Mesenchymal stem cells reprogram host macrophages to attenuate obliterative bronchiolitis in murine orthotopic tracheal transplantation

International Immunopharmacology 15 (2013) 726–734

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Mesenchymal stem cells reprogram host macrophages to attenuate obliterative bronchiolitis in murine orthotopic tracheal transplantation Zhixiang Guo a, 1, Xiaohui Zhou a, b, 1, Jing Li a, b, Qingshu Meng a, b, Hao Cao a, Le Kang a, Yinkai Ni a, Huimin Fan a, b,⁎, Zhongmin Liu a, b,⁎ a b

Department of Cardiovascular and Thoracic Surgery, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China Research Center for Translational Medicine, Tongji University School of Medicine, Shanghai 200120, China

a r t i c l e

i n f o

Article history: Received 10 December 2012 Received in revised form 4 February 2013 Accepted 1 March 2013 Available online 15 March 2013 Keywords: Obliterative bronchiolitis Tracheal transplantation Mesenchymal stem cells Macrophage PGE2 IL-10

a b s t r a c t After lung transplantation, obliterative bronchiolitis (OB) is one of the major limitations for the long-term survival of allografts. At present, effective treatment to prevent this phenomenon remains elusive. Mesenchymal stem cells (MSCs) are capable of modulating the immune system through the interaction with a wide range of immune cells. Here, we found that treatment of mice with bone marrow derived MSCs prevents the development of airway occlusion and increased IL-10 levels in trachea grafts, which was eliminated by the depletion of macrophages. Mechanistically, MSCs-derived PGE2, through the receptors EP2 and EP4, promoted the release of IL-10 and inhibited the production of IL-6 and TNF-α by macrophages. These results suggest that MSCs can both decrease the innate inflammatory responses and prevent allograft rejection by down-regulating the levels of IL-6 and TNF-α and increasing IL-10 production respectively. For easy availability and immune privilege, MSC-based treatment of OB provides an effective strategy for regulation of immune responses in lung transplantation. © 2013 Elsevier B.V. All rights reserved.

1. Introduction For many end-stage pulmonary diseases, the only available therapy is lung transplantation. However, long-term allograft survival in lung transplantation is hampered by chronic graft dysfunction, of which the greatest risk is caused by OB and bronchiolitis obliterans syndrome (BOS). After transplantation, 49% recipients developed BOS in 5 years and 75% in 10 years [1]. Despite advances in early diagnosis and care of lung transplant recipients, there is little improvement on total disease-related mortality [2], where acute cellular rejection remains a risk factor for the development of BOS [3]. The causes of post-transplant OB are not fully understood. Recent studies have shown that the absence of immunosuppressive macrophages may increase the susceptibility of human lung allografts to

Abbreviations: MSCs, mesenchymal stem cells; OB, obliterative bronchiolitis; OAD, obliterative airway disease; BOS, bronchiolitis obliterans syndrome; SPF, specific pathogen free; C57BL/6, C57BL/6 mouse; BALB/c, BALB/c mouse; PGE2, prostaglandin E2; LPS, lipopolysaccharide; IL, interleukin; IgG, goat isotype immunoglobulin; FACS, fluorescence-activated cell sorting; Elisa, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; POD, postoperative day; OTT, orthotopic tracheal transplantation; HTT, hereorthotopic tracheal transplantation. ⁎ Corresponding authors at: Department of Cardiovascular and Thoracic Surgery, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China. E-mail addresses: [email protected] (H. Fan), [email protected] (Z. Liu). 1 Contributed equally to this work. 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.03.002

the rejection process [4]. Macrophages secrete both proinflammatory and anti-inflammatory mediators, playing a critical role in innate immune responses associated to OB [5–7]. IL-10, an anti-inflammatory cytokine from macrophages [8,9], has been shown attenuating acute immune-complex-induced alveolitis in the rat lung [10]. Consistent with its immunosuppressive role, IL-10 also appears to play a protective role in transplant models [11,12]. Mesenchymal stem cells (MSCs), multipotent progenitor cells derived from bone marrow, contribute to the maintenance and regeneration of various connective tissues including bone, adipose, cartilage and muscle [13]. On the other hand, MSCs also exert potent immunomodulatory effects in many pulmonary conditions [14], and decrease tissue damage and/or increase repair in response to injury when migrating to the lung [15–17]. Interestingly, MSCs can markedly suppress the production of the inflammatory cytokines TNF-α, IL-6, and IFN-γ from murine macrophages stimulated with LPS, while increase the production of IL-10 [18]. To date, several animal models were established for the study of OB after lung transplantation, including orthotopic lung transplantation, orthotopic tracheal transplantation (OTT) and heterotopic tracheal transplantation(HTT) [19]. The mouse model of OTT is an easier procedure with high reproducibility in which a process of epithelial damage was associated with tissue remodeling, resulting in OB. Orthotopic tracheal grafts retain airway connections to the environment and generate a form of obstructive airway disease (OAD). OTT may therefore serve as a better clinical model of lung transplantation.

