Lung DOI 10.1007/s00408-014-9654-x
Infusion of Mesenchymal Stem Cells Protects Lung Transplants from Cold Ischemia-Reperfusion Injury in Mice Weijun Tian • Yi Liu • Bai Zhang • Xiangchen Dai Guang Li • Xiaochun Li • Zhixiang Zhang • Caigan Du • Hao Wang
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Received: 9 July 2014 / Accepted: 7 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Cold ischemia-reperfusion injury (IRI) is a major cause of graft failure in lung transplantation. Despite therapeutic benefits of mesenchymal stem cells (MSCs) in attenuating acute lung injury, their protection of lung transplants from cold IRI remains elusive. The present study was to test the efficacy of MSCs in the prevention of cold IRI using a novel murine model of orthotopic lung transplantation. Methods Donor lungs from C57BL/6 mice were exposed to 6 h of cold ischemia before transplanted to syngeneic recipients. MSCs were isolated from the bone marrows of
W. Tian B. Zhang X. Dai Z. Zhang H. Wang (&) Department of General Surgery, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin 300052, China e-mail: [email protected] Y. Liu G. Li Department of Biology, Tianjin Medical University, Tianjin, China X. Li Department of Cardiology, Tianjin Medical University General Hospital, Tianjin, China Z. Zhang H. Wang Tianjin General Surgery Institute, Tianjin, China C. Du Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, Vancouver, BC, Canada C. Du (&) Department of Urologic Sciences, The University of British Columbia, VGH-Jack Bell Research Centre, 2660 Oak Street, Vancouver, BC V6H 3Z6, Canada e-mail: [email protected]
C57BL/6 mice for recipient treatment. Gas exchange was determined by the measurement of blood oxygenation, and lung injury and inflammation were assessed by histological analyses. Results Intravenously delivered MSC migration/trafficking to the lung grafts occurred within 4-hours post-transplantation. As compared to untreated controls, the graft arterial blood oxygenation (PaO2/FiO2) capacity was significantly improved in MSC-treated recipients as early as 4 h post-reperfusion and such improvement continued over time. By 72 h, oxygenation reached normal level that was not seen in controls. MSCs treatment conferred significant protection of the grafts from cold IRI and cell apoptosis, which is correlated with less cellular infiltration, a decrease in proinflammatory cytokines (TNF-a, IL-6) and toll-like receptor 4, and an increase in anti-inflammatory TSG-6 generation. Conclusions MSCs provide significant protection against cold IRI in lung transplants, and thus may be a promising strategy to improve outcomes after lung transplantation. Keywords Mesenchymal stem cells Orthotopic lung transplantation Cold ischemia-reperfusion injury Mice Abbreviations ANOVA Analysis of variance eGFF Enhanced green fluorescence protein H&E Hematoxylin & eosin MSCs Mesenchymal stem cells IL Interleukin IRI Ischemia-reperfusion injury ROS Reactive oxygen species SEM Standard error of mean TNF Tumor necrosis factor TLR Toll-like receptor
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TSG TUNEL
Tumor necrosis factor-a stimulated gene/ protein Terminal deoxynucleotyl transferase dUTP nick end labeling
transplants from cold IRI has not been investigated as of yet. The novelty of the present study can be seen: (i) by the establishment of a novel murine model of orthotopic, single-lung transplantation, in which lung transplants experienced 6 h-cold ischemia to mimic cold IRI in clinical lung transplantation; and (ii) in the findings of the therapeutic efficacy of intravenously infused MSCs in the prevention of the cold IRI specifically.
Introduction In lung transplantation, cold ischemia-reperfusion injury (IRI) from cold preservation and subsequent warm reperfusion causes significant transplant damage [1–3]. Pulmonary IRI usually appears within 72 h post-transplantation in the lung parenchyma, gaseous exchange system, and pulmonary circulation, which significantly contribute to the immediate graft dysfunction or chronic function loss [3]. A number of studies have indicated that IRI causes progressive damage to lung grafts, in large part, by eliciting a severe inflammatory reaction and the release of reactive oxygen species (ROS) [4–6]. Except of neutrophils infiltration/activation, a variety of proinflammatory factors including tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and IL-6 are the parts of this pathological cascade and induce endothelium injury and inflammatory cells recruitment [3, 7]. Strategies targeting against the generation and propagation of inflammation and ROS release may provide therapeutic potential, accordingly. Mesenchymal stem cells (MSCs) are multipotent and non-immunogenic cells that reside mainly in bone marrow and other post-natal tissues and organs, and their potential of differentiation and replacement properties was initially ascribed to regenerative medicine [8–11]. Later, many studies have found that the most of the positive effects of MSC engraftment are actually due to the paracrine release of cytokines, and other soluble factors that modulate the inflammation or immune reactions [9–11]. To date, it has been well documented that MSCs can secrete a variety of bioactive molecules that confer their immunomodulatory function and reconstruction of a suitable microenvironment for tissue repair. We and others have demonstrated that MSCs are able to migrate to sites suffering injury and inflammation [12–16], suggesting that MSCs may become a promising candidate for cell-based therapy to treat IRI in lung transplants. It has been reported that MSC administration can effectively attenuate inflammation, alleviate reperfusion injury and improve graft outcomes in the prevention of pulmonary IRI [5, 7]. However, these studies were performed in a model of warm lung ischemia with the left pulmonary hilum clamped, which does not completely mimic the exposure of lung transplants to cold IRI. The efficacy and mechanisms of MSCs in the protection of lung
