Hepatocyte growth factor enhances the inflammation-alleviating effect of umbilical cord–derived mesenchymal stromal cells in a bronchiolitis obliterans model

Cytotherapy, 2016; 18: 402–412

UMBILICAL CORD CELLS

Hepatocyte growth factor enhances the inflammation-alleviating effect of umbilical cord–derived mesenchymal stromal cells in a bronchiolitis obliterans model

XIAO-PEI CAO1,2, DONG-MEI HAN3, LI ZHAO4, ZI-KUAN GUO2, FENG-JUN XIAO2, YI-KUN ZHANG2, XIAO-YAN ZHANG2, LI-SHENG WANG2, HENG-XIANG WANG3 & HUA WANG2 1

HeBei North University, Zhangjiakou, China, 2Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing, China, 3Department of Hematology, Air Force General Hospital, Beijing, China, and 4 Department of Experimental Pathology, Beijing Institute of Radiation Medicine, Beijing, China Abstract Background aims. Specific and effective therapy for prevention or reversal of bronchiolitis obliterans (BO) is lacking. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF) gene modified mesenchymal stromal cells (MSCs) on BO. Methods. A mouse model of experimental BO was established by subcutaneously transplanting the tracheas from C57BL/6 mice into Balb/C recipients, which were then administered saline, Ad-HGF-modified human umbilical cord-MSCs (MSCs-HGF) or Ad-Null-modified MSCs (MSCs-Null). The therapeutic effects of MSCs-Null and MSCsHGF were evaluated by using fluorescence-activated cell sorting (FACS) for lymphocyte immunophenotype of spleen, enzymelinked immunosorbent assay (ELISA) and real-time polymerase chain reaction (rt-PCR) for cytokine expression, and histopathological analysis for the transplanted trachea. Results. The histopathologic recovery of allograft tracheas was improved significantly after MSCs-Null and MSCs-HGF treatment and the beneficial effects were particularly observed in MSCs-HGF–treated mice. Furthermore, the allo-transplantation–induced immunophenotype disorders of the spleen, including regulatory T (Treg),T helper (Th)1,Th2 and Th17, were attenuated in both cell-treated groups. MSCs-HGF treatment reduced expression and secretion of inflammation cytokines interferon-gamma (IFN-γ), and increased expression of antiinflammatory cytokine interleukin (IL)-4 and IL-10. It also decreased the expression level of the profibrosis factor transforming growth factor (TGF)-β. Conclusion. Treatment of BO with HGF gene modified MSCs results in reduction of local inflammation and promotion in recovery of allograft trachea histopathology. These findings might provide an effective therapeutic strategy for BO. Key Words: Bronchiolitis obliterans, Hepatocyte growth factor, Th cells, Treg cells, Umbilical cord–derived mesenchymal stromal cells

Introduction Allogeneic transplantation has become a widely accepted treatment for hematologic malignancies. Despite new and better treatments for infectious complications and better immunosuppressive drug treatments, pulmonary complications are still the main cause of longterm morbidity and mortality following hematopoietic stem cell transplantation (HSCT) [1–3]. Bronchiolitis obliterans (BO) is a serious, progressive, noninfec-

tious, and fatal pulmonary complication.The underlying pathogenic mechanisms are poorly understood and chronic graft-versus-host disease (cGVHD) has been suggested to be involved in the development of BO [4,5]. At present, the treatment and prevention of BO remain a difficult issue. Furthermore, success in establishing a BO animal model has been limited by the lack of the pathogenesis of BO. A technically simple model of allograft airway transplantation established by Hertz et al. is one effective strategy used to

Correspondence: Heng-Xiang Wang, MD, Department of Hematology, Air Force General Hospital, Beijing 100142, China. E-mail: wanghengxiang123@ aliyun.com; Hua Wang, PhD, Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850, China. E-mail: [email protected] (Received 31 January 2015; accepted 26 December 2015) ISSN 1465-3249 Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.12.006