Z. Guo et al. / International Immunopharmacology 15 (2013) 726–734

Here, we report that adoptive transfer of MSCs ameliorated the development of OB through regulating the function of macrophages in a murine model of OTT. We found that PGE2 and its down-stream pathways play a key role in MSCs' regulation of airway rejective responses. These data provide novel therapeutic strategies to promote the long term allograft survival of lung transplants. 2. Materials and methods 2.1. Animal maintenance Specific pathogen-free, female and male mice (C57BL/6; BALB/c) were purchased from Shanghai Laboratory Animal Company (Shanghai, China). 6–10 week-old mice were maintained on a 12-h light/dark cycle at 25 °C and were provided free access to commercial rodent food and tap water before the experiments. All experiments using mice were performed in accordance with protocols approved by the institutional animal care and use committee of Tongji University. 2.2. Trachea transplantation OTT was performed as previously described [20]. Briefly, seven or eight rings of donor trachea were implanted end-to-end into the recipient via a midline cervical incision. Mice that died due to surgical technical failures within the first 24 h following transplantation were excluded in the study. 2.3. Experimental groups OTT was performed in a syngeneic (C57BL6-to-C57BL6; syngeneic group) and allogeneic setting (BALB/c-to-C57BL6; allogeneic group, MSC group and MSCs + indomethacin group). Recipients of allografts were randomly assigned to receive physiological saline. 1 million bone marrow-derived MSCs were given to mice intravenously with or without indomethacin (10 mg/kg, subcutaneously, Cayman Chemicals, USA) in the treatment group, and 300 μl of physiological saline in control group 1 h prior to transplantation. Seven grafts of each group were harvested on POD 7, POD 14 and POD 30 (see Table 1). 2.4. Mouse MSC cultures MSCs were isolated and cultured using standard protocols [21]. Bone marrow cells from 6 week-old C57BL/6 mice were collected by flushing the femurs and tibias in aseptic conditions with Alpha-MEM (Gibco, USA) medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gibco, USA). Erythrocyte-depleted bone marrow cells were plated at a density of 4 × 106 cells per cm2 in Alpha-MEM medium supplemented with 10% FBS, 1% L-glutamine (Gibco, USA), 100 IU/ml penicillin and 100 mg/ml streptomycin (Gibco, USA) and cultured at 37 °C in 5% CO2. Culture medium was changed at day 2 to remove

Table 1 Study groups. Strains

Syngeneic group

C57BL6– C57BL6 Allogeneic group BALB/c– C57BL6 MSCs group BALB/c– C57BL6 MSCs + indomethacin BALB/c– group C57BL6

Treatment

Numbers POD 7

POD 14

POD 30

Physiological saline

6

6

6

Physiological saline

6

6

6

MSCs

6

6

6

MSCs + indomethacin 6

6

6

727

non-adherent cells. Whole medium was subsequently replaced every 3 days. The cells were grown for 7 days until almost confluent. Adherent cells were then detached using 0.25% trypsin-EDTA and replated at a 1:3 dilution until passage 2. Subsequent passaging and seeding of the cells were performed at a density of 5000 cells per cm2. To exclude macrophage contamination, we used magnetic cell sorting (Miltenyi Biotech, Auburn, Invitrogen, USA) to deplete the CD11b (macrophage marker) positive cells (Biolegend, USA). Osteogenic and adipogenic differentiation assays were performed as previously described [22]. For standard osteogenic differentiation, confluent monolayers of MSCs were incubated in medium supplemented with 10−8 M dexamethasone (SERVA, Germany), 50 μM L-ascorbic acid (Sigma-Aldrich, USA), and 10 mM β-glycerol phosphate (SigmaAldrich, USA) with changes of medium every 3 days. After 21 days the cultures were fixed with 70% cold ethanol for 1 h at room temperature, and incubated with Alizarin Red S (Sigma-Aldrich, USA) (1% aqueous solution, pH 4.0, adjusted with ammonium hydroxide) for 5 min. Excess stain was removed by washing four times with PBS. For standard adipogenic differentiation, confluent monolayers of MSCs were incubated in medium supplemented with 10−8 M dexamethasone, 50 μg/ml indomethacine (Sigma-Aldrich, USA) and 10−4 M L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA) with changes of medium every 3 days. After 21 days, cultures were fixed in 4% paraformaldehyde in PBS for 10 min and stained with Oil Red O (Sigma-Aldrich, USA). The phenotype of MSCs was analyzed by flow cytometry on a FACScan flowcytometer (BD Biosciences, NJ, USA). The following mAbs were used: fluorescein isothiocyanate (FITC)-labeled, anti-CD11b, anti-CD45, antiSCA-1, (APC)-labeled, anti-CD34, phycoerythrin (PE)-labeled antiCD105, and anti-CD29 (Biolegend, USA). Isotype controls were used in all cases.

2.5. Co-culture of macrophages and mesenchymal stromal cells The MH-s murine alveolar macrophage cell line was purchased from the American Type Culture Collection, maintained as a continuous culture as previously described [23]. MSCs were plated in 48-well flat-bottom plates, 5 × 10 5 MH-s cells were added to each well (MSCs: MH-s ratio 1:8). For the cyclooxygenase (COX) activity assay, cells were grown in T-75 flasks, and 25 million cells were assayed for each condition. Controls included MH-s and MSCs cultured alone. Cells were cultured 24 h with or without 300 ng/ml LPS (Sigma, USA). When performing inhibitor/neutralizing antibody experiments, compounds or antibodies were added at the initiation of the coculture.

