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Adipose-derived stem cells induced dendritic cells undergo tolerance and inhibit Th1 polarization
Cellular Immunology 278 (2012) 152–157
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Adipose-derived stem cells induced dendritic cells undergo tolerance and inhibit Th1 polarization Wei Peng a,b, Tianjun Gao b, Zai-liang Yang a, Shi-chang Zhang a, Ming-liang Ren a, Zheng-guo Wang a, Bo Zhang a,⇑ a b
Department 4, Research Institute of Field Surgery, The 3rd Military Medical University, State Key Lab of Trauma, Burns and Combined Injury, 400042 Chongqing, China 309th Hospital of PLA, 100091, Beijing, China
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Article history: Received 21 June 2012 Accepted 23 July 2012 Available online 10 August 2012 Keywords: Adipose-derived stem cells Dendritic cells Tolerance Th1 polarization
a b s t r a c t Adipose tissue-derived stem cells (ADSC) have been shown to possess stem cell properties such as transdifferentiation, self-renewal and therapeutic potential. However, the property of ADSC to accommodate immune system is still unknown. In this study, ADSC were cocultured with allogenetic dendritic cells (DC), and then treated DC were mixed with allogenetic CD4+ T cells. The results demonstrated that ADSC could downregulate costimulatory molecules, including CD80, CD83, CD86, and cytokine secretion such as interleukin (IL)-12 and tumor necrosis factor (TNF)-a, while upregulate indoleamine-2,3-dioxygenase (IDO) of allogenetic DC. In addition, treated DC could inhibit CD4+ Tcell activation and naïve T cells toward Th1 polarization. The results suggest that ADSC could negatively modulate immunity and induce immune tolerance, which provide a promising strategy in transplantation or autoimmune disease. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Adipose-derived stem cells (ADSC) are able to differentiate into the classical mesodermal tissues like bone, fat, nerve, cardiomyocytes, hepatocytes and pancreatic cells [1–4]. Therefore, they represent a promising source for cell therapy, especially as their isolation is less invasive compared to bone marrow extractions and their expansion in culture is quite easy [5–7]. In addition, stem-cell transplantation has emerged as a new treatment modality for patients with refractory, severe autoimmune disease [8–10]. Its rationale is based on eliminating autoaggressive lymphocytes by lympho- or myeloablative conditioning followed by stem-cell rescue [11–13]. Preclinical studies in animal models of autoimmune disease and observations in patients with rheumatoid arthritis (RA) who were cured after allogeneic bone marrow transplantation for concomitant hematologic malignancy have provided support for the concept [14–16]. However, the immune property of ADSC is still unknown. We wondered whether ADSC could also modulate immune response, To identify it, we isolated ADSC from human fat tissue, and explored its immune function. The results demonstrated that ADSC could negatively modulate immunity and induce immune tolerance by regulating DC.
2.1. Preparation of ADSC
⇑ Corresponding author. Fax: +86 23 68757443. E-mail address: [email protected] (B. Zhang). 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.07.008
In brief, the subcutaneous adipose tissue was dissected from omental fat who underwent abdominal surgery. The volunteer were healthy with the age of 30–50 years. The isolated adipose tissue was washed extensively with phosphate-buffered saline (PBS) to remove contaminating debris and blood cells. The 50 mL adipose tissue was minced and digested with 50 mL collagenase I (2 mg/mL; Worthington Biochemical) at 37 °C for 30 min. Collagenase activity was neutralized by DMEM-F12 (HyClone) containing 15% fetal bovine serum (FBS; HyClone). Digested adipose tissue was filtered twice with a 100-lm and then with a 25-lm nylon membrane to eliminate the undigested fragments. The cellular suspension was centrifuged at 1000 g for 10 min. The cell pellets were re-suspended in cell culture medium (CCM) and cultivated for 24 h at 37 °C in 5% CO2. Unattached cells and debris were removed, and fresh CCM containing 15% FBS was added to the adherent cells, which were cultured at 37 °C in 5% CO2 until 70–80% confluent. Identity of the isolated cells was determined by assessing their surface markers using flow cytometric analysis with FITC-conjugated antibodies against CD29, CD44, CD105, and FITC-conjugated isotype-matched control antibodies (BD).
