology of DCs in different groups on day 7. (A) Immature DCs (control group) (400). (B) Mature DCs induced by LPS (400). The suspension of mature DCs was shown as extensive dendrite formation and large cell clusters. (C) Immature DCs (butyrate-induced DCs on later stage) (400). The DCs were shown that only a few cells acquired multiple cytoplasmic projections and few cell clusters. (D) Immature DCs (butyrate-induced DCs on early stage) (400). The DCs were shown that few small cytoplasmic projections on the cells, without changes to the volume or formation of cell clusters.
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Fig. 2. Effect of butyrate on the phenotypic characterization of DCs. Monocytes were cultured for 6 days with GM-CSF (1000 U/mL) and IL-4 (500 U/mL). Subsequently, the immature DCs were activated with LPS (1 mg/L) or butyrate (1 mmol/L) for 24 h. In addition, to study the influence of butyrate on early stage of DC differentiation, monocytes were cultured in the presence of butyrate (1 mmol/L). Then the LPS (1 mg/L) was added into the culture after the butyrate was depleted on day 6. DCs were harvested 24 h later to detect the phenotypes of each group. Light curves were represented as control, and dark curve were represented as fluorescence intensity of DC’s surface markers (CD80, CD83, CD86, CD1a, and HLA-DR) in different induction groups. Numbers calculated by flow cytometry indicated the mean fluorescence intensity (MFI) of each sample. Control (group A); LPS (group B); Na-B (group C); Na-B + LPS (group D). Shown are the means ± SD (n = 5). ⁄P < 0.05; ⁄⁄P < 0.01.
by means of different CFSE signal peaks. The majority of the DCs induced by LPS underwent several cell divisions, visualized by the respective CFSE peaks, which had subsequently lost half of their CFSE signal with each division round. The results showed mDCs exhibited remarkable divisions of T lymphocyte, and there were little division of T lymphocyte in other groups. From histograms
on each plot (Fig. 4B), the division cycles could be visualized by using Proliferation kinetics model. The peak in dark grey represented non-divided CFSE-labeled responder lymphocyte (mother cell populations) whereas the different peaks in light grey indicated daughter cell populations. Next, the proliferation index was calculated by the proportions that progeny cells occupied progen-
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Fig. 3. Effect of butyrate on the phagocytic activity of DCs. Immature DCs were generated from monocytes cultured with GM-CSF (1000 U/mL) and IL-4 (500 U/mL), in the absence or presence of 1 mmol/L butyrate. On day 7, after using the different stimuli, the antigen capture capacity of DCs was evaluated using flow cytometry to assess single cell engulfment of FITC-labeled dextran. Light curves were represented as control, and dark curve were represented as fluorescence intensity of DC’s engulfment of FITClabeled dextran in different induction groups. Numbers calculated by flow cytometry indicated the mean fluorescence intensity (MFI) of each sample. Control (group A); LPS (group B); Na-B (group C); Na-B + LPS (group D). Shown are the means ± SD (n = 5). ⁄P < 0.05; ⁄⁄P < 0.01.
