Effects of Hypoxia on the Immunomodulatory Properties of Human Gingiva

Abstract

The environment of bone marrow mesenchymal stem cells (MSCs) is hypoxic, which plays an important role in maintaining their self-renewal potential and undifferentiated state. MSCs have been proven to possess immunomodulatory properties and have been used clinically to treat autoimmune diseases. Here, we tested the effects of hypoxia on the immunomodulatory properties of MSCs and examined its possible underlying mechanisms. We found that hypoxic stimulation promoted the immunomodulatory properties of human gingiva–derived mesenchymal stem cells (hGMSCs) by enhancing the suppressive effects of hGMSCs on peripheral blood mononuclear cells (PBMCs). The proliferation of PBMCs was significantly inhibited, while the apoptosis of PBMCs was increased, which was associated with the Fas ligand (FasL) expression of hGMSCs. The in vivo study showed that systemically infused hGMSCs could enhance skin wound repair, and 24-h hypoxic stimulation significantly promoted the reparative capacity of hGMSCs. For mechanism, hGMSC treatment inhibited the local inflammation of injured skin by suppressing the inflammatory cells, reducing the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), and increasing anti-inflammatory cytokine interleukin-10 (IL-10), which was promoted by hypoxia. Hypoxia preconditioning may be a good optimizing method to promote the potential of MSCs for the future cell-based therapy.

Keywords

immunomodulation Fas ligand protein interleukin-10 apoptosis cell proliferation oxygen

Introduction

Due to their multipotent capacities, mesenchymal stem cells (MSCs) have been widely used in tissue engineering and wound healing (Yamada et al. 2004; Schwarz et al. 2014). Studies have shown that MSCs possess immunomodulatory properties, which has laid a foundation for the clinical use of MSCs in the treatment of some autoimmune diseases (Abdi et al. 2008; Nemeth et al. 2010). However, some restrictions still inhibit the application of MSCs in tissue engineering and autoimmune disease treatments, such as the high demand for these cells when systematically administrated, their unstable functional status, low level of biological safety, and the potential for vascular thrombosis (Bang et al. 2005; Kondziolka et al. 2005; Furlani et al. 2009). To resolve these issues, it is necessary to explore methods to improve the potential of MSCs for tissue engineering and immunomodulation.

Physical stimulation and the application of cytokines are the major methods used to optimize the tissue engineering and immunomodulatory properties of MSCs. Hypoxia is a simple stimulus used by researchers to promote the applicable characteristics in various types of MSCs, such as embryonic stem cells, induced pluripotent stem cells, and some adult somatic stem cells (Grayson et al. 2007; Das et al. 2010; Hung et al. 2012). Hu et al. (2008) adopted hypoxia to optimize the activities of MSCs and found that hypoxic preconditioning enhanced the capacity of MSCs to repair infarcted myocardium. It has been reported that hypoxia enhances proliferation and maintains stemness of adipose tissue–derived stromal cells (ASCs), and hypoxia- preconditioned ASCs enhance angiogenic potential via secretion of increased vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) (Liu et al. 2013; Yamamoto et al. 2013). Moreover, the normal microenvironment of bone marrow MSCs is hypoxic, which plays an important role in maintaining their self-renewal potential and undifferentiated state (Némos et al. 2012). Hypoxia may therefore be useful for promoting the immunomodulatory properties of MSCs.

Bone marrow mesenchymal stem cells (BMMSCs) are a major source of MSCs, and based on the work of many researchers, MSCs can also be obtained from other tissues, including adipose tissue, muscle, liver, lung, and others (Williams et al. 1999; Campagnoli et al. 2001). Human gingiva–derived mesenchymal stem cells (hGMSCs) are a population of MSCs derived from human gingiva that are also capable of self-renewal, multipotent differentiation, and immunomodulation (Zhang et al. 2009). Gingiva can easily be obtained in the clinic, and the isolation of hGMSCs is simple, making hGMSCs good candidates for tissue regeneration and cell-based therapy. Recently, the immunomodulatory properties of MSCs were thoroughly studied. Akiyama et al. (2012) reported that MSC-mediated immunomodulation might be associated with the expression of the Fas ligand (FasL), which induces T-cell apoptosis, triggering immune tolerance. Zhao et al. (2012) found that dental pulp stem cells (DPSCs) also express FasL, which regulates their immunomodulatory properties. In 2013, Xu et al. first reported that cranial neural crest cell–derived gingiva–derived mesenchymal stem cells (GMSCs) possess superior immunoregulatory function by expression of a high level of FasL, and knockdown of FasL in these cells showed a significant reduction in their immunomodulatory capacity. Some anti-inflammatory cytokines also participate in the hGMSC-mediated immunomodulation, such as interleukin-10 (IL-10), which could inhibit the pro-inflammatory function of activated T cells (Zhang et al. 2009).