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Materials and Methods Animals Both strains of male C57BL/6 (10–12 week old, H-2b) and enhanced green fluorescence protein (eGFP?)-transgenic C57BL/6 mice (6–8 week old, H-2b) were purchased from the Jackson Laboratories (Bar Harbor, MA). Animals were housed in the Animal Care Facility of the University of Western Ontario (London, ON, Canada), and experiments were performed following the protocol approved by the Animal Use Subcommittee and conducted according to the guidelines of the Canadian Council on Animal Care. Preparation of MSCs MSCs were isolated from eGFP?-transgenic C57BL/6 mice (6–8 weeks old) according to a standard protocol as reported previously [16]. Briefly, the whole bone marrow cells were collected from femurs and tibias, followed by washing and culturing in low-glucose DMEM medium supplemented with 2 % fetal bovine serum, penicillin/streptomycin (100 U/mL; Invitrogen, Burlington, ON, Canada), and epidermal growth factor and platelet-derived growth factor (both at 10 ng/mL; R&D Systems, Minneapolis, MN). After 48 h of incubation, the nonadherent cells were removed by aspiration. When the remaining/adherent cells reached 90 % culture confluence, they were trypsinized with 0.25 % trypsin solution (Sigma-Aldrich, Burlington, ON, Canada) and subcultured in a density of 5,000 cells/mL. After 2–3 similar passages, CD45? cells were depleted using CD45positive selection (Miltenyi Biotec Inc. Auburn, CA) and the remaining CD45-negative cells were confirmed to be MSCs. Orthotopic, Single-Lung Transplantation, and MSC Infusion Left orthotopic vascularized lung transplantation was performed between C57BL/6 (H-2b) mice. Mice were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/ kg) administered (i.m. injection) and ventilated with room air by a rodent ventilator. Both donor and recipient operations were performed under a microscope with 10–409
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magnification (Leica, Buffalo Grove, IL). Donor lungs were prepared following protocols as described previously [17]. Briefly, the left lung of the donor mice was exposed through a median sternotomy, followed by being slowly perfused in situ with 1.0 mL of cold heparinized Ringer’s lactate solution through the inferior vena cava and aorta before the retrieval. After the donor lungs were carefully harvested and preserved in Ringer’s solution at 4 °C for 6 h, orthotopic left lung allograft transplantation was performed by anastomosis of pulmonary vein and bronchus using suitable cuffs and suturing of the pulmonary artery by 11-0 silk suture. The chest incisions were closed until confirming that the grafts had been successfully reperfused and finally ventilated. Two hours before syngeneic transplantation, eGFP?MSCs (1 9 106) in a total volume of 0.3 mL were infused through the tail vein. Saline injection was used as untreated control. Lung transplant recipients received MSCs or saline were examined at 4, 24, and 72 h (n = 6 in each group at each time point) following reperfusion. Determination of Oxygen Saturation Prior to sample collection, the gas exchange of lung transplants was tested. The blood from the left pulmonary vein cuff was sampled, and the blood gas was measured using a conventional analyzer (CG 8? Cartridge, iSTAT Portable Clinical Analyzer; iSTAT Corp, East Windsor, NJ). Organ Harvest Mice were euthanized at 4, 24, and 72 h, and then lungs, hearts, kidneys, and livers were harvested for histological analyses. These samples were either fixed in 10 % formaldehyde solution for histological analysis by a routine manner or embedded in Tissue-Tek OCT (Skura Finetek, Torrance, CA). The tissues in OCT were cut into 5 lmthick cryosections for MSC tracking.
Fig. 1 The appearance and blood gas analysis of lung grafts. a Transplanted lung showed normal appearance without pulmonary edemas and collapses at 24 h post-reperfusion. b Blood gas assay of lung grafts. MSC administration significantly improved the PaO2/ FiO2 compared to untreated mice, and pulmonary function increased over the time period of this experiment. *P \ 0.0001 versus untreated group
Infused MSC Tracking The injected MSCs were tracked by eGFP fluorescence in tissue cryosections of grafted lungs, and native lung, hearts, livers, and kidneys under a fluorescence microscope (ZEISS, Oberkochen, Germany). Graft Histology Graft sections were stained with hematoxylin & eosin (H&E) for evaluating and determining IRI score in a blinded manner. Pulmonary injury was scored using a 7-point scale according to the combined assessments of
alveolar congestion, hemorrhage, alveolar/interstitial edema, and inflammatory cells infiltration in the airspace and vessel wall in accordance with previous reports [5, 18]. Apoptosis Assay Cell apoptosis in lung grafts 72 h after reperfusion was determined by terminal deoxynucleotyl transferase dUTP nick end labeling (TUNEL) staining according to the instruction of the kit (R&D system, Minneapolis, MN). Apoptotic cells with severe DNA damage would be labeled brown.
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Immunohistological Examination The protein expression in tissue sections by 72 h was detected using an avidin–biotin immunoperoxidase method (Vector Laboratories, Burlingame, CA) with hematoxylin showing nucleus. The primary antibodies rat anti-mouse CD68 (macrophage marker), myeloperoxidase (MPO, activated neutrophil marker), CD4 and CD8 monoclonal antibodies, IL-6, TNF-a, Toll-like receptor-4 (TLR4) were purchased from BD Biosciences (Mississauga, ON, Canada) and anti- tumor necrosis factor-a stimulated gene/protein 6 (TSG-6) antibody from R&D system (Minneapolis, MN). Statistical Analysis All data were presented as mean ± standard error of mean (SEM). Comparison between two groups was performed by two-tailed t test or analysis of variance (ANOVA). P value of less than 0.05 was considered statistically significant.