W. Peng et al. / Cellular Immunology 278 (2012) 152–157
2.2. Dendritic cell generation Briefly, PBMCs were isolated from healthy donors by FicollHypaque density gradient centrifugation and then seeded into culture flasks in RPMI-1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 lg/mL), and 10% FBS. After monocytes adhered (incubation for 2 h), the adherent cells were cultured for 5 days in RPMI-1640 containing 1000 U/mL of gpokemonulocyte-macrophage colony-stimulating factor (R&D Systems, Inc., Minneapolis, MN) and interleukin-4 (IL-4; R&D Systems, Inc.), and were the cultured for an additional 2 days in the presence of 1000 U/mL of tumor necrosis factor a (R&D Systems, Inc.) to induce final maturation. After 7 days of culture, the mature DCs were harvested and analyzed for DC typical phenotypes by FACS analysis using primary antibodies against CD80, CD83 and CD86. 2.3. ADSC and DC mixed reactions ADSC (2 105) and DC (2 105) cells were added to roundbottom 96-well plates to a final volume of 200 ll RPMI 1640 with 10% FCS. After 3 days, co-cultured DC were harvested by microbeads (Miltenyi Biotech, Bergisch, Gladbach, Germany) to detect the expression of costimulatory molecules by flow cytometry. Cells were collected and pelleted with 100 lL PBS and incubated for 30 min at 4 °C in the dark using primary antibodies against CD80, CD83 and CD86. Flow cytometric data were analyzed by using CellQuest software (BD Biosciences, San Jose, CA). In addition, supernatants were removed and further analyzed for cytokine production (IL-12 and TNF-a) with ELISA. 2.4. Treated DC and CD4+ T cells mixed reactions CD4+ T cells (>95%) were positively selected from nonadherent cells of PBMC using microbeads. MLR were conducted in 96-well flat-bottom culture plates. ADSC-treated DC (2 105) were co-cultured with 2 105 allogeneic CD4+ cells. After 3 days, co-cultured
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T cells were harvested to detect the expression of activation markers (CD25 and CD69) by flow cytometry. In addition, supernatants were removed and further analyzed for cytokine production (IFN-c and IL-2) with ELISA. 2.5. Treated DC and naïve T cells mixed reactions Allogeneic CD4+CD45RA+ naïve T cells were isolated from PBMC by immunomagnetic purification. Briefly, untouched CD4+ Tcells were obtained through a CD4+ T cell isolation kit, according to the manufacturer’s instructions. Next, CD45RA+ cells were positively selected by CD45RA-coated immunomagnetic microbeads. CD4+CD45RA+ naïve T cells (2 105) were co-cultured with ADSC -treated DC (4 104), at a ratio of 5:1 for 5 days. On day 6, cells were restimulated with plate-bound anti-CD3 (5 lg/mL in PBS, overnight, 4 °C) and anti-CD28 (l lg/mL) antibody. Supernatants were removed and further analyzed for cytokine production (IFNc and IL-4) with ELISA. 2.6. Western blot analysis ADSC-treated DC were collected and washed twice by cold PBS, and each well was treated with 50 lL lysis buffer (2 mmol/L TrisHCl pH 7.4, 50 mmol/L NaCl, 25 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, supplemented with protease inhibitors 1 mmol/L phenylmethylsulfonylfluoride, 10 mg/L pepstatin, 10 mg/L aprotinin, and 5 mg/L leupeptin) (all from Sigma). Protein concentrations were determined using the bicinchoninic acid protein assay. Equal amounts of protein (40 lg) were separated on a 15% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond C, Amersham, Freiburg, Germany). Membranes were blocked in 5% nonfat dry milk in TBS for 1 h at room temperature and probed with rabbit anti-IDO antibodies (1:500, Santa Cruz Biotechnology, USA) overnight at 4 °C. After 3 times washing with TBS containing 0.1% Tween 20, membranes were incubated with antirabbit IgGhorseradish-peroxidase (1:5000, Santa Cruz Biotechnology, USA),
Fig. 1. Morphologic and phenotypic characteristics of ADSC. The subcutaneous adipose tissue was dissected from omental fat. The adipose tissue was minced and digested with collagenase I. The cell pellets were re-suspended in cell culture medium (CCM) and cultivated for 24 h at 37 °C in 5% CO2. (A). Morphologic characteristics of ADSC (200). (B). The surface marker expression of ADSC using flow cytometric analysis with FITC-conjugated antibodies and FITC-conjugated isotype-matched control antibodies. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
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and developed by luminol mediated chemiluminescence (Appylgen Technologies Inc, China). To confirm equal protein loading, membranes were reprobed with a 1:1000 dilution of an anti-actin antibody (Santa Cruz Biotechnology, USA). Densitometric analyses were performed using Scion Image software. 2.7. Statistical analysis The statistical significance of differential findings between experimental groups and controls were determined by t-test and were considered significant for P < 0.05. 3. Results 3.1. Morphologic and phenotypic characteristics of of ADSC At 24 h of primary culture in medium containing 10% fetal bovine serum, ADSC adhered to the culture dish sparsely and the majority of cells displayed a spindle-like shape. As shown in Fig. 1A, after 14 days, ADSCs commonly achieved 80% confluence with a rather homogeneous population of fibroblast-like morphology. In addition, as shown in Fig. 1B, flow cytometric analysis showed that the ADSC expressed high level of CD29, CD44 and CD105. These results revealed that cultured ADSC were homogeneous and did not contain hematopoietic lineages, consistent with the previous report. 3.2. Morphologic and phenotypic characteristics of DC On day 9 of the cell culture, the mature DC displaying typical characteristics of morphology were harvested from monocytes cultured in the medium containing GM-CSF, IL-4, and tumor necrosis factor-a. As shown in Fig. 2A, these mature cells were loosely suspended, exhibited irregular cell shape, and displayed many fine processes at their edges. The mature denditric cells were also analyzed for phenotype by flow cytometry. As shown in Fig. 2B, the results showed that these mature DC expressed high levels of CD80, CD83 and CD86.