2.6. Analysis of cytokine production by ELISA MH-s, MSCs, and MH-s plus MSCs were cultured for 24 h with or without LPS and the following agents in the supernatants were evaluated by ELISA: IL-10 (Biolegend, USA) and PGE2 (Cayman Chemical Company, USA), following the manufacturer's instructions. The homogenate supernatants were harvested and total protein concentrations in the supernatants were measured and analyzed for the expression of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A and IL-10 by ELISA (Biolegend, USA).

2.7. Phagocytosis of zymosan particles by macrophages MH-s were cultured in the absence or presence of MSCs (MH-s: MSCs ratio = 8:1) with or without LPS (300 ng/ml) for 24 h. FITCzymosan particles (200 μg/ml) were added to the co-culture system and incubated for another 1 h. Then, phagocytosis of MH-s was evaluated on flow cytometer.

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2.8. Analysis of cytokine production by intracellular staining and flow cytometry

3. Results 3.1. MSC characterization

The production of IL-10 was analyzed by intracellular staining and flow cytometry. MH-s, MSCs, and MH-s plus MSCs were incubated for 18 h with or without LPS, inomycin (500 ng/ml) and PMA (50 ng/ml) were added. Monesin (1000×) (Sigma, USA) was added at 19 h of culture to inhibit the release of cytokines. After 24 h of culture, cells were then stained with FITC-labeled antibodies directed to cell surface F4/80, and stained with PE-labeled antibodies directed to intracellular IL-10 (BD Biosciences, USA).

2.9. Nitrite assay Nitrite was measured colorimetrically. Nitrite concentrations were estimated by comparing to a standard curve prepared with sodium nitrite in complete medium [24].

In flow cytometric analysis, MSCs demonstrated expression of the typical surface markers, Sca-1 (stem cell antigen-1), CD105 and CD29, and the absence of hematopoietic and endothelial markers, such as CD11b, CD45 and CD34 (Fig. 1A). Morphologically, these cells had a spindled, fibroblast appearance after expansion (Fig. 1B). We then tested the differentiation capacity of the isolated MSCs. Upon induction of the MSCs in adipocyte-differentiation media, the MSCs readily differentiated into cells containing lipid droplets indicated by colorant Oil Red O staining, while culture of MSCs in osteogenic-differentiation media resulted in the formation of calcium containing precipitates visualized by Alizarin Red S staining (Fig. 1C, D). These experiments confirmed the typical property of MSCs that were utilized for our downstream experiments. 3.2. Evaluation of the transplantation models

2.10. Real-time PCR mRNA of cyclooxygenase-2 (COX2) expression levels were quantified by real time quantitative PCR. RT-PCR was performed as described previously [25].

2.11. Ex vivo studies on isolated lung macrophages We sacrificed OTT mice 24 h after operation with or without intravenous injection of 1 million MSCs. We removed the lungs, minced them into small pieces and then incubated the pieces in RPMI 1640 medium with 1% penicillin–streptomycin and 1% L-glutamine for 30 min at 37 °C 5% CO2 in the presence of collagenase type 1 (300 U ml −1) and DNase I (50 U ml −1) (Worthington Biochemicals). After the incubation, we filtered the cell suspension through a 70-mm cell strainer, and then cells were washed with complete RPMI medium. We incubated the resulting cells for 15 min at 4 °C with anti-CD11b (Biolegend, USA) magnetic beads and subsequently applied them to MS columns (Miltenyi) for the positive selection of CD11b cells. We then plated the CD11b negative cells into 96-well plates at a concentration of 50,000 cells per well per 200 μl medium containing 10 μg/ml LPS. After incubating the cells for 17–18 h we added 500 ng/ml ionomycin and 50 ng/ml PMA for 6 h and then collected the supernatants for the detection of IL-10 by ELISA.

2.12. Preparation of liposomal encapsulated clodronate and depletion of macrophages Multilamellar liposomes were prepared as previously described [26,27]. For the depletion of macrophages, liposomal clodronate (50 mg/kg and 30 mg/kg) was injected intravenously at 48 and 24 h before tracheal transplantation. Plain liposomes were injected into a group of tracheal transplantation animals as a control.

