Crosstalk between bone marrow-derived mesenchymal stem cells and regulatory T cells through a glucocorticoid-induced leucine zipper/developmental endothelial locus-1-dependent mechanism

Abstract

Bone marrow is a reservoir for regulatory T (T_reg) cells, but how T_reg cells are regulated in that environment remains poorly understood. We show that expression of glucocorticoid (GC)-induced leucine zipper (GILZ) in bone marrow mesenchymal lineage cells or bone marrow-derived mesenchymal stem cells (BMSCs) increases the production of T_reg cells via a mechanism involving the up-regulation of developmental endothelial locus-1 (Del-1), an endogenous leukocyte-endothelial adhesion inhibitor. We found that the expression of Del-1 is increased ∼4-fold in the bone tissues of GILZ transgenic (Tg) mice, and this increase is coupled with a significant increase in the production of IL-10 (2.80 vs. 0.83) and decrease in the production of IL-6 (0.80 vs. 2.33) and IL-12 (0.25 vs. 1.67). We also show that GILZ-expressing BMSCs present antigen in a way that favors T_reg cells. These results indicate that GILZ plays a critical role mediating the crosstalk between BMSCs and Treg in the bone marrow microenvironment. These data, together with our previous findings that overexpression of GILZ in BMSCs antagonizes TNF-α-elicited inflammatory responses, suggest that GILZ plays important roles in bone-immune cell communication and BMSC immune suppressive functions.—Yang, N., Baban, B., Isales, C. M., Shi, X.-M. Crosstalk between bone marrow-derived mesenchymal stem cells and regulatory T cells through a glucocorticoid-induced leucine zipper/developmental endothelial locus-1-dependent mechanism.

Glucocorticoid (GC)-induced leucine zipper (GILZ) is an endogenous GC anti-inflammatory effect mediator and exerts its anti-inflammation action via interaction with, and disruption of, the transcriptional activities of NF-κB and activator protein 1 (1–3), the 2 principal inflammation-signaling mediators. The expression of GILZ is induced rapidly by all forms of steroid hormone GCs (4, 5) in almost all cell types tested, including bone marrow mesenchymal stem cells (BMSCs) (1, 6–8). Bone marrow is the site where adult hematopoiesis takes place and thus has a direct impact on the immune system. In contrast, the immune system also has a profound impact on bone. For example, in autoimmune disease the immune system is constantly activated by soluble factors such as TNF-α, IL-1β, and IL-6 that are secreted from antigen-stimulated immune cells. However, studies on the interactions or crosstalks between the bone cells and immune cells in the bone marrow are sparse. Developmental endothelial locus 1 (Del-1), also known as endothelial growth factor-like repeats and discoidin I-like domains 3, was identified from endothelial cells as a negative regulator of neutrophil extravasation (9). Recent studies show that Del-1 is also expressed in tissues such as the brain, eye, gingiva, and lung (10) and that it inhibits inflammatory bone loss (9, 11). Evidence also showed that Del-1 expression is down-regulated by inflammatory factors such as TNF-α, LPS, and IL-17 (9, 11). In an effort to study the effect of GILZ on bone formation, we found unexpectedly that the expression of Del-1 is elevated significantly in bone tissues of GILZ Tg mice in which the expression of GILZ is under the control of a 3.6 kb type I collagen promoter (12). This finding, together with our previous studies showing that overexpression of GILZ in BMSCs inhibits proinflammatory cytokine TNF-α-induced cyclooxygenase-2 expression (3) and antagonizes TNF-α inhibition of osteogenic differentiation (13), led us to hypothesize that Del-1, induced by GILZ in BMSCs, plays a critical role in bone and immune system communication.

Regulatory T (T_reg) cells are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and control autoimmune disorders. Studies show that high levels of functional CD4⁺Foxp3⁺ T_reg cells exist in bone marrow (14–16) and play important roles regulating bone (15). BMSCs are multipotent progenitor cells and have therapeutic value in regenerative medicine for a range of acute and chronic diseases (17–19). It is noteworthy that evidence now show that the beneficial effects of BMSCs are achieved primarily through their ability of releasing soluble mediators, which are capable of reducing inflammation, promoting angiogenesis, and increasing cell survival at the sites of injury, rather than their ability of differentiating into the type of cells of that tissue and repair. For example, BMSCs exert diverse and potent modulatory effects on T cells either through direct cell-cell contact or through releasing factors such as indoleamine-2,3-dioxygenase (20), NO (21), IL-27 (22), and TGF-β (23). However, controversies remain regarding the range of effects that BMSCs can exert on individual T-cell effector subsets. In this study, we investigated the roles of GILZ in T_reg cell regulation and function using GILZ Tg mice in which the expression of GILZ is under the control of a bone marrow mesenchymal lineage cell-specific promoter and the BMSCs that are transduced with a GILZ-expressing retrovirus.