3.3. ADSC induced DC undergo tolerance To explore the effect of ADSC to DC, after ADSC and DC mixed reactions, we analyzed the costimulatory molecules, including CD80, CD83, CD86, cytokine secretion such as IL-12 and TNF-a, and IDO of allogenetic DC. As shown in Fig. 3, the results demonstrated that ADSC could downregulate CD80, CD83 and CD86 expression, inbibit IL-12 and TNF-a secretion, and induce IDO expression. 3.4. Treated DC inhibited CD4+ T cell activation We wonder whether ADSC treated DC could inhibit CD4+ T cell activation. We also cocultured treated DC and CD4+ T cells together, and analyzed the expression of CD4+ T cell activation markers of CD25 and CD69, and cytokine secretion of IFN-c and IL-2. As shown in Fig. 4, treated DC could downregulate CD25 and CD69 expression of CD4+ T cells, and inhibit cytokine secretion of IFNc and IL-2. these results demonstrated that treated DC could inhibit CD4+ T cell activation. 3.5. Treated DC inhibited naïve T cells toward Th1 polarization The CD4+CD45RA+ naïve T cells were then co-cultured with treated DC, and after 5 days, the T cells were recovered and restimulated with anti-CD3 (5 lg/mL) and anti-CD28 (1 l/mL) antibodies in combination. As shown in Fig. 5, the production of the cytokine was measured by ELISA and results indicated that the secretion of IL-4 was increased, whereas IFN-c production was decreased in the culture as compared with control group. Accordingly, these results indicate that treated DC inhibited CD4+CD45RA+ naïve T cells to a Th1 polarization. 4. Discussion Soft-tissue loss presents an ongoing challenge in plastic and reconstructive surgery [17,18]. Standard approaches to soft-tissue reconstruction include autologous tissue flaps, autologous fat
Fig. 2. Morphologic and phenotypic characteristics of DC. PBMCs were isolated from healthy donors. After monocytes adhered, the cells were cultured for 5 days in RPMI1640 containing 1000 U/mL of GM-CSF and IL-4, and were the cultured for an additional 2 days in the presence of 1000 U/mL of TNF-a. After 7 days of culture, (A). Morphologic characteristics of DC (200). (B). The surface marker expression of DC using flow cytometric analysis with FITC-conjugated antibodies and FITC-conjugated isotype-matched control antibodies. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
W. Peng et al. / Cellular Immunology 278 (2012) 152–157
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Fig. 3. ADSC induced DC undergo tolerance. ADSC (2 105) and DC (2 105) cells were added to round-bottom 96-well plates to a final volume of 200 ll RPMI 1640 with 10% FCS. (A).After 3 days, co-cultured DC were harvested by microbeads and pelleted with 100 lL PBS and incubated for 30 min at 4 °C in the dark using primary antibodies against CD80, CD83 and CD86. Flow cytometric data were analyzed by using CellQuest software. (B). After 3 days, supernatants were removed and further analyzed for cytokine production (IL-12 and TNF-a) with ELISA. (C). After 3 days, co-cultured DC were harvested by microbeads and washed twice by cold PBS, and DC were treated with lysis buffer to further analyze the IDO expression with Western blot assay. 1: PBS; 2: untreated DC; 3: treated DC. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
Fig. 4. Treated DC inhibited CD4+ T cell activation. CD4+ T cells (>95%) were positively selected from nonadherent cells of PBMC using microbeads. MLR were conducted in 96-well flat-bottom culture plates. ADSC-treated DC (2 105) were co-cultured with 2 105 allogeneic CD4+ cells. (A).After 3 days, co-cultured T cells were harvested to detect the expression of activation markers (CD25 and CD69) by flow cytometry. (B). After 3 days, supernatants were removed and further analyzed for cytokine production (IFN-c and IL-2) with ELISA. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