We first performed syngeneic and allogeneic OTT in mice and most recipient mice survived throughout the study period (>94%). Airway stenosis was approximately 46.19 ± 5.46% on postoperative day (POD) 7, 39.40 ± 4.36% on POD 14 and 32.57 ± 4.52% on POD 30 in the allogeneic group. On PODs 2–3, the allo-recipients began to show signs of shortness of breath, mild stridor and reduced activity. From POD 7, these symptoms improved gradually. The syngeneic-recipients had almost no symptoms. No surgery-related complications, such as anastomosis dehiscence, were observed during the course of the study. Gross examination at harvest showed patent lumens of all tracheal segments in all the groups. 3.3. MSCs attenuate airway stenosis in allograft transplant recipients In order to assess the effects of MSC treatment on acute allotracheal rejection, tracheas were harvested on PODs 7, 14 and 30, fixed in formalin and then paraffinized and sectioned for staining with H&E. We next analyzed the histological changes of the graft by H&E staining to evaluate the development and severity of OB. Syngeneic tracheal grafts did not develop luminal obliteration, and they were morphologically indistinguishable from naive tracheas. There were signs of a large number of inflammatory cell infiltrates and the epithelial cells appeared flattened without ciliation in the allogeneic grafts (Fig. 2A). We observed that a markedly higher proportion of physiologic, ciliated, columnar epithelia were present in allografts treated with MSCs compared with the control. Adoptive transfer of MSCs markedly attenuated the development of luminal stenosis caused by submucous fibroproliferation that consisted of circularly oriented cells and connective tissue fibers. Allografts in animals showed increased endomembrane thickness, while MSC treatment attenuated submucous fibroproliferation significantly (32.34 ± 3.60% vs. 45.21% ± 5.45% on POD 7, 25.64 ± 4.36% vs. 39.40% ± 4.61% on POD 14, 20.87 ± 4.52% vs. 32.57% ± 2.99% on POD 30) (P b 0.05 vs. allogeneic group or MSCs + indomethacin group) (Fig. 2B). 3.4. Effect of MSC on intragraft cytokine concentrations

2.13. Statistical analyses Data are presented as mean ± standard deviation. Comparisons between the groups were carried out by independent sample t-tests or analysis of variance between the groups with the Tukey's Multiple Comparison test, where appropriate. Probability values (P) of less than 0.05 were considered significant. Statistical analysis was performed on the SPSS statistical software package 17.0 for Windows (SPSS Inc., Chicago, IL).

We hypothesized that MSCs might down-regulate the inflammatory immune response; therefore, we analyzed the expression of several cytokines in trachea grafts. 7 days after injection of MSCs, mice were sacrificed, tracheas were harvested and homogenate supernatants were harvested and analyzed for the expression of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A and IL-10 by ELISA. IL-10 levels in intragrafts from the MSC treatment (MSC group) increased significantly (P b 0.05), while IL-2 (P b 0.05), IL-6 (P b 0.05), IFN-γ (P b 0.05) and TNF (P b 0.05) production decreased, compared to the allogeneic group.

Z. Guo et al. / International Immunopharmacology 15 (2013) 726–734

729

A

control

CD105

CD11b

CD45

B

CD29

Sca-1

CD34

C

D

Fig. 1. Characterization of bone marrow-derived mesenchymal stromal cells (MSCs). MSCs were isolated from bone marrow of adult C57BL/6J and cultured using standard protocols. (A) Analysis of the phenotype of MSCs by flow cytometry. (B) Morphology of MSCs (×200). (C) Culture of MSCs in adipocyte-differentiation media showing cells containing drops of fat revealed by Oil Red O (×200). (D) Culture of MSCs in osteogenic-differentiation media showing the formation of calcium containing precipitates stained by Alizarin Red S. A representative experiment is shown (×200).

Day 7 (400X)

Syngeneic

Allogeneic

MSC

MSC indometacin

B 60

luminal obliteration (%) Sy A ng l e lo n SC ge ei c in do nei m MS c Sy eta C c A nge in M SC llo ne ge i c in do nei m MS c Sy eta C n c A ge in M SC llo ne ge ic in do nei m MS c et C ac in

A

day 7

40

Day 14 (400X)

day 14 day 30

*

*

*

20

0

M

Day 30 (400X)

Day 7 (100X)

Fig. 2. Adoptive transfer of MSCs attenuated the pathology of trachea allograft. (A) H&E staining of representative graft sections at indicated time points (n = 6 per group per time point). Panel A showed the representative data of 3 separated experiments. Magnification was ×100 or ×400. (B) Statistical analysis of luminal obliteration of orthotopic tracheal graft from MSCs or control treated groups at the indicated time points (n = 6 per group per time point, P b 0.05*, MSCs vs. control group).