Treatment of BO with HGF modified MSCs investigate the pathogenesis and evaluate the treatment of experimental BO [6]. The histopathologic features of this experimental BO model largely reproduce the changes in human recipients who develop BO after allogeneic HSCT. By using this model, investigations have been performed to testify various new strategies for the treatment of BO, such as interleukin (IL)-10 (DNAX Corp.)[7], Tacrolimus (Astellas Pharma) [4] and Pirfenidone (Marnac, Inc., Dallas, TX) [8]. Mesenchymal stromal cells (MSCs) are adult stem cells characterized by their immune-regulation properties. Prochymal, an allogeneic stem cell therapy based on MSCs derived from the bone marrow of adult donors, is the first therapy officially approved in Canada for the treatment of acute graft-versus-host disease (GVHD) [9]. However, little is known about the therapeutic effect of MSCs on BO. Hepatocyte growth factor (HGF) is a multifunctional factor with the target cells from a variety of tissues. In animal models of bleomycin-induced lung fibrosis, HGF could modulate the epithelial-mesenchymal transition and reduce the fibrin deposition at least partially by inhibition of transforming growth factor (TGF)-β1 secretion, exerting its anti-fibrotic and antiinflammation effect [10]. However, the short half-life of HGF protein in vivo requires multiple dosing and intravenous drug delivery can not guarantee the deposition of HGF in the injured tissues. In the present study, the therapeutic effect of Ad-HGF modified MSCs on transplant-related BO was observed and the mechanisms were investigated.

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Balb/C and C57BL/6 were used as recipients and donors, respectively.Tracheal transplantation was performed as described by Hertz et al. [6]. Briefly, the donor mice were humanely killed by general anesthesia with sodium pentobarbital, and the trachea was revealed after an incision in the neck and separated from the subcutaneous tissue, cervical muscles and esophagus.The excised and isolated trachea was placed in ice-cold Euro-Collins solution for trachea preservation. The recipient mice were intraperitoneally anesthetized with sodium pentobarbital, and the trachea was implanted subcutaneously into the back of the mouse. The recipient mice were then injected with 1 × 106 human umbilical cord mesenchymal stromal cells (hUCMSCs) infected with Ad-Null or Ad-HGF of 150 multiplicity of infection (MOI) or saline via the tail vein 4 h after trachea implantation. At day 10 or 21 post-transplantation, 8 mice per group were euthanized and the serum was collected for determination of cytokines. The spleens were collected for lymphocyte phenotype analysis. The implanted trachea was removed for RNA isolation and histopathologic analysis. Histopathology evaluation Tracheas from C57BL/6 mice were implanted into the sub-cutaneous tissue of BALB/c mice, and the transplanted trachea was evaluated morphologically for airway rejection changes on days 10 and 21 post-operation. The transplanted tracheas were fixed in 10% formalin, embedded in paraffin, stained using hematoxylin and eosin (H-E) and analyzed with light microscopy.

Methods Isolation and culture of umbilical cord–derived MSCs Human umbilical cords were obtained after an informed consent from the Beijing Hospital of Chinese Traditional and Western Medicine in accordance with the Guidelines for the Use of Human Subjects. Umbilical cord–derived mesenchymal stromal cells (UC-MSCs) were isolated and culture expanded as described previously [11]. The cells at passages 3–5 were used in the experiments that follow. All of the procedures were also approved by the Ethics Committee of Beijing Institute of Radiation Medicine.

Computerized morphometry Images of HE-stained tracheal sections were taken with a high-resolution video camera attached to a Leica DM6000B microscope. Luminal occlusion was evaluated by determining the reduction in luminal area using Leica Qwin 3.4 software. The percentage of luminal obstruction in transplanted trachea was measured by outlining the inner surface of the cartilage and residual lumen. The percentage of airway obstruction was then calculated as follows: Area within the cartilage − area within residual lumen ×100% Area within the cartilage

Animal care Male C57BL/6 mice and female Balb/C mice (6–8 weeks old, 18–20 g) were obtained from the Academy of Military Medical Sciences and were housed and cared for in the pathogen-free facility. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals approved by the Committee of the Beijing Institute of Radiation Medicine.