transplantation and alloplastic implants [19–21]. All of these approaches have disadvantages, including donor-site morbidity, implant migration and absorption and foreign body reaction [22– 24]. Stem cell application has recently been suggested as a possible novel therapy [25–27]. Adipose-derived stem cells (ADSC) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue, which is an easily accessible and abundant source of putative stem cells for translational clinical research [28,29]. Their therapeutic use in pre-clinical studies and experimental clinical trials has been well documented [4,30–32]. Previous studies proved that human bone marrow mesenchymal stem cells (BM-MSC) are multipotent progenitor cells that have transient immunomodulatory properties on Natural Killer (NK) cells, Dendritic Cells (DC), and T cells [33,34]. BM-MSC modulate hematopoiesis and can exert immune regulatory functions, both in vivo and in vitro [35,36]. The immunomodulatory properties of BM-MSC require cell-cell contact as well as soluble factors, including interleukin (IL)-6, hepatocyte growth factor (HGF), nitric oxide (NO), prostaglandin (PGE)-2, indolamine 2,3 dioxygenase (IDO), and HLA-G [36]. BM-MSC transplantation was trailed in a number of autoimmune diseases and represented a promising strategy [37–39]. The evidences revealed that cell specific differences at transcriptional and proteomic levels between both ADSC and BM-MSC types according to their tissue origin as well as functional differences in their differentiation processes towards adipogenic, osteogenic and chondrogenic programs. Nevertheless, in vitro as well as in vivo ADSC displayed the same ability than MSC to differentiate towards chondrocytes/osteoblasts, comforting the status of both cell sources as promising regenerative cells [40]. However, the mechanism and therapeutical potential of ADSC in autoimmune diseases is still known. To identify that, we isolated and cultured ADSC, and explored the immune property. Firstly, we isolated ADSC from omental fat. After 14 days of culture, ADSC dis-
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Fig. 5. Treated DC inhibited naïve T cells toward Th1 polarization. The CD4+CD45RA+ naïve T cells were co-cultured with treated DC, and after 5 days, the T cells were restimulated with anti-CD3 (5 lg/mL) and anti-CD28 (1 l/mL) antibodies in combination. The production of the cytokines (IL-4 and IFN-c) were measured with ELISA. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
played a rather homogeneous population of fibroblast-like morphology and expressed high level of CD29, CD44 and CD105. In addition, we also generated DC from the PBMCs. On day 9 of the cell culture, DC displayed typical characteristics of morphology and expressed high level of CD80, CD83 and CD86. Secondly, to identify the function of ADSC to DC, the key contributor to the induction of primary and secondary immune responses, we cocultured ADSC with allogenetic DC together and analyzed the costimulatory molecule expression and cytokine secretion. The results demonstrated that ADSC could downregulate costimulatory molecules, including CD80, CD83, CD86, and cytokine secretion such as IL-12 and TNF-a while upregulate IDO of DC. Thirdly, to study the effect of treated DC to CD4+ T cell activation or naïve T cell polarization, we cocultured treated DC with allogenetic CD4+ T cells or CD4+CD45RA+ naïve T cells together, and analyzed the activation markers and cytokine production. The results demonstrated that treated DC could could downregulate CD25 and CD69 expression of CD4+ T cells, and inhibit cytokine secretion of IFN-c and IL-2. Additionly, treated DC inhibited CD4+CD45RA+ naïve T cells to a Th1 polarization through upregulate IL-4 production while downregulate IFN-c production. The results demonstrated that leading to the immunomodulatory interference of ADSC on DC might by regulating cell membrane molecular expression and cytokine secretion. In conclusion, our finding demonstrated that ADSC could induce DC tolerance, result in CD4+ T cell activation inhibition and naïve T cells toward Th1 polarization. The results might provide ADSC a theoretic foundation and therapeutical strategy for autoimmune disease. Acknowledgment This work was supported by 2011CB964701 of China. References [1] H. Nakagami, R. Morishita, K. Maeda, Y. Kikuchi, T. Ogihara, Y. Kaneda, Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy, J. Atheroscler.Thromb. 13 (2) (2006 Apr) 77–81. [2] A. Schaffler, C. Buchler. Concise review: adipose tissue-derived stromal cellsbasic and clinical implications for novel cell-based therapies. Stem Cells 2007 Apr; 25(4):818–827. [3] Y. Kamada, Y. Yoshida, Y. Saji, J. Fukushima, S. Tamura, S. Kiso, et al., Transplantation of basic fibroblast growth factor-pretreated adipose tissuederived stromal cells enhances regression of liver fibrosis in mice, Am. J. Physiol. 296 (2) (2009 Feb) G157–G167.