Results MSC Treatment Achieves Normal Appearance and Function of Lung Grafts
The blood oxygenation (PaO2/FiO2, mmHg) as a biomarker for the pulmonary function of gas exchange was examined at 4, 24, and 72 h following reperfusion. At as early as 4 h post-transplantation, the blood oxygenation in MSC-treated mice showed significant improvement compared with that of untreated group (153.6 ± 4.1 vs. 46.0 ± 5.2 mmHg, P \ 0.0001, t test, n = 6) (Fig. 1b). At 24 h post-transplantation, graft oxygenation capacity continued to elevate owing to MSC infusion (216.3 ± 5.5 vs. 90.0 ± 1.0 mmHg, P \ 0.0001, t test, n = 6) (Fig. 1b). By 72 h post-transplantation, oxygenation reached to the normal levels in MSC-treated group, which were not seen in controls (309.0 ± 3.6 vs. 111.0 ± 8.5 mmHg, P \ 0.0001, t test, n = 6) (Fig. 1b). The two-way ANOVA also showed that the blood oxygenation in MSCs-treated group was significantly higher than that in control group at all of time points (P \ 0.0001, n = 6). Thus, MSC infusion remarkably improved the gas exchange of the grafts and such effect was further enhanced over the time period of this experiment. Intravenous Infused MSCs Mainly Locate in Grafted Lungs
As shown in Fig. 1a, grafted lungs harvested from MSCtreated recipients 24 h post-transplantation, displayed normal morphology and well congestion without pulmonary edemas, collapses or necrosis, which were different from the lung grafts in untreated controls with severe tissue damage (data not shown).
We tracked the fate of MSCs with eGFP fluorescence at the same time points as gas exchange detection. In coincidence with previous reports, substantial numbers of eGFP?-MSCs were localized in grafted lungs at as early as 4 h but not many in the naı¨ve lung (Fig. 2), and such MSCs homing to injured sites almost remained unchanged until 72 h after transplantation (data not shown). While a few of eGFP? MSCs were seen in the native lungs, but none of MSCs were
Fig. 2 The dislocation of infused MSCs in the organs of the recipients. The fluorescent images (9400) displayed the location of eGFP?-MSCs in each organ at 4 h post-transplantation. Most eGFP
positive MSCs located in grafted lungs (a), a few in native lungs (b) but none were observed in native hearts, livers, or kidneys (c). Data were a typical microscopic view of organ sections in each group
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Lung Fig. 3 Histological analysis of lung grafts. a Sections of transplanted lungs harvested 24 h following transplantation were stained with hematoxylin and eosin (9200), and showed severe lung injury characterized by obvious alveolar congestion and collapse in untreated mice, while MSC treatment ameliorated this injury. b The IRI score of lung grafts was evaluated based on the pathological performance. The graft injury score was significantly higher in untreated group compared with that of MSC-treated mice. *P \ 0.05 versus untreated group
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found in native hearts, livers, or kidneys of the recipients (Fig. 2), indicating that intravenously delivered MSCs indeed migrated and homed to grafted lungs and then exerted their biological effects. MSC Delivery Confers Protection of the Lung Grafts Histopathology of lung transplants in untreated mice showed typical features of IRI with interstitial hemorrhage, edema, alveolar congestion, and collapse (Fig. 3a). By contrast, MSC treatment clearly attenuated these changes at the same time point, which was evidenced by that the alveolar structure was intact and free of edema and fluid (Fig. 3a). These histological observations were further confirmed by the injury scores, representing the total grades of alveolar congestion, hemorrhage, edema, and infiltration of inflammatory cells. The injury score in MSC-treated group was much lower than that of untreated controls (Fig. 3b, 4-hour reperfusion: untreated 3.5 ± 0.6 versus MSC-treated 1.6 ± 0.5; 24-hour reperfusion: untreated 5.6 ± 0.5 versus MSC-treated 4.2 ± 0.4; 72-hour reperfusion: untreated 4.5 ± 0.6 versus MSC-treated 2.6 ± 0.5, P \ 0.05), suggesting that MSC treatment significantly improved the pathological performance of grafted lungs. Infused MSCs Attenuate Cell Apoptosis in the Lung Grafts TUNEL assays of lung grafts by 72 h post-transplantation showed more apoptotic cell death in untreated controls than
Untreated
Fig. 5 Inflammatory cell infiltration in lung transplants at 72 h post- c transplantation. a Massive infiltration of macrophages, neutrophils, and CD8? T cells was found in lung grafts of untreated controls, while MSC infusion markedly reduced intragraft inflammatory cell infiltration. b Decreased expression of TNF-a and IL-6 was detected in lung grafts of MSC-treated mice as compared with those of untreated controls. Data were a representative of six determinants. Magnification 9200
that of MSC-treated animals (Fig. 4). Similar changes were also found in lung grafts at both 4 and 24 h post-transplantation (data not shown). These results suggested that MSC delivery effectively prevented cell apoptosis postreperfusion, which was beneficial to the gaseous exchange of lung grafts. MSC Therapy Suppresses Intragraft Inflammation The infiltrating cells comprising macrophages, neutrophils, CD4?, and CD8? T cells were examined by immunohistochemistry staining 72 h post-reperfusion. As shown in Fig. 5a, much more positively staining of CD68 (macrophages), MPO (neutrophils), and CD8 (CD8? T cells) was seen in lung grafts from untreated animals as compared to fewer of these infiltrates in MSC-treated grafts (Fig. 5a). While, only a few CD4? T cells were observed in lung grafts (data not shown). These data indicate that MSC infusion could effectively attenuate inflammatory immune response to the transplanted lungs after cold IRI in this model. In consistence with the levels of inflammatory cell infiltration, a high level of intragraft expressions of
MSC-treated
Fig. 4 Cell apoptosis in lung grafts at 72 h post-transplantation. Intragraft apoptotic cell death was found in untreated group. In contrast, MSC administration prevented cell apoptosis in lung grafts. Data were a representative of six determinants
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Macrophages
Neutrophils
CD8+ cells
MSC-treated
Untreated
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MSC-treated
TNF-α
IL-6
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Lung Fig. 6 MSCs reduce intragraft inflammatory cytokines and TLR Expression, but increase TSG-6 expression at 72 h following Lung transplantation. a The level of TLR-4 expression in lung grafts of untreated animals was much higher than that of MSC-treated animals. b MSC infusion markedly increased TSG-6 level in lung grafts as compared to untreated controls. Data were a representative of six determinants. Magnification 9200
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Untreated
MSC-treated
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proinflammatory cytokine TNF-a and IL-6 was also detected in untreated controls (Fig. 5b) 72 h following transplantation, whereas MSC infusion markedly decreased the cytokine expression at the same time point (Fig. 5b), implying that the suppression of intragraft inflammation achieved by MSCs could contribute to the improvement of survival and function of lung grafts.