MATERIALS AND METHODS

Chemicals and antibodies

All chemicals were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA) or Sigma-Aldrich (St. Louis, MO, USA) except where specified. Antibodies were purchased from eBioscience (San Diego, CA, USA) except where specified.

Animals

All animal procedures were performed in accordance with the approval of the Institutional Animal Care and Use Committee at the Georgia Regents University. Animals were housed in the Laboratory Animal Service facility under a 12-h dark-light cycle and provided with standard rodent chow and water ad libitum. Controls [8- to 12-wk-old wild-type (WT) mice] and GILZ Tg mice were used in this study. Generation and characterization of GILZ Tg mice (Col3.6-GILZ) were described previously (12).

Mouse BMSCs

Isolation, characterization, and infection of BMSCs were described previously (24). In brief, BMSCs were isolated from 18-mo-old male C57BL/6 mice using a negative immunodepletion (using magnetic beads conjugated with anti-mouse CD11b, CD45R/B220, and Pan DC) and positive immunoselection (using anti-Sca-1 beads). For GILZ overexpression, BMSCs were infected with retroviruses expressing GILZ [or green fluorescent protein (GFP) as a control]. The viral infection efficiency (>90%) and levels of GILZ expression in the infected cells were confirmed by Western blot and immunofluorescence microscopy as described previously (3).

RNA extraction and real-time qRT-PCR

Total cellular RNA was isolated using TRIzol reagent according to the manufacturer's instructions (Invitrogen Corporation, Grand Island, NY, USA). Equal amounts of total RNA (2 µg) were reverse transcribed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), and the mRNA levels of the indicated genes were analyzed in triplicate using SYBR Green Master Mix and a Chromo-4 real-time RT-PCR instrument. The mRNA levels were normalized to β-actin (internal control), and gene expression was presented as fold changes. The primer sequences used in the PCR reactions were as follows: 5′-CCTGTGAGATAAGCGAAGC-3′ (forward) and 5′-GAGCTCGGTGAGTAGATG-3′ (reverse) for Del-1 (accession no.: NC_000079.6), 5′-CTGGCACCACACCTTCTACA-3′ (forward) and 5′-GGTACGACCAGAGGCATACA-3′ (reverse) for β-actin (accession no.: NM_007393), and 5′-GCTGCACAATTTCTCCACCT-3′ (forward) and 5′-GCTCACGAATCTGCTCCTTT-3′ (reverse) for GILZ (accession no.: AF024519).

Flow cytometry

GILZ Tg mice and their WT littermates were killed to collect bone marrow and blood cells. Both tibias and femurs were dissected and bone marrow cells flushed with RPMI 1640 medium + 10% fetal bovine serum (FBS). Blood was collected using heparinized capillary tubes from hearts. Cells were first incubated with antibodies against cell surface markers CD4, CD8, CD11b, CD11c, CD45, CD31, CD34, and Sca1 and then fixed and permeabilized using fixed/permeabilized concentrate (eBioScience) before incubation with antibodies for intracellular labeling of Del-1, FOXP3, IL-17, IL-12, IL-10, IL-6 (BD BioSciences, Bedford, MA, USA). After one wash, cells were run through a 4-color flow cytometer (FACSCalibur, BD Biosciences, San Diego, CA, USA), and data were collected using CellQuest software (BD Biosciences, San Jose, CA, USA) as described previously (25). As a gating strategy, for each sample, isotype-matched controls were analyzed to set the appropriate gates. For each marker, samples were analyzed in duplicate measurements. To minimize false-positive events, the number of double-positive events detected with the isotype controls was subtracted from the number of double-positive cells stained with corresponding antibodies (not isotype control), respectively. Cells expressing a specific marker were reported as a percentage of the number of gated events. Statistical analysis was performed using Prism 6.0 software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry and immunocytochemistry

For immunohistochemistry, left femurs of male mice were decalcified in EDTA, embedded in paraffin, and sectioned at 4–6 μm. For immunocytochemistry, bone marrow cells were flushed from right femurs using DMEM supplemented with 10% FBS, single cell suspensions were prepared, and 1 × 10⁵ cells were applied to slide using a cytospin and then fixed and stained. Immunostaining was carried out utilizing primary antibodies against FOXP3, IL-10, GILZ, and Del-1 according to previously described protocols (26).