Cutaneous wound healing represents a highly coordinated process to achieve tissue homeostasis, which involves complex interactions of different types of resident cells and infiltrating immune cells as well as their secreted soluble mediators (Eming et al. 2007). It is a good model to study the immunomodulatory capacities of MSCs and to compare different intervention methods. Here, we used the skin wound as an in vivo model to test the effects of hypoxia on the immunomodulatory properties of hGMSCs, tested the hypothesis that hypoxia could promote hGMSC-mediated immunomodulation, and examined some possible underlying mechanisms.

Materials and Methods

Animals

C57BL/6J mice (male, 8–10 weeks old) were obtained from the Experimental Animal Center of Sichuan University. All animal care and experiments were performed in accordance with the institutional guidelines of the Medical Ethics Committee, West China Hospital of Stomatology, Sichuan University.

Antibodies and Reagents

Neutralizing antibodies for human FasL or Trail and IL-10 were purchased from R&D Systems (Minneapolis, MN, USA). Human recombinant IL-10 was purchased from PeproTech (Rocky Hill, NJ, USA). Ficoll-Paque Plus was purchased from GE Healthcare (London, UK). Horseradish peroxidase (HRP)–conjugated secondary antibody and GAPDH were purchased from ZSGB-BIO (Beijing, China). TRIzol was purchased from Invitrogen (Carlsbad, CA, USA).

Cell Cultures

hGMSCs were isolated by digesting gingival tissues in sterile phosphate-buffered saline (PBS) containing 4 mg/mL collagenase IV as described in previous studies (Zhang et al. 2009). The healthy gingival tissues, which were remnants of tissues that were discarded following routine dental procedures, were obtained from the West China Hospital of Stomatology (Sichuan, China). The 5 donors had a mean age of 30 y. Whole blood was drawn from 5 healthy female volunteers (mean age, 25 y) under a protocol approved by the Medical Ethics Committee, West China Hospital of Stomatology, Sichuan University (SN: 2010019). The hGMSCs from second to sixth passages were used in the experiments. Peripheral blood mononuclear cells (PBMCs) were cultured as described in the Appendix.

Exposure to Hypoxia

Hypoxic stimulation was achieved using a finely controlled ProOx-C-chamber system (Thermo Fisher Scientific, Waltham, MA, USA) with 2% oxygen, 93% N₂, and 5% CO₂. The hGMSCs were divided into the 3 following groups according to different hypoxia periods: the normal oxygen group (H0), the 12-h hypoxia group (H12), and the 24-h hypoxia group (H24).

Coculture of hGMSCs and PBMCs

In the coculture system, hGMSCs exposed to hypoxia for different time periods were used as the stimulators, whereas human PBMCs were used as the responders, in a ratio of 3:1. PBMCs were activated with 10 µg/mL ConA and cocultured with the hGMSCs for 3 d.

PBMC Proliferation Assay

To prevent hGMSC proliferation in the PBMC proliferation assays, the hGMSCs were inactivated by treatment with mitomycin C (15 µg/mL) for 2 h. A PBMC group cultured without ConA was applied as a positive control. The proliferation of PBMCs was analyzed after 3 d of coculture using an MTS assay (Promega, Madison, WI, USA), according to the manufacturer’s protocol.

In other experiments, coculture supernatants, human recombinant IL-10 (60 pg/mL), and neutralizing antibody for human IL-10 (10 µg/mL) were added into the coculture systems. All experiments were performed in triplicate and repeated at least twice.

Flow Cytometry Analysis

After different periods of hypoxia, the hGMSCs were trypsinized, washed with PBS, and fixed in 70% ethanol at 4°C. The fixed cells were stained with 20 µg/mL RNase A and 50 µg/mL PI at room temperature in the dark for 30 min. The cells were then analyzed by flow cytometry (FACSAria; Becton Dickinson, Franklin Lakes, NJ, USA). The same procedure was used to analyze the cell cycle stages of PBMCs that were cocultured with the hGMSCs.

The role of FasL and Trail in the apoptotic death of PBMCs cocultured with hGMSCs was analyzed using an anti-FasL neutralizing antibody (1 µg/mL; R&D Systems) and an anti-Trail neutralizing antibody (0.06 µg/mL; R&D Systems). After 3 d of coculture, the PBMCs were collected for apoptosis assays. The FITC Annexin V Apoptosis Detection Kit I (R&D Systems) was used according to the manufacturer’s instructions to evaluate apoptosis of PBMCs in the different experimental groups.