Z. Guo et al. / International Immunopharmacology 15 (2013) 726–734

3.5. MSCs protect allograft survival by increasing macrophage-derived IL-10 expression

5

20

4

15

*

10

3 2

MH-s cell line was used as a macrophage model to explore the molecular basis of the MSCs–macrophage interaction. Co-cultures were carried out in the presence of 300 ng/ml LPS. An antibody against macrosialin (CD68) (Biolegend, USA) was used to identify the presence of activated macrophages. CD68+ cells were found in all of the samples that were tested (Fig. 5A, B). CD68 was present at a higher level in MSC/ MH-s co-cultures (P b 0.05). However, CD68 expression was lower in the control samples and co-cultures treated with an iNOS inhibitor (L-NAME iNOS inhibitor, 1 mM) (Sigma-Aldrich, USA) (Fig. 5A, B). We then analyzed whether MH-s cells cultured in the presence of MSCs displayed higher phagocyte ability. Experiments were performed by incubating MH-s and MSCs (MH-s: MSC ratio = 8:1) with FITClabeled zymosan particles (250 μg/ml), for 1 h at 37 °C. Phagocytosis was evaluated by flow cytometer. MH-s cells cultured with MSCs showed no changes in their ability to phagocytoze zymosan particles compared to MH-s only (P > 0.05) (Fig. 5C). These data showed that MSCs could potentially increase the activation of macrophages but does not affect their ability to phagocytoze zymosan particles. Next we decided to examine whether MSCs could modulate the production of cytokines by MH-s cells. MH-s cells were cultured overnight in the absence or presence of MSCs (MH-s: MSCs ratio = 8:1) and with or without LPS (300 ng/ml). The presence of IL-10 was then evaluated in the supernatants by ELISA (Fig. 5D). Addition of LPS stimulates the production of IL-10 slightly (P > 0.05) by MH-s cells, but IL-10 production was triggered when MH-s cells were co-cultured with MSCs (Fig. 5D). To evaluate the possible contribution of MSCs to the production of cytokines observed in co-cultures of MH-s and MSCs stimulated by LPS, the presence of IL-10 was analyzed by intracellular staining and flow cytometry. Activation by LPS and ionomycin and PMA for 6 h, and monesin for 5 h resulted in the stimulation of IL-10 production by MH-s but not by MSCs (Fig. 5E, F) and MSC/MH-s coculture with LPS enhance the production of IL-10 (Fig. 5E, F). These data suggest that MSCs mediated the increase of IL-10 production by MH-s cells. Then, we explored the potential molecular basis of the interaction between MSCs and macrophages. MSCs cocultured with MH-s in the presence of LPS can induce the production of nitric oxide (NO) [31]. The latter plays a critical role in the release of PGE2 by direct activation of COX [35]. MSCs have been shown to produce prostaglandin E2 and possibly affect other immune cells via EP1–EP4 receptors

1

60

20

0

0

80

10

30

60

8

20 0

*

6 4 2 0

*

40

0

40

100

80

IL-10(pg/ml)

TNF(pg/ml)

5

IL-4(pg/ml)

25

IL-17A(pg/ml)

IL-2(pg/ml)

Since IL-10 levels in trachea grafts increased significantly in OTT mice that were treated with MSCs compared to that in the control group, we asked whether anti-inflammatory cytokine IL-10 might be important for the protective function of MSCs. IL-10 are known to be mainly produced by subsets of macrophages [28], So we postulated that MSCs may affect macrophages to increase IL-10 level. As PGE2 has been reported to mediate multiple immune suppressive effects of MSCs [29–34], we treated mice with indomethacin, a COX inhibitor, which could inhibit the release of PGE2. We found that the ability of MSCs to down-regulate the production of inflammatory cytokines and up-regulate the production of IL-10 was strongly Inhibited (Fig. 3A, B). Allografts in mice that received indomethacin also showed increased endomembrane thickness (Fig. 2A), Airway obliteration was approximately 43.36% ± 4.52% on POD 7, 35.38% ± 4.55% on POD 14, 30.48% ± 4.23% on POD 30 (Fig. 2B). To evaluate the significance of macrophage-dependent IL-10 production in vivo, we depleted macrophages in mice by clodronateloaded liposome. The numbers of macrophages in spleen and lung increased greatly in allogeneic OTT mice compared to syngeneic OTT mice (data not show). This increase was completely eliminated when macrophages were depleted with clodronate-filled liposomes (Fig. 4A, B). IL-10 levels in trachea graft, as detected by ELISA, was significantly lower in MSCs plus clodronate liposome group than in MSCs with empty liposome group (P b 0.05) (Fig. 4C). Allografts in mice that received MSCs plus clodronate liposome showed increased endomembrane thickness (Fig. 4D). The beneficial effect of MSCs was eliminated by macrophage depletion. This shows that macrophages are required for the increased IL-10 production mediated by MSCs. To assess the macrophage-dependent IL-10 production in OTT mice, macrophages (CD11b+) were isolated from the lungs of MSCtreated and untreated mice, then placed in culture wells and stimulated with LPS for 24 h, and ionomycin and PMA for 6 h. Macrophages from MSC-treated OTT mice produced more IL-10 than the untreated mice (P b 0.05) (Fig. 4E), showing that MSC treatment increased the IL-10 production by macrophages in vivo.

3.6. Molecular basis of the MSCs–macrophage interaction

IFN-γ(pg/ml)

However, MSC delivery did not affect the production of IL-17 or IL-4 in trachea graft significantly on POD 7 (Fig. 3).

IL-6(pg/ml)

730

80 60

*

40 20 0

*

20

10

0

Fig. 3. Intragraft cytokine expression profiles by ELISA. Homogenate supernatants were harvested on POD 7 and analyzed for the expression of IL-2, IL-4, IL-6, IFN-γ, TNF, IL-17A and IL-10, measured by ELISA. Intragraft cytokine expression differences between the MSC group vs. allogeneic group or MSCs + indomethacin group were compared using one-way analysis of variance followed by the Tukey's Multiple Comparison test. P b 0.05* was considered significant.