Western blot analysis

Western blot analyses were performed as described previously (3). In brief, retrovirus-infected mesenchymal stem cells (MSCs) were harvested in lysis buffer. Equal amounts of total protein were separated on 12% SDS-PAGE, transferred onto nitrocellulose membrane, and blocked in 5% nonfat dry milk for 2 h at room temperature. The membranes then were incubated with the indicated primary antibodies for at least 1 h at room temperature. After several washes, the membrane was incubated with IRDye 680 secondary antibody and scanned using an Odyssey Infrared Imaging System (Li-Cor Biotechnology, Lincoln, NE, USA).

Mixed lymphocyte reaction

The mixed lymphocyte reaction is a commonly used functional assay to determine the proliferative capacity of T lymphocytes in response to antigen presentation. This assay requires the use of responder cells (splenic T cells) and stimulating cells (in this case, MSC-GILZ or MSC-GFP cells) from animals with different genetic backgrounds. The responder cells and stimulator cells were set up in triplicate wells in a RPMI 1640 medium, which was supplemented with FBS, penicillin, streptomycin, l-glutamine, and 2-mercaptoethanol. Responder T cells were initially enriched using magnetic assorted cell sorting and used at 1 × 10⁴ cells/well. MSC-GILZ or MSC-GFP cells (as stimulators) were used at 5 × 10⁴/well. After 72–96 h of incubation at 37°C in a humidified 5% CO₂ environment, all cells were harvested into flow cytometry tubes. After one wash with PBS, all samples were then incubated with anti-rat CD71-phycoerythrin-conjugated antibody (a marker for activated and dividing T cells) for 20 min in dark on ice. Samples were then washed with PBS and T-cell proliferation was measured by using flow cytometry analysis for CD71 expression. The average of the triplicate samples was recorded for each group.

RESULTS

Del-1 expression increased in the bone marrow of GILZ Tg mice

During the course of another study investigating the role of GILZ in bone formation (12), we accidently found that Del-1, a central regulator of immune homeostasis that has also been shown to inhibit inflammatory bone loss (11), is highly expressed in bone tissues of GILZ Tg mice, in which the expression of GILZ is under the control of a 3.6 kb type I collagen promoter fragment (Col3.6). This led us to hypothesize that GILZ may have a role mediating the communications between bone and immune cells through Del-1 because GILZ expression is restricted in bone marrow mesenchymal lineage cells or BMSCs in our GILZ Tg mice (12). To test this hypothesis, we first collected bone marrow and bone samples of GILZ Tg and their age- and gender-mated WT littermate control mice and performed flow cytometry and immunohistochemistry studies. Results showed that the percentage of GILZ-positive cells was significantly higher in the bone marrow of GILZ Tg mice than that in control mice (Fig. 1A). The percentage of GILZ-positive cells in peripheral blood, however, was not changed significantly because of the mesenchymal cell lineage-specific GILZ expression, as demonstrated by qRT-PCR analysis of RNA samples isolated from BMSCs and hematopoietic lineage cells (bone marrow monocyte/macrophage) of the GILZ Tg mice (Fig. 1B). In line with this result, a high-level expression of Del-1 mRNA (Fig. 1C) and a large increase in the numbers of Del-1-expressing cells were detected in the bone marrow of GILZ Tg mice (Fig. 1D). To identify the type of cells in which Del-1 expression is up-regulated, we sorted bone marrow cells using antibodies against cell surface marker CD11b, Sca1, CD34, and CD31. Results showed that the percentages of Del-1-positive cell populations in whole bone marrow, hematopoietic stem cell (HSC), and MSC lineages are all increased remarkably in GILZ Tg mice compared with that in WT mice (Fig. 1E). However, Del-1 expression in endothelial progenitor cells (EPCs) remained unchanged. We also noticed that the percentage of Del-1-positive cells is extremely low in HSCs (∼0.01–0.02%) compared with that in MSCs (∼0.1–0.3%) or EPCs (∼0.45%) in both GILZ Tg and WT mice, indicating that increased expression of Del-1 in MSCs may contribute the most to the observed phenotype in GILZ Tg mice. Immunohistochemical studies performed on decalcified femoral bone sections showed a much stronger Del-1 expression in the bone marrow compartment of the GILZ Tg mice compared with that in WT mice (Fig. 1F), and more importantly, the expression of Del-1 and GILZ seemed to colocalize in the same cells (Fig. 1G). To demonstrate that the elevated Del-1 expression is caused by increased GILZ in mesenchymal lineage cells or BMSCs, we performed immunocytochemistry and Western blot analyses using purified BMSCs that were subsequently engineered to overexpress GILZ (or GFP as a control) from a retrovirus vector (Fig. 2A) (27). Again, the results showed a significant increase of Del-1 mRNA and protein expression in GILZ-overexpressing cells (MSC-GILZ) (Fig. 2B–D). Together, these results confirmed that overexpression of GILZ in BMSCs up-regulates Del-1 expression.