Western Blot Analysis

Western blot was performed as described in the Appendix.

Reverse Transcriptase Polymerase Chain Reaction

Reverse transcriptase polymerase chain reaction (RT-PCR) was performed as described in the Appendix.

Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay was performed as described in the Appendix.

Skin Wound and Treatment with Hypoxia- Preconditioned hGMSCs

Skin wound experiments were performed in male mice. The detailed methods are described in the Appendix. For hGMSC treatment, 2 × 10⁶ of P3 hGMSCs were infused intravenously into mice (n = 20 each group) at 1 d after skin excision. The results are representative of 3 independent experiments.

Statistics

The quantitative data were analyzed using SPSS 11.5 software (SPSS, Inc., an IBM Company, Chicago, IL, USA) with either an analysis of variance (ANOVA) or t test and expressed as mean ± SD values. Values were considered significantly different if P < 0.05.

Results

Isolation and Cell Culture of hGMSCs and PBMCs

We isolated the hGMSCs according to the methods of Zhang et al. (2009), who were the first to identify hGMSCs. Flow cytometric analysis showed that the cultured cells were positive for mesenchymal stem cell surface markers (CD29, CD90, and CD105) and negative for hematologic markers (CD45) (Fig. 1A).

Hypoxia Promotes the Suppressive Effect of hGMSCs on the Proliferation of PBMCs

To study the effects of hypoxia, we exposed hGMSCs to hypoxic conditions for different periods (0 h, 12 h, and 24 h) and subsequently cocultured them with PBMCs at a ratio of 3:1 for 3 d. After different periods of hypoxia, the cell cycle stage of hGMSCs was examined using flow cytometry. We found that hypoxia promoted the proliferation of hGMSCs and that the percentage of cells in the S and G2-M stages was increased in the 12-h hypoxia group compared with the normal oxygen group, with the highest percentage observed in the 24-h hypoxia group (P < 0.01) (Fig. 1B).

After coculture, the MTS assays showed that hypoxia significantly promoted the suppressive effect of hGMSCs on the proliferation of PBMCs. As shown in Fig. 1C, PBMCs were significantly stimulated by ConA, and the SI was 7.00 times (P < 0.001) that of the nonstimulated PBMCs. Both normoxic hGMSCs and hypoxic hGMSCs exhibited suppressive effects on the proliferation of PBMCs, with SI values of 5.14 (0 h), 3.99 (12 h), and 1.57 (24 h), respectively. As the time of exposure to hypoxia was prolonged, the inhibitory effect was significantly enhanced.

An examination of the cell cycles of PBMCs after coculture showed that the proliferation of PBMCs was significantly prohibited by hGMSCs (8.73%) compared with the control group (15.33%, P < 0.01), which confirmed that hypoxia enhanced the inhibitory effect of hGMSCs on PBMC proliferation (Fig. 1D, E).

FasL and IL-10 Contribute to the Promotion of hGMSC-Based Immunomodulation by Hypoxia

We examined the apoptosis of PBMCs after 3 d of coculture with hypoxia-preconditioned hGMSCs using the fluorescent dye annexin V-FITC. Fig. 2A, B showed the percentage of apoptotic PBMCs. Normoxic hGMSCs induced significant PBMC apoptosis, and hypoxic hGMSCs induced apoptosis even more strongly, yielding 36.61% (12 h) and 60.10% (24 h) apoptotic PBMCs. These data suggested that hypoxia could strengthen the effect of hGMSC-mediated apoptosis of PBMCs in a time-dependent manner.

Recent findings suggest that dental pulp stem cells and neural crest cell–derived GMSCs exert their immunomodulatory properties via the Fas/FasL pathway. We evaluated FasL expression in hGMSCs exposed to hypoxia to determine whether Fas/FasL was involved in the hGMSC-mediated induction of PBMC apoptosis. Western blotting showed that hypoxia increased the FasL expression of hGMSCs in a time-dependent manner, which was consistent with the results obtained using RT-PCR (Fig. 2C, D).

According to the results above, we hypothesized that FasL might contribute to the enhancement of immunomodulatory function of hGMSCs exposed to hypoxia. To test this hypothesis, we added an anti-FasL antibody to the coculture system to neutralize the FasL expressed by hGMSCs and found that PBMC apoptosis was decreased when FasL was neutralized. Thus, FasL neutralization hampered the hGMSC-induced PBMC apoptosis promoted by hypoxia (Fig. 2E).