Z. Guo et al. / International Immunopharmacology 15 (2013) 726–734

A

OTT+pbs

OTT+MSC+empty liposome

B

OTT+MSC+clodronate liposome

9.53

Spleen

35.80

38.96

16.79

lung

pbs MSC+empty liposome MSC+clodronate liposome

40 30

*

20 10 0 50

macrophages

43.53

macrophages

50

38.90

731

pbs MSC+empty liposome MSC+clodronate liposome

40 30

*

20 10 0

CD11b

D

C pbs MSC+empty liposome MSC+clodronate liposome

250

IL-10(pg/ml)

IL-10(pg/ml)

40

E

30 20

*

200 150

* normal Isograft Allograft+PBS Allograft+MSC

100

10

50

0

0

Fig. 4. Quantification of IL-10 was detected after macrophage was isolated or depleted. Macrophages were isolated from lungs and spleen of allogeneic mice OTT mice with PBS treatment, MSCs + empty clodronate treatment or MSCs + clodronate liposome treatment and detected by FACScan flowcytometer, M reduced notably (MSCs + clodronate liposome vs. MSCs + empty clodronate, P b 0.05) (A, B). Then intragraft IL-10 of these mouse was detected by ELISA, quantification of IL-10 was lower (MSCs + clodronate liposome vs. MSCs + empty clodronate, P b 0.05) (C) and allografts in animals that received MSCs + clodronate liposomes showed increased endomembrane thickness, magnification was ×100 or ×400 (D). 24 h after the operation of mice OTT with or without MSC treatment, lung macrophages were isolated (four mice per group), cultured and treated ex vivo with LPS for 24 h, inomycin and PMA for 6 h, IL-10 production of these isolated macrophages was shown by ELISA, quantification of IL-10 was higher in allograft MSC group (allograft MSC group vs. allograft P b 0.05) (E).

[36,37]. Macrophage expresses EP receptors [38]. So we examined the NO productions in MH-s or co-culture system with MSCs after LPS stimulation. Supernatants from co-culture systems were collected and analyzed after 24 h. We found that the amount of NO was significantly increased (Fig. 6A) in the MH-s/MSC coculture systems after LPS stimulation (P b 0.05). Furthermore, inducible nitric oxide synthase iNOS inhibitor decreased the production of NO. NO directly activate COX, and then mediate the COX down-stream signaling pathway. COX2 produces the substrate for prostaglandin synthase enzymes. The expression of COX2 increased significantly in MSCs after 24 h LPS stimulation (P b 0.05) (Fig. 6B), while iNOS inhibition resulted in a significant reduction in COX2 enzyme activity and the production of IL-10 after 24 h LPS stimulation (Fig. 6D). Then, PGE2 levels were assessed in co-culture systems with or without specific inhibitors. Supernatants from MSC/MH-s co-cultures of 24 h duration (with LPS stimulation) were analyzed for the presence of PGE2 (Fig. 6C). Neither MSCs nor MH-s cells cultured alone generated high levels of PGE2. In contrast, MSC/MH-s co-cultures had a significant accumulation of PGE2 after 24 h (P b 0.05) (Fig. 6C). Interestingly, PGE2 production also declined upon treatment with indomethacin (5 μM) and iNOS inhibitor. As it seemed that PGE2 might mediate the effect of the up-regulation of IL-10 by macrophages, we asked whether specific EP receptors could be involved in transducing this prostaglandin signal. To answer this question, we used prostaglandin receptor antagonists. Both EP2 (AH 6809 EP2 antagonist, 10 μM) and EP4 (GW 627368X EP4 antagonist, Cayman Chemical Company, USA) receptor antagonists prevented the increase of IL-10 in the MH-s/MHC cocultures supplemented with LPS.

The increase of IL-10 was also eliminated when MSCs were treated with iNOS inhibitor or indomethacin containing cultures (Fig. 6D). This suggests that MSCs require nitric oxide to achieve their effect on the production of IL-10 in these co-cultures. Thus, on the basis of our in vitro studies, our predicted model was that MSCs may respond to the presence of inflammatory agents by increasing prostaglandin E2 synthesis and secretion, which subsequently activates prostaglandin E2 and E4 receptors on macrophages resulting in IL-10 induction. 4. Discussion OB remains a major obstacle to long-term allograft survival after pulmonary transplantation. After lung transplantation, almost 75% of transplant patients eventually develop a chronic syndrome involving progressive airway narrowing known as BOS [1]. Here, we sought to investigate the role of MSCs in the occurrence and development of OB in a mouse model of OTT. Although murine orthotopic lung transplant models mimic the surgical procedure of the human transplant, their use for OB studies show several limitations because of the time-consuming, technical demanding, and high histological variability in the development of airway lesions. The mouse OTT model is an easier procedure with high reproducibility and has been used extensively to study the immune aspects of airway obstruction and the process of epithelial damage associated with tissue remodeling that results in OB [19]. Our previous studies also showed that OTT undergo acute rejection and accurately models transplantation-associated changes in the large airways in rat models [39,40].