Figure 1.

Expression of GILZ and Del-1 in WT and GILZ Tg mice. A) Flow cytometry analysis showing percentage of GILZ-expressing cells in bone marrow and peripheral blood cells of WT vs. Tg mice. B) qRT-PCR analysis showing levels of GILZ mRNA in bone marrow mesenchymal stem cells (MSC) vs. hematopoietic lineage cells [bone marrow monocyte/macrophage (BMM)]. C) qRT-PCR analysis showing the relative levels of Del-1 mRNA in bone tissues of both WT and Tg mice. D) Representative flow cytometry profiles showing Del-1-expressing cell numbers in whole bone marrow cells of WT and Tg mice. E) Representative flow cytometry dot plots showing Del-1 expression by bone marrow EPCs, bone marrow MSCs and bone marrow HSCs. F) Representative immunohistochemical staining images showing Del-1 expression in the femoral bone sections of both WT and GILZ Tg mice. G) Representative immunofluorescent images of cytospin preparations of bone marrow cells showing Del-1 expression in both WT and GILZ Tg mice. A–C) Data are presented as the means of 4 experiments. D, E) Data represent the results from a pool of 3 animals in each group. F, G) Data are representative results from 3 independent experiments. Statistical analysis was performed using Prism 6.0 software. Two-tailed unpaired Student’s t test was used for statistical comparisons. Graphs represent means ± sd. *P < 0.05, **P < 0.01.

Figure 2.

Effects of GILZ on Del-1 expression in BMSCs. A) Immunocytochemical staining showing the efficiency and expression of GFP (green) and GILZ (red) in retrovirus-infected BMSCs. B) Representative immunocytochemical staining images showing the expression of Del-1 in GILZ- and GFP-overexpressing BMSCs (MSC-GILZ and MSC-GFP, respectively). C) Western blot analysis showing levels of Del-1 and GILZ protein expression in MSC-GILZ and MSC-GFP cells. D) Quantitative result of C (from 3 independent experiments) showing relative level of Del-1 and GILZ protein. Statistical analysis was performed using Prism 6.0 software. Two-tailed unpaired Student’s t test was used for statistical comparisons. Graphs represent means ± sd. **P < 0.01.

GILZ modulates T-lymphocyte differentiation

To determine the effects of GILZ on immunity in vivo, we collected bone marrow and peripheral blood cells from GILZ Tg and WT mice and analyzed immune cell profile. Results showed that in bone marrow the percentage of CD4⁺, CD8⁺, CD11b⁺, and CD11c⁺ cells had no difference between GILZ Tg and WT mice (Fig. 3). In peripheral blood, however, the percentage of CD8⁺ cells increased significantly in GILZ Tg mice (P < 0.05). The percentage of CD4⁺ cells showed a trend of decrease in Tg mice although this decrease was not statistically significant (Fig. 3A, B). No significant difference was detected in the percentages of CD11b⁺ and CD11c⁺ cells both in bone marrow and in blood (Fig. 3C–F).

Figure 3.