As described earlier, secretory factors play an important role in the immunomodulatory properties of MSCs and hGMSCs, and IL-10 is an essential anti-inflammatory cytokine involved in this process. We next examined potential soluble factors involved in the hGMSC-mediated apoptosis of PBMCs promoted by hypoxia. Our results showed that an increased level of IL-10 was present in the supernatants of PBMCs cocultured with hGMSCs treated with hypoxia compared with that in the coculture system with hGMSCs treated with normal oxygen. The highest level of IL-10 secretion was observed in the 24-h hypoxia group (P < 0.01) (Fig. 3A). To test the effects of IL-10 in the supernatants, we added human recombinant IL-10 and cocultured supernatants to the PBMC culture medium and found that cocultured supernatants and IL-10 could significantly suppress the PBMC proliferation, and the strongest inhibitory effect was observed in the supernatant of the 24-h hypoxia group (P < 0.01). There was no significant difference between the 24-h hypoxia group and human recombinant IL-10 group (PBMCs + ConA + IL-10) (P > 0.05) (Fig. 3B). In addition, treatment with IL-10 neutralizing antibody could reverse the inhibitory effects of hypoxia-preconditioned hGMSCs on PBMC proliferation (Fig. 3C). These results suggest that IL-10 contributes to the hGMSC-mediated immunomodulation enhanced by hypoxia.

Hypoxia Enhances hGMSC-Treated Skin Wound Healing in Mice

To examine the immunomodulatory properties of hGMSCs stimulated by hypoxia, we used an excisional skin-healing model in mice. Fig. 4A, B showed that mice receiving a systematic infusion of hGMSCs stimulated by 24-h hypoxia (H24) displayed accelerated skin wound closure compared with the mice treated with hGMSCs nonstimulated by hypoxia (H0). Histologic analysis showed less recruitment of inflammatory cells and a more organized granulation tissue at the wound site in the H24 group compared with the H0 and PBS groups, but there was no significant difference between H12 and H0 groups (Fig. 4C). ELISA analysis showed that hypoxia promoted the inhibitory effects of hGMSCs on the production of pro-inflammatory cytokine tumor necrosis factor–α (TNF-α) and increased the production of anti-inflammatory cytokine IL-10 (Fig. 4D, E). These results suggested that 24-h hypoxia enhanced hGMSC-treated skin wound healing and that 12-h hypoxia stimulation was not enough to promote the hGMSC-treated skin wound healing compared with the normal oxygen stimulation.

Discussion

The present study explored a novel preconditioning strategy for enhancing the immunomodulatory properties of hGMSCs and their underlying mechanisms. We have shown that 2% hypoxia increased the IL-10 production and FasL expression of hGMSCs, thereby enhancing their prohibitory effect on PBMC proliferation and promoting their induction of PBMC apoptosis. Hypoxia has been used by other researchers as a simple means of optimizing cell function (Rosová et al. 2008). It is known that hypoxia promotes the proliferation and stemness of MSCs and enhances their proangiogenic properties, which is accompanied by an increased expression of VEGF, hepatocyte growth factor, platelet-derived growth factor, and bFGF proteins (Crisostomo et al. 2008; Chacko et al. 2010; Busletta et al. 2011). In these studies, the oxygen concentration and the duration of hypoxia were different, which may have influenced the functional status of the MSCs. We used 2% oxygen and 12-h/24-h hypoxic preconditioning periods in our study, which promoted the immunomodulatory properties of hGMSCs. If the oxygen concentration is too low or the hypoxic time is too long, the functions of MSCs may be abrogated (Potier et al. 2007).

Recently, serial in vitro and in vivo studies suggested that hGMSCs possess immunomodulatory properties, and they have been applied in several inflammation-related disease models (Ding et al. 2010; Jung et al. 2011; Davies et al. 2012). Researchers have reported that hGMSCs exhibit potent suppressive effects on the proliferation and activation of PBMCs stimulated either phytohemagglutinin (PHA) or allogenic lymphocytes in mixed lymphocyte reactions (MLRs) (Zhang et al. 2009). Zhang et al. and Davies et al. reported that IL-10 and indoleamine 2,3-dioxygenase (IDO) expressed by hGMSCs could dampen the pro-inflammatory function of activated T cells (Zhang et al. 2009; Lee et al. 2012). It is reported that hypoxia modulates the chemokine profile of MSCs, stimulating the production of inflammatory cytokines, such as IL-10, by MSCs (Li et al. 2010), which shifts the cytokines toward the anti-inflammatory mediators (Chen et al. 2010). In this study, we found that hypoxia promoted the suppressive effects of hGMSCs, which may be partially due to the secretion of IL-10 and the proliferation of hGMSCs. In vivo, we used a full-thickness skin wound–healing model to test the effects of hypoxia on the immunomodulatory properties of hGMSCs and found that after hGMSC treatment, the level of pro-inflammatory cytokine TNF-α decreased, while the anti-inflammatory cytokine IL-10 increased during the wound-healing process. Altogether, IL-10 is a fundamental element in the immunomodulatory properties of hGMSCs to guide the normal wound repair.