Immunohistochemistry Paraffin-embedded implanted trachea sections were used for immunohistochemistry. For terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) of the tracheas, we used the DeadEnd Fluorometric TUNEL System (Promega Corporation) according to the manufacturer’s instructions. For TUNEL staining, positive cells show Buffy within

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Table I. Primers of cytokines. Name mβ-actin forward primer mβ-actin reverse primer mIL-10 forward primer mIL-10 reverse primer mIFN-γ forward primer mIFN-γ reverse primer mTGF-β forward primer mTGF-β reverse primer mIL-4 forward primer mIL-4 reverse primer

Sequences 5′-tttccagccttccttctt-3′ 5′-gtctttacggatgtcaacg-3′ 5′-aataagagcaaggcagtggag-3′ 5′-tgtatgcttctatgcagttgatga-3′ 5′-actaccttcttcagcaacagcaa-3′ 5′-ctggtggaccactcggatga-3′ 5′-ggcggtgctcgctttgta-3′ 5′-ctcatagatggcgttgttgc-3′ 5′-acggagatggatgtgccaaac-3′ 5′-agcaccttggaagccctacaga-3′

nucleus, while nucleus of negative cells turns light blue. And, the positive cells in stained tissue sections were quantified from five random ×400 images per group. The percentage of apoptosis was shown as the ratio of positive cells to total cells. Measurement of cytokine expression using enzyme-linked immunosorbent assay and real-time polymerase chain reaction At days 10 and 21 post-operation, blood was collected and serum was isolated, aliquoted and stored at −80°C for enzyme-linked immunosorbent assay (ELISA) detection. The expression levels of TGF-β, IL-10, IL-4 and interferon (IFN)-γ were measured via ELISA according to the manufacturer’s guidelines (R&D Systems; RayBiotech, Inc., catalog number ELM-IL10, ELM-IL4, ELM-IFNg). Real-time polymerase chain reaction (RT-PCR) was performed to determine the expression levels of IL-4, IL-10, IFN-γ and TGF-β in the transplanted tracheas. Total RNA was extracted using TRIzol reagent (Invitrogen/Life Technologies, catalog number 15596018) and complementary DNA (cDNA) was synthesized with RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific, catalog number K1622) according to the manufacturer’s instructions.The messenger RNA (mRNA) expression of cytokines was quantified using 7500 Fast Real-Time PCR System (Applied Biosystems) and SYBR Premix Ex Taq II.The expression levels were normalized by β-actin. The sequences of primers are shown in Table I. Immunophenotype detection of spleen lymphocytes At days 10 or 21 post-operation, the spleens were harvested and cell suspensions were prepared. The cells were stimulated with BD Golgiplug leukocyte activation cocktail (catalog number 550583) and BD GolgiStop Protein Transport Inhibitor (catalog number 554724) for 5 h, and then were labeled with Mouse Regulatory T cell Staining Kit (eBioscience, catalog

number 88–8118) and T helper (Th)1/Th2 /Th17 Phenotyping Kit (BD, catalog number 51-9006631). Flow cytometry was performed to analyze the T-cell subpopulations of Th1, Th2 and Th17 cells. The data analysis was conducted with Flowjo 7.6.1 software. Statistical analysis All data are expressed as mean ± SD. One-way analysis of variance was used to compare the means of two or more experimental groups, followed by the Dunnett post hoc test. Statistical differences between groups were considered to be significant at P < 0.05. Results MSCs-based HGF gene therapy alleviated the histopathologic changes and tracheal occlusion in BO mice Histopathologic examination of implanted tracheas obtained at days 10 and 21 showed that tracheal allograft lumen changes ranged from minimal encroachment to near-total occlusion by fibrovascular connective tissue.This was accompanied by epithelial cell changes, necrosis with desquamation of epithelial cells, destruction of basement membrane, formation of granulation tissue and scattered accumulations of inflammatory cells (Figure 1A). Compared with untreated controls, the histopathologic changes and the percentage of airway obstruction were alleviated in the treatment group. Luminal obstruction of the untreated control group was 92.42 ± 9.66%, whereas it reduced to 50.81 ± 10.53% and 17.7 ± 15% in MSCs-Null and MSCs-HGF groups, respectively, 10 days after treatment. At day 21 following transplantation, the tracheal occlusion was 97.19 ± 2.63% in the control group and 55.96 ± 14.73% and 24.07 ± 14.34% in MSCs-Null group and MSCsHGF group, respectively (Figure 1B). The difference between control group and MSCs-Null group was statistically significant (P < 0.001). Furthermore, the difference between MSCs-Null group and MSCsHGF group was significant (P < 0.001 or P < 0.01). MSC-HGF protected tracheal epithelial cell and cartilage cell from apoptosis It is well-known that apoptosis in the transplanted tissues or organs is a common phenomenon that affects the functional activity of the grafts. In this study, it was found that apoptotic cells were readily observed within the transplanted tracheal epithelium cell and cartilage, and both MSCs-HGF and MSCs-Null could inhibit allogenic. graft-induced apoptosis (Figure 1C). However, quantitative analysis showed that the percentages of apoptotic cells decreased significantly in MSC-HGF and MSC-Null mice at day 10 and day