itor cells. The results (Fig. 4C) showed that butyrate-induced DCs on later stage for 24 h exhibited reduced allostimulatory capability, and the allogeneic proliferation index was 6-fold lower than that of mDCs. Furthermore, the proliferation index of butyrate-induced DCs on early stage was 12-fold lower than that of in the mDCs, and 2-fold lower than that of butyrate-induced DCs on later stage. These results indicate that the phenotypic changes induced by butyrate led to changes in lymphocyte proliferation. 3.5. Butyrate modulates IL-12, IL-10, and INF-c production of DCs IL-12 p40, IL-10, and IFN-c were assessed 24 h after the stimulation of each group to investigate the effects of butyrate on the regulation of the cytokine production of DCs. Compared with the mDCs, butyrate-induced DCs on later stage exhibited a nearly 3-fold decrease in IL-12 p40 secretion. Butyrate-induced DCs on early stage exhibited a similar IL-12 p40 secretion capacity to that of butyrate-induced DCs on later stage. Next, IFN-c was detected in each group. The results showed that IFN-c production of mDCs was nearly 5-fold higher than IFN-c production of butyrate-induced DCs. In contrast, IL-10 secretion by DCs of butyrate-induced DCs was nearly 11- and 7.5-fold higher than IL-10 secretion of mDCs (Fig. 5). 4. Discussion The short-chain fatty acid molecule butyrate, an HDAC inhibitor, has been widely applied induce stem cells differentiation and affect carcinoma cells invasion or metastasis [17,18]. However, very little research on the ability of butyrate to regulate DC function has been done. In this study, we provide morphological,
phenotypical, and functional evidence that butyrate suppresses the differentiation of monocyte-derived DCs in vitro. Our data showed that butyrate impaired DC function, which may represent a new approach in immunosuppression. The distinctive morphology of mDCs is dendrite formation, but we observed that DCs induced by butyrate exhibited a different morphology compared to LPS-induced DCs. Butyrate inhibited dendritic morphology formation, including the formation of clusters and increases in volume, causing the DCs to have a morphology similar to that of imDCs. Furthermore, the influence of butyrate was unaffected by the addition of LPS to early stage DCs. The inhibitory effects of butyrate in the early stages of DC maturation were more remarkable than the effects of adding butyrate on day 6. This suggests that butyrate could interfere with the differentiation and maturation process of DCs in early stages, keeping the cells in a stable, immature morphology. When stimulated with an inflammatory factor (LPS), the imDCs would convert into the mature phenotype, characterized by increased expression of MHC molecules, costimulatory molecules, and increased T-cell stimulating capacity [2,12]. We observed that butyrate down-regulated MHC molecules, costimulatory molecules (CD80), classical DC-associated molecules (CD1a), and DCspecific maturation molecules (CD83), which provide the cells with optimal T-cell stimulatory capacity. CD83 is a characteristic marker that is highly expressed on mDCs [19]; hence, the low expression of CD83 on butyrate-induced cells indicates a suppression of the DC maturation process. The marker CD80 has been shown to provide critical costimulatory signals through interaction with the receptor CD28 found on T cells [20]. Therefore, low CD80 expression may be an effective mechanism to down-regulate the T cell response. Furthermore our data show that butyrate does not remarkably down-regulate expression of the costimulatory
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Fig. 4. Effects of butyrate on the T cell allostimulatory capacity of DCs. Immature DCs were generated from monocytes cultured with GM-CSF (1000 U/mL) and IL-4 (500 U/ mL) in the absence or in the presence of 1 mmol/L butyrate. On day 6, immature DCs were further incubated with LPS or butyrate. After 24 h, the cells were harvested and used as stimulators of allogeneic T cell proliferation. The allogeneic lymphocytes (responder cells) were resuspended in PBS containing 0.25 lmol/L CFDA-SE and incubated at 37 °C for 15 min. Then the stimulator DCs were added to allogeneic CFSE-labeled lymphocytes at a ratio of 1:10. CFSE-labeled lymphocytes without stimulator DCs served as the control. The cells were cultured for 96 h and FACS detection was conducted. Flow histograms of CFSE fluorescence for allogeneic lymphocyte of different groups were shown in (A). Discrete division cycles could be visualized by means of different CFSE signal peaks, which had subsequently lost half of their CFSE signal with each division round. Proliferation kinetics model of lymphocytes were shown in (B). The peak in dark grey represented non-divided CFSE-labeled responder lymphocyte whereas the peak in light grey indicated daughter cell populations. The proliferation indexes of different groups were shown in (C). Control (group A); LPS (group B); Na-B (group C); Na-B + LPS (group D). Shown are the means ± SD (n = 5). ⁄⁄P < 0.01.
