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Mesenchymal stem cells attenuated PLGA-induced inflammatory responses by inhibiting host DC maturation and function
Biomaterials 53 (2015) 688e698
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Mesenchymal stem cells attenuated PLGA-induced inflammatory responses by inhibiting host DC maturation and function Heng Zhu a, ***, 1, Fei Yang b, 1, Bo Tang a, Xi-Mei Li a, Ya-Nan Chu a, Yuan-Lin Liu a, Shen-Guo Wang b, De-Cheng Wu b, **, Yi Zhang a, * a
Department of Cell Biology, Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, PR China BNLMS, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun No. 1 Street, Haidian District, Beijing 100190, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 December 2014 Received in revised form 27 February 2015 Accepted 4 March 2015 Available online 24 March 2015
The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGADC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Poly (lactide-co-glycolide) Dendritic cells Mesenchymal stem cells Host inflammatory responses
The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects
* Corresponding author. Tel.: þ86 10 66931320; fax: þ86 10 68213039. ** Corresponding author. Tel.: þ86 10 82611492; fax: þ86 10 82611492. *** Corresponding author. Tel.: þ86 10 66930913; fax: þ86 10 68213039. E-mail addresses: [email protected] (H. Zhu), [email protected] (D.-C. Wu), [email protected] (Y. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.03.005 0142-9612/© 2015 Elsevier Ltd. All rights reserved.
of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGA-DC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the
H. Zhu et al. / Biomaterials 53 (2015) 688e698
inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair. 1. Introduction Poly lactic-co-glycolic acid (PLGA) has been used in a variety of preclinical studies and clinical applications for tissue repair [1,2]. Most notable among these applications are the PLGA scaffolds that are made for filling large tissue defects [3,4], building threedimensional cell-scaffold constructs for tissue regeneration [5,6], or for preparing drug and cytokine-coated grafts for localized delivery [7,8]. However, growing evidence has demonstrated that implanted PLGA scaffolds cause serious host inflammatory responses. A retrospective analysis regarding clinical cases demonstrated that patients who underwent bone fixation with PLGA scaffolds developed cystic swelling underneath their scalps [9]. Asawa Y et al. reported that the early stage foreign body reactions against PLGA scaffolds diminished tissue regeneration during the autologous transplantation of tissue-engineered cartilage in a canine model [10]. Another study demonstrated that subcutaneously implanted PLGA films triggered acute inflammatory response and caused remarkable macrophage infiltration in a rodent model [11]. To reduce these inflammatory responses, a number of techniques have been developed for preparing scaffolds, including impregnating demineralized bone particles into the PLGA [12], incorporating SDF-1a [13], anti-inflammatory siRNA [14] or masitinib [15] into PLGA scaffolds and coating a thin surface of calcium phosphate onto PLGA scaffolds [16]. However, the techniques described in these reports are complex and not easily standardized. Most importantly, the role of antigen presenting cells, which initiate host immune responses, was overlooked. Dendritic cells (DC) are the most potent professional antigen presenting cells [17,18]. By taking up and processing innate antigens or invading pathogens, DCs rapidly present antigens to antigenspecific naïve T cells. The process is also accompanied by the expression of co-stimulatory molecules, including MHC-II, CD80 and CD86, and the secretion of immune cytokines, including interleukin-12 (IL-12) and tumor necrosis factor-a (TNF-a). These cell surface molecules and immune factors are indispensable for DCs to induce T cell activation and thereafter to initiate specific responses [17,18]. Interestingly, recent studies revealed that PLGA is capable of promoting DC maturation in vitro [19e25]. The treatment of DCs with PLGA microparticles or films led to the maturation of these cells through increased co-stimulatory molecule expression and pro-inflammatory cytokine secretion [19e21]. Moreover, co-culturing DCs with PLGA enhanced the allostimulatory capacity of the DCs [24]. Thus, we suggest that DC may be a cell target to reduce PLGA-induced host inflammatory responses. Mesenchymal stem cells (MSC) were originally isolated from bone marrow and now have been successfully isolated from adipose tissue, compact bone, placenta and other connective tissues [25e27]. Under certain conditions, MSCs can differentiate into numerous tissue cell types and promote tissue repair [28,29]. Moreover, MSCs express a low level of co-stimulatory molecules, which indicates that they have low antigenicity [25e27]. Most importantly, MSCs are able to suppress immune cell development in a dose dependent manner by means of secretary factors and/or direct contact [30,31]. Therefore, MSCs have been successfully used for the treatment of numerous diseases, including osteogenesis imperfecta, acute graft-versus-host disease, and rheumatoid arthritis [32e34].
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Given the fundamental role of DCs in host immune responses and the potent immunosuppressive properties of MSCs, they may be an effective strategy for alleviating PLGA-elicited inflammatory reactions via targeting DCs. In the current study, we explored the effects of MSCs on PLGA-induced DCs (PLGA-DC) and the subsequent host immune responses. Moreover, the cellular and molecular mechanisms regarding the capacity for MSCs to regulate PLGADCs were also investigated. 2. Materials and methods 2.1. PLGA film preparation PLGA was prepared according to a previously described protocol with minor modifications [35]. Briefly, L-lactide and glycolide were purchased from PURAC (the Netherlands) and purified by recrystallization in ethyl acetate (Beijing Tonghua fine chemicals company, China), twice. PLGA was synthesized by ring-opening polymerization of the L-lactide and glycolide (mol. Ratio 50:50) under 65 Pa in sealed glass ampoules at 180 C for 20 h in the presence of stannous octoate, which was used as a catalyst (0.05 wt%). The raw PLGA (50/50) was dissolved in chloroform and then purified by ethanol precipitation. It was then dried in a vacuum at room temperature for 48 h. The molecular weight of PLGA (50/50) (Mw ¼ 100 kDa) was determined with GPCmax VE-2001(viscotek) gel-permeation chromatography (GPC). PLGA was further fabricated into porous films. Briefly, PLGA was dissolved in dioxane to form a 10% concentration solution. Then, presieved NaCl particles (diameter of 0.18e0.25 mm) were weighed and then added into the solution to form a slurry. The slurry was fed into a flat box shaped mold without cover with length and width of 120 mm both, and then the mold was cooled down to 20 C for more than 2 h. After this, the frozen solution was freeze-dried at 50 C for more than 24 h. Then NaCl particles in the scaffolds were eliminated by washing several times in pure water with stir until the NaCl can not be detected by AgNO3. The wet scaffolds were vacuum dried in room temperature for 24 h. The PLGA scaffolds were stored under dry vacuum conditions. The porous PLGA films were prepared using a hole puncher (8 mm in diameter and 3 mm in thickness) and were used for in vivo and in vitro experiments unless otherwise described. 2.2. Animals Normal inbred two-week-old and eight-week-old C57BL/6 (H2b) mice, as well as eight-week-old BALB/c mice, were purchased from the Academy of Military Medical Sciences of China Laboratory Animal Center (Beijing). All experiments in this study were performed in accordance with the Academy of Military Medical Sciences Guide for Laboratory Animals. 2.3. Murine MSC isolation and MSC-PLGA construct preparation Murine MSCs were prepared from compact bones by virtue of high cell yield and low contamination of hematopoietic cells according to a previous protocol [26]. Briefly, long bones from two-week-old C57BL/6 mice were dissected, and the bone marrow cells were flushed out. Next, the chopped compact bones were digested by collagenase II (Sigma) and the bone chips were cultured in MSC culture medium (aMEM supplemented with 10% fetal bovine serum). The adherent cells, at passages 3e6, were used for in vivo and in vitro experiments unless otherwise described. For the preparing MSC-PLGA constructs, MSCs that were resuspended in cultured medium were seeded onto sterilized PLGA films to allow the MSCs adhere to the films. Then, a whole volume of culture medium was added 2 h later. The MSC-PLGA constructs were cultured in MSC culture medium for 48 h before they were cocultured with DCs or used in implantation experiments unless other described. 2.4. The cell morphology and growth kinetics of the MSCs on the PLGA films The cell morphology of the MSCs on the PLGA films was observed with scanning electron microscopy (SEM; JSM-6700F, JEOL) at day 3 after the cells were loaded. The growth kinetics of the MSCs on the PLGA was determined using the Cell Counting Kit 8 (CCK-8, Dojindo, Japan) according to the manufacturer's protocol. In brief, a total of 2 103 MSCs were seeded onto the sterile PLGA films, the MSC-PLGA constructs were cultured in 24-well plates and were added to the CCK-8 solution at a ratio of 100 mL/1 mL, and the plates were incubated at 37 C for 1 h. Absorbance was then measured at a wavelength of 450 nm using a microplate reader. Five wells at each time point were assayed. The CCK-8 experiments were performed at days 1, 3, 6, 9, 12, 15, 18, and 21. 2.5. Murine DC generation Murine DCs were prepared from bone marrow progenitors [36]. In brief, bone marrow mononuclear cells were prepared from 8-week-old C57BL/6 or BALB/c mice by red cell depletion. The DCs were generated with RPMI 1640 medium that was supplemented with 10% fetal bovine serum (FBS), 10 ng/ml of mouse granulocytemacrophage colony stimulating factor (GM-CSF), and 1 ng/ml of mouse
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interleukin-4 (IL-4) (Peprotech) for five days. Loose and non-adherent cells were harvested, and CD11cþ cells were purified with a MACS CD11cþ isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD11c cells positive were identified as immature DCs (iDCs) and used for subsequent experiments. In some co-culture experiments, trans-well chamber systems were used to separate DCs from MSCPLGA constructs or PLGA films. 2.6. Cell morphology and ultra-structure of DCs A total of 2 104 MSCs were seeded onto each sterile PLGA films, and they were co-cultured with 2 105 iDCs in 6-well plates for 48 h. The cell morphology of the PLGA- or MSC-PLGA construct-induced DCs was observed under a phase contrast microscope (Nikon TE2000-U). For the transmission electron microscopy ultrastructural observations (TEM, HITACHI, Tokyo, Japan), PLGA- or MSC-PLGA construct-induced DCs were collected and fixed for 4 h at 4 C in 5% glutaraldehyde, washed 3 times in 0.1 mol/L phosphate buffered saline (PBS), post-fixed for 2 h at 4 C in 2% osmium tetroxide, dehydrated in a graded series of ethanol, embedded in Epon 812, cut into ultrathin sections (75 nm), and then stained with uranyl acetate and lead citrate. The sections were finally viewed and recorded with a HITACHI H-600 electron microscope at 80 KV.
2.8. Immune factor determination by real-time PCR and ELISA To assess the effect of MSCs on immune cytokine expression in DCs and T cells in vitro, PLGA- or MSC-PLGA constructs were co-cultured with iDCs at different ratios for 48 h. The MSC:iDC ratios were 1:100, 1:50, 1:20 and 1:10. CD3þ T lymphocytes were purified from healthy mouse spleens using a MACS CD3 isolation kit (Miltenyi Biotec) and were further primed with PLGA- or MSC-educated PLGA-DCs before the immune factors were detected. For the in vivo analyses, recipient nucleated splenocytes were collected at days 3 and 7 post-PLGA film implantation. Total RNA was extracted with TRIZOL reagent (Invitrogen) and reverse transcribed using the mRNA Selective PCR kit (TaKaRa). Mouse TNF-a, IL-12, IFN-g, IL-4, IL-17A and Foxp3 cDNA were amplified by real-time PCR using the SYBR Green PCR kit (Sigma). The primer sequences used for the realtime PCR are shown in Table S1. Meanwhile, the culture supernatants from the in vitro DCs and DC-T cell culture systems were collected by centrifugation and filtered to deplete the cellular components. Mouse TNF-a, IL-12, IFN-g, IL-4 and IL-17A concentrations in the supernatants were determined according to the reagent protocols of the quantitative determination kit (R&D Systems, Minneapolis). 2.9. The DC endocytosis properties and T cell priming capacity
2.7. Immunophenotyping of the DCs and T cells To detect the effect of the MSCs on the DC immuno-phenotype in vitro, PLGA- or MSC-PLGA constructs were co-cultured with iDCs at different ratios for 48 h. The MSCs:iDC ratios were 1:100, 1:50, 1:20 and 1:10. To determine the DC phenotypes and the T cell subpopulations in vivo, recipient nucleated splenocytes were collected at days 3 and 7 post-PLGA film implanting. To learn the signaling pathway effects on the DC surface markers, the chemical inhibitors, SB203580HCL, PD98059 and JNK inhibitor II (20 mm/ml of each), which are specific inhibitors of the p38/MAPK, ERK/ MAPK and JNK/MAPK pathways, respectively, were added to the PLGA film and DC co-culture system (2 106 iDCs each sterile PLGA films). The DCs and T cells were stained with FITC, PE or Allophycocyanin (APC) conjugated monoclonal antibodies against mouse CD3, CD69, CD80, CD86, CD11c and Ia (MHC-II molecular) (eBio-Science, San Diego). For intercellular cytokine staining, recipient splenocytes were harvested and stimulated with 50 ng/ml phorbol 12myristate 13-acetate (PMA), 500 ng/ml ionomycin, and 3 mg/ml brefeldin A (SigmaeAlrich) for six hours. The splenocytes were then washed and labeled for CD3, and CD4 as well as intracellular interferon-g (IFN-g), interleukin-17A (IL-17A), or IL-4. For analysis of regulatory T cells (Tregs), the anti-mouse intranuclear forkhead box P3 (FoxP3) kit (eBioscience, San Diego, CA) was used according to the manufacturer's protocol. Events were collected by flow cytometry with a FACScalibur system (BectoneDickinson), and data analysis was conducted with the WinMDI 2.9 software.