Effects of GILZ on T-lymphocyte profile. A) Representative flow cytometry dot plots showing the expression of CD4 and CD8 on T lymphocytes in the bone marrow and peripheral blood of GILZ Tg and WT mice. B) Quantitative measure of frequency of (A). C, D) Representative flow cytometry dot plots depicting the gating of CD11b⁺ T-lymphocyte cells in bone marrow (C) and peripheral blood (D). The gated CD11b⁺ cells were then analyzed for CD11c. E, F) Summary for the frequency of CD11b⁺ and CD11c⁺ T lymphocytes in the bone marrow and peripheral blood. Data are pooled from 2 independent experiments (n = 6 mice per group). Statistical analysis was performed using Prism 6.0 software. Two-tailed unpaired Student’s t test was used for statistical comparisons. Graphs represent means ± sd. *P < 0.05.

Next, we examined whether expression of GILZ in BMSCs can regulate the production of Treg in vivo. Fluorescence-activated cell sorting analysis results showed that the percentages of FOXP3⁺ and CD25⁺ cells are increased significantly in both blood (Fig. 4A) and bone marrow (Fig. 4B) of the GILZ Tg mice. This data were further confirmed by immunohistochemistry studies performed on femoral bone sections of the GILZ Tg mice, which showed a significantly higher intensity of Foxp3-positive signals (Fig. 4C). Taking together, these results demonstrated that overexpression of GILZ in bone marrow mesenchymal lineage cells is capable of modulating the functions of T lymphocytes and the conversion of T-helper to T_reg cells.

Figure 4.

Effects of GILZ on T_reg cell production. A, B) Flow cytometry analysis showing Foxp3⁺ and CD25⁺ cells in blood (A) and bone marrow (B) of GILZ Tg and WT mice. A, B) Quantitative results are shown as bar graphs (right panels). C) Representative immunohistochemical staining results showing IL-10 and Foxp3 expression in femoral bone sections of GILZ Tg and WT mice. A, B) Data are presented as mean of 6 experiments. C) Data are representative of 3 independent experiments. Statistical analysis was performed using Prism 6.0 software. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data are presented as the means ± sd. *P < 0.05, **P < 0.01.

GILZ alters cytokine production profile

Because GILZ increased Treg and IL-10-expressing cells (Fig. 4C), we examined whether inflammatory cytokine production profile is altered in GILZ Tg mice. Flow cytometry analysis showed that in bone marrow the population of IL-10-positive cells is increased, but the population of IL-12-positive population is decreased significantly in GILZ Tg mice (Fig. 5A). To support this result, we challenged the mice with LPS (100 µg/mouse, i.p.) and examined IL-6 cell population. The reason that the mice were challenged is that the IL-6-producing T lymphocytes are difficult to detect in naïve mice. Twenty-four hours after LPS injection, mice were sacrificed and bone marrow cells collected and analyzed. Results showed that LPS treatment increased the populations of IL-6- and IL-12-positive cells dramatically in WT mice. In contrast, this increase was not detected in GILZ Tg mice (Fig. 5B, C), suggesting that the modulatory effect of GILZ on immune system observed in GILZ Tg mice is mediated through GILZ regulation of Del-1 expression and T-lymphocyte differentiation.

Figure 5.

Effects of GILZ on cytokine production. A) Flow cytometry analysis showing IL-10- and IL-12-producing cell frequency in the bone marrow of GILZ Tg and WT mice. B) IL-6-producing cell frequency in the bone marrow of LPS-challenged GILZ Tg and WT mice. C) IL-12-producing cell frequency in the bone marrow of LPS-challenged GILZ Tg and WT mice. Data are presented as the means of 3 experiments. Statistical analysis was performed using Prism 6.0 software. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data are presented as the means ± sd. *P < 0.1, **P < 0.05.

Crosstalk between BMSCs and T lymphocytes

To demonstrate functionally the effect of GILZ on T lymphocytes, we performed mixed lymphocytes reaction assays and examined the rate of T-lymphocyte proliferation. Cultures of splenocytes isolated from CD-1 mice were stimulated with BMSCs that were isolated from C57BL/6 mice and subsequently engineered to stably express GILZ or GFP (as a control) as described previously (27). Results showed that after 72 h of incubation, MSC-GFP control cells induced 16% of the lymphocytes (CD71⁺ population) to proliferate. In contrast, MSC-GILZ cells induced only 4.3% of the lymphocytes to proliferate (Fig. 6A, B). In line with the lymphocyte proliferation results, stimulation with MSC-GILZ cells resulted in 2-fold higher Treg (Foxp3⁺ population) induction activity than that of the MSC-GFP control cells (Fig. 6C, D). Together, these results indicated that GILZ-expressing BMSCs can act as “antigen-presenting-like” cells and inhibit lymphocyte proliferation with greater immunosuppressive activities.