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Fig. 5. Effects of butyrate on DCs cytokine release. Immature DCs were generated from monocytes in the absence or presence of 1 mmol/L butyrate and stimulated with LPS (1 mg/L) or butyrate (1 mmol/L) for 24 h. Then IL-12, IL-10, and IFN-c were measured by ELISA using matched-pair antibodies. Wells from which IL-12 p40, IL-10, and IFN-c were collected contained 1 mL culture medium with 1 106 DCs. Control (group A); LPS (group B); Na-B (group C); Na-B + LPS (group D). Shown are the means ± SD (n = 5). ⁄ P < 0.05.
molecule CD86, compared with other surface markers of butyrateinduced DCs, especially CD80. It is different from previous reports [21,22]. There is still some controversy about role of CD80 and CD86 in triggering costimulation [23,24]. Both molecules are capable of binding CD28 on T cells, but some studies have shown that CD80 costimulation is superior to CD86 costimulation [25,26]. This would suggest that in our model, highly expression of CD86 alone was not sufficient to provide effective costimulation [25,27]. We observed a decrease in the expression of various surface molecules known to play important roles in the process of T cell activation by mediating intercellular contact and delivering essential costimulatory activity [28,29]. Our studies also showed that butyrate markedly reduced antigen-specific T cell proliferation. Therefore, the impaired ability of DCs to stimulate T cell proliferation function was most likely due to the down-regulation of MHC molecules and costimulatory molecules (CD80) on DCs [30,31]. Importantly, butyrate modulated cytokine production by DCs. The major Th1-skewing factors IL-12 and IFN-c were substantially suppressed by butyrate, which might critically influence the development of a subsequent T cell response. These results were consistent with previous reports [21,32,33]. Our study found that the anti-inflammatory cytokine IL-10 was increased in butyrate-induced DCs, similar to Millard’s research [34]. As a suppressive effect of the cytokine, IL-10 strongly inhibited proliferation of both CD4+ and CD8+ T cell, and shifted the T-helper cell (Th) response toward a predominant Th2 profile [35,36]. The alteration of DC-derived cytokine production by butyrate contributed to impairment of the DC immunostimulatory capacity, consistent with phenotypic changes and MLR results described above. Another notable characteristic of DCs induced with butyrate was that imDCs had an increased endocytic capacity. Moreover, the endocytic capacity of DCs could be further enhanced when butyrate was added in the early stage of differentiation. Furthermore, DC differentiation was inhibited by the addition of butyrate to DCs in early stages, keeping the cells in a stable morphology. Besides the morphological differences between DCs at different stages, the notable changes following butyrate addition to early stage DCs were the maturation of the phenotype and the continuous down-regulation of T-cell stimulatory activity. Importantly, the immature state of DCs could not be activated to a mature state by a stimulus such as LPS. This suggests that butyrate keeps the DCs in a stable, immature stage, which may be better than other inducing methods. The changes observed in cytokine production were similar with the addition of butyrate both in early stage and later stage, and the enhanced endocytic capacity of DCs would benefit the induction of Ag-specific immunological tolerance [37,38]. These data suggest that butyrate plays an important role in the early stage of DC differentiation by blocking the process of maturation from monocytic precursors into mDCs, and thus potentially impairing the normal function of DCs. Millard et al. [34] and Säemann et al. [22] combined butyrate and LPS on DC’s induction, and found butyrate could decrease the maturation markers of DCs and down-regulate their ability to prime