To determine the effect of the MSCs on DC endocytosis, iDCs were exposed to PLGAor MSC-PLGA constructs at different MSC:iDC ratios (1:100,1:50,1:20,1:10) for 48 h. DC endocytosis was measured as the cellular uptake of FITC-dextran and was quantified by flow cytometry as previously described [37]. Briefly, 1 106 DCs per sample were incubated in medium containing FITC-dextran (1 mg/ml; molecular weight 40,000; Sigma, St Louis, MO) for 1 h. After incubation, the cells were washed twice with cold phosphate buffered saline (PBS) to stop endocytosis and remove the excess dextran. The quantitative endocytosis capacity of the DCs was determined by the fold change of the mean fluorescence intensity (MFI) values relative to that of the PLGA-DCs. To investigate the effect of MSCs on the DC priming capacity, the PLGA-DCs and MSC-treated PLGA-DCs from the above co-culture system were harvested and irradiated (15 Gy). Next, the DCs were cultured in triplicate at 2 104 cells in 200 mL per well in 96-well flat-bottomed plates with 2 105 allogeneic CD3þ T cells. After four days, the cells were pulsed for 16 h with 3H-thymidine (1 mCi/well [0.037 MBq/ well]). Thymidine incorporation was measured by standard liquid scintillation counting. The results are expressed in counts per minute and shown as the mean ± SD of triplicate values. 2.10. Western-blotting PLGA films or MSC-PLGA constructs (1 105 cells) were co-cultured with iDCs (1 106 cells/well). The DCs were collected after 0, 5, 10, 30, 60, 120, and 240 min of
Fig. 1. The morphological characteristics of PLGA film and MSC-PLGA construct. The gross morphology of PLGA film and MSC-PLGA construct are shown in Fig. 1A. The ultra morphology was shown by scanning electron microscopy (Fig. 1B and C). Additionally, the growth kinetics of MSCs on PLGA film was determined by CCK-8 assay (Fig. 1D). Bars in Fig. 1AeC represent 1 cm, 200 mm, and 50 mm, respectively.
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Fig. 2. MSCs inhibited PLGA-induced DC maturation in vitro. In the MSC/DC co-culture system, MSCs suppress the dendrite formation of PLGA-DCs (Fig. 2A). In addition, the expression of CD11c, CD80, CD86 and Ia in PLGA-DCs were decreased by MSCs in a dose dependent manner (Fig. 2B and C). Further, MSCs down-regulated the transcription and secretion level of IL-12 and TNF-a (Fig. 2D and E). Bars in Fig. 2A upper row and lower row represent 200 mm and 50 mm, respectively.
co-culture. Protein lysis buffer (BioRad, Hercules, CA) was added, and the thawed lysates were vortexed and centrifuged. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked by incubation with 5% wt/vol nonfat dry milk. The membranes were then incubated with anti-p38, anti-phosphop38 (P-p38), anti-ERK, anti-phospho-ERK (P-ERK), anti-JNK, anti-phospho-JNK (PJNK), and b-actin antibodies (Cell Signal Technology) at the appropriate dilutions overnight at 4 C. After incubation, the membranes were washed in Tris buffered saline with Tween-20 (TBST). Horseradish peroxidase-conjugated secondary antibodies were added to the membranes in 5% nonfat dry milk in TBST. 2.11. Implantation of the MSC-PLGA constructs in the murine model The MSC-PLGA constructs were collected and gently washed in PBS for the transplantation experiment. Thirty normal inbred 8-week-old BALB/c mice received a dorsal subcutaneous implantation of an MSC-PLGA construct. Mice implanted with blank PLGA films served as controls. Five mice were sacrificed at days 3 and 7 postimplantation. The DC and T cell immunophenotypes and inflammatory cytokine expression were determined as described above. The proliferation of splenic corpuscles in the spleens and the immune cell filtration around the implants were observed using pathological examinations. The specimens were fixed in 10% neutralbuffered formalin, imbedded in paraffin, cut into 5-mm-thick sections and stained with hematoxylin and eosin (H&E). The sections were observed using inverted microscopy (Nikon TE2000-U). 2.12. Statistical analysis Data are represented as the mean values with standard deviations. Statistical significance was analyzed using Student's t test. P values less than 0.05 were considered to be significant.
3. Results 3.1. MSCs inhibited PLGA-induced DC maturation in vitro The gross morphology of the PLGA films and MSC-PLGA constructs are shown in Fig. 1A. The scanning electron microscopy data showed that the MSCs grew robustly in the porous PLGA films (Fig. 1B and C). The results of the CCK-8 assay (Fig. 1D) demonstrated that the MSCs maintained a high proliferation capacity on the PLGA film for at least 3 weeks. To investigate whether the MSCs affected the DC morphology, the PLGA-DCs and MSC-treated PLGA-DCs were observed with phase contrast and transmission electron microscopes. As shown in Fig. 2A, the PLGA-DCs displayed protruding veils with abundant cytoplasm, while the MSC-educated PLGA-DCs were round and lack the veiled appearance. Cell surface molecules and immune factors are indispensable for DCs to initiate specific responses. Compared with PLGA-DCs, the MSC-treated PLGA-DCs had decreased CD11c, CD80, CD86 and Ia expression (Fig. 2B). Importantly, the inhibitory effect was dosedependent. With a low MSC/DC ratio of 1:50, the CD11c, CD86 and Ia expression was significantly reduced (Fig. 2C. *, P < 0.05; **, P < 0.01).
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Fig. 3. MSCs enhanced the phagocytic capacity but suppressed T cell stimulatory properties of PLGA-DCs. MSC-treated PLGA-DCs uptook more FITC labeled Dextran particles. The data were collected by FACS and the results were indicated as the fold change of mean fluorescence intensity values relative to that of PLGA-DCs (Fig. 3A). In addition, the T cell proliferation that triggered by MSC-treated PLGA-DCs declined in a MSC dose dependent manner (Fig. 3B). Moreover, the expression of T cell derived IL-4, INF-g, IL-17 and Foxp3 were regulated by different DCs (Fig. 3C and D).
Further investigations showed that MSCs also suppressed the expression of key immune factors in the PLGA-DCs. The real-time PCR and ELISA results demonstrated that MSCs suppressed the expression of IL-12 and TNF-a in the PLGA-DCs (Fig. 2D and E). Additionally, a low MSC/DC ratio (1:50) resulted in significant suppression of the transcription and secretion of IL-12 and TNF-a (Fig. 2D and E) (*, P < 0.05; **, P < 0.01), implying that a small number of MSCs is capable of blocking PLGA-DC maturation. 3.2. MSCs modulated the phagocytic capacity and T cell stimulatory property of PLGA-DCs Up to this point, we determined that MSCs potently suppressed the maturation of PLGA-DCs. However, it remained unknown whether MSCs affected the immune function of the PLGA-DCs. By using FITC-labeled dextran endocytosis and H3 incorporation assays, we further determined the effects of MSCs on the phagocytic capacity and T cell stimulatory property of the PLGA-DCs. The phagocytic capacity of the MSC-treated PLGA-DCs was indicated as the fold change in the mean fluorescence intensity values relative to the PLGA-DCs. As shown in Fig. 3A, the MSCeducated PLGA-DCs exerted greater phagocytic capacity than the PLGA-DCs. Moreover, the modulatory effect of the MSCs on DC endocytosis was dose-dependent. The phagocytic capacity of the MSC-treated PLGA-DCs increased significantly at an MSC: DC ratio of 1:20 (Fig. 3A). (*, P < 0.05). Meanwhile, we investigated the T cell priming capacity of the DCs. Compared with the PLGA-DCs stimulated T cells, the T cell proliferation triggered by MSC-treated PLGA-DCs was remarkably
dampened (Fig. 3B) (*, P < 0.05, **, P < 0.01). Additionally, the priming effects of the PLGA-DCs were reduced with increased MSC number (Fig. 3B). Most importantly, we found that MSC-educated PLGA-DCs promoted Th2 and regulatory T cell (Treg) differentiation but suppressed Th1 and Th17 cell differentiation. Treating PLGA-DCs with MSCs led to an increase of IL-4 and Foxp3 but caused a decrease of INF-g and IL-17A in T lymphocytes (Fig. 3C and D) (*, P < 0.05, **, P < 0.01), indicating that MSC-treated PLGA-DCs play a role in shifting the balance from Th1 and Th17 to Th2 and Treg responses. 3.3. MSCs suppress PLGA-DC maturation without intercellular contact at a low MSC/DC ratio To explore whether the inhibition of MSCs on PLGA-DCs requires intercellular contact, we used a Transwell chamber system to separate DCs (upper compartment) from the MSC-PLGA constructs or PLGA films (lower compartment) (Fig. 4A). We found that PLGA films could trigger DC dendrite formation and induce CD11c, CD80, CD86 and Ia expression without direct contact with DCs, which indicated that PLGA might induce DC maturation via a contact independent mechanism. Interestingly, we found that MSCs significantly prevented DC maturation in the Transwell chamber system, implicating that soluble factors from the MSCs were involved in the observed suppression (Fig. 4B). Further, we defined what factors in the supernatant were involved in the inhibitory effects by utilizing neutralization antibodies. IL-6 and M-CSF were investigated because numerous studies have demonstrated that they are pivotal cytokines in the
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Fig. 4. MSCs suppressed PLGA-DC maturation via secreted cytokines dependent manner. A transwell co-culture in vitro model was setup to analyze whether the inhibition of MSCs on PLGA-DCs require intercellular contact. MSCs were capable of inhibit dendri formation of PLGA-DCs without direct contact (Fig. 4A). Additionally, the MSC derived soluble factor decreased the expression of CD11c, CD80, CD86 and Ia in PLGA-DCs (Fig. 4B). Furthermore, the inhibitory effects of MSCs on PLGA-DC immunophenotype were partially reverted by the neutralization of IL-6 and M-CSF in MSC supernatants (Fig. 4C). Bars in Fig. 4A represent 200 mm.
control of DC maturation. In the current study, the expression of CD11c, CD80, CD86 and Ia were significant reduced by direct supplement of anti-IL-6 (200 ng/ml) and/or anti-M-CSF (200 ng/ml) neutralizing antibodies (Fig. 4C). The data suggested that MSCs might maintain the observed immature DC phenotype via IL-6 and M-CSF secretion. 3.4. MSCs suppressed PLGA-induced DC maturation partially by inactivation of the p38/MAPK and ERK/MAPK pathways To examine the intracellular signaling cascades, we investigated MAPK pathway protein phosphorylation in the PLGA-DCs because the activation of MAPKs signals is critical for DC development [38e40]. The results showed that the p38/MAPK and ERK/MAPK pathways in the DCs were markedly activated by the PLGA films in a time-dependent manner (Fig. 5A); however, no obvious phosphorylation of the JNK/SAPK pathway proteins was detected in the PLGA-DCs (Fig. 5A). Most importantly, exposure of the PLGA-DCs to MSCs without any exogenous cytokine or chemical inhibitor addition caused significant inactivation of the p38/MAPK and ERK/ MAPK pathways in the DCs (Fig. 5B).