MSC Treatment Reduces TLR-4 but Enhances TSG-6 Expression in Lung Transplants Toll-like receptors (TLRs), TLR-4 in particular, play a pivotal role in the inflammatory process of pulmonary IRI, and are an important functional biomarker for the local inflammation [19, 20]. We examined TLR-4 protein in lung
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grafts after 72 h of reperfusion, and found that MSC infusion greatly suppressed intragraft TLR-4 expression compared with untreated group (Fig. 6a), which was in line with its suppression of inflammatory factors as presented in Fig. 5. Tumor necrosis factor-a stimulated gene/protein (TSG6) secreted by MSCs has been reported to have multipotency in anti-inflammation, and plays important roles upon tissue injury [21, 22]. In the present study, TSG-6 expression was assessed in lung grafts 72 h post-transplantation. The results showed that MSC treatment was obviously associated with promoted intragraft TSG-6 expression as compared with untreated controls (Fig. 6b), indicating that TSG-6 production could be one of the inherent mechanisms for anti-inflammatory efficacy of MSCs in the present study.
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Discussion The present study was designed to investigate the efficacy of autologous bone marrow-derived MSCs in the prevention of pulmonary cold IRI post-lung transplantation using a murine model of syngeneic lung transplantation. The lung is vulnerable to pathological damage, and any condition impairing the alveolar oxygenation or pulmonary blood flow will finally result in lung ischemia [3]. The rigorous anoxic ischemia due to the interruption of both pulmonary bloodstream and ventilation for up to several hours are inevitable in lung transplantation. The subsequent reperfusion of grafted lungs usually leads to alveolar damage, pulmonary edema, and hypoxemia within 72 h, which is the common risk factor for graft dysfunction and chronic function loss following lung transplantation. Animal studies have prompted the development of pulmonary IRI therapy post-lung transplantation. In the recent years, investigations have been focusing on the benefits of MSCs in the prevention of lung IRI; however, most of these studies used a warm ischemia model with pulmonary hilum clamped by a clip [5, 7, 18, 23]. Actually in clinic, IRI following lung transplantation is inevitable during the surgical process. The applications of rat and other large animal implant models based on cuff technique greatly contribute to the understanding of pathogenesis of lung IRI [24]. Although the mouse model of orthotopic lung transplantation subjects to high microsurgical demands [25, 26], mice models offer a broader research scope because of knockout and transgenic backgrounds. In the present study, we developed the orthotopic, single-lung transplantation model in mice by anastomosis of pulmonary vein and bronchus using appropriate cuffs and pulmonary artery sutured with 11-0 silk suture, which could effectively prevent thrombosis. With 6-hour lung cold ischemia, we tested the role of intravenously infused MSCs on the prevention of lung cold I/R injury at 4, 24, and 72 h following transplantation. Data presented here indicated that grafted lungs in MSC-treated mice showed near normal appearance without any visible edemas or collapses at 24 h. The gas exchange assay demonstrated that infused MSCs significantly improved the blood oxygenation (PaO2/FiO2) of lung grafts at as early as 4 h after reperfusion, with further amelioration that approached normal values by 72 h post-transplantation. By contrast, the gas exchange of mouse recipients received saline (as untreated controls) never reached to the level that was achieved by MSC treatment, indicating that intravenously delivered MSCs effectively improved the function of lung grafts. It has been widely accepted that MSC-based cell therapy has been a promising strategy for the treatment of degenerating and immunological disorders, and animal studies
confirm the long-term regenerative and immunomodulatory function of MSCs, while the fate and distribution of MSCs in vivo post infusion remain to be elucidated. Undoubtedly, intravenous administration is the most suitable route for MSC delivery in potential clinical application. Although MSCs have the properties of migrating and homing to injured sites, some reports concerning cell tracking have indicated that the majority of MSCs localizes to the lungs post intravenous infusion with only a small portion homing to the target organs along with circulation [13, 14]. In fact, this provides a real benefit to the treatment of pulmonary injury. In the current study, we used eGFP-transgenic MSCs for IRI treatment and tracked the migration in a short term. As expected, infused MSCs mainly homed to grafted lungs rather than other native organs of recipient mice, providing important preconditions for their therapeutic effect. Histological assessment indicated that MSC infusion effectively protected the alveolar network and reduced interstitial hemorrhage, edema, alveolar congestion and collapse 24 h post-transplantation. The injury score analysis based on the pathological features demonstrated that MSC treatment significantly decreased the injury score compared with controls at each time point. In addition, we did wet/dry ratio assay and found less fluid inside lung grafts of MSC-treated mice as compared to those of untreated recipients (data not shown). Apoptosis is the main mechanism of cell death observed in lung