alloreactive naïve T cells. The effect of butyrate on the differentiation of DCs might be weakened when combined with stimulus such as LPS, which could not accurately reflect its suppression effects. Therefore, only butyrate was added to induce DCs final differentiation in our experiments and found butyrate had the suppressive effects on the later stage of DCs. Next, we also examined the effect of butyrate on early stage of DCs’ differentiation and found the suppressive effect persisted, even after butyrate was depleted and LPS was added on day 6 to induce final maturation. The results demonstrated that imDCs induced by butyrate on early stage of cultivation could resist extraneous stimulus, and maintain the immaturation stage of DCs. However, in Wang’s and Säemann’s studies, DCs were induced by GM-CSF and IL-4 for 5 or 7 days and then LPS for 2 days to explore effects of butyrate on early stage of DCs’ differentiation [21,22]. Butyrate was added throughout the whole culture period, which was different from our experiments. The depletion of butyrate could better explain the persistent suppressive effects on DCs. Our studiy found that butyrate could suppress the maturation procedure of DCs, which was similar to Säemann’s study. But Wang et al. [21] reported the opposite results. Wang reported that butyrate did not up-regulate maturation markers (CD83, CD86, MHC class I, and MHC class II molecules) of DCs, and there was no significant difference between butyrate-induced DCs and control DCs in MLR. It is probably due to the different concentration of butyrate in culture. The 0.5 mmol/L concentration of butyrate in Wang’s study might be too low to resist the maturation promoting effect of LPS, and our preliminary experiments showed that immunosuppression effect of 1 mmol/L butyrate were better than 0.5 mmol/L butyrate in our culture condition. Our results, demonstrating impairment of DC differentiation by butyrate, represent important evidence necessary to understand the mechanism of butyrate on immunological tolerance and support further interest in butyrate as an immunotherapeutic reagent. Because DCs are a crucial factor in the initiation of an effective immune response, the use of butyrate to interfere with DC maturation and differentiation may substantially modify the outcome of an immune response. Acknowledgments This study was supported by research funding from the National Natural Science Foundation of China (No. 81001306). References [1] W.J. Mayer, U.M. Irschick, P. Moser, M. Wurm, H.P. Huemer, N. Romani, E.U. Irschick, Characterization of antigen-presenting cells in fresh and cultured human corneas using novel dendritic cell markers, Invest. Ophthalmol. Vis. Sci. 48 (2007) 4459–4467. [2] M. Bros, F. Jährling, A. Renzing, N. Wiechmann, N.A. Dang, A. Sutter, R. Ross, J. Knop, S. Sudowe, A.B. Reske-Kunz, A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid, Blood 109 (2007) 3820–3829.
L. Liu et al. / Cellular Immunology 277 (2012) 66–73 [3] S.M. Barratt-Boyes, A.W. Thomson, Dendritic cells: tools and targets for transplant tolerance, Am. J. Transplant. 5 (2005) 2807–2813. [4] L. Rober, F.N. Wan, M.S. Ralph, Dendritic cells in transplantation – friend or foe, Immunity 14 (2001) 357–368. [5] N. Tochiki, M. Narita, Z. Zheng, C. Lu, A. Saitoh, N. Watanabe, N. Satoh, T. Furukawa, K. Toba, I. Fuse, Y. Aizawa, M. Takahashi, Induction of recipient cellspecific donor T-cell anergy by UV-C-irradiated recipient immature monocytederived dendritic cells, Bone Marrow Transplant. 