To further clarify the association of the MAPK pathways and the PLGA-DCs, specific chemical inhibitors, including SB203580 (for p38/MAPK), PD98059 (for ERK/MAPK), and JNK inhibitor II (for JNK/ SAPK) were added to the PLGA-DC culture system. As shown in Fig. 5C, SB203580 and PD98059 markedly suppressed dendrite development, whereas the JNK inhibitor II exerted mild effects on the DC morphology. Furthermore, SB203580 and PD98059 significantly reduced the CD11c, CD80, CD86 and Ia expression in the PLGA-DCs (Fig. 5D) (*, P < 0.05). These data suggest that the p38/ MAPK and ERK/MAPK pathways play essential roles in PLGA-DC maturation. Collectively, we suggest that the PLGA films enhanced DC maturation by activating the p38/MAPK and ERK/MAPK pathways, while the MSCs exhibited an inhibitory effect on the PLGA-DCs by partially dampening the activation of those pathways. 3.5. MSCs suppressed DC maturation in a PLGA film implantation model Although in vitro studies have clearly shown that MSCs are capable of suppressing PLGA-DC maturation and functions, it
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Fig. 5. MSCs suppressed the PLGA-induced DC maturation partially by inactivation of the p38/MAPK and ERK/MAPK pathway. PLGA film remarkable activated the p38/MAPK and ERK/MAPK pathways in DCs (Fig. 5A), while MSCs dampened the phosphorylation of signal pathways (Fig. 5B). Further, specific blockage of the pathway by chemical inhibitors suppressed the dendrite formation and immunophenotype of PLGA-DCs (Fig. 5C and D). These data suggest that p38/MAPK and ERK/MAPK pathway play essential roles in the maturation of PLGA-DCs. Bars in Fig. 5C upper row and lower row represent 200 mm and 50 mm, respectively.
remains unknown whether this is the case in vivo. Therefore, the phenotype and the pro-inflammatory cytokine expression of DCs were determined at days 3 and 7 post-PLGA film implantation. Promisingly, we found that the MSC co-implantation significantly reduced the proportion of mature DCs (CD11cþCD80þ, CD11cþCD86þ, and CD11cþIaþ) among the host splenocytes (Fig. 6AeC) (*, P < 0.05; **, P < 0.01). Furthermore, the expression of pivotal cytokines for DC maturation was also inhibited by the MSCs (Fig. 6B and C). Compared with the PLGA film-implanted mice, the IL-12 mRNA levels dropped more than 50% and the TNF-a mRNA levels decreased more than 70% at day 3 in the MSC-PLGA construct-implanted mouse splenocytes (Fig. 6B) (*, P < 0.05; **, P < 0.01). Similar effects were observed at day 7 (Fig. 6C). 3.6. MSCs regulated the T cell subpopulations and attenuated PLGAinduced host inflammatory reactions in vivo To explore the influence of the MSCs on the DC priming properties in vivo, we first assessed T cell activation in the host spleens. CD69 expression is a prerequisite for T cell proliferation [41,42]; therefore, the CD3þCD69þ proportions in the recipient spleens were determined. The results showed that the MSCs significantly down-regulated the CD3þCD69þ T lymphocyte proportion in the host spleens in a time-dependent manner (Fig. 7A) (*, P < 0.05). Because the in vitro experiments indicated that the MSC-treated PLGA-DCs caused a shift in the T cell balance from Th1 and Th17 to Th2 and Treg, further in vivo experiments were performed to validate our findings. The proportions of Th1 cells (CD4þIFN-gþ), Th2 cells (CD4þIL-4þ), Th17 cells (CD4þIL-17Aþ) and Tregs
(CD4þCD25þFoxp3þ) in the recipient spleens were determined by intracellular cytokine staining and analyzed by FACS at day 7. The results showed that the MSCs significantly decreased the proportion of Th1 and Th17 cells but increased the proportion of Th2 cells and Tregs in the recipients (Fig. 7B) (*, P < 0.05). Consistently, the real-time PCR results showed that the MSCs down-regulated IFN-g and IL-17A expression but up-regulated IL-4 and Foxp3 expression (Fig. 7C). (*, P < 0.05; **, P < 0.01). These results implicate that MSCPLGA co-implantation induces functional alterations in splenic lymphocytes in vivo. Histological analysis of the recipient spleens was performed at day 7 and we found that the spleen nodules in the MSC-PLGA construct-implanted recipients were smaller than those in the PLGA film-implanted recipients (Fig. 7D). The mouse spleen nodules showed active hyperplasia, which suggested that the engrafted PLGA films had antigenicity and activated lymphocytes in the spleens, whereas the MSCs ameliorated the immune responses (Fig. 7D). Immune cell infiltration around the implants were observed at day 21, as shown in Fig. 7E. The implanted-PLGA film activated inflammatory cells in vivo and a fibrotic capsule formed at the implantation sites; however, no remarkable immune cell infiltration near the MSC-PLGA constructs was observed (Fig. 7E). 4. Discussion In the current study, we demonstrated the suppressive effects of MSCs on the maturation and immune functions of PLGA-DCs in vivo and in vitro. Additionally, we found that the MSCs were capable of
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Fig. 6. MSCs suppressed DC maturation in a PLGA film implantation model. The immunophenotype and the pro-inflammatory cytokine expression of DCs were determined at day 3 and day 7 post-PLGA film implantations. MSC co-implantation significantly reduced the proportion of mature DCs (CD11cþCD80þ, CD11cþCD86þ, and CD11cþIaþ) among host splenocytes (Fig. 6A). Additionally, MSCs decreased the transcription of T cell derived IL-12 and TNF-a in the splenocytes of MSC-PLGA construct-implanted mice (Fig. 6B and C).
attenuating the PLGA-induced host inflammatory responses in a mouse model. Although PLGA is known to trigger inflammatory responses toward implants, the underlying mechanisms remain unclear. For a long time, much of the in vitro and in vivo studies have mostly focused on the involvement of neutrophils and lymphocytes until Yoshida et al. [19] reported that PLGA could directly promote iDC differentiation into mature DCs in vitro. As is well known, DCs are the most potent antigen presenting cells. The DC activation inevitably initiates the host immune response to a specific antigen; thus, infiltrating inflammatory cells may impair tissue regeneration surrounding the implanted PLGA scaffolds. Therefore, we assume that DCs are a potential cellular target for preventing PLGA-induced host inflammatory responses. DC differentiation is greatly dependent upon stromal environments [43e45]. Zhang et al. reported splenic endothelial cells drive mature DCs to trans-differentiate into regulatory DCs [45]. Our previous study demonstrated that MSCs were capable of suppressing human monocyte differentiation into DCs in vitro [37]. Thus, we explored the possibility of PLGA-induced host inflammatory response control by MSCs. We found that mouse MSCs could suppress CD11c, CD80, CD86 and Ia expression and that the MSC-treated PLGA-DCs were phenotypically similar to iDCs. Functionally, the MSCs educated the PLGA-DCs turn to promote Th2 and regulatory T cell differentiation. This observation indicates that MSCs might drive PLGA-DCs into a new type of DCs with suppressive immune functions. In fact, the modulatory effect on MSCs on PLGA-DCs is in line with previous findings that MSCs act as hematopoiesis and immune modulators [25]. Myeloid DCs are specialized cells derived from the monocyte hematopoietic lineage that develop in bone marrow niches [17]. As one of the cells that creates the bone marrow niche,
MSCs supply numerous cytokines and cell surface signals that modulate hematopoietic cell development and differentiation. MCSF is constitutively released by MSCs and promotes DC precursor differentiation into macrophages, which reduces the DC number [46,47]. Moreover, MSCs secrete high IL-6 level, which is involved in the suppressive mechanism induced by MSCs [48e50]. Previous reports have shown that this inhibition may require direct cellecell interactions of the DC precursors with neighboring stromal cells [37,51]. However, in our study, the PLGA-DCs generated in the transwell chamber system were only slightly decreased. Therefore, we suggest that MSCs exhibit suppressive effect on DCs mainly by secreting a cocktail of higher immune cytokine amounts instead of direct cellecell contact. To understand the molecular mechanism underlying the suppressive effect of the MSCs on the PLGA-DCs, the MAPK pathway was chosen to investigate as it was reported to be involved in DC maturation in inflammatory microenvironments [38e40]. We observed that the PLGA film elicited a remarkable activation of the p38/MAPK and ERK/MAPK pathways in the DCs. Moreover, inhibition of these pathways with chemical inhibitors changed the PLGADC cell morphology and phenotypes, proving that the p38/MAPK and ERK/MAPK pathways are involved in the PLGA stimulatory effects. To the best of our knowledge, many investigations were conducted to understand the mechanism behind PLGA stimulation, such as on integrin-based DC adhesion, NF-kB signaling and tolllike receptor signaling cascades; however, the role of MAPK pathways remained unknown [21,23,52]. In this study, we found that PLGA induced DC maturation via activation of the p38/MAPK and ERK/MAPK pathways. Most importantly, the observed MSCs dramatically suppressed PLGA film-triggered MAPK signal transduction in the DCs. Additionally, we noticed that a single pathway inhibitor failed to completely suppress iDC differentiation into
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Fig. 7. MSCs influenced T cell subpopulation and attenuated PLGA-induced host inflammatory reaction in vivo. The proportion of CD3þCD69þ T lymphocytes in PLGA implantation mice was decreased by MSCs in a time-dependent manner (Fig. 7A). Moreover, the FACS data showed that the MSCs significantly decreased the proportion of Th1 (CD4þIFN-gþ) and Th17 (CD4þIL-17Aþ) but increased the proportion of Th2 (CD4þIL-4þ) and Tregs (CD4þCD25þFoxp3þ) in recipients (Fig. 7B). In addition, MSCs suppressed the transcription of IFN-g and IL-17 but up-regulated IL-4 and Foxp3 in splenocytes (Fig. 7C). The results of histological analysis performed at day 7 showed that spleen nodules in MSC-PLGA constructimplanted recipients were smaller than that of PLGA film-implanted recipients (Fig. 7D). Furthermore, the immune cell filtration was observed around the implanted PLGA film and a fibrotic capsule formed at the implantation sites at day 21. No remarkable immune cell filtration near the MSC-PLGA construct was observed (Fig. 7E). Bars represent 500 mm and 100 mm in Fig. 7D and E, respectively.
PLGA-DCs, which suggests that some other mechanisms are also involved in PLGA stimulation. Until now, our knowledge regarding the stimulating effects of PLGA on DCs mainly was based on in vitro observations. Here, our findings demonstrated that PLGA did stimulate DC maturation in vivo. Most importantly, the MSCs were capable of inhibiting processing, which implicates the potential application of MSCs to reduce unexpected inflammatory responses. Compared with previous reports, MSC co-implantation possesses some advantages in controlling PLGA-induced host immune responses. First, MSCs do not release degradation products, which reduce the potential disadvantages of preparing a new complex. Additionally, MSCs exert immunosuppressive effects in a paracrine manner and avoid whole body infusion. Further, MSCs are characterized with low immunogenicity and multi-differentiation potency, which indicates a tissue repairing property of MSCs in the MSC-PLGA constructs. Armed with a PLGA film implantation murine model, we found that MSC co-delivery significantly suppressed DC maturation and attenuated the host immune defense in vivo. The CD3þCD69þ activated T lymphocyte proportion was dramatically reduced in the recipient spleens, which supported the finding that MSCs impaired the stimulatory activity of the PLGA-DCs. Additionally, histological results confirmed that MSCs inhibited local lymphocyte proliferation in spleens by reducing the number and size of the spleen nodules and attenuated the immune infiltration around the implants. It was reported that IL-12 cytokine production by DCs correlates with T lymphocyte proliferation and a Th1-type response
[53,54]. Consistent with this in vitro data, the MSCs significantly reduced the DC IL-12 expression in the mouse model. Furthermore, it was observed that the INF-g and IL-17A expression decreased while IL-4 expression increased in T lymphocytes of recipient spleens, which supported a shift from Th1 and Th17 cells to Th2 cells in vivo [55,56]. The subpopulation of CD4þCD25þFoxp3þ Tregs was first identified based on its crucial role in the control of autoimmune processes [57,58]. In this study, we found that the MSC-PLGA construct co-implantation significantly increased the proportion of Tregs among the splenocytes. As it is well known, the generation of Tregs is greatly dependent upon immune cytokines. Previous studies proved that IL-4 is an indispensable inducer of peripheral CD4þCD25 naïve T cell differentiation into CD4þCD25þFoxp3þ Tregs [59,60]. Moreover, upon stimulation with IL-4, Tregs are more potent in suppressing activation and IFN-g expression in T lymphocytes [49,50]. Consistent with previous studies, we found that IL-4 secretion and transcription were upregulated in the T cells that were activated by the MSC-treated PLGA-DCs.
5. Conclusions We demonstrated that MSCs suppress PLGA-DC maturation and immune functions in vivo and in vitro. These findings reveal a novel role for MSCs in controlling the PLGA-induced host immune response by targeting DCs and may provide an alternative and accessible strategy to improve the biocompatibility of PLGA bioscaffolds for tissue engineering.
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Acknowledgments This study was supported by the Beijing Natural Sciences Grants (No. 7132133), and National Natural Science Foundation (31070996, 31171084, 81101342, 81371945). The authors declare no competing financial interests. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.03.005.