Figure 6.

Effects of GILZ on lymphocyte proliferation and T_reg cell induction in vitro. Splenocytes were isolated from CD-1 mice and stimulated with MSC-GILZ or MSC-GFP isolated from C57BL/6 mice. A, B) Representative flow cytometry profile and quantitative results showing the number of CD71-positive splenocytes and CD71-positive cell frequency in mixed lymphocyte reaction assays. C, D) Representative flow cytometry profile and quantitative results showing the number of Foxp3-positive splenocytes. n = 3. **P < 0.01.

DISCUSSION

Bone marrow harbors high level of functional T_reg cells, which undergo active expansion in the bone marrow under pathologic circumstance (15). Microenvironment cues, such as cytokines, chemokines, and cell surface markers expressed on adjacent cells, play important roles in T_reg cell regulation. In this study, we showed that GILZ expressed in BMSCs can increase the production of T_reg cells (Fig. 4) and that this regulation is mediated, at least in part, through GILZ up-regulation of Del-1 expression (Fig. 1, 2). This finding provides new insight into the molecular mechanisms underlying bone and immune system interaction and explains, at least in part, how BMSCs exert their anti-inflammatory actions.

Naive T cells, upon T-cell receptor activation by certain extracellular stimuli, such as TGF-β, can express Foxp3 and become Treg (28). In the presence of IL-6, however, TGF-β-induced T_reg cell development is abrogated and the naive T cells diverted into T_h17 developmental pathway (29, 30). Studies have shown that the production of IL-12 is associated with functional reprogramming of T_reg cells into IFN-γ-producing Th1-like cells (31) and knockdown of GILZ in dendritic cells increases the secretion of IL-12 and T-cell induction (32). These results are in line with our data that up-regulation of Tregs by GILZ through Del-1 decreases the production of IL-6 and IL-12 (Fig. 5). Further, in their studies, Ciucurel et al. (33, 34) showed that Del-1 has a bimodal effect on inflammatory cell recruitment, depending on the biologic context. Consistent with their conclusion, our data suggest that Del-1 acts as an immunomodulator by launching the balance between inflammatory responses and immune-regulatory mechanisms to re-establish and maintain the homeostatic mode of the microenvironment of the tissues. In fact, this could be potentially a partial answer to the question raised by Khader (35) of whether Del-1 is a universal negative regulator of IL-17 in different types of tissues. Our data strongly suggest that Del-1 may insert its regulatory effect in containing IL-17 function through GILZ by promoting the conversion of TH-17 cells into Tregs. Furthermore, while in the process of reporting this work, 2 relevant studies were published: the first study by Zhao et al. (36) showed that lentivirus-mediated overexpression of Del-1 in MSCs can effectively alleviate LPS-induced lung injury, decrease the number of neutrophils in bronchoalveolar lavage, and decrease serum levels of TNF-α and IL-6 in mice, and the second study by Luz-Crawford et al. (37) showed that BMSCs deficient of GILZ (cells isolated from GILZ knockout mice) have reduced immune suppressive functions. Our data bridge these 2 studies together and thus establishes GILZ-Del-1 axis as a critical pathway facilitating the communications between mesenchymal and hematopoietic lineage cells in the bone microenvironment and shed new lights on our current understanding of the mechanisms by which MSCs exert their potent anti-inflammatory actions (Fig. 7).

Figure 7.

Proposed schematic model illustrating how MSC may regulate T_reg cell production via GILZ-Del-1 axis. GILZ expressed in MSCs increases the expression and release of Del-1, which in turn increases the production of IL-10 and decreases IL-6 and IL-12, thus promoting T_reg cell production.

Glossary

BMSC

bone marrow-derived mesenchymal stem cell

Del-1

developmental endothelial locus-1

EPC

endothelial progenitor cell

FBS

fetal bovine serum

GC

glucocorticoid

GILZ

glucocorticoid-induced leucine zipper

HSC

hematopoietic stem cell

MSC

mesenchymal stem cell

Tg

transgenic

T_reg

regulatory T

WT

wild-type