41 (2008) 1037–1045. [6] Y. Ikeda, F. Akbar, H. Matsui, M. Onji, Characterization of antigen-presenting dendritic cells in the peripheral blood and colonic mucosa of patients with ulcerative colitis, Eur. J. Gastroenterol. Hepatol. 13 (2001) 841–850. [7] P.T. Coates, F.J. Duncan, B.L. Colvin, Z. Wang, A.F. Zahorchak, W.J. Shufesky, A.E. Morelli, A.W. Thomson, In vivo-mobilized kidney dendritic cells are functionally immature, subvert alloreactive T-cell responses, and prolong organ allograft survival, Transplantation 77 (2004) 1080–1089. [8] F. Geissmann, P. Revy, A. Regnault, Y. Lepelletier, M. Dy, N. Brousse, S. Amigorena, O. Hermine, A. Durandy, TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells, J. Immunol. 162 (1999) 4567–4575. [9] M.B. Lutz, R.M. Suri, M. Niimi, A.L. Ogilvie, N.A. Kukutsch, S. Rössner, G. Schuler, J.M. Austyn, Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo, Eur. J. Immunol. 30 (2000) 1813–1822. [10] Z. Zheng, M. Narita, M. Takahashi, A. Liu, T. Furukawa, K. Toba, Y. Aizawa, Induction of T cell anergy by the treatment with IL-10-treated dendritic cells, Comp. Immunol. Microbiol. Infect. Dis. 27 (2004) 93–103. [11] R.M. Gorczynski, J. Bransom, M. Cattral, X. Huang, J. Lei, L. Xiaorong, W.P. Min, Y. Wan, J. Gauldie, Synergy in induction of increased renal allograft survival after portal vein infusion of dendritic cells transduced to express TGF-b and IL10, along with administration of CHO cells expressing the regulatory molecule OX2, Clin. Immunol. 95 (2000) 182–189. [12] A. Cignetti, A. Vallario, I. Roato, P. Circosta, B. Allione, L. Casorzo, P. Ghia, F. Caligaris-Cappio, Leukemia-derived immature dendritic cells differentiate into functionally competent mature dendritic cells that efficiently stimulate T cell responses, J. Immunol. 173 (2004) 2855–2865. [13] L. Wang, H.S. Luo, H. Xia, Sodium butyrate induces human colon carcinoma HT-29 cell apoptosis through a mitochondrial pathway, J. Int. Med. Res. 37 (2009) 803–811. [14] G. Liang, O. Taranova, K. Xia, Y. Zhang, Butyrate promotes induced pluripotent stem cell generation, J. Biol. Chem. 285 (2010) 25516–25521. [15] S.K. Jackson, A. DeLoose, K.M. Gilbert, The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors, Immunology 106 (2003) 486–495. [16] K.M. Gilbert, S.R. Boger, P. Price, E.K. Fifer, T cell tolerance induced by histone deacetylase inhibitor is mediated by P21cip1, Immunopharmacol. Immunotoxicol. 27 (2005) 545–564. [17] R. Joachimiak, A. Kaznica, T. Drewa, Influence of sodium butyrate on hepatocellular carcinoma (hepG2) and glioblastoma (C6) cell lines in vitro, Acta Pol. Pharm. 64 (2007) 561–563. [18] M. Zhou, P. Li, L. Tan, S. Qu, Q.L. Ying, H. Song, Differentiation of mouse embryonic stem cells into hepatocytes induced by a combination of cytokines and sodium butyrate, J. Cell. Biochem. 109 (2010) 606–614. [19] L.J. Zhou, T.F. Tedder, Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily, J. Immunol. 154 (1995) 3821– 3835. [20] A. Mondino, M.K. Jenkins, Surface proteins involved in T cell costimulation, J. Leukoc. Biol. 55 (1994) 805–815. [21] B. Wang, A. Morinobu, M. Horiuchi, J. Liu, S. Kumagai S, Butyrate inhibits functional differentiation of human monocyte-derived dendritic cells, Cell. Immunol. 253 (2008) 54–58. [22] M.D. Säemann, O. Parolini, G.A. Böhmig, P. Kelemen, P.M. Krieger, J. Neumuller, K. Knarr, W. Kammlander, W.H. Horl, C. Diakos, K. Stuhlmeier, G.J. Zlabinger,
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
73