References [1] Athanasiou KA, Niederauer GG, Agrawal CM. Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers. Biomaterials 1996;17:93e102. [2] Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. Eur Cell Mater 2008;15:100e14. [3] Harada N, Watanabe Y, Sato K, Abe S, Yamanaka K, Sakai Y, et al. Bone regeneration in a massive rat femur defect through endochondral ossification achieved with chondrogenically differentiated MSCs in a degradable scaffold. Biomaterials 2014;35:7800e10. [4] Martin JR, Gupta MK, Page JM, Yu F, Davidson JM, Guelcher SA, et al. A porous tissue engineering scaffold selectively degraded by cell-generated reactive oxygen species. Biomaterials 2014;35:3766e76. [5] Chang NJ, Lam CF, Lin CC, Chen WL, Li CF, Lin YT, et al. Transplantation of autologous endothelial progenitor cells in porous PLGA scaffolds create a microenvironment for the regeneration of hyaline cartilage in rabbits. Osteoarthr Cartil 2013;21:1613e22. [6] Park JS, Yang HN, Woo DG, Jeon SY, Park KH. Multilineage differentiation of human-derived dermal fibroblasts transfected with genes coated on PLGA nanoparticles plus growth factors. Biomaterials 2013;34:582e97. [7] Lin CY, Chang YH, Li KC, Lu CH, Sung LY, Yeh CL, et al. The use of ASCs engineered to express BMP2 or TGF-b3 within scaffold constructs to promote calvarial bone repair. Biomaterials 2013;34:9401e12. [8] Lee JM, Kim JD, Oh EJ, Oh SH, Lee JH, Im GI. PD98059-impregnated functional PLGA scaffold for direct tissue engineering promotes chondrogenesis and prevents hypertrophy from mesenchymal stem cells. Tissue Eng A 2014;20: 982e91. [9] Eppley BL, Morales L, Wood R, Pensler J, Goldstein J, Havlik RJ, et al. Resorbable PLLA-PGA plate and screw fixation in pediatric craniofacial surgery: clinical experience in 1883 patients. Plast Reconstr Surg 2004;114:850e6. discussion 857. [10] Asawa Y, Sakamoto T, Komura M, Watanabe M, Nishizawa S, Takazawa Y, et al. Early stage foreign body reaction against biodegradable polymer scaffolds affects tissue regeneration during the autologous transplantation of tissue-engineered cartilage in the canine model. Cell Transpl 2012;21(7): 1431e42. [11] McBane JE, Sharifpoor S, Cai K, Labow RS, Santerre JP. Biodegradation and in vivo biocompatibility of a degradable, polar/hydrophobic/ionic polyurethane for tissue engineering applications. Biomaterials 2011;32: 6034e44. [12] Yoon SJ, Kim SH, Ha HJ, Ko YK, So JW, Kim MS, et al. Reduction of inflammatory reaction of poly(D,L-lactic-co-glycolic acid) using demineralized bone particles. Tissue Eng A 2008;14:539e47. [13] Thevenot PT, Nair AM, Shen J, Lotfi P, Ko CY, Tang L. The effect of incorporation of SDF-1alpha into PLGA scaffolds on stem cell recruitment and the inflammatory response. Biomaterials 2010;31:3997e4008. [14] Mountziaris PM, Tzouanas SN, Sing DC, Kramer PR, Kasper FK, Mikos AG. Intra-articular controlled release of anti-inflammatory siRNA with biodegradable polymer microparticles ameliorates temporomandibular joint inflammation. Acta Biomater 2012;8:3552e60. [15] Avula MN, Rao AN, McGill LD, Grainger DW, Solzbacher F. Modulation of the foreign body response to implanted sensor models through device based delivery of the tyrosine kinase inhibitor, masitinib. Biomaterials 2013;34: 9737e46. [16] Lickorish D, Chan J, Song J, Davies JE. An in-vivo model to interrogate the transition from acute to chronic inflammation. Eur Cell Mater 2004;8:12e9. discussion 20. [17] Dalod M, Chelbi R, Malissen B, Lawrence T. Dendritic cell maturation: functional specialization through signaling specificity and transcriptional programming. EMBO J 2014;16(33):1104e16. ML, Vargas P, Chabaud M, Le Berre M, Liu YJ, Collin O, et al. Migration of [18] Heuze dendritic cells: physical principles, molecular mechanisms, and functional implications. Immunol Rev 2013;256:240e54. [19] Yoshida M, Babensee JE. Poly(lactic-co-glycolic acid) enhances maturation of human monocyte-derived dendritic cells. J Biomed Mater Res A 2004;71: 45e54.
697
[20] Yoshida M, Mata J, Babensee JE. Effect of poly(lactic-co-glycolic acid) contact on maturation of murine bone marrow-derived dendritic cells. J Biomed Mater Res A 2007;80:7e12. [21] Park J, Babensee JE. Differential functional effects of biomaterials on dendritic cell maturation. Acta Biomater 2012;8:3606e17. [22] Bennewitz NL, Babensee JE. The effect of the physical form of poly(lactic-coglycolic acid) carriers on the humoral immune response to co-delivered antigen. Biomaterials 2005;26:2991e9. [23] Rogers TH, Babensee JE. The role of integrins in the recognition and response of dendritic cells to biomaterials. Biomaterials 2011;32:1270e9. [24] Schliehe C, Redaelli C, Engelhardt S, Fehlings M, Mueller M, van Rooijen N, et al. CD8-dendritic cells and macrophages cross-present poly(D,L-lactate-coglycolate) acid microsphere-encapsulated antigen in vivo. J Immunol 2011;187:2112e21. [25] Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008;8:726e36. [26] Zhu H, Guo ZK, Jiang XX, Li H, Wang XY, Yao HY, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc 2010;5:550e60. [27] Frenette PS, Pinho S, Lucas D, Scheiermann C. Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu Rev Immunol 2013;31:285e316. [28] Zhu H, Jiang XX, Guo ZK, Li H, Su YF, Yao HY, et al. Tumor necrosis factor-alpha alters the modulatory effects of mesenchymal stem cells on osteoclast formation and function. Stem Cells Dev 2009;18:1473e84. [29] Xu FF, Zhu H, Li XM, Yang F, Chen JD, Tang B, et al. Intercellular adhesion molecule-1 inhibits osteogenic differentiation of mesenchymal stem cells and impairs bio-scaffold-mediated bone regeneration in vivo. Tissue Eng A 2014;20:2768e82. lvez AB, Cobo M, Cuevas-Ocan ~ a S, Gutie rrez-Guerrero A, S [30] Carrillo-Ga anchezGilabert A, Bongarzone P, et al. Mesenchymal stromal cells express GARP/ LRRC32 on their surface: effects on their biology and immunomodulatory capacity. Stem Cells 2015;33(1):183e95. http://dx.doi.org/10.1002/stem.1821. [31] Kuçi Z, Seiberth J, Latifi-Pupovci H, Wehner S, Stein S, Grez M, et al. Clonal analysis of multipotent stromal cells derived from CD271þ bone marrow mononuclear cells: functional heterogeneity and different mechanisms of allosuppression. Haematologica 2013;98:1609e16. [32] Pereira RF, O'Hara MD, Laptev AV, Halford KW, Pollard MD, Class R, et al. Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta. Proc Natl Acad Sci U S A 1998;95:1142e7. [33] Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008;371:1579e86. [34] Augello A, Tasso R, Negrini SM, Cancedda R, Pennesi G. Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis. Arthritis Rheum 2007;56:1175e86. [35] Yang F, Qu X, Cui W, Bei J, Yu F, Lu S, et al. Manufacturing and morphology structure of polylactide-type microtubules orientation-structured scaffolds. Biomaterials 2006;27:4923e33. [36] Zhang M, Tang H, Guo Z, An H, Zhu X, Song W, et al. Splenic stroma drives mature dendritic cells to differentiate into regulatory dendtritic cells. Nat Immunol 2004;5:1124e33. [37] Jiang XX, Zhang Y, Liu B, Zhang SX, Wu Y, Yu XD, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005;105:4120e6. [38] Escors D, Lopes L, Lin R, Hiscott J, Akira S, Davis RJ, et al. Targeting dendritic cell signaling to regulate the response to immunization. Blood 2008;111: 3050e61. [39] Jarnicki AG, Conroy H, Brereton C, Donnelly G, Toomey D, Walsh K, et al. Attenuating regulatory t cell induction by TLR agonists through inhibition of p38 MAPK signaling in dendritic cells enhances their efficacy as vaccine adjuvants and cancer immunotherapeutics. J Immunol 2008;180:3797e806. [40] Al-Huseini LM, Aw Yeang HX, Sethu S, Alhumeed N, Hamdam JM, Tingle Y, et al. Nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) modulates dendritic cell immune function through regulation of p38 MAPK-cAMPresponsive element binding protein/activating transcription factor 1 signaling. J Biol Chem 2013;288:22281e8. [41] de la Fuente H, Cruz-Adalia A, Martinez Del Hoyo G, Cibri an-Vera D, Bonay P, rez- Herna ndez D, et al. The leukocyte activation receptor CD69 controls t Pe cell differentiation through its interaction with galectin-1. Mol Cell Biol 2014;34:2479e87. [42] Sancho D, Santis AG, Alonso-Lebrero JL, Viedma F, Tejedor R, S anchezMadrid F. Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors. J Immunol 2000;165:3868e75. [43] Klebanoff CA, Spencer SP, Torabi-Parizi P, Grainger JR, Roychoudhuri R, Ji Y, et al. Retinoic acid controls the homeostasis of pre-cDC-derived splenic and intestinal dendritic cells. J Exp Med 2013;210:1961e76. [44] Tuna H, Avdiushko RG, Sindhava VJ, Wedlund L, Kaetzel CS, Kaplan AM, et al. Regulation of the mucosal phenotype in dendritic cells by PPARg: role of tissue microenvironment. J Leukoc Biol 2014;95:471e85. [45] Globisch T, Steiner N, Fülle L, Lukacs-Kornek V, Degrandi D, Dresing P, et al. Cytokine-dependent regulation of dendritic cell differentiation in the splenic microenvironment. Eur J Immunol 2014;44:500e10.
698
H. Zhu et al. / Biomaterials 53 (2015) 688e698
[46] Spaggiari GM, Abdelrazik H, Becchetti F, Moretta L. MSCs inhibit monocytederived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2. Blood 2009;113:6576e83. [47] Ulivi V, Tasso R, Cancedda R, Descalzi F. Mesenchymal stem cell paracrine activity is modulated by platelet lysate: induction of an inflammatory response and secretion of factors maintaining macrophages in a proinflammatory phenotype. Stem Cells Dev 2014;23:1858e69. [48] Djouad F, Charbonnier LM, Bouffi C, Louis-Plence P, Bony C, Apparailly F, et al. Mesenchymal stem cells inhibit the differentiation of dendritic cells through an interleukin-6-dependent mechanism. Stem Cells 2007;25:2025e32. [49] Li YP, Paczesny S, Lauret E, Poirault S, Bordigoni P, Mekhloufi F, et al. Human mesenchymal stem cells license adult CD34þ hemopoietic progenitor cells to differentiate into regulatory dendritic cells through activation of the Notch pathway. J Immunol 2008;180:1598e608. [50] Deng Y, Yi S, Wang G, Cheng J, Zhang Y, Chen W, et al. Umbilical cord-derived mesenchymal stem cells instruct dendritic cells to acquire tolerogenic phenotypes through the IL-6-mediated upregulation of SOCS1. Stem Cells Dev 2014;23:2080e92. [51] Aldinucci A, Rizzetto L, Pieri L, Nosi D, Romagnoli P, Biagioli T, et al. Inhibition of immune synapse by altered dendritic cell actin distribution: a new pathway of mesenchymal stem cell immune regulation. J Immunol 2010;185: 5102e10. [52] Shokouhi B, Coban C, Hasirci V, Aydin E, Dhanasingh A, Shi N, et al. The role of multiple toll-like receptor signalling cascades on interactions between biomedical polymers and dendritic cells. Biomaterials 2010;31:5759e71.
[53] Domínguez PM, Ardavín C. Differentiation and function of mouse monocytederived dendritic cells in steady state and inflammation. Immunol Rev 2010;234:90e104. [54] Nizzoli G, Krietsch J, Weick A, Steinfelder S, Facciotti F, Gruarin P, et al. Human CD1cþ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T-cell responses. Blood 2013;122:932e42. [55] Liu Z, Fan H, Jiang S. CD4(þ) T-cell subsets in transplantation. Immunol Rev 2013;252:183e91. [56] Pradhan P, Qin H, Leleux JA, Gwak D, Sakamaki I, Kwak LW, et al. The effect of combined IL10 siRNA and CpG ODN as pathogen-mimicking microparticles on Th1/Th2 cytokine balance in dendritic cells and protective immunity against B cell lymphoma. Biomaterials 2014 Jul;35:5491e504. [57] Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic selftolerance maintained by activated T cells expressing IL-2 receptor alphachains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995;155:1151e64. [58] Graca L, Thompson S, Lin CY, Adams E, Cobbold SP, Waldmann H. Both CD4(þ) CD25(þ) and CD4(þ)CD25() regulatory cells mediate dominant transplantation tolerance. J Immunol 2002;168:5558e65. [59] Maerten P, Shen C, Bullens DM, Van Assche G, Van Gool S, Geboes K, et al. Effects of interleukin 4 on CD25þCD4þ regulatory T cell function. J Autoimmun 2005;25:112e20. [60] Skapenko A, Kalden JR, Lipsky PE, Schulze-Koops H. The IL-4 receptor alphachain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD25þCD4þ regulatory T cells from CD25-CD4þ precursors. J Immunol 2005;175:6107e16.