transplantation-triggered IRI. Donor lungs are unavoidably confronted parenchyma cell apoptotic death during the preservation period ex vivo, and cell apoptosis in lung grafts will be worsened following blood reperfusion [2, 27]. TUNEL detection indeed showed that the amount of apoptotic cells located in grafted lungs of untreated mice 72 h post-transplantation, which got markedly reduced in that of MSC-treated mice. We believe that this reduction could contribute to the protective role of MSCs in pulmonary function. Experimental studies regarding the mechanism of IRI highlight that it is a pathologically inflammatory course involves a neutrophil predominating immunocyte infiltration and proinflammatory cytokine extravasation [3]. We also checked the intragraft cellular infiltration and inflammation-related molecular expression by 72 h. The results showed that much more macrophages, neutrophils, and CD8? T cells located in lung grafts of untreated recipients, while MSC administration greatly reduced intragraft inflammatory cell recruitment. It is worth mentioning that we did not find infiltrating CD4? T cells in both MSCtreated and untreated control groups at 72 h, which was different from some earlier reports [7, 25], possibly due to the different detection time points and the different transplant models. In addition, cytokine examination indicated
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that both IL-6 and TNF-a expressions were effectively attenuated in lung grafts of MSC-treated mice compared with controls. The marked decrease of inflammatory cells and cytokines suggested the diminished intragraft inflammation achieved by MSC infusion, which benefited to the protection of lung grafts and restored the pulmonary function. As the first line of defense, the innate immune system is activated with the initial reperfusion in minutes posttransplantation. TLR signaling, a key innate immune pathway, has been demonstrated to contribute to pulmonary IRI-associated inflammatory response [28, 29]. In particular, TLR4 activation plays critical roles in initial edema formation and onset of downstream signal pathways concerning inflammation process of lung IRI in pulmonary hilum clamping model [19, 20, 30]. Thus, in current study, we evaluated the TLR4 level 72 h post-transplantation and found that the intragraft TLR4 expression in the untreated control group was much higher than that of MSC-treated mice. In other words, MSC infusion effectively inhibited TLR4 signaling pathway, which might make important contributions to the low proinflammatory cytokine production, since IL-6 and TNF-a are both downstream molecules in TLR4 pathway [31]. Considerable efforts have been made to explain the therapeutic potentials of MSCs in many disorder treatments. The damage repair and function improvement observed in subsequent investigations showed much more close connection with the paracrine secretions or cell-tocell contacts [32, 33]. It is now believed that MSCs cultured in vitro secrets a large number of cytokines and expresses high levels of additional beneficial factors after engrafting into injured sites [32]. Among these factors, TSG-6 is a multipotent anti-inflammatory protein and makes contributions to the immuno-modulatory function of MSCs [13, 22]. Therefore, we examined intragraft level of TSG-6 and did find TSG-6 expression in lung grafts of MSC-treated recipients, but hardly found this protein in lung grafts of untreated mice. The tissue repair and function restore achieved by MSC infusion could be partly ascribed to the TLR-4 attenuation together with TSG-6 secretion.
Conclusion Owing to the important role of MSCs in the suppression of inflammatory response and improvement of pulmonary function, our data suggest that MSC infusion could be used as a potential strategy for preventing cold IRI following lung transplantation. In addition, the modified mouse orthotopic lung transplantation model used in this study would provide a useful tool to the further mechanism and
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efficacy studies in achieving long-term lung transplant acceptance. Acknowledgments We are grateful to Dr. Bertha Garcia at Western University, London, Canada for her critical review of the histological slides, and to Drs. Shuhua Luo and Weihua Liu for their technical assistance. This work was supported by Grant to H.W. from National Natural Science Foundation of China (No. 81273257), and National High Technology Research and Development Program 863 (2012AA021003). C.D. was supported by grants from the Kidney Foundation of Canada and the Canadian Institutes of Health Research. Conflict of interest disclose.
The authors have no conflicts of interest to