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Z. Guo et al. / International Immunopharmacology 15 (2013) 726–734

A

B CD68

20

15.63

*

7.66

15

CD68 %

5.60

MSC+MH-S

MH-S

D

MH-S

2.4

MH-S+LPS

2.3

5

500

IL-10(pg/ml)

0.3

10

0

F4/80 MSC+MH-S+iNOS inhibitor

C

MH-S MSC+MH-S MSC+MH-S+iNOS inhibitor

400 300

MH-S MSC MSC+MHS MHS+LPS MSC+LPS MSC+MHS+LPS

*

200 100 0

beads MSC+MH-S+LPS

0.00

0.44

1.79

97.76

0.00

0.52

8. 89

90.59

0.02

4.80

6.91

88.27

0.04

5.56

13.16

81.24 F4/80

MH-S

MH-S+LPS

MSC+MH-S

percentage of IL-10+ / F4/80+ cells

F IL-10

E

20 15

MH-S MH-S+LPS MSC+MH-S MSC+MH-S+LPS

*

10 5 0

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Fig. 5. The presence of CD68+ macrophages and the production of IL-10 by macrophages. MH-s were cultured overnight in the absence or presence of MSCs (MH-s: MSCs ratio 8:1) or iNOS inhibitor and stimulated with LPS. The dot plots indicate the percentages of CD68+ macrophages (MSCs + MH-s vs. MH-s or MSCs + MH-S + iNOS inhibitor, P b 0.05) (A, B). MH-s were cultured 24 h in the absence or presence of MSCs MH-s: MSC ratio = 8:1) with or without LPS (300 ng/ml). FITC-zymosan particles (200 μg/ml) were added to the co-culture system and incubated for another 1 h. Then, phagocytosis of MH-s was evaluated on flow cytometer (P > 0.05) (C). MH-s were cultured 24 h in the absence or presence of MSCs (MH-s: MSC ratio = 8:1) with or without LPS (300 ng/ml), and cytokines were analyzed in cell-supernatants by ELISA (D) or by intracellular staining and flow cytometry (E, F). MSC/MH-s coculture with LPS enhances the production of IL-10 (D, E, F). (E) IL-10 was produced by MH-S but not by MSCs. Panel F indicates the percentages of IL-10 producing cells among F4/80+ cells (macrophages).

Although little is known about the in vivo behavior of MSCs, in many clinical studies, MSCs, autologous or allogeneic, have been shown to be potentially efficacious for treatment of a wide variety of clinical conditions. MSCs have emerged as a promising therapeutic tool in cell therapy and have been introduced in the clinic with encouraging preliminary results [29,41]. MSCs were first reported to have a potent immunosuppressive effect in vivo in humans in 2004, a patient with severe treatment-resistant grade IV acute graftversus-host disease of the gut and liver, disease was successfully treated with transplanted haploidentical mesenchymal stem cells [42]. Then, cases of hemorrhagic cystitis, pneumomediastinum and peritonitis were treated with allogeneic mesenchymal stem cells. The mechanisms in which MSCs mediate the therapeutic properties have not been clearly defined. Studies suggest that the therapeutic abilities of MSCs in a variety of clinical settings are due to their anti-inflammatory and immunomodulatory properties, which have been validated in vitro and in vivo in a number of animal models related to either alloreactive immunity, autoimmunity or anti-tumor immunity [29,41,43–45]. MSCs are known to inhibit T cell proliferation [46] and modulate B cell function [47]. Our observations showed that adoptive transfer of bone-marrow derived MSCs significantly upregulates IL-10 production, while decreased levels of inflammatory cytokines including IL-6, IFN-γ and TNF-α in trachea grafts in OTT mice. Endogenous IL-10 were proved to play a key role in regulating the development

of OB [11]. Therefore, the beneficial effect of MSCs may dependent upon the IL-10 production. Recently, we found subsets of macrophages play important roles in the initiation and development of OB in mouse OTT models (data not shown). Upon activation, macrophages undergo differentiation and polarization into different subsets producing either anti-inflammatory cytokines or inflammatory cytokines according to microenvironmental stimuli. Large amounts of IL-10 are known to be produced by subsets of macrophages [28]. Based on previous studies, we hypothesized that MSCs would modulate the rejection response and inhibit the development of OB via regulating the function of macrophages. Macrophages are widely distributed in many different tissues in the human body and are key components of innate immunity. Macrophages display remarkable plasticity and can dramatically change their physiology in response to environmental stimuli. Surface expression of CD68 is one of the activation markers for macrophages [48]. Our data showed low levels of CD68 expression on the plasma membrane of resident MH-s. When MH-s was co-cultured with MSCs plus LPS, CD68 expression increased significantly. This provided clear evidence that MSCs promote the activation of MH-s in the presence of LPS. Clearance of apoptotic cells by macrophages plays a critical role in the resolution of inflammatory processes [49], so we explored whether MSCs increase the phagocytose of MH-s. In our phagocytose system, MSCs did not increase the uptake of zymosan particles by macrophages. This is consistent with previous work [18] that co-culture of macrophage and MSCs showed