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Mesenchymal stem cells attenuated PLGA-induced inflammatory responses by inhibiting host DC maturation and function Heng Zhu a, ***, 1, Fei Yang b, 1, Bo Tang a, Xi-Mei Li a, Ya-Nan Chu a, Yuan-Lin Liu a, Shen-Guo Wang b, De-Cheng Wu b, **, Yi Zhang a, * a
Department of Cell Biology, Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, PR China BNLMS, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun No. 1 Street, Haidian District, Beijing 100190, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 December 2014 Received in revised form 27 February 2015 Accepted 4 March 2015 Available online 24 March 2015
The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGADC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Poly (lactide-co-glycolide) Dendritic cells Mesenchymal stem cells Host inflammatory responses
The poly lactic-co-glycolic acid (PLGA) bio-scaffold is a biodegradable scaffold commonly used for tissue repair. However, implanted PLGA scaffolds usually cause serious inflammatory responses around grafts. To improve PLGA scaffold-based tissue repair, it is important to control the PLGA-mediated inflammatory responses. Recent evidence indicated that PLGA induce dendritic cell (DC) maturation in vitro, which may initiate host immune responses. In the present study, we explored the modulatory effects
* Corresponding author. Tel.: þ86 10 66931320; fax: þ86 10 68213039. ** Corresponding author. Tel.: þ86 10 82611492; fax: þ86 10 82611492. *** Corresponding author. Tel.: þ86 10 66930913; fax: þ86 10 68213039. E-mail addresses: [email protected] (H. Zhu), [email protected] (D.-C. Wu), [email protected] (Y. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.03.005 0142-9612/© 2015 Elsevier Ltd. All rights reserved.
of mesenchymal stem cells (MSC) on PLGA-induced DCs (PLGA-DC). We found that mouse MSCs inhibited PLGA-DC dendrite formation, as well as co-stimulatory molecule and pro-inflammatory factor expression. Functionally, MSC-educated PLGA-DCs promoted Th2 and regulatory T cell differentiation but suppressed Th1 and Th17 cell differentiation. Mechanistically, we determined that PLGA elicited DC maturation via inducing phosphorylation of p38/MAPK and ERK/MAPK pathway proteins in DCs. Moreover, MSCs suppressed PLGA-DCs by partially inactivating those pathways. Most importantly, we found that the MSCs were capable of suppressing DC maturation and immune function in vivo. Also, the proportion of mature DCs in the mice that received MSC-PLGA constructs greatly decreased compared with that of their PLGA-film implantation counterparts. Additionally, MSCs co-delivery increased regulatory T and Th2 cells but decreased the Th1 and Th17 cell numbers in the host spleens. Histological analysis showed that MSCs alleviated the
H. Zhu et al. / Biomaterials 53 (2015) 688e698
inflammatory responses around the grafted PLGA scaffolds. In summary, our findings reveal a novel function for MSCs in suppressing PLGA-induced host inflammatory response and suggest that DCs are a new cellular target in improving PLGA scaffold-based tissue repair. 1. Introduction Poly lactic-co-glycolic acid (PLGA) has been used in a variety of preclinical studies and clinical applications for tissue repair [1,2]. Most notable among these applications are the PLGA scaffolds that are made for filling large tissue defects [3,4], building threedimensional cell-scaffold constructs for tissue regeneration [5,6], or for preparing drug and cytokine-coated grafts for localized delivery [7,8]. However, growing evidence has demonstrated that implanted PLGA scaffolds cause serious host inflammatory responses. A retrospective analysis regarding clinical cases demonstrated that patients who underwent bone fixation with PLGA scaffolds developed cystic swelling underneath their scalps [9]. Asawa Y et al. reported that the early stage foreign body reactions against PLGA scaffolds diminished tissue regeneration during the autologous transplantation of tissue-engineered cartilage in a canine model [10]. Another study demonstrated that subcutaneously implanted PLGA films triggered acute inflammatory response and caused remarkable macrophage infiltration in a rodent model [11]. To reduce these inflammatory responses, a number of techniques have been developed for preparing scaffolds, including impregnating demineralized bone particles into the PLGA [12], incorporating SDF-1a [13], anti-inflammatory siRNA [14] or masitinib [15] into PLGA scaffolds and coating a thin surface of calcium phosphate onto PLGA scaffolds [16]. However, the techniques described in these reports are complex and not easily standardized. Most importantly, the role of antigen presenting cells, which initiate host immune responses, was overlooked. Dendritic cells (DC) are the most potent professional antigen presenting cells [17,18]. By taking up and processing innate antigens or invading pathogens, DCs rapidly present antigens to antigenspecific naïve T cells. The process is also accompanied by the expression of co-stimulatory molecules, including MHC-II, CD80 and CD86, and the secretion of immune cytokines, including interleukin-12 (IL-12) and tumor necrosis factor-a (TNF-a). These cell surface molecules and immune factors are indispensable for DCs to induce T cell activation and thereafter to initiate specific responses [17,18]. Interestingly, recent studies revealed that PLGA is capable of promoting DC maturation in vitro [19e25]. The treatment of DCs with PLGA microparticles or films led to the maturation of these cells through increased co-stimulatory molecule expression and pro-inflammatory cytokine secretion [19e21]. Moreover, co-culturing DCs with PLGA enhanced the allostimulatory capacity of the DCs [24]. Thus, we suggest that DC may be a cell target to reduce PLGA-induced host inflammatory responses. Mesenchymal stem cells (MSC) were originally isolated from bone marrow and now have been successfully isolated from adipose tissue, compact bone, placenta and other connective tissues [25e27]. Under certain conditions, MSCs can differentiate into numerous tissue cell types and promote tissue repair [28,29]. Moreover, MSCs express a low level of co-stimulatory molecules, which indicates that they have low antigenicity [25e27]. Most importantly, MSCs are able to suppress immune cell development in a dose dependent manner by means of secretary factors and/or direct contact [30,31]. Therefore, MSCs have been successfully used for the treatment of numerous diseases, including osteogenesis imperfecta, acute graft-versus-host disease, and rheumatoid arthritis [32e34].
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Given the fundamental role of DCs in host immune responses and the potent immunosuppressive properties of MSCs, they may be an effective strategy for alleviating PLGA-elicited inflammatory reactions via targeting DCs. In the current study, we explored the effects of MSCs on PLGA-induced DCs (PLGA-DC) and the subsequent host immune responses. Moreover, the cellular and molecular mechanisms regarding the capacity for MSCs to regulate PLGADCs were also investigated. 2. Materials and methods 2.1. PLGA film preparation PLGA was prepared according to a previously described protocol with minor modifications [35]. Briefly, L-lactide and glycolide were purchased from PURAC (the Netherlands) and purified by recrystallization in ethyl acetate (Beijing Tonghua fine chemicals company, China), twice. PLGA was synthesized by ring-opening polymerization of the L-lactide and glycolide (mol. Ratio 50:50) under 65 Pa in sealed glass ampoules at 180 C for 20 h in the presence of stannous octoate, which was used as a catalyst (0.05 wt%). The raw PLGA (50/50) was dissolved in chloroform and then purified by ethanol precipitation. It was then dried in a vacuum at room temperature for 48 h. The molecular weight of PLGA (50/50) (Mw ¼ 100 kDa) was determined with GPCmax VE-2001(viscotek) gel-permeation chromatography (GPC). PLGA was further fabricated into porous films. Briefly, PLGA was dissolved in dioxane to form a 10% concentration solution. Then, presieved NaCl particles (diameter of 0.18e0.25 mm) were weighed and then added into the solution to form a slurry. The slurry was fed into a flat box shaped mold without cover with length and width of 120 mm both, and then the mold was cooled down to 20 C for more than 2 h. After this, the frozen solution was freeze-dried at 50 C for more than 24 h. Then NaCl particles in the scaffolds were eliminated by washing several times in pure water with stir until the NaCl can not be detected by AgNO3. The wet scaffolds were vacuum dried in room temperature for 24 h. The PLGA scaffolds were stored under dry vacuum conditions. The porous PLGA films were prepared using a hole puncher (8 mm in diameter and 3 mm in thickness) and were used for in vivo and in vitro experiments unless otherwise described. 2.2. Animals Normal inbred two-week-old and eight-week-old C57BL/6 (H2b) mice, as well as eight-week-old BALB/c mice, were purchased from the Academy of Military Medical Sciences of China Laboratory Animal Center (Beijing). All experiments in this study were performed in accordance with the Academy of Military Medical Sciences Guide for Laboratory Animals. 2.3. Murine MSC isolation and MSC-PLGA construct preparation Murine MSCs were prepared from compact bones by virtue of high cell yield and low contamination of hematopoietic cells according to a previous protocol [26]. Briefly, long bones from two-week-old C57BL/6 mice were dissected, and the bone marrow cells were flushed out. Next, the chopped compact bones were digested by collagenase II (Sigma) and the bone chips were cultured in MSC culture medium (aMEM supplemented with 10% fetal bovine serum). The adherent cells, at passages 3e6, were used for in vivo and in vitro experiments unless otherwise described. For the preparing MSC-PLGA constructs, MSCs that were resuspended in cultured medium were seeded onto sterilized PLGA films to allow the MSCs adhere to the films. Then, a whole volume of culture medium was added 2 h later. The MSC-PLGA constructs were cultured in MSC culture medium for 48 h before they were cocultured with DCs or used in implantation experiments unless other described. 2.4. The cell morphology and growth kinetics of the MSCs on the PLGA films The cell morphology of the MSCs on the PLGA films was observed with scanning electron microscopy (SEM; JSM-6700F, JEOL) at day 3 after the cells were loaded. The growth kinetics of the MSCs on the PLGA was determined using the Cell Counting Kit 8 (CCK-8, Dojindo, Japan) according to the manufacturer's protocol. In brief, a total of 2 103 MSCs were seeded onto the sterile PLGA films, the MSC-PLGA constructs were cultured in 24-well plates and were added to the CCK-8 solution at a ratio of 100 mL/1 mL, and the plates were incubated at 37 C for 1 h. Absorbance was then measured at a wavelength of 450 nm using a microplate reader. Five wells at each time point were assayed. The CCK-8 experiments were performed at days 1, 3, 6, 9, 12, 15, 18, and 21. 2.5. Murine DC generation Murine DCs were prepared from bone marrow progenitors [36]. In brief, bone marrow mononuclear cells were prepared from 8-week-old C57BL/6 or BALB/c mice by red cell depletion. The DCs were generated with RPMI 1640 medium that was supplemented with 10% fetal bovine serum (FBS), 10 ng/ml of mouse granulocytemacrophage colony stimulating factor (GM-CSF), and 1 ng/ml of mouse
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interleukin-4 (IL-4) (Peprotech) for five days. Loose and non-adherent cells were harvested, and CD11cþ cells were purified with a MACS CD11cþ isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD11c cells positive were identified as immature DCs (iDCs) and used for subsequent experiments. In some co-culture experiments, trans-well chamber systems were used to separate DCs from MSCPLGA constructs or PLGA films. 2.6. Cell morphology and ultra-structure of DCs A total of 2 104 MSCs were seeded onto each sterile PLGA films, and they were co-cultured with 2 105 iDCs in 6-well plates for 48 h. The cell morphology of the PLGA- or MSC-PLGA construct-induced DCs was observed under a phase contrast microscope (Nikon TE2000-U). For the transmission electron microscopy ultrastructural observations (TEM, HITACHI, Tokyo, Japan), PLGA- or MSC-PLGA construct-induced DCs were collected and fixed for 4 h at 4 C in 5% glutaraldehyde, washed 3 times in 0.1 mol/L phosphate buffered saline (PBS), post-fixed for 2 h at 4 C in 2% osmium tetroxide, dehydrated in a graded series of ethanol, embedded in Epon 812, cut into ultrathin sections (75 nm), and then stained with uranyl acetate and lead citrate. The sections were finally viewed and recorded with a HITACHI H-600 electron microscope at 80 KV.
2.8. Immune factor determination by real-time PCR and ELISA To assess the effect of MSCs on immune cytokine expression in DCs and T cells in vitro, PLGA- or MSC-PLGA constructs were co-cultured with iDCs at different ratios for 48 h. The MSC:iDC ratios were 1:100, 1:50, 1:20 and 1:10. CD3þ T lymphocytes were purified from healthy mouse spleens using a MACS CD3 isolation kit (Miltenyi Biotec) and were further primed with PLGA- or MSC-educated PLGA-DCs before the immune factors were detected. For the in vivo analyses, recipient nucleated splenocytes were collected at days 3 and 7 post-PLGA film implantation. Total RNA was extracted with TRIZOL reagent (Invitrogen) and reverse transcribed using the mRNA Selective PCR kit (TaKaRa). Mouse TNF-a, IL-12, IFN-g, IL-4, IL-17A and Foxp3 cDNA were amplified by real-time PCR using the SYBR Green PCR kit (Sigma). The primer sequences used for the realtime PCR are shown in Table S1. Meanwhile, the culture supernatants from the in vitro DCs and DC-T cell culture systems were collected by centrifugation and filtered to deplete the cellular components. Mouse TNF-a, IL-12, IFN-g, IL-4 and IL-17A concentrations in the supernatants were determined according to the reagent protocols of the quantitative determination kit (R&D Systems, Minneapolis). 2.9. The DC endocytosis properties and T cell priming capacity
2.7. Immunophenotyping of the DCs and T cells To detect the effect of the MSCs on the DC immuno-phenotype in vitro, PLGA- or MSC-PLGA constructs were co-cultured with iDCs at different ratios for 48 h. The MSCs:iDC ratios were 1:100, 1:50, 1:20 and 1:10. To determine the DC phenotypes and the T cell subpopulations in vivo, recipient nucleated splenocytes were collected at days 3 and 7 post-PLGA film implanting. To learn the signaling pathway effects on the DC surface markers, the chemical inhibitors, SB203580HCL, PD98059 and JNK inhibitor II (20 mm/ml of each), which are specific inhibitors of the p38/MAPK, ERK/ MAPK and JNK/MAPK pathways, respectively, were added to the PLGA film and DC co-culture system (2 106 iDCs each sterile PLGA films). The DCs and T cells were stained with FITC, PE or Allophycocyanin (APC) conjugated monoclonal antibodies against mouse CD3, CD69, CD80, CD86, CD11c and Ia (MHC-II molecular) (eBio-Science, San Diego). For intercellular cytokine staining, recipient splenocytes were harvested and stimulated with 50 ng/ml phorbol 12myristate 13-acetate (PMA), 500 ng/ml ionomycin, and 3 mg/ml brefeldin A (SigmaeAlrich) for six hours. The splenocytes were then washed and labeled for CD3, and CD4 as well as intracellular interferon-g (IFN-g), interleukin-17A (IL-17A), or IL-4. For analysis of regulatory T cells (Tregs), the anti-mouse intranuclear forkhead box P3 (FoxP3) kit (eBioscience, San Diego, CA) was used according to the manufacturer's protocol. Events were collected by flow cytometry with a FACScalibur system (BectoneDickinson), and data analysis was conducted with the WinMDI 2.9 software.