Bacterial metabolite interference with maturation of human monocytederived dendritic cells, J. Leukoc. Biol. 71 (2002) 238–246. K.S. Hathcock, G. Laszlo, C. Pucillo, P. Linsley, R.J. Hodes, Comparative analysis of B7–1 and B7–2 costimulatory ligands: expression and function, J. Exp. Med. 180 (1994) 631–640. S. Salek-Ardakani, R. Arens, R. Flynn, A. Sette, S.P. Schoenberger, M. Croft, Preferential use of B7.2 and not B7.1 in priming of vaccinia virus-specific CD8 T cells, J. Immunol. 182 (2009) 2909–2918. U. Matulonis, C. Dosiou, G. Freeman, C. Lamont, P. Mauch, L.M. Nadler, J.D. Griffin, B7–1 is superior to B7–2 costimulation in the induction and maintenance of T cell-mediated antileukemia immunity. Further evidence that B7–1 and B7–2 are functionally distinct, J. Immunol. 156 (1996) 1126– 1131. P. Rajavelu, S.D. Das, Expression of co-stimulatory molecules B7.1 & B7.2 on macrophages infected with various strains of Mycobacterium tuberculosis & its influence on T-cell apoptosis Indian, J. Med. Res. 127 (2008) 388–394. G.A. Bohmig, P.M. Krieger, M.D. Saemann, C. Wenhardt, E. Pohanka, G.J. Zlabinger, N-butyrate downregulates the stimulatory function of peripheral blood-derived antigen-presenting cells: a potential mechanism for modulating T-cell responses by short-chain fatty acids, Immunology 92 (1997) 234–243. A. Gruber, T. Brocker, MHC class I-positive dendritic cells (DC) control CD8 T cell homeostasis in vivo: T cell lymphopenia as a prerequisite for DC-mediated homeostatic proliferation of naive CD8 T cells, J. Immunol. 175 (2005) 201– 206. S.Y. Tseng, M. Otsuji, K. Gorski, X. Huang, J.E. Slansky, S.I. Pai, A. Shalabi, T. Shin, D.M. Pardoll, H. Tsuchiya, B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells, J. Exp. Med. 193 (2001) 839–846. F. Fu, Y. Li, S. Qian, L. Lu, F. Chambers, T.E. Starzl, J.J. Fung, A.W. Thomson, Costimulatory molecule-deficient dendritic cell progenitors (MHC class II+, CD80dim, CD86-) prolong cardiac allograft survival in nonimmunosuppressed recipients, Transplantation 62 (1996) 659–665. L. Lu, D. McCaslin, T.E. Starzl, A.W. Thomson, Bone marrow-derived dendritic cell progenitors (NLDC 145+, MHC class II+, B7–1dim, B7–2-) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes, Transplantation 60 (1995) 1539–1545. F. Miro, C. Nobile, N. Blanchard, M. Lind, O. Filipe-Santos, C. Fieschi, A. Chapgier, G. Vogt, L. de Beaucoudrey, D.S. Kumararatne, F. Le Deist, J.A. Casanova, S. Amigorena, C. Hivroz, T cell-dependent activation of dendritic cells requires IL-12 and IFN-gamma signaling in T cells, J. Immunol. 177 (2006) 3625–3634. D. Messmer, I. Hatsukari, N. Hitosugi, I.G. Schmidt-Wolf, P.C. Singhal, Morphine reciprocally regulates IL-10 and IL-12 production by monocytederived human dendritic cells and enhances T cell activation, Mol. Med. 12 (2006) 284–290. A.L. Millard, P.M. Mertes, D. Ittelet, F. Villard, P. Jeannesson, J. Bernard, Butyrate affects differentiation, maturation and function of human monocyte-derived dendritic cells and macrophages, Clin. Exp. Immunol. 130 (2002) 245–255. J. Peguet-Navarro, C. Moulon, C. Caux, C. Dalbiez-Gauthier, J. Banchereau, D. Schmitt, Interleukin-10 inhibits the primary allogeneic T cell response to human epidermal Langerhans cells, Eur. J. Immunol. 24 (1994) 884–891. A.M. Stax, C. Crul, S.W. Kamerling, N. Schlagwein, R.N. van der Geest, A.M. Woltman, C. van Kooten, CD40L stimulation of rat dendritic cells specifically favors the IL-12/IL-10 ratio resulting in a strong T cell stimulatory capacity, Mol. Immunol. 45 (2008) 2641–2650. H. Toyokawa, A. Nakao, R.J. Bailey, M.A. Nalesnik, T. Kaizu, J.L. Lemoine, A. Ikeda, K. Tomiyama, G.D. Papworth, L. Huang, A.J. Demetris, T.E. Starzl, N. Murase, Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation, Liver Transpl. 14 (2008) 346–357. M. Nouri-Shirazi, E. Guinet, Direct and indirect cross-tolerance of alloreactive T cells by dendritic cells retained in the immature stage, Transplantation 74 (2002) 1035–1044.