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Contents lists available at SciVerse ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Adipose-derived stem cells induced dendritic cells undergo tolerance and inhibit Th1 polarization Wei Peng a,b, Tianjun Gao b, Zai-liang Yang a, Shi-chang Zhang a, Ming-liang Ren a, Zheng-guo Wang a, Bo Zhang a,⇑ a b
Department 4, Research Institute of Field Surgery, The 3rd Military Medical University, State Key Lab of Trauma, Burns and Combined Injury, 400042 Chongqing, China 309th Hospital of PLA, 100091, Beijing, China
a r t i c l e
i n f o
Article history: Received 21 June 2012 Accepted 23 July 2012 Available online 10 August 2012 Keywords: Adipose-derived stem cells Dendritic cells Tolerance Th1 polarization
a b s t r a c t Adipose tissue-derived stem cells (ADSC) have been shown to possess stem cell properties such as transdifferentiation, self-renewal and therapeutic potential. However, the property of ADSC to accommodate immune system is still unknown. In this study, ADSC were cocultured with allogenetic dendritic cells (DC), and then treated DC were mixed with allogenetic CD4+ T cells. The results demonstrated that ADSC could downregulate costimulatory molecules, including CD80, CD83, CD86, and cytokine secretion such as interleukin (IL)-12 and tumor necrosis factor (TNF)-a, while upregulate indoleamine-2,3-dioxygenase (IDO) of allogenetic DC. In addition, treated DC could inhibit CD4+ Tcell activation and naïve T cells toward Th1 polarization. The results suggest that ADSC could negatively modulate immunity and induce immune tolerance, which provide a promising strategy in transplantation or autoimmune disease. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
Adipose-derived stem cells (ADSC) are able to differentiate into the classical mesodermal tissues like bone, fat, nerve, cardiomyocytes, hepatocytes and pancreatic cells [1–4]. Therefore, they represent a promising source for cell therapy, especially as their isolation is less invasive compared to bone marrow extractions and their expansion in culture is quite easy [5–7]. In addition, stem-cell transplantation has emerged as a new treatment modality for patients with refractory, severe autoimmune disease [8–10]. Its rationale is based on eliminating autoaggressive lymphocytes by lympho- or myeloablative conditioning followed by stem-cell rescue [11–13]. Preclinical studies in animal models of autoimmune disease and observations in patients with rheumatoid arthritis (RA) who were cured after allogeneic bone marrow transplantation for concomitant hematologic malignancy have provided support for the concept [14–16]. However, the immune property of ADSC is still unknown. We wondered whether ADSC could also modulate immune response, To identify it, we isolated ADSC from human fat tissue, and explored its immune function. The results demonstrated that ADSC could negatively modulate immunity and induce immune tolerance by regulating DC.
2.1. Preparation of ADSC
⇑ Corresponding author. Fax: +86 23 68757443. E-mail address: [email protected] (B. Zhang). 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.07.008
In brief, the subcutaneous adipose tissue was dissected from omental fat who underwent abdominal surgery. The volunteer were healthy with the age of 30–50 years. The isolated adipose tissue was washed extensively with phosphate-buffered saline (PBS) to remove contaminating debris and blood cells. The 50 mL adipose tissue was minced and digested with 50 mL collagenase I (2 mg/mL; Worthington Biochemical) at 37 °C for 30 min. Collagenase activity was neutralized by DMEM-F12 (HyClone) containing 15% fetal bovine serum (FBS; HyClone). Digested adipose tissue was filtered twice with a 100-lm and then with a 25-lm nylon membrane to eliminate the undigested fragments. The cellular suspension was centrifuged at 1000 g for 10 min. The cell pellets were re-suspended in cell culture medium (CCM) and cultivated for 24 h at 37 °C in 5% CO2. Unattached cells and debris were removed, and fresh CCM containing 15% FBS was added to the adherent cells, which were cultured at 37 °C in 5% CO2 until 70–80% confluent. Identity of the isolated cells was determined by assessing their surface markers using flow cytometric analysis with FITC-conjugated antibodies against CD29, CD44, CD105, and FITC-conjugated isotype-matched control antibodies (BD).