To determine the effect of the MSCs on DC endocytosis, iDCs were exposed to PLGAor MSC-PLGA constructs at different MSC:iDC ratios (1:100,1:50,1:20,1:10) for 48 h. DC endocytosis was measured as the cellular uptake of FITC-dextran and was quantified by flow cytometry as previously described [37]. Briefly, 1 106 DCs per sample were incubated in medium containing FITC-dextran (1 mg/ml; molecular weight 40,000; Sigma, St Louis, MO) for 1 h. After incubation, the cells were washed twice with cold phosphate buffered saline (PBS) to stop endocytosis and remove the excess dextran. The quantitative endocytosis capacity of the DCs was determined by the fold change of the mean fluorescence intensity (MFI) values relative to that of the PLGA-DCs. To investigate the effect of MSCs on the DC priming capacity, the PLGA-DCs and MSC-treated PLGA-DCs from the above co-culture system were harvested and irradiated (15 Gy). Next, the DCs were cultured in triplicate at 2 104 cells in 200 mL per well in 96-well flat-bottomed plates with 2 105 allogeneic CD3þ T cells. After four days, the cells were pulsed for 16 h with 3H-thymidine (1 mCi/well [0.037 MBq/ well]). Thymidine incorporation was measured by standard liquid scintillation counting. The results are expressed in counts per minute and shown as the mean ± SD of triplicate values. 2.10. Western-blotting PLGA films or MSC-PLGA constructs (1 105 cells) were co-cultured with iDCs (1 106 cells/well). The DCs were collected after 0, 5, 10, 30, 60, 120, and 240 min of
Fig. 1. The morphological characteristics of PLGA film and MSC-PLGA construct. The gross morphology of PLGA film and MSC-PLGA construct are shown in Fig. 1A. The ultra morphology was shown by scanning electron microscopy (Fig. 1B and C). Additionally, the growth kinetics of MSCs on PLGA film was determined by CCK-8 assay (Fig. 1D). Bars in Fig. 1AeC represent 1 cm, 200 mm, and 50 mm, respectively.
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Fig. 2. MSCs inhibited PLGA-induced DC maturation in vitro. In the MSC/DC co-culture system, MSCs suppress the dendrite formation of PLGA-DCs (Fig. 2A). In addition, the expression of CD11c, CD80, CD86 and Ia in PLGA-DCs were decreased by MSCs in a dose dependent manner (Fig. 2B and C). Further, MSCs down-regulated the transcription and secretion level of IL-12 and TNF-a (Fig. 2D and E). Bars in Fig. 2A upper row and lower row represent 200 mm and 50 mm, respectively.
co-culture. Protein lysis buffer (BioRad, Hercules, CA) was added, and the thawed lysates were vortexed and centrifuged. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked by incubation with 5% wt/vol nonfat dry milk. The membranes were then incubated with anti-p38, anti-phosphop38 (P-p38), anti-ERK, anti-phospho-ERK (P-ERK), anti-JNK, anti-phospho-JNK (PJNK), and b-actin antibodies (Cell Signal Technology) at the appropriate dilutions overnight at 4 C. After incubation, the membranes were washed in Tris buffered saline with Tween-20 (TBST). Horseradish peroxidase-conjugated secondary antibodies were added to the membranes in 5% nonfat dry milk in TBST. 2.11. Implantation of the MSC-PLGA constructs in the murine model The MSC-PLGA constructs were collected and gently washed in PBS for the transplantation experiment. Thirty normal inbred 8-week-old BALB/c mice received a dorsal subcutaneous implantation of an MSC-PLGA construct. Mice implanted with blank PLGA films served as controls. Five mice were sacrificed at days 3 and 7 postimplantation. The DC and T cell immunophenotypes and inflammatory cytokine expression were determined as described above. The proliferation of splenic corpuscles in the spleens and the immune cell filtration around the implants were observed using pathological examinations. The specimens were fixed in 10% neutralbuffered formalin, imbedded in paraffin, cut into 5-mm-thick sections and stained with hematoxylin and eosin (H&E). The sections were observed using inverted microscopy (Nikon TE2000-U). 2.12. Statistical analysis Data are represented as the mean values with standard deviations. Statistical significance was analyzed using Student's t test. P values less than 0.05 were considered to be significant.
3. Results 3.1. MSCs inhibited PLGA-induced DC maturation in vitro The gross morphology of the PLGA films and MSC-PLGA constructs are shown in Fig. 1A. The scanning electron microscopy data showed that the MSCs grew robustly in the porous PLGA films (Fig. 1B and C). The results of the CCK-8 assay (Fig. 1D) demonstrated that the MSCs maintained a high proliferation capacity on the PLGA film for at least 3 weeks. To investigate whether the MSCs affected the DC morphology, the PLGA-DCs and MSC-treated PLGA-DCs were observed with phase contrast and transmission electron microscopes. As shown in Fig. 2A, the PLGA-DCs displayed protruding veils with abundant cytoplasm, while the MSC-educated PLGA-DCs were round and lack the veiled appearance. Cell surface molecules and immune factors are indispensable for DCs to initiate specific responses. Compared with PLGA-DCs, the MSC-treated PLGA-DCs had decreased CD11c, CD80, CD86 and Ia expression (Fig. 2B). Importantly, the inhibitory effect was dosedependent. With a low MSC/DC ratio of 1:50, the CD11c, CD86 and Ia expression was significantly reduced (Fig. 2C. *, P < 0.05; **, P < 0.01).
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Fig. 3. MSCs enhanced the phagocytic capacity but suppressed T cell stimulatory properties of PLGA-DCs. MSC-treated PLGA-DCs uptook more FITC labeled Dextran particles. The data were collected by FACS and the results were indicated as the fold change of mean fluorescence intensity values relative to that of PLGA-DCs (Fig. 3A). In addition, the T cell proliferation that triggered by MSC-treated PLGA-DCs declined in a MSC dose dependent manner (Fig. 3B). Moreover, the expression of T cell derived IL-4, INF-g, IL-17 and Foxp3 were regulated by different DCs (Fig. 3C and D).
Further investigations showed that MSCs also suppressed the expression of key immune factors in the PLGA-DCs. The real-time PCR and ELISA results demonstrated that MSCs suppressed the expression of IL-12 and TNF-a in the PLGA-DCs (Fig. 2D and E). Additionally, a low MSC/DC ratio (1:50) resulted in significant suppression of the transcription and secretion of IL-12 and TNF-a (Fig. 2D and E) (*, P < 0.05; **, P < 0.01), implying that a small number of MSCs is capable of blocking PLGA-DC maturation. 3.2. MSCs modulated the phagocytic capacity and T cell stimulatory property of PLGA-DCs Up to this point, we determined that MSCs potently suppressed the maturation of PLGA-DCs. However, it remained unknown whether MSCs affected the immune function of the PLGA-DCs. By using FITC-labeled dextran endocytosis and H3 incorporation assays, we further determined the effects of MSCs on the phagocytic capacity and T cell stimulatory property of the PLGA-DCs. The phagocytic capacity of the MSC-treated PLGA-DCs was indicated as the fold change in the mean fluorescence intensity values relative to the PLGA-DCs. As shown in Fig. 3A, the MSCeducated PLGA-DCs exerted greater phagocytic capacity than the PLGA-DCs. Moreover, the modulatory effect of the MSCs on DC endocytosis was dose-dependent. The phagocytic capacity of the MSC-treated PLGA-DCs increased significantly at an MSC: DC ratio of 1:20 (Fig. 3A). (*, P < 0.05). Meanwhile, we investigated the T cell priming capacity of the DCs. Compared with the PLGA-DCs stimulated T cells, the T cell proliferation triggered by MSC-treated PLGA-DCs was remarkably
dampened (Fig. 3B) (*, P < 0.05, **, P < 0.01). Additionally, the priming effects of the PLGA-DCs were reduced with increased MSC number (Fig. 3B). Most importantly, we found that MSC-educated PLGA-DCs promoted Th2 and regulatory T cell (Treg) differentiation but suppressed Th1 and Th17 cell differentiation. Treating PLGA-DCs with MSCs led to an increase of IL-4 and Foxp3 but caused a decrease of INF-g and IL-17A in T lymphocytes (Fig. 3C and D) (*, P < 0.05, **, P < 0.01), indicating that MSC-treated PLGA-DCs play a role in shifting the balance from Th1 and Th17 to Th2 and Treg responses. 3.3. MSCs suppress PLGA-DC maturation without intercellular contact at a low MSC/DC ratio To explore whether the inhibition of MSCs on PLGA-DCs requires intercellular contact, we used a Transwell chamber system to separate DCs (upper compartment) from the MSC-PLGA constructs or PLGA films (lower compartment) (Fig. 4A). We found that PLGA films could trigger DC dendrite formation and induce CD11c, CD80, CD86 and Ia expression without direct contact with DCs, which indicated that PLGA might induce DC maturation via a contact independent mechanism. Interestingly, we found that MSCs significantly prevented DC maturation in the Transwell chamber system, implicating that soluble factors from the MSCs were involved in the observed suppression (Fig. 4B). Further, we defined what factors in the supernatant were involved in the inhibitory effects by utilizing neutralization antibodies. IL-6 and M-CSF were investigated because numerous studies have demonstrated that they are pivotal cytokines in the
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Fig. 4. MSCs suppressed PLGA-DC maturation via secreted cytokines dependent manner. A transwell co-culture in vitro model was setup to analyze whether the inhibition of MSCs on PLGA-DCs require intercellular contact. MSCs were capable of inhibit dendri formation of PLGA-DCs without direct contact (Fig. 4A). Additionally, the MSC derived soluble factor decreased the expression of CD11c, CD80, CD86 and Ia in PLGA-DCs (Fig. 4B). Furthermore, the inhibitory effects of MSCs on PLGA-DC immunophenotype were partially reverted by the neutralization of IL-6 and M-CSF in MSC supernatants (Fig. 4C). Bars in Fig. 4A represent 200 mm.
control of DC maturation. In the current study, the expression of CD11c, CD80, CD86 and Ia were significant reduced by direct supplement of anti-IL-6 (200 ng/ml) and/or anti-M-CSF (200 ng/ml) neutralizing antibodies (Fig. 4C). The data suggested that MSCs might maintain the observed immature DC phenotype via IL-6 and M-CSF secretion. 3.4. MSCs suppressed PLGA-induced DC maturation partially by inactivation of the p38/MAPK and ERK/MAPK pathways To examine the intracellular signaling cascades, we investigated MAPK pathway protein phosphorylation in the PLGA-DCs because the activation of MAPKs signals is critical for DC development [38e40]. The results showed that the p38/MAPK and ERK/MAPK pathways in the DCs were markedly activated by the PLGA films in a time-dependent manner (Fig. 5A); however, no obvious phosphorylation of the JNK/SAPK pathway proteins was detected in the PLGA-DCs (Fig. 5A). Most importantly, exposure of the PLGA-DCs to MSCs without any exogenous cytokine or chemical inhibitor addition caused significant inactivation of the p38/MAPK and ERK/ MAPK pathways in the DCs (Fig. 5B).