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Figure 1. Effect of MSC-based HGF gene therapy on histopathologic changes and tracheal occlusion in BO mice. To evaluate the therapeutic effect of MSCs-Null and MSCs-HGF, local transplanted trachea was isolated. (A) H-E staining of trachea from BO mice (scale bar 100 μm). Representative images of H-E–stained trachea from normal, untreated control, MSCs-Null and MSCs-HGF groups are shown. On post-transplantation days 10 and 21, tracheas from each group were fixed with 4% paraformaldehyde, embedded in paraffin and cut into 5-μm thick sections before staining. The image of the control group displayed lack of mucosal epithelial cells and basement membrane damage, inflammatory cell infiltration, cartilage necrosis and fall off and fibrous connective tissue hyperplasia. The abovementioned pathological changes in the MSC treatment group were obviously improved. (B) Luminal occlusion of H-E–stained tracheal sections derived from control, MSCs-Null and MSCs-HGF groups were evaluated by determining the reduction in luminal area using Leica Qwin 3.4 software. (C) The apoptosis in the tracheal epithelial cell and cartilage cell was analyzed using TUNEL. (D) The apoptotic cells in stained tissue sections were quantified on the basis of five random ×400 images per group. Data are shown as means ± SD. Group comparisons were analyzed using one-way analysis of variance (ANOVA) with Dunnett post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

21 and they were lower in the MSC-HGF group than that in the MSC-Null group (Figure 1D). MSCs-HGF therapy reduced inflammatory and fibrotic responses in BO mice To evaluate the anti-inflammatory and antifibrotic effect of MSCs-HGF on BO mice, the protein levels of IFNγ, IL-4, IL-10 and TGF-β in peripheral blood were measured using ELISA at day 10 and 21 posttransplantation. As shown in Figure 2A, the expression level of IFN-γ and TGF-β in the allograft group were higher than the normal group, and MSCs treatment could reduce the serum levels of inflammatory cytokine IFN-γ and fibrotic cytokine TGF-β. HGF

modification could enhance the function of MSCs to decrease the expression level of these two cytokines; the difference between the MSCs-Null group and the MSCs-HGF group was significantly different (P < 0.05) at day 10. Meanwhile the level of the antiinflammatory/regulatory cytokine IL-4 and IL-10 was higher than that of the saline control group. The mRNA expression levels of IFN-γ, IL-4, IL10 and TGF-β in the transplanted tracheas were also detected using real-time PCR. Compared with the untreated control group, MSCs-Null could reduce the mRNA expression levels of IFN-γ and TGF-β, and increased the expression of IL-4 and IL-10 (Figure 2B). The changes were particularly evident in HGF-MSC– treated mice.