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attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-kappaB signaling in resident macrophages. Blood 118(2):330–338. doi:10.1182/blood-2010-12-327353 Sun CK, Yen CH, Lin YC, Tsai TH, Chang LT, Kao YH, Chua S, Fu M, Ko SF, Leu S, Yip HK (2011) Autologous transplantation of adipose-derived mesenchymal stem cells markedly reduced acute ischemia-reperfusion lung injury in a rodent model. J Transl Med 9:118. doi:10.1186/1479-5876-9-118 Reis A, Giaid A, Serrick C, Shennib H (1995) Improved outcome of rat lung transplantation with modification of the nonsuture external cuff technique. J Heart Lung Transplant 14(2):274–279 Okazaki M, Krupnick AS, Kornfeld CG, Lai JM, Ritter JH, Richardson SB, Huang HJ, Das NA, Patterson GA, Gelman AE, Kreisel D (2007) A mouse model of orthotopic vascularized aerated lung transplantation. Am J Transplant 7(6):1672–1679. doi:10.1111/j.1600-6143.2007.01819.x Krupnick AS, Lin X, Li W, Okazaki M, Lai J, Sugimoto S, Richardson SB, Kornfeld CG, Garbow JR, Patterson GA, Gelman AE, Kreisel D (2009) Orthotopic mouse lung transplantation as experimental methodology to study transplant and tumor biology. Nat Protoc 4(1):86–93. doi:10.1038/nprot.2008.218 Sugimoto R, Tanaka Y, Noda K, Kawamura T, Toyoda Y, Billiar TR, McCurry KR, Nakao A (2012) Preservation solution supplemented with biliverdin prevents lung cold ischaemia/reperfusion injury. Eur J Cardiothorac Surg 42(6):1035–1041. doi:10. 1093/ejcts/ezs298 Kreisel D, Goldstein DR (2013) Innate immunity and organ transplantation: focus on lung transplantation. Transpl Int 26(1):2–10. doi:10.1111/j.1432-2277.2012.01549.x Diamond JM, Wigfield CH (2013) Role of innate immunity in primary graft dysfunction after lung transplantation. Curr Opin Organ Transplant. doi:10.1097/MOT.0b013e3283651994 Kaczorowski DJ, Nakao A, Mollen KP, Vallabhaneni R, Sugimoto R, Kohmoto J, Tobita K, Zuckerbraun BS, McCurry KR, Murase N, Billiar TR (2007) Toll-like receptor 4 mediates the early inflammatory response after cold ischemia/reperfusion. Transplantation 84(10):1279–1287. doi:10.1097/01.tp.0000287597.87571.17 Ben DF, Yu XY, Ji GY, Zheng DY, Lv KY, Ma B, Xia ZF (2012) TLR4 mediates lung injury and inflammation in intestinal ischemia-reperfusion. J Surg Res 174(2):326–333. doi:10.1016/j. jss.2010.12.005 Uccelli A, Moretta L, Pistoia V (2008) Mesenchymal stem cells in health and disease. Nat Rev Immunol 8(9):726–736. doi:10. 1038/nri2395 Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, Epstein SE (2004) Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res 94(5):678–685. doi:10.1161/01.RES.0000118601. 37875.AC
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Infusion of Mesenchymal Stem Cells Protects Lung Transplants from Cold Ischemia-Reperfusion Injury in Mice Weijun Tian • Yi Liu • Bai Zhang • Xiangchen Dai Guang Li • Xiaochun Li • Zhixiang Zhang • Caigan Du • Hao Wang
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Received: 9 July 2014 / Accepted: 7 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Cold ischemia-reperfusion injury (IRI) is a major cause of graft failure in lung transplantation. Despite therapeutic benefits of mesenchymal stem cells (MSCs) in attenuating acute lung injury, their protection of lung transplants from cold IRI remains elusive. The present study was to test the efficacy of MSCs in the prevention of cold IRI using a novel murine model of orthotopic lung transplantation. Methods Donor lungs from C57BL/6 mice were exposed to 6 h of cold ischemia before transplanted to syngeneic recipients. MSCs were isolated from the bone marrows of
W. Tian B. Zhang X. Dai Z. Zhang H. Wang (&) Department of General Surgery, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin 300052, China e-mail: [email protected] Y. Liu G. Li Department of Biology, Tianjin Medical University, Tianjin, China X. Li Department of Cardiology, Tianjin Medical University General Hospital, Tianjin, China Z. Zhang H. Wang Tianjin General Surgery Institute, Tianjin, China C. Du Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, Vancouver, BC, Canada C. Du (&) Department of Urologic Sciences, The University of British Columbia, VGH-Jack Bell Research Centre, 2660 Oak Street, Vancouver, BC V6H 3Z6, Canada e-mail: [email protected]
C57BL/6 mice for recipient treatment. Gas exchange was determined by the measurement of blood oxygenation, and lung injury and inflammation were assessed by histological analyses. Results Intravenously delivered MSC migration/trafficking to the lung grafts occurred within 4-hours post-transplantation. As compared to untreated controls, the graft arterial blood oxygenation (PaO2/FiO2) capacity was significantly improved in MSC-treated recipients as early as 4 h post-reperfusion and such improvement continued over time. By 72 h, oxygenation reached normal level that was not seen in controls. MSCs treatment conferred significant protection of the grafts from cold IRI and cell apoptosis, which is correlated with less cellular infiltration, a decrease in proinflammatory cytokines (TNF-a, IL-6) and toll-like receptor 4, and an increase in anti-inflammatory TSG-6 generation. Conclusions MSCs provide significant protection against cold IRI in lung transplants, and thus may be a promising strategy to improve outcomes after lung transplantation. Keywords Mesenchymal stem cells Orthotopic lung transplantation Cold ischemia-reperfusion injury Mice Abbreviations ANOVA Analysis of variance eGFF Enhanced green fluorescence protein H&E Hematoxylin & eosin MSCs Mesenchymal stem cells IL Interleukin IRI Ischemia-reperfusion injury ROS Reactive oxygen species SEM Standard error of mean TNF Tumor necrosis factor TLR Toll-like receptor
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TSG TUNEL
Tumor necrosis factor-a stimulated gene/ protein Terminal deoxynucleotyl transferase dUTP nick end labeling
transplants from cold IRI has not been investigated as of yet. The novelty of the present study can be seen: (i) by the establishment of a novel murine model of orthotopic, single-lung transplantation, in which lung transplants experienced 6 h-cold ischemia to mimic cold IRI in clinical lung transplantation; and (ii) in the findings of the therapeutic efficacy of intravenously infused MSCs in the prevention of the cold IRI specifically.