To further clarify the association of the MAPK pathways and the PLGA-DCs, specific chemical inhibitors, including SB203580 (for p38/MAPK), PD98059 (for ERK/MAPK), and JNK inhibitor II (for JNK/ SAPK) were added to the PLGA-DC culture system. As shown in Fig. 5C, SB203580 and PD98059 markedly suppressed dendrite development, whereas the JNK inhibitor II exerted mild effects on the DC morphology. Furthermore, SB203580 and PD98059 significantly reduced the CD11c, CD80, CD86 and Ia expression in the PLGA-DCs (Fig. 5D) (*, P < 0.05). These data suggest that the p38/ MAPK and ERK/MAPK pathways play essential roles in PLGA-DC maturation. Collectively, we suggest that the PLGA films enhanced DC maturation by activating the p38/MAPK and ERK/MAPK pathways, while the MSCs exhibited an inhibitory effect on the PLGA-DCs by partially dampening the activation of those pathways. 3.5. MSCs suppressed DC maturation in a PLGA film implantation model Although in vitro studies have clearly shown that MSCs are capable of suppressing PLGA-DC maturation and functions, it
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Fig. 5. MSCs suppressed the PLGA-induced DC maturation partially by inactivation of the p38/MAPK and ERK/MAPK pathway. PLGA film remarkable activated the p38/MAPK and ERK/MAPK pathways in DCs (Fig. 5A), while MSCs dampened the phosphorylation of signal pathways (Fig. 5B). Further, specific blockage of the pathway by chemical inhibitors suppressed the dendrite formation and immunophenotype of PLGA-DCs (Fig. 5C and D). These data suggest that p38/MAPK and ERK/MAPK pathway play essential roles in the maturation of PLGA-DCs. Bars in Fig. 5C upper row and lower row represent 200 mm and 50 mm, respectively.
remains unknown whether this is the case in vivo. Therefore, the phenotype and the pro-inflammatory cytokine expression of DCs were determined at days 3 and 7 post-PLGA film implantation. Promisingly, we found that the MSC co-implantation significantly reduced the proportion of mature DCs (CD11cþCD80þ, CD11cþCD86þ, and CD11cþIaþ) among the host splenocytes (Fig. 6AeC) (*, P < 0.05; **, P < 0.01). Furthermore, the expression of pivotal cytokines for DC maturation was also inhibited by the MSCs (Fig. 6B and C). Compared with the PLGA film-implanted mice, the IL-12 mRNA levels dropped more than 50% and the TNF-a mRNA levels decreased more than 70% at day 3 in the MSC-PLGA construct-implanted mouse splenocytes (Fig. 6B) (*, P < 0.05; **, P < 0.01). Similar effects were observed at day 7 (Fig. 6C). 3.6. MSCs regulated the T cell subpopulations and attenuated PLGAinduced host inflammatory reactions in vivo To explore the influence of the MSCs on the DC priming properties in vivo, we first assessed T cell activation in the host spleens. CD69 expression is a prerequisite for T cell proliferation [41,42]; therefore, the CD3þCD69þ proportions in the recipient spleens were determined. The results showed that the MSCs significantly down-regulated the CD3þCD69þ T lymphocyte proportion in the host spleens in a time-dependent manner (Fig. 7A) (*, P < 0.05). Because the in vitro experiments indicated that the MSC-treated PLGA-DCs caused a shift in the T cell balance from Th1 and Th17 to Th2 and Treg, further in vivo experiments were performed to validate our findings. The proportions of Th1 cells (CD4þIFN-gþ), Th2 cells (CD4þIL-4þ), Th17 cells (CD4þIL-17Aþ) and Tregs
(CD4þCD25þFoxp3þ) in the recipient spleens were determined by intracellular cytokine staining and analyzed by FACS at day 7. The results showed that the MSCs significantly decreased the proportion of Th1 and Th17 cells but increased the proportion of Th2 cells and Tregs in the recipients (Fig. 7B) (*, P < 0.05). Consistently, the real-time PCR results showed that the MSCs down-regulated IFN-g and IL-17A expression but up-regulated IL-4 and Foxp3 expression (Fig. 7C). (*, P < 0.05; **, P < 0.01). These results implicate that MSCPLGA co-implantation induces functional alterations in splenic lymphocytes in vivo. Histological analysis of the recipient spleens was performed at day 7 and we found that the spleen nodules in the MSC-PLGA construct-implanted recipients were smaller than those in the PLGA film-implanted recipients (Fig. 7D). The mouse spleen nodules showed active hyperplasia, which suggested that the engrafted PLGA films had antigenicity and activated lymphocytes in the spleens, whereas the MSCs ameliorated the immune responses (Fig. 7D). Immune cell infiltration around the implants were observed at day 21, as shown in Fig. 7E. The implanted-PLGA film activated inflammatory cells in vivo and a fibrotic capsule formed at the implantation sites; however, no remarkable immune cell infiltration near the MSC-PLGA constructs was observed (Fig. 7E). 4. Discussion In the current study, we demonstrated the suppressive effects of MSCs on the maturation and immune functions of PLGA-DCs in vivo and in vitro. Additionally, we found that the MSCs were capable of
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Fig. 6. MSCs suppressed DC maturation in a PLGA film implantation model. The immunophenotype and the pro-inflammatory cytokine expression of DCs were determined at day 3 and day 7 post-PLGA film implantations. MSC co-implantation significantly reduced the proportion of mature DCs (CD11cþCD80þ, CD11cþCD86þ, and CD11cþIaþ) among host splenocytes (Fig. 6A). Additionally, MSCs decreased the transcription of T cell derived IL-12 and TNF-a in the splenocytes of MSC-PLGA construct-implanted mice (Fig. 6B and C).
attenuating the PLGA-induced host inflammatory responses in a mouse model. Although PLGA is known to trigger inflammatory responses toward implants, the underlying mechanisms remain unclear. For a long time, much of the in vitro and in vivo studies have mostly focused on the involvement of neutrophils and lymphocytes until Yoshida et al. [19] reported that PLGA could directly promote iDC differentiation into mature DCs in vitro. As is well known, DCs are the most potent antigen presenting cells. The DC activation inevitably initiates the host immune response to a specific antigen; thus, infiltrating inflammatory cells may impair tissue regeneration surrounding the implanted PLGA scaffolds. Therefore, we assume that DCs are a potential cellular target for preventing PLGA-induced host inflammatory responses. DC differentiation is greatly dependent upon stromal environments [43e45]. Zhang et al. reported splenic endothelial cells drive mature DCs to trans-differentiate into regulatory DCs [45]. Our previous study demonstrated that MSCs were capable of suppressing human monocyte differentiation into DCs in vitro [37]. Thus, we explored the possibility of PLGA-induced host inflammatory response control by MSCs. We found that mouse MSCs could suppress CD11c, CD80, CD86 and Ia expression and that the MSC-treated PLGA-DCs were phenotypically similar to iDCs. Functionally, the MSCs educated the PLGA-DCs turn to promote Th2 and regulatory T cell differentiation. This observation indicates that MSCs might drive PLGA-DCs into a new type of DCs with suppressive immune functions. In fact, the modulatory effect on MSCs on PLGA-DCs is in line with previous findings that MSCs act as hematopoiesis and immune modulators [25]. Myeloid DCs are specialized cells derived from the monocyte hematopoietic lineage that develop in bone marrow niches [17]. As one of the cells that creates the bone marrow niche,
MSCs supply numerous cytokines and cell surface signals that modulate hematopoietic cell development and differentiation. MCSF is constitutively released by MSCs and promotes DC precursor differentiation into macrophages, which reduces the DC number [46,47]. Moreover, MSCs secrete high IL-6 level, which is involved in the suppressive mechanism induced by MSCs [48e50]. Previous reports have shown that this inhibition may require direct cellecell interactions of the DC precursors with neighboring stromal cells [37,51]. However, in our study, the PLGA-DCs generated in the transwell chamber system were only slightly decreased. Therefore, we suggest that MSCs exhibit suppressive effect on DCs mainly by secreting a cocktail of higher immune cytokine amounts instead of direct cellecell contact. To understand the molecular mechanism underlying the suppressive effect of the MSCs on the PLGA-DCs, the MAPK pathway was chosen to investigate as it was reported to be involved in DC maturation in inflammatory microenvironments [38e40]. We observed that the PLGA film elicited a remarkable activation of the p38/MAPK and ERK/MAPK pathways in the DCs. Moreover, inhibition of these pathways with chemical inhibitors changed the PLGADC cell morphology and phenotypes, proving that the p38/MAPK and ERK/MAPK pathways are involved in the PLGA stimulatory effects. To the best of our knowledge, many investigations were conducted to understand the mechanism behind PLGA stimulation, such as on integrin-based DC adhesion, NF-kB signaling and tolllike receptor signaling cascades; however, the role of MAPK pathways remained unknown [21,23,52]. In this study, we found that PLGA induced DC maturation via activation of the p38/MAPK and ERK/MAPK pathways. Most importantly, the observed MSCs dramatically suppressed PLGA film-triggered MAPK signal transduction in the DCs. Additionally, we noticed that a single pathway inhibitor failed to completely suppress iDC differentiation into
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Fig. 7. MSCs influenced T cell subpopulation and attenuated PLGA-induced host inflammatory reaction in vivo. The proportion of CD3þCD69þ T lymphocytes in PLGA implantation mice was decreased by MSCs in a time-dependent manner (Fig. 7A). Moreover, the FACS data showed that the MSCs significantly decreased the proportion of Th1 (CD4þIFN-gþ) and Th17 (CD4þIL-17Aþ) but increased the proportion of Th2 (CD4þIL-4þ) and Tregs (CD4þCD25þFoxp3þ) in recipients (Fig. 7B). In addition, MSCs suppressed the transcription of IFN-g and IL-17 but up-regulated IL-4 and Foxp3 in splenocytes (Fig. 7C). The results of histological analysis performed at day 7 showed that spleen nodules in MSC-PLGA constructimplanted recipients were smaller than that of PLGA film-implanted recipients (Fig. 7D). Furthermore, the immune cell filtration was observed around the implanted PLGA film and a fibrotic capsule formed at the implantation sites at day 21. No remarkable immune cell filtration near the MSC-PLGA construct was observed (Fig. 7E). Bars represent 500 mm and 100 mm in Fig. 7D and E, respectively.
PLGA-DCs, which suggests that some other mechanisms are also involved in PLGA stimulation. Until now, our knowledge regarding the stimulating effects of PLGA on DCs mainly was based on in vitro observations. Here, our findings demonstrated that PLGA did stimulate DC maturation in vivo. Most importantly, the MSCs were capable of inhibiting processing, which implicates the potential application of MSCs to reduce unexpected inflammatory responses. Compared with previous reports, MSC co-implantation possesses some advantages in controlling PLGA-induced host immune responses. First, MSCs do not release degradation products, which reduce the potential disadvantages of preparing a new complex. Additionally, MSCs exert immunosuppressive effects in a paracrine manner and avoid whole body infusion. Further, MSCs are characterized with low immunogenicity and multi-differentiation potency, which indicates a tissue repairing property of MSCs in the MSC-PLGA constructs. Armed with a PLGA film implantation murine model, we found that MSC co-delivery significantly suppressed DC maturation and attenuated the host immune defense in vivo. The CD3þCD69þ activated T lymphocyte proportion was dramatically reduced in the recipient spleens, which supported the finding that MSCs impaired the stimulatory activity of the PLGA-DCs. Additionally, histological results confirmed that MSCs inhibited local lymphocyte proliferation in spleens by reducing the number and size of the spleen nodules and attenuated the immune infiltration around the implants. It was reported that IL-12 cytokine production by DCs correlates with T lymphocyte proliferation and a Th1-type response
[53,54]. Consistent with this in vitro data, the MSCs significantly reduced the DC IL-12 expression in the mouse model. Furthermore, it was observed that the INF-g and IL-17A expression decreased while IL-4 expression increased in T lymphocytes of recipient spleens, which supported a shift from Th1 and Th17 cells to Th2 cells in vivo [55,56]. The subpopulation of CD4þCD25þFoxp3þ Tregs was first identified based on its crucial role in the control of autoimmune processes [57,58]. In this study, we found that the MSC-PLGA construct co-implantation significantly increased the proportion of Tregs among the splenocytes. As it is well known, the generation of Tregs is greatly dependent upon immune cytokines. Previous studies proved that IL-4 is an indispensable inducer of peripheral CD4þCD25 naïve T cell differentiation into CD4þCD25þFoxp3þ Tregs [59,60]. Moreover, upon stimulation with IL-4, Tregs are more potent in suppressing activation and IFN-g expression in T lymphocytes [49,50]. Consistent with previous studies, we found that IL-4 secretion and transcription were upregulated in the T cells that were activated by the MSC-treated PLGA-DCs.
5. Conclusions We demonstrated that MSCs suppress PLGA-DC maturation and immune functions in vivo and in vitro. These findings reveal a novel role for MSCs in controlling the PLGA-induced host immune response by targeting DCs and may provide an alternative and accessible strategy to improve the biocompatibility of PLGA bioscaffolds for tissue engineering.
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Acknowledgments This study was supported by the Beijing Natural Sciences Grants (No. 7132133), and National Natural Science Foundation (31070996, 31171084, 81101342, 81371945). The authors declare no competing financial interests. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2015.03.005.