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Figure 2. Expression levels of pro-inflammatory, pro-fibrotic and anti-inflammatory cytokines in peripheral blood and allograft trachea. (A) The samples of peripheral blood were collected from BO mice at days 10 and 21 after MSC-based therapy. The expression levels of the pro-inflammatory cytokines IFN-γ, anti-inflammatory IL-4 and IL-10 and pro-fibrotic cytokines TGF-β were measured using ELISA. (B) Total RNA was extracted from allograft trachea of BO mice at days 10 and 21 after therapy, and mRNA expression of IFN-γ, IL-4, IL-10 and TGF-β were measured using RT-PCR. Data are shown as means ± SD. Group comparisons were analyzed using one-way ANOVA with Dunnett post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Treatment of BO with HGF modified MSCs The phenotypical features of spleen lymphocytes in BO mice after MSC treatment Lymphocytes of the spleen from different groups were isolated and stained with different antibodies and detected using fluorescence-activated cell sorting (FACS). As shown in Figure 3, tracheal allografts could induce the increase of CD4+CD25+FOXP3+ regulatory T (Treg) cells (Figure 3A and B), Th17 cells (Figure 3E and F) and Th1 to Th2 cell ratios (Figure 3C and D) 10 and 21 days post-transplantation. MSCs-Null or MSCs-HGF treatment could significantly lower the percentage of the above-mentioned cells in BO mice. Compared with the MSCs-Null group, the percentage of Treg cells and the ratio of Th1 to Th2 cells at 10 day post-transplantation were significantly lower in the MSCs-HGF group; the difference between the two groups was significantly different (P < 0.01 or P < 0.05). Discussion BO is a serious pulmonary complication, which is a progressive, insidious, non-infectious, high-mortality disease, especially after allogeneic HSCT and lung transplantation. Respiratory epithelial cell injury, peribronchial inflammation, and proliferation of fibrovascular connective tissue are responsible for the airway occlusion observed in BO. Although the morphology of BO is wellknown, the mechanisms are still not fully understood and the effective therapeutic strategies are lacking [1]. MSCs have emerged as a promising therapeutic tool in cell therapy. Previous experimental and clinical studies suggested that the therapeutic abilities of MSCs in a variety of clinical settings are due to their antiinflammatory and immunomodulatory properties. Meanwhile, MSCs can also reduce collagen deposition within the lumen [12,13].To further enhance the anti-inflammatory and anti-fibrotic function of MSCs on BO, various cytokines were used to modify MSCs. HGF is a pleiotropic growth factor that has mitogenic, morphogenic, angiogenitic and anti-apoptotic activities in a wide variety of cells. The study by Faehling et al. indicated that HGF could suppress TGF-β–induced actin expression to prevent the myofibroblast transformation [14]. Our previous study [15] also suggested that HGF-modified MSCs reduce pulmonary inflammation and fibrosis in radiation- or bleomycin-induced lung injury and fibrosis. In this study, we used a mouse heterotopic tracheal graft model as first described by Hertz et al. [6]. The present study supports that the heterotopic tracheal transplantation model shows that the abovementioned key features are similar to human BO. In this study, we chose male C57BL/6 mice and female Balb/C mice. There were two reasons: one is that the BO model described here could be established more

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easily after transplantation of tracheas from different strains of mice by inducing stronger rejection reactions. The other is that we found in clinical practice that the occurrence percentage of graft rejection between different genders is higher than that of the same gender. MSC-based gene therapy could ameliorate the development of BO compared with control groups, as shown by the histological observation on day 10 or day 21 after tracheal transplantation. And, at least in part, MSCs could reduce epithelial loss, inflammatory cell infiltration and luminal fibroobliteration. HGF gene modification could further alleviate the histopathologic changes.The tracheal occlusion in the MSCs-HGF group was obviously milder than the MSCs-Null group. Although the luminal occlusion slightly increased in the MSC-null group and MSC-HGF after 21 days compared with the 10 days results, the difference of luminal occlusion between 21 days and 10 days had no significant difference. It might be due to the fact that the duration of administrated MSCs was not long enough so the effect was not persistent.The fate of the transplanted MSCs might determine the persistence of action.These implied that giving MSC as a second dose maybe could increase the follow-up time. Occlusion of the airway lumen was caused by fibrous tissue over-growth, which is the phenomenon of the myofibroblast transformation induced by TGF-β. A number of recent investigations indicate that HGF prevents epithelial-esenchymal transition (EMT) by interfering with TGF-β signaling [8,16–18]. Our study also verified that HGF-modified MSCs could alleviate tracheal occlusion partly through decreasing the expression level of TGF-β. Inflammatory cytokines play important roles in BO formation and progress. IL-10 is an anti-inflammatory cytokine that could reduce natural killer cell and Th1 cell expression of IFN-γ and modulate cellular and hormonal immunity against an allograft by suppressing the expression of IL-2, IL-4 and IFN-γ [7,19]. Also, IL-10 may play a potential role in the development of experimental BO. In a murine model of allotropic tracheal transplantation, MSCs could induce an increase in IL-10 levels [20]. Our study showed that MSC treatment could increase the expression level of IL-10 that was down-regulated by heterograft not only in the transplanted trachea but also in the peripheral blood. As to IL-4, another anti-inflammatory cytokine, its expression change was similar to that of IL-10. IFN-γ has been proven to be an immune response cytokine associated with rejection. Elevated IFN-γ levels in serum could signify a greater risk for BO [21,22]. MSC-based treatment could greatly decrease the IFN-γ expression level in serum and local tracheal graft. HGF modification could enhance the immune-modulation ability of MSCs.