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2.2. Dendritic cell generation Briefly, PBMCs were isolated from healthy donors by FicollHypaque density gradient centrifugation and then seeded into culture flasks in RPMI-1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 lg/mL), and 10% FBS. After monocytes adhered (incubation for 2 h), the adherent cells were cultured for 5 days in RPMI-1640 containing 1000 U/mL of gpokemonulocyte-macrophage colony-stimulating factor (R&D Systems, Inc., Minneapolis, MN) and interleukin-4 (IL-4; R&D Systems, Inc.), and were the cultured for an additional 2 days in the presence of 1000 U/mL of tumor necrosis factor a (R&D Systems, Inc.) to induce final maturation. After 7 days of culture, the mature DCs were harvested and analyzed for DC typical phenotypes by FACS analysis using primary antibodies against CD80, CD83 and CD86. 2.3. ADSC and DC mixed reactions ADSC (2 105) and DC (2 105) cells were added to roundbottom 96-well plates to a final volume of 200 ll RPMI 1640 with 10% FCS. After 3 days, co-cultured DC were harvested by microbeads (Miltenyi Biotech, Bergisch, Gladbach, Germany) to detect the expression of costimulatory molecules by flow cytometry. Cells were collected and pelleted with 100 lL PBS and incubated for 30 min at 4 °C in the dark using primary antibodies against CD80, CD83 and CD86. Flow cytometric data were analyzed by using CellQuest software (BD Biosciences, San Jose, CA). In addition, supernatants were removed and further analyzed for cytokine production (IL-12 and TNF-a) with ELISA. 2.4. Treated DC and CD4+ T cells mixed reactions CD4+ T cells (>95%) were positively selected from nonadherent cells of PBMC using microbeads. MLR were conducted in 96-well flat-bottom culture plates. ADSC-treated DC (2 105) were co-cultured with 2 105 allogeneic CD4+ cells. After 3 days, co-cultured
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T cells were harvested to detect the expression of activation markers (CD25 and CD69) by flow cytometry. In addition, supernatants were removed and further analyzed for cytokine production (IFN-c and IL-2) with ELISA. 2.5. Treated DC and naïve T cells mixed reactions Allogeneic CD4+CD45RA+ naïve T cells were isolated from PBMC by immunomagnetic purification. Briefly, untouched CD4+ Tcells were obtained through a CD4+ T cell isolation kit, according to the manufacturer’s instructions. Next, CD45RA+ cells were positively selected by CD45RA-coated immunomagnetic microbeads. CD4+CD45RA+ naïve T cells (2 105) were co-cultured with ADSC -treated DC (4 104), at a ratio of 5:1 for 5 days. On day 6, cells were restimulated with plate-bound anti-CD3 (5 lg/mL in PBS, overnight, 4 °C) and anti-CD28 (l lg/mL) antibody. Supernatants were removed and further analyzed for cytokine production (IFNc and IL-4) with ELISA. 2.6. Western blot analysis ADSC-treated DC were collected and washed twice by cold PBS, and each well was treated with 50 lL lysis buffer (2 mmol/L TrisHCl pH 7.4, 50 mmol/L NaCl, 25 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, supplemented with protease inhibitors 1 mmol/L phenylmethylsulfonylfluoride, 10 mg/L pepstatin, 10 mg/L aprotinin, and 5 mg/L leupeptin) (all from Sigma). Protein concentrations were determined using the bicinchoninic acid protein assay. Equal amounts of protein (40 lg) were separated on a 15% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond C, Amersham, Freiburg, Germany). Membranes were blocked in 5% nonfat dry milk in TBS for 1 h at room temperature and probed with rabbit anti-IDO antibodies (1:500, Santa Cruz Biotechnology, USA) overnight at 4 °C. After 3 times washing with TBS containing 0.1% Tween 20, membranes were incubated with antirabbit IgGhorseradish-peroxidase (1:5000, Santa Cruz Biotechnology, USA),
Fig. 1. Morphologic and phenotypic characteristics of ADSC. The subcutaneous adipose tissue was dissected from omental fat. The adipose tissue was minced and digested with collagenase I. The cell pellets were re-suspended in cell culture medium (CCM) and cultivated for 24 h at 37 °C in 5% CO2. (A). Morphologic characteristics of ADSC (200). (B). The surface marker expression of ADSC using flow cytometric analysis with FITC-conjugated antibodies and FITC-conjugated isotype-matched control antibodies. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
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and developed by luminol mediated chemiluminescence (Appylgen Technologies Inc, China). To confirm equal protein loading, membranes were reprobed with a 1:1000 dilution of an anti-actin antibody (Santa Cruz Biotechnology, USA). Densitometric analyses were performed using Scion Image software. 2.7. Statistical analysis The statistical significance of differential findings between experimental groups and controls were determined by t-test and were considered significant for P < 0.05. 3. Results 3.1. Morphologic and phenotypic characteristics of of ADSC At 24 h of primary culture in medium containing 10% fetal bovine serum, ADSC adhered to the culture dish sparsely and the majority of cells displayed a spindle-like shape. As shown in Fig. 1A, after 14 days, ADSCs commonly achieved 80% confluence with a rather homogeneous population of fibroblast-like morphology. In addition, as shown in Fig. 1B, flow cytometric analysis showed that the ADSC expressed high level of CD29, CD44 and CD105. These results revealed that cultured ADSC were homogeneous and did not contain hematopoietic lineages, consistent with the previous report. 3.2. Morphologic and phenotypic characteristics of DC On day 9 of the cell culture, the mature DC displaying typical characteristics of morphology were harvested from monocytes cultured in the medium containing GM-CSF, IL-4, and tumor necrosis factor-a. As shown in Fig. 2A, these mature cells were loosely suspended, exhibited irregular cell shape, and displayed many fine processes at their edges. The mature denditric cells were also analyzed for phenotype by flow cytometry. As shown in Fig. 2B, the results showed that these mature DC expressed high levels of CD80, CD83 and CD86.