The Fas-FasL cell death pathway mediates the apoptosis of many cell types. Akiyama et al. (2012) reported that MSCs induced T-cell apoptosis via the Fas-FasL pathway. In this study, we found that hGMSCs expressed FasL, which was consistent with a recently reported study (Xu et al. 2013), and the level of FasL expression increased as the duration of hypoxia increased; thus, PBMC apoptosis was elevated. In 2014, Chen et al. reported that telomerase improved immunomodulatory properties of MSCs by upregulating FasL expression. Another study reported that dental follicle cells and cementoblasts induced apoptosis of ameloblast-lineage cells, as well as Hertwig’s epithelial root sheath (HERS)/epithelial rests of Malassez (ERM) cells, through the Fas-FasL pathway during tooth development (Ding et al. 2010). The TNF-related apoptosis-inducing ligand (Trail), also referred to as Apo-2 ligand, is a member of the TNF/nerve growth factor (NGF) superfamily and acts via several receptors thought to induce or block apoptosis (Pitti et al. 1996). It has been reported that Trail possesses pronounced immunoregulatory properties that are important in immunologic tumor surveillance (Ashkenazi et al. 1999). To determine whether Trail is involved in hGMSC-mediated immunomodulation, we added a Trail-neutralizing antibody to the coculture system and found that Trail was not involved in the hGMSC-mediated promotion of PBMC apoptosis. Altogether, we could conclude that FasL regulated the immunomodulatory properties of hGMSCs, and hypoxia promoted these properties. In addition to the FasL pathway, other pathways may be involved in hGMSC-mediated immunomodulation which should be the focus of future research.

In conclusion, we have demonstrated that hypoxia promoted the expression of FasL and the production of IL-10 of hGMSCs, contributing to the promotion of hGMSC-based immunomodulation. Hypoxic preconditioning is a good method to optimize the potential of hGMSCs for tissue regeneration and cell-based therapy.

Author Contributions

C.M. Jiang, J. Wang, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; J. Liu, contributed to conception and design, drafted and critically revised the manuscript; J.Y. Zhao, L. Xiao, A. Shu, contributed to data acquisition and interpretation, critically revised the manuscript; Y.C. Gou, contributed to data acquisition, critically revised the manuscript; H.X. Quan, contributed to data acquisition, drafted the manuscript; Q. Cheng, Y.L. Zhang, W. He, contributed to data acquisition, drafted the manuscript; Y.T. Wang, contributed to data acquisition, analysis, and interpretation, drafted the manuscript; W.J. Yu, Y.F. Huang, contributed to data acquisition and analysis, drafted manuscript; Y.T. Yi, Y. Chen, contributed to data analysis, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Footnotes

Acknowledgements

We thank B. Han (Department of Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, China) for gingival tissue.

This work was supported by grants from the National Natural Science Foundation of China (No. 31070825, 81030034) and from the Sichuan Provincial Foundation of China (No. 2010JY0011, 2010JY0010).

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

A supplemental appendix to this article is published electronically only at .

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Supplementary Material

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Effects of Hypoxia on the Immunomodulatory Properties of Human Gingiva–Derived Mesenchymal Stem Cells

Abstract

Keywords

Introduction

Materials and Methods

Animals

Antibodies and Reagents

Cell Cultures

Exposure to Hypoxia

Coculture of hGMSCs and PBMCs

PBMC Proliferation Assay

Flow Cytometry Analysis

Western Blot Analysis

Reverse Transcriptase Polymerase Chain Reaction

Enzyme-Linked Immunosorbent Assay

Skin Wound and Treatment with Hypoxia- Preconditioned hGMSCs

Statistics

Results

Isolation and Cell Culture of hGMSCs and PBMCs

Hypoxia Promotes the Suppressive Effect of hGMSCs on the Proliferation of PBMCs

FasL and IL-10 Contribute to the Promotion of hGMSC-Based Immunomodulation by Hypoxia

Hypoxia Enhances hGMSC-Treated Skin Wound Healing in Mice

Discussion

Author Contributions

Footnotes

Acknowledgements

References

Supplementary Material