Introduction In lung transplantation, cold ischemia-reperfusion injury (IRI) from cold preservation and subsequent warm reperfusion causes significant transplant damage [1–3]. Pulmonary IRI usually appears within 72 h post-transplantation in the lung parenchyma, gaseous exchange system, and pulmonary circulation, which significantly contribute to the immediate graft dysfunction or chronic function loss [3]. A number of studies have indicated that IRI causes progressive damage to lung grafts, in large part, by eliciting a severe inflammatory reaction and the release of reactive oxygen species (ROS) [4–6]. Except of neutrophils infiltration/activation, a variety of proinflammatory factors including tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and IL-6 are the parts of this pathological cascade and induce endothelium injury and inflammatory cells recruitment [3, 7]. Strategies targeting against the generation and propagation of inflammation and ROS release may provide therapeutic potential, accordingly. Mesenchymal stem cells (MSCs) are multipotent and non-immunogenic cells that reside mainly in bone marrow and other post-natal tissues and organs, and their potential of differentiation and replacement properties was initially ascribed to regenerative medicine [8–11]. Later, many studies have found that the most of the positive effects of MSC engraftment are actually due to the paracrine release of cytokines, and other soluble factors that modulate the inflammation or immune reactions [9–11]. To date, it has been well documented that MSCs can secrete a variety of bioactive molecules that confer their immunomodulatory function and reconstruction of a suitable microenvironment for tissue repair. We and others have demonstrated that MSCs are able to migrate to sites suffering injury and inflammation [12–16], suggesting that MSCs may become a promising candidate for cell-based therapy to treat IRI in lung transplants. It has been reported that MSC administration can effectively attenuate inflammation, alleviate reperfusion injury and improve graft outcomes in the prevention of pulmonary IRI [5, 7]. However, these studies were performed in a model of warm lung ischemia with the left pulmonary hilum clamped, which does not completely mimic the exposure of lung transplants to cold IRI. The efficacy and mechanisms of MSCs in the protection of lung
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Materials and Methods Animals Both strains of male C57BL/6 (10–12 week old, H-2b) and enhanced green fluorescence protein (eGFP?)-transgenic C57BL/6 mice (6–8 week old, H-2b) were purchased from the Jackson Laboratories (Bar Harbor, MA). Animals were housed in the Animal Care Facility of the University of Western Ontario (London, ON, Canada), and experiments were performed following the protocol approved by the Animal Use Subcommittee and conducted according to the guidelines of the Canadian Council on Animal Care. Preparation of MSCs MSCs were isolated from eGFP?-transgenic C57BL/6 mice (6–8 weeks old) according to a standard protocol as reported previously [16]. Briefly, the whole bone marrow cells were collected from femurs and tibias, followed by washing and culturing in low-glucose DMEM medium supplemented with 2 % fetal bovine serum, penicillin/streptomycin (100 U/mL; Invitrogen, Burlington, ON, Canada), and epidermal growth factor and platelet-derived growth factor (both at 10 ng/mL; R&D Systems, Minneapolis, MN). After 48 h of incubation, the nonadherent cells were removed by aspiration. When the remaining/adherent cells reached 90 % culture confluence, they were trypsinized with 0.25 % trypsin solution (Sigma-Aldrich, Burlington, ON, Canada) and subcultured in a density of 5,000 cells/mL. After 2–3 similar passages, CD45? cells were depleted using CD45positive selection (Miltenyi Biotec Inc. Auburn, CA) and the remaining CD45-negative cells were confirmed to be MSCs. Orthotopic, Single-Lung Transplantation, and MSC Infusion Left orthotopic vascularized lung transplantation was performed between C57BL/6 (H-2b) mice. Mice were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/ kg) administered (i.m. injection) and ventilated with room air by a rodent ventilator. Both donor and recipient operations were performed under a microscope with 10–409
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magnification (Leica, Buffalo Grove, IL). Donor lungs were prepared following protocols as described previously [17]. Briefly, the left lung of the donor mice was exposed through a median sternotomy, followed by being slowly perfused in situ with 1.0 mL of cold heparinized Ringer’s lactate solution through the inferior vena cava and aorta before the retrieval. After the donor lungs were carefully harvested and preserved in Ringer’s solution at 4 °C for 6 h, orthotopic left lung allograft transplantation was performed by anastomosis of pulmonary vein and bronchus using suitable cuffs and suturing of the pulmonary artery by 11-0 silk suture. The chest incisions were closed until confirming that the grafts had been successfully reperfused and finally ventilated. Two hours before syngeneic transplantation, eGFP?MSCs (1 9 106) in a total volume of 0.3 mL were infused through the tail vein. Saline injection was used as untreated control. Lung transplant recipients received MSCs or saline were examined at 4, 24, and 72 h (n = 6 in each group at each time point) following reperfusion. Determination of Oxygen Saturation Prior to sample collection, the gas exchange of lung transplants was tested. The blood from the left pulmonary vein cuff was sampled, and the blood gas was measured using a conventional analyzer (CG 8? Cartridge, iSTAT Portable Clinical Analyzer; iSTAT Corp, East Windsor, NJ). Organ Harvest Mice were euthanized at 4, 24, and 72 h, and then lungs, hearts, kidneys, and livers were harvested for histological analyses. These samples were either fixed in 10 % formaldehyde solution for histological analysis by a routine manner or embedded in Tissue-Tek OCT (Skura Finetek, Torrance, CA). The tissues in OCT were cut into 5 lmthick cryosections for MSC tracking.
Fig. 1 The appearance and blood gas analysis of lung grafts. a Transplanted lung showed normal appearance without pulmonary edemas and collapses at 24 h post-reperfusion. b Blood gas assay of lung grafts. MSC administration significantly improved the PaO2/ FiO2 compared to untreated mice, and pulmonary function increased over the time period of this experiment. *P \ 0.0001 versus untreated group
Infused MSC Tracking The injected MSCs were tracked by eGFP fluorescence in tissue cryosections of grafted lungs, and native lung, hearts, livers, and kidneys under a fluorescence microscope (ZEISS, Oberkochen, Germany). Graft Histology Graft sections were stained with hematoxylin & eosin (H&E) for evaluating and determining IRI score in a blinded manner. Pulmonary injury was scored using a 7-point scale according to the combined assessments of
alveolar congestion, hemorrhage, alveolar/interstitial edema, and inflammatory cells infiltration in the airspace and vessel wall in accordance with previous reports [5, 18]. Apoptosis Assay Cell apoptosis in lung grafts 72 h after reperfusion was determined by terminal deoxynucleotyl transferase dUTP nick end labeling (TUNEL) staining according to the instruction of the kit (R&D system, Minneapolis, MN). Apoptotic cells with severe DNA damage would be labeled brown.