3.3. ADSC induced DC undergo tolerance To explore the effect of ADSC to DC, after ADSC and DC mixed reactions, we analyzed the costimulatory molecules, including CD80, CD83, CD86, cytokine secretion such as IL-12 and TNF-a, and IDO of allogenetic DC. As shown in Fig. 3, the results demonstrated that ADSC could downregulate CD80, CD83 and CD86 expression, inbibit IL-12 and TNF-a secretion, and induce IDO expression. 3.4. Treated DC inhibited CD4+ T cell activation We wonder whether ADSC treated DC could inhibit CD4+ T cell activation. We also cocultured treated DC and CD4+ T cells together, and analyzed the expression of CD4+ T cell activation markers of CD25 and CD69, and cytokine secretion of IFN-c and IL-2. As shown in Fig. 4, treated DC could downregulate CD25 and CD69 expression of CD4+ T cells, and inhibit cytokine secretion of IFNc and IL-2. these results demonstrated that treated DC could inhibit CD4+ T cell activation. 3.5. Treated DC inhibited naïve T cells toward Th1 polarization The CD4+CD45RA+ naïve T cells were then co-cultured with treated DC, and after 5 days, the T cells were recovered and restimulated with anti-CD3 (5 lg/mL) and anti-CD28 (1 l/mL) antibodies in combination. As shown in Fig. 5, the production of the cytokine was measured by ELISA and results indicated that the secretion of IL-4 was increased, whereas IFN-c production was decreased in the culture as compared with control group. Accordingly, these results indicate that treated DC inhibited CD4+CD45RA+ naïve T cells to a Th1 polarization. 4. Discussion Soft-tissue loss presents an ongoing challenge in plastic and reconstructive surgery [17,18]. Standard approaches to soft-tissue reconstruction include autologous tissue flaps, autologous fat
Fig. 2. Morphologic and phenotypic characteristics of DC. PBMCs were isolated from healthy donors. After monocytes adhered, the cells were cultured for 5 days in RPMI1640 containing 1000 U/mL of GM-CSF and IL-4, and were the cultured for an additional 2 days in the presence of 1000 U/mL of TNF-a. After 7 days of culture, (A). Morphologic characteristics of DC (200). (B). The surface marker expression of DC using flow cytometric analysis with FITC-conjugated antibodies and FITC-conjugated isotype-matched control antibodies. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
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Fig. 3. ADSC induced DC undergo tolerance. ADSC (2 105) and DC (2 105) cells were added to round-bottom 96-well plates to a final volume of 200 ll RPMI 1640 with 10% FCS. (A).After 3 days, co-cultured DC were harvested by microbeads and pelleted with 100 lL PBS and incubated for 30 min at 4 °C in the dark using primary antibodies against CD80, CD83 and CD86. Flow cytometric data were analyzed by using CellQuest software. (B). After 3 days, supernatants were removed and further analyzed for cytokine production (IL-12 and TNF-a) with ELISA. (C). After 3 days, co-cultured DC were harvested by microbeads and washed twice by cold PBS, and DC were treated with lysis buffer to further analyze the IDO expression with Western blot assay. 1: PBS; 2: untreated DC; 3: treated DC. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
Fig. 4. Treated DC inhibited CD4+ T cell activation. CD4+ T cells (>95%) were positively selected from nonadherent cells of PBMC using microbeads. MLR were conducted in 96-well flat-bottom culture plates. ADSC-treated DC (2 105) were co-cultured with 2 105 allogeneic CD4+ cells. (A).After 3 days, co-cultured T cells were harvested to detect the expression of activation markers (CD25 and CD69) by flow cytometry. (B). After 3 days, supernatants were removed and further analyzed for cytokine production (IFN-c and IL-2) with ELISA. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