References [1] Athanasiou KA, Niederauer GG, Agrawal CM. Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers. Biomaterials 1996;17:93e102. [2] Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. Eur Cell Mater 2008;15:100e14. [3] Harada N, Watanabe Y, Sato K, Abe S, Yamanaka K, Sakai Y, et al. Bone regeneration in a massive rat femur defect through endochondral ossification achieved with chondrogenically differentiated MSCs in a degradable scaffold. Biomaterials 2014;35:7800e10. [4] Martin JR, Gupta MK, Page JM, Yu F, Davidson JM, Guelcher SA, et al. A porous tissue engineering scaffold selectively degraded by cell-generated reactive oxygen species. Biomaterials 2014;35:3766e76. [5] Chang NJ, Lam CF, Lin CC, Chen WL, Li CF, Lin YT, et al. Transplantation of autologous endothelial progenitor cells in porous PLGA scaffolds create a microenvironment for the regeneration of hyaline cartilage in rabbits. Osteoarthr Cartil 2013;21:1613e22. [6] Park JS, Yang HN, Woo DG, Jeon SY, Park KH. Multilineage differentiation of human-derived dermal fibroblasts transfected with genes coated on PLGA nanoparticles plus growth factors. Biomaterials 2013;34:582e97. [7] Lin CY, Chang YH, Li KC, Lu CH, Sung LY, Yeh CL, et al. The use of ASCs engineered to express BMP2 or TGF-b3 within scaffold constructs to promote calvarial bone repair. Biomaterials 2013;34:9401e12. [8] Lee JM, Kim JD, Oh EJ, Oh SH, Lee JH, Im GI. PD98059-impregnated functional PLGA scaffold for direct tissue engineering promotes chondrogenesis and prevents hypertrophy from mesenchymal stem cells. Tissue Eng A 2014;20: 982e91. [9] Eppley BL, Morales L, Wood R, Pensler J, Goldstein J, Havlik RJ, et al. Resorbable PLLA-PGA plate and screw fixation in pediatric craniofacial surgery: clinical experience in 1883 patients. Plast Reconstr Surg 2004;114:850e6. discussion 857. [10] Asawa Y, Sakamoto T, Komura M, Watanabe M, Nishizawa S, Takazawa Y, et al. Early stage foreign body reaction against biodegradable polymer scaffolds affects tissue regeneration during the autologous transplantation of tissue-engineered cartilage in the canine model. Cell Transpl 2012;21(7): 1431e42. [11] McBane JE, Sharifpoor S, Cai K, Labow RS, Santerre JP. Biodegradation and in vivo biocompatibility of a degradable, polar/hydrophobic/ionic polyurethane for tissue engineering applications. Biomaterials 2011;32: 6034e44. [12] Yoon SJ, Kim SH, Ha HJ, Ko YK, So JW, Kim MS, et al. Reduction of inflammatory reaction of poly(D,L-lactic-co-glycolic acid) using demineralized bone particles. Tissue Eng A 2008;14:539e47. [13] Thevenot PT, Nair AM, Shen J, Lotfi P, Ko CY, Tang L. The effect of incorporation of SDF-1alpha into PLGA scaffolds on stem cell recruitment and the inflammatory response. Biomaterials 2010;31:3997e4008. [14] Mountziaris PM, Tzouanas SN, Sing DC, Kramer PR, Kasper FK, Mikos AG. Intra-articular controlled release of anti-inflammatory siRNA with biodegradable polymer microparticles ameliorates temporomandibular joint inflammation. Acta Biomater 2012;8:3552e60. [15] Avula MN, Rao AN, McGill LD, Grainger DW, Solzbacher F. Modulation of the foreign body response to implanted sensor models through device based delivery of the tyrosine kinase inhibitor, masitinib. Biomaterials 2013;34: 9737e46. [16] Lickorish D, Chan J, Song J, Davies JE. An in-vivo model to interrogate the transition from acute to chronic inflammation. Eur Cell Mater 2004;8:12e9. discussion 20. [17] Dalod M, Chelbi R, Malissen B, Lawrence T. Dendritic cell maturation: functional specialization through signaling specificity and transcriptional programming. EMBO J 2014;16(33):1104e16. ML, Vargas P, Chabaud M, Le Berre M, Liu YJ, Collin O, et al. Migration of [18] Heuze dendritic cells: physical principles, molecular mechanisms, and functional implications. Immunol Rev 2013;256:240e54. [19] Yoshida M, Babensee JE. Poly(lactic-co-glycolic acid) enhances maturation of human monocyte-derived dendritic cells. J Biomed Mater Res A 2004;71: 45e54.
697
[20] Yoshida M, Mata J, Babensee JE. Effect of poly(lactic-co-glycolic acid) contact on maturation of murine bone marrow-derived dendritic cells. J Biomed Mater Res A 2007;80:7e12. [21] Park J, Babensee JE. Differential functional effects of biomaterials on dendritic cell maturation. Acta Biomater 2012;8:3606e17. [22] Bennewitz NL, Babensee JE. The effect of the physical form of poly(lactic-coglycolic acid) carriers on the humoral immune response to co-delivered antigen. Biomaterials 2005;26:2991e9. [23] Rogers TH, Babensee JE. The role of integrins in the recognition and response of dendritic cells to biomaterials. Biomaterials 2011;32:1270e9. [24] Schliehe C, Redaelli C, Engelhardt S, Fehlings M, Mueller M, van Rooijen N, et al. CD8-dendritic cells and macrophages cross-present poly(D,L-lactate-coglycolate) acid microsphere-encapsulated antigen in vivo. J Immunol 2011;187:2112e21. [25] Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008;8:726e36. [26] Zhu H, Guo ZK, Jiang XX, Li H, Wang XY, Yao HY, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc 2010;5:550e60. [27] Frenette PS, Pinho S, Lucas D, Scheiermann C. Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu Rev Immunol 2013;31:285e316. [28] Zhu H, Jiang XX, Guo ZK, Li H, Su YF, Yao HY, et al. Tumor necrosis factor-alpha alters the modulatory effects of mesenchymal stem cells on osteoclast formation and function. Stem Cells Dev 2009;18:1473e84. [29] Xu FF, Zhu H, Li XM, Yang F, Chen JD, Tang B, et al. Intercellular adhesion molecule-1 inhibits osteogenic differentiation of mesenchymal stem cells and impairs bio-scaffold-mediated bone regeneration in vivo. Tissue Eng A 2014;20:2768e82. lvez AB, Cobo M, Cuevas-Ocan ~ a S, Gutie rrez-Guerrero A, S [30] Carrillo-Ga anchezGilabert A, Bongarzone P, et al. Mesenchymal stromal cells express GARP/ LRRC32 on their surface: effects on their biology and immunomodulatory capacity. Stem Cells 2015;33(1):183e95. http://dx.doi.org/10.1002/stem.1821. [31] Kuçi Z, Seiberth J, Latifi-Pupovci H, Wehner S, Stein S, Grez M, et al. Clonal analysis of multipotent stromal cells derived from CD271þ bone marrow mononuclear cells: functional heterogeneity and different mechanisms of allosuppression. Haematologica 2013;98:1609e16. [32] Pereira RF, O'Hara MD, Laptev AV, Halford KW, Pollard MD, Class R, et al. Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta. Proc Natl Acad Sci U S A 1998;95:1142e7. [33] Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008;371:1579e86. [34] Augello A, Tasso R, Negrini SM, Cancedda R, Pennesi G. Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis. Arthritis Rheum 2007;56:1175e86. [35] Yang F, Qu X, Cui W, Bei J, Yu F, Lu S, et al. Manufacturing and morphology structure of polylactide-type microtubules orientation-structured scaffolds. Biomaterials 2006;27:4923e33. [36] Zhang M, Tang H, Guo Z, An H, Zhu X, Song W, et al. Splenic stroma drives mature dendritic cells to differentiate into regulatory dendtritic cells. Nat Immunol 2004;5:1124e33. [37] Jiang XX, Zhang Y, Liu B, Zhang SX, Wu Y, Yu XD, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005;105:4120e6. [38] Escors D, Lopes L, Lin R, Hiscott J, Akira S, Davis RJ, et al. Targeting dendritic cell signaling to regulate the response to immunization. Blood 2008;111: 3050e61. [39] Jarnicki AG, Conroy H, Brereton C, Donnelly G, Toomey D, Walsh K, et al. Attenuating regulatory t cell induction by TLR agonists through inhibition of p38 MAPK signaling in dendritic cells enhances their efficacy as vaccine adjuvants and cancer immunotherapeutics. J Immunol 2008;180:3797e806. [40] Al-Huseini LM, Aw Yeang HX, Sethu S, Alhumeed N, Hamdam JM, Tingle Y, et al. Nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) modulates dendritic cell immune function through regulation of p38 MAPK-cAMPresponsive element binding protein/activating transcription factor 1 signaling. J Biol Chem 2013;288:22281e8. [41] de la Fuente H, Cruz-Adalia A, Martinez Del Hoyo G, Cibri an-Vera D, Bonay P, rez- Herna ndez D, et al. The leukocyte activation receptor CD69 controls t Pe cell differentiation through its interaction with galectin-1. Mol Cell Biol 2014;34:2479e87. [42] Sancho D, Santis AG, Alonso-Lebrero JL, Viedma F, Tejedor R, S anchezMadrid F. Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors. J Immunol 2000;165:3868e75. [43] Klebanoff CA, Spencer SP, Torabi-Parizi P, Grainger JR, Roychoudhuri R, Ji Y, et al. Retinoic acid controls the homeostasis of pre-cDC-derived splenic and intestinal dendritic cells. J Exp Med 2013;210:1961e76. [44] Tuna H, Avdiushko RG, Sindhava VJ, Wedlund L, Kaetzel CS, Kaplan AM, et al. Regulation of the mucosal phenotype in dendritic cells by PPARg: role of tissue microenvironment. J Leukoc Biol 2014;95:471e85. [45] Globisch T, Steiner N, Fülle L, Lukacs-Kornek V, Degrandi D, Dresing P, et al. Cytokine-dependent regulation of dendritic cell differentiation in the splenic microenvironment. Eur J Immunol 2014;44:500e10.
698
H. Zhu et al. / Biomaterials 53 (2015) 688e698
[46] Spaggiari GM, Abdelrazik H, Becchetti F, Moretta L. MSCs inhibit monocytederived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2. Blood 2009;113:6576e83. [47] Ulivi V, Tasso R, Cancedda R, Descalzi F. Mesenchymal stem cell paracrine activity is modulated by platelet lysate: induction of an inflammatory response and secretion of factors maintaining macrophages in a proinflammatory phenotype. Stem Cells Dev 2014;23:1858e69. [48] Djouad F, Charbonnier LM, Bouffi C, Louis-Plence P, Bony C, Apparailly F, et al. Mesenchymal stem cells inhibit the differentiation of dendritic cells through an interleukin-6-dependent mechanism. Stem Cells 2007;25:2025e32. [49] Li YP, Paczesny S, Lauret E, Poirault S, Bordigoni P, Mekhloufi F, et al. Human mesenchymal stem cells license adult CD34þ hemopoietic progenitor cells to differentiate into regulatory dendritic cells through activation of the Notch pathway. J Immunol 2008;180:1598e608. [50] Deng Y, Yi S, Wang G, Cheng J, Zhang Y, Chen W, et al. Umbilical cord-derived mesenchymal stem cells instruct dendritic cells to acquire tolerogenic phenotypes through the IL-6-mediated upregulation of SOCS1. Stem Cells Dev 2014;23:2080e92. [51] Aldinucci A, Rizzetto L, Pieri L, Nosi D, Romagnoli P, Biagioli T, et al. Inhibition of immune synapse by altered dendritic cell actin distribution: a new pathway of mesenchymal stem cell immune regulation. J Immunol 2010;185: 5102e10. [52] Shokouhi B, Coban C, Hasirci V, Aydin E, Dhanasingh A, Shi N, et al. The role of multiple toll-like receptor signalling cascades on interactions between biomedical polymers and dendritic cells. Biomaterials 2010;31:5759e71.
[53] Domínguez PM, Ardavín C. Differentiation and function of mouse monocytederived dendritic cells in steady state and inflammation. Immunol Rev 2010;234:90e104. [54] Nizzoli G, Krietsch J, Weick A, Steinfelder S, Facciotti F, Gruarin P, et al. Human CD1cþ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T-cell responses. Blood 2013;122:932e42. [55] Liu Z, Fan H, Jiang S. CD4(þ) T-cell subsets in transplantation. Immunol Rev 2013;252:183e91. [56] Pradhan P, Qin H, Leleux JA, Gwak D, Sakamaki I, Kwak LW, et al. The effect of combined IL10 siRNA and CpG ODN as pathogen-mimicking microparticles on Th1/Th2 cytokine balance in dendritic cells and protective immunity against B cell lymphoma. Biomaterials 2014 Jul;35:5491e504. [57] Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic selftolerance maintained by activated T cells expressing IL-2 receptor alphachains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995;155:1151e64. [58] Graca L, Thompson S, Lin CY, Adams E, Cobbold SP, Waldmann H. Both CD4(þ) CD25(þ) and CD4(þ)CD25() regulatory cells mediate dominant transplantation tolerance. J Immunol 2002;168:5558e65. [59] Maerten P, Shen C, Bullens DM, Van Assche G, Van Gool S, Geboes K, et al. Effects of interleukin 4 on CD25þCD4þ regulatory T cell function. J Autoimmun 2005;25:112e20. [60] Skapenko A, Kalden JR, Lipsky PE, Schulze-Koops H. The IL-4 receptor alphachain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD25þCD4þ regulatory T cells from CD25-CD4þ precursors. J Immunol 2005;175:6107e16.