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Figure 3. Immunophenotype changes of spleen lymphocytes after transplantation. Lymphocytes of spleen were collected on days 10 and 21 after MSC-based therapy. The lymphocyte immunophenotypes were labeled with fluorescence antibody and detected using fluorescenceactivated cell sorting (FACS). Tregs cells (A and B) were labeled with a regulatory T-cell staining kit. Percentage of Th1 cells, Th2 cells (C and D) and Th17 cells (E and F) were labeled with Th1/Th2/Th17 Phenotyping Kit. Representative images of FACS were shown. Results are shown as means ± SD. Group comparisons were analyzed using one-way ANOVA with Dunnett post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

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Figure 3. (continued).

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Figure 3. (continued).

CD4+ T lymphocytes have an important role in adaptive immune system. CD4+ T cells differentiate into an array of functionally different effector Th and Treg-cell subsets. Treg cells are critical for immune regulation and immune protection against various infections. Treg cells also maintain immune tolerance, suppress inflammation and prevent allograft rejection. In transplant rejection, the proportion of Treg cells is increased. Our experiment proved that the percentage of spleen-derived Treg was increased in the graft model, however, it decreased in the MSCtreated groups. As a signature cytokine, Th1 cells selectively produce large amounts of IFN-γ and Th2 cells selectively secrete IL-4 and TL-10. Th1 cells are important in host defense against intracellular pathogens, whereas Th2 cells mediate protection against extracellular parasites but may also cause harmful allergic responsiveness to develop. A recent study showed that the Th1 response against tracheal allografts is

likely to be critical in the development of BO [19]. In this study, we observed an increased ratio of Th1 to Th2 in hetero-transplantation model, especially in the early stage after graft. So, it is important to decrease Th1 expression or increase Th2 expression to prevent the formation of BO. Our study proved that MSCs could meet the need to decrease the Th1 to Th2 ratio. At day 10 after treatment, the difference between the MSCs-HGF group and the MSCs-Null group had significant difference. The results showed that HGF modification had better immune-modulation activity. Th17 cells are also involved in rejection after organ transplantation by secreting IL-17, which can promote neutrophil proliferation and migration. Furthermore, IL-17 can affect endothelial cell activation and fibroblast activation and proliferation, which are important steps in graft rejection.The study by Nakagiri et al. proved that IL-17 secreted from Th17 cells

Treatment of BO with HGF modified MSCs mediates the fibroblast proliferation process, in a mouse BO model of ectopic tracheal transplantation, into the subcutaneous tissue [23]. Thus, the decrease of Th17 cells will mitigate the destruction of the transplanted tracheae. The work of Obermajer et al. suggested that the conversion of Th17 into Treg cells plays an important role when MSCs were used to induce longterm acceptance of allogeneic heart grafts in mice [24]. But till now, whether Th17 cells are involved in the alleviation of MSCs in BO remains elusive. Our findings showed that the percentage of spleen-derived Th17 cells was increased in allogaft BO mice and decreased after MSC-based therapy. But the percentages of Th17 cells in the MSCs-HGF group and the MSCs-Null group were comparable, suggesting that it was MSCs that played the modulation role, and HGF genes had little regulatory effect on Th17 cells. In conclusion, we have shown that HGF-modified MSCs can prevent the formation of the airway fibroproliferative lesion associated with tracheal graft rejection by means of modulating the inflammation and fibrosis process. The present study needs further clinical trials to evaluate the role of this approach in the prevention of BO.

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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (number 81200405 and number 81372924).

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Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jcyt.2015.12.006.