transplantation and alloplastic implants [19–21]. All of these approaches have disadvantages, including donor-site morbidity, implant migration and absorption and foreign body reaction [22– 24]. Stem cell application has recently been suggested as a possible novel therapy [25–27]. Adipose-derived stem cells (ADSC) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue, which is an easily accessible and abundant source of putative stem cells for translational clinical research [28,29]. Their therapeutic use in pre-clinical studies and experimental clinical trials has been well documented [4,30–32]. Previous studies proved that human bone marrow mesenchymal stem cells (BM-MSC) are multipotent progenitor cells that have transient immunomodulatory properties on Natural Killer (NK) cells, Dendritic Cells (DC), and T cells [33,34]. BM-MSC modulate hematopoiesis and can exert immune regulatory functions, both in vivo and in vitro [35,36]. The immunomodulatory properties of BM-MSC require cell-cell contact as well as soluble factors, including interleukin (IL)-6, hepatocyte growth factor (HGF), nitric oxide (NO), prostaglandin (PGE)-2, indolamine 2,3 dioxygenase (IDO), and HLA-G [36]. BM-MSC transplantation was trailed in a number of autoimmune diseases and represented a promising strategy [37–39]. The evidences revealed that cell specific differences at transcriptional and proteomic levels between both ADSC and BM-MSC types according to their tissue origin as well as functional differences in their differentiation processes towards adipogenic, osteogenic and chondrogenic programs. Nevertheless, in vitro as well as in vivo ADSC displayed the same ability than MSC to differentiate towards chondrocytes/osteoblasts, comforting the status of both cell sources as promising regenerative cells [40]. However, the mechanism and therapeutical potential of ADSC in autoimmune diseases is still known. To identify that, we isolated and cultured ADSC, and explored the immune property. Firstly, we isolated ADSC from omental fat. After 14 days of culture, ADSC dis-
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Fig. 5. Treated DC inhibited naïve T cells toward Th1 polarization. The CD4+CD45RA+ naïve T cells were co-cultured with treated DC, and after 5 days, the T cells were restimulated with anti-CD3 (5 lg/mL) and anti-CD28 (1 l/mL) antibodies in combination. The production of the cytokines (IL-4 and IFN-c) were measured with ELISA. Experiments performed in triplicate showed consistent results. Compared with controls, P < 0.05.
played a rather homogeneous population of fibroblast-like morphology and expressed high level of CD29, CD44 and CD105. In addition, we also generated DC from the PBMCs. On day 9 of the cell culture, DC displayed typical characteristics of morphology and expressed high level of CD80, CD83 and CD86. Secondly, to identify the function of ADSC to DC, the key contributor to the induction of primary and secondary immune responses, we cocultured ADSC with allogenetic DC together and analyzed the costimulatory molecule expression and cytokine secretion. The results demonstrated that ADSC could downregulate costimulatory molecules, including CD80, CD83, CD86, and cytokine secretion such as IL-12 and TNF-a while upregulate IDO of DC. Thirdly, to study the effect of treated DC to CD4+ T cell activation or naïve T cell polarization, we cocultured treated DC with allogenetic CD4+ T cells or CD4+CD45RA+ naïve T cells together, and analyzed the activation markers and cytokine production. The results demonstrated that treated DC could could downregulate CD25 and CD69 expression of CD4+ T cells, and inhibit cytokine secretion of IFN-c and IL-2. Additionly, treated DC inhibited CD4+CD45RA+ naïve T cells to a Th1 polarization through upregulate IL-4 production while downregulate IFN-c production. The results demonstrated that leading to the immunomodulatory interference of ADSC on DC might by regulating cell membrane molecular expression and cytokine secretion. In conclusion, our finding demonstrated that ADSC could induce DC tolerance, result in CD4+ T cell activation inhibition and naïve T cells toward Th1 polarization. The results might provide ADSC a theoretic foundation and therapeutical strategy for autoimmune disease. Acknowledgment This work was supported by 2011CB964701 of China. References [1] H. Nakagami, R. Morishita, K. Maeda, Y. Kikuchi, T. Ogihara, Y. Kaneda, Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy, J. Atheroscler.Thromb. 13 (2) (2006 Apr) 77–81. [2] A. Schaffler, C. Buchler. Concise review: adipose tissue-derived stromal cellsbasic and clinical implications for novel cell-based therapies. Stem Cells 2007 Apr; 25(4):818–827. [3] Y. Kamada, Y. Yoshida, Y. Saji, J. Fukushima, S. Tamura, S. Kiso, et al., Transplantation of basic fibroblast growth factor-pretreated adipose tissuederived stromal cells enhances regression of liver fibrosis in mice, Am. J. Physiol. 296 (2) (2009 Feb) G157–G167.
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