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Fig. 6. Summary of studies of the molecular pathways involved in the interaction between MSCs and macrophages. MH-s were cultured 24 h in the absence or presence of MSCs (MH-s: MSC ratio = 8:1) with or without LPS (300 ng/ml, A, C, D all with LPS). Nitrite assays (A) and cytokines were analyzed in cell-supernatants by real-time PCR (B) and ELISA (C, D). MH-s + LPS (MSCs + LPS) group of Figure B were separated culture 24 h and added TRIZOL, then put them together to be quantified by Real time PCR. (C, D) Results are expressed in pg/ml. MSC/MH-s coculture enhances the production of NO, NO induces the production of COX2 (INOS inhibitor can block the process), resulting in increased production and release of PGE2 (COX non-selective inhibitor-indomethacin can restrain the process). PGE2 binds to EP2 and EP4 receptors on the macrophage, increasing its IL-10 secretion and reducing inflammation. MSC/MH-s coculture enhances the production of IL-10.

no enhancement of their ability to phagocytize zymosan particles. So, MSCs could potentially promote the activation of macrophages. Coculture system showed that MSCs promote the IL-10 production by MH-s, consistent with the finding that MSC-educated macrophages displayed a function of producing anti-inflammatory cytokines [31]. Interestingly, macrophages isolated from MSC-treated OTT mice produce significantly higher amounts of IL-10 than those from non-treated mice. Moreover, adoptive transfer of bone-marrow derived MSCs significantly upregulates IL-10 production, while decreased the levels of inflammatory cytokines including IL-6, IFN-γ and TNF-α in trachea grafts in OTT mice. These results suggest that MSCs switch the macrophages to an anti-inflammatory profile by suppressing the production of inflammatory cytokines. Previous data showed that injected MSCs interact with circulating and tissue (mostly lung) macrophages and reprogram them [31]. Of note, we found that macrophages treated with MSCs produce large amounts of NO and IL-10. The observation that the macrophages isolated from treated OTT mice produce significantly higher amounts of IL-10 than those from non-treated mice suggests a temporary reprogramming of macrophage function in vivo. Since IL-10 can be produced by variety of cell types, we depleted the macrophages by clodronate-loaded liposomes to evaluate the significance of macrophage-dependent IL-10 production in vivo. The beneficial effect of MSCs was eliminated by macrophage depletion. This shows that macrophages are the major source of increased IL-10 production mediated by MSCs. As airway obliteration is a progressive disease, we evaluated the progression of OB on day 7, day 14, and day 30 post-transplantation. Graft obliteration and fibrotication in orthotopic tracheal transplants were assessed after MSC injection by histological evaluation. Date showed that recipient mice that received MSCs had attenuated clinical and histological lung allograft rejection. Notably, allografts in the indomethacin plus MSC group showed more severe pathological changes than in the MSCs and syngeneic groups. Therefore, MSCs may prevent allograft rejection and the development of OB by increasing IL-10 production by macrophages. PGE2 is known to modulate immune responses and is widely viewed as a general immunosuppressant. Our data revealed that

macrophages treated with MSCs produce large amounts of NO. NO has been shown to mediate the release of PGE2 by direct activation of COX [35] and its down-stream signaling pathway. COX2 produces the substrate for prostaglandin synthase enzymes. In in vitro coculture system, MSC-derived PGE2 promoted the release of IL-10 and inhibited the production of IL-6 and TNF-α by macrophages. iNOS inhibition resulted in a significant reduction in COX2 enzyme activity and the production of IL-10. Furthermore, COX inhibitor eliminated the up-regulation of IL-10 by MSCs in in vivo and in vitro experiments. EP2 and EP4 receptors, among the four receptors (EP1–EP4 receptors) for PGE2 recognized so far, are mainly involved in the immunosuppressive effect of PGE2. EP2 and EP4 agonists significantly prolonged allograft survival compared with controls [50,51]. Our results showed that both EP2 and EP4 receptor antagonists prevented the increase of IL-10 in the MH-s/MHC co-cultures supplemented with LPS. The increase of IL-10 was also eliminated when MSCs were treated with iNOS inhibitor or indomethacin. This suggests that MSCs require nitric oxide and EP2, and EP4 to achieve their effect on the production of IL-10 in these co-culture systems. In conclusion, this study showed that MSCs inhibit the development of airway obstruction in a mouse model of OTT largely through the modulation of macrophages. The beneficial effect stems from MSC derived-prostaglandin E2, in EP2 and EP4 receptor dependent manner, act on macrophages to stimulate the production and release of IL-10, an anti-inflammatory cytokine. This information adds to the growing body of knowledge of the pathogenesis of OB in lung transplantation and raises the possibility of the future utilization of MSCs in the prevention of this devastating complication. Funding This work was supported by the National Natural Science Foundation of China (81070073, 81273263), Shanghai Municipal Science and Technology Commission of international cooperation projects (11410702000), Shanghai Municipal Program on Key Basic Research Project (11JC1410800, 801), 973 Program (2012CB526605), Shanghai Municipal Natural Science Foundation (11ZR1429600) and Shanghai

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Science and Technology Development Funds (12QA1402900). Key Disciplines Group Construction Project of Pudong Health Bureau of Shanghai (PWzxkq2010-01) and Outstanding Leaders Training Program of Pudong Health Bureau of Shanghai (PWR12011-01).

[22]

Conflict of interest

[24]

None declared.

[23]

[25]

Acknowledgments [26]

We thank Tsun Andy at Key Laboratory of Molecular Virology & Immunology, Unit of Molecular Immunology, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences for his critical comments on this manuscript.

[27]

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