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Immunohistological Examination The protein expression in tissue sections by 72 h was detected using an avidin–biotin immunoperoxidase method (Vector Laboratories, Burlingame, CA) with hematoxylin showing nucleus. The primary antibodies rat anti-mouse CD68 (macrophage marker), myeloperoxidase (MPO, activated neutrophil marker), CD4 and CD8 monoclonal antibodies, IL-6, TNF-a, Toll-like receptor-4 (TLR4) were purchased from BD Biosciences (Mississauga, ON, Canada) and anti- tumor necrosis factor-a stimulated gene/protein 6 (TSG-6) antibody from R&D system (Minneapolis, MN). Statistical Analysis All data were presented as mean ± standard error of mean (SEM). Comparison between two groups was performed by two-tailed t test or analysis of variance (ANOVA). P value of less than 0.05 was considered statistically significant.
Results MSC Treatment Achieves Normal Appearance and Function of Lung Grafts
The blood oxygenation (PaO2/FiO2, mmHg) as a biomarker for the pulmonary function of gas exchange was examined at 4, 24, and 72 h following reperfusion. At as early as 4 h post-transplantation, the blood oxygenation in MSC-treated mice showed significant improvement compared with that of untreated group (153.6 ± 4.1 vs. 46.0 ± 5.2 mmHg, P \ 0.0001, t test, n = 6) (Fig. 1b). At 24 h post-transplantation, graft oxygenation capacity continued to elevate owing to MSC infusion (216.3 ± 5.5 vs. 90.0 ± 1.0 mmHg, P \ 0.0001, t test, n = 6) (Fig. 1b). By 72 h post-transplantation, oxygenation reached to the normal levels in MSC-treated group, which were not seen in controls (309.0 ± 3.6 vs. 111.0 ± 8.5 mmHg, P \ 0.0001, t test, n = 6) (Fig. 1b). The two-way ANOVA also showed that the blood oxygenation in MSCs-treated group was significantly higher than that in control group at all of time points (P \ 0.0001, n = 6). Thus, MSC infusion remarkably improved the gas exchange of the grafts and such effect was further enhanced over the time period of this experiment. Intravenous Infused MSCs Mainly Locate in Grafted Lungs
As shown in Fig. 1a, grafted lungs harvested from MSCtreated recipients 24 h post-transplantation, displayed normal morphology and well congestion without pulmonary edemas, collapses or necrosis, which were different from the lung grafts in untreated controls with severe tissue damage (data not shown).
We tracked the fate of MSCs with eGFP fluorescence at the same time points as gas exchange detection. In coincidence with previous reports, substantial numbers of eGFP?-MSCs were localized in grafted lungs at as early as 4 h but not many in the naı¨ve lung (Fig. 2), and such MSCs homing to injured sites almost remained unchanged until 72 h after transplantation (data not shown). While a few of eGFP? MSCs were seen in the native lungs, but none of MSCs were
Fig. 2 The dislocation of infused MSCs in the organs of the recipients. The fluorescent images (9400) displayed the location of eGFP?-MSCs in each organ at 4 h post-transplantation. Most eGFP
positive MSCs located in grafted lungs (a), a few in native lungs (b) but none were observed in native hearts, livers, or kidneys (c). Data were a typical microscopic view of organ sections in each group
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Lung Fig. 3 Histological analysis of lung grafts. a Sections of transplanted lungs harvested 24 h following transplantation were stained with hematoxylin and eosin (9200), and showed severe lung injury characterized by obvious alveolar congestion and collapse in untreated mice, while MSC treatment ameliorated this injury. b The IRI score of lung grafts was evaluated based on the pathological performance. The graft injury score was significantly higher in untreated group compared with that of MSC-treated mice. *P \ 0.05 versus untreated group
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found in native hearts, livers, or kidneys of the recipients (Fig. 2), indicating that intravenously delivered MSCs indeed migrated and homed to grafted lungs and then exerted their biological effects. MSC Delivery Confers Protection of the Lung Grafts Histopathology of lung transplants in untreated mice showed typical features of IRI with interstitial hemorrhage, edema, alveolar congestion, and collapse (Fig. 3a). By contrast, MSC treatment clearly attenuated these changes at the same time point, which was evidenced by that the alveolar structure was intact and free of edema and fluid (Fig. 3a). These histological observations were further confirmed by the injury scores, representing the total grades of alveolar congestion, hemorrhage, edema, and infiltration of inflammatory cells. The injury score in MSC-treated group was much lower than that of untreated controls (Fig. 3b, 4-hour reperfusion: untreated 3.5 ± 0.6 versus MSC-treated 1.6 ± 0.5; 24-hour reperfusion: untreated 5.6 ± 0.5 versus MSC-treated 4.2 ± 0.4; 72-hour reperfusion: untreated 4.5 ± 0.6 versus MSC-treated 2.6 ± 0.5, P \ 0.05), suggesting that MSC treatment significantly improved the pathological performance of grafted lungs. Infused MSCs Attenuate Cell Apoptosis in the Lung Grafts TUNEL assays of lung grafts by 72 h post-transplantation showed more apoptotic cell death in untreated controls than
Untreated
Fig. 5 Inflammatory cell infiltration in lung transplants at 72 h post- c transplantation. a Massive infiltration of macrophages, neutrophils, and CD8? T cells was found in lung grafts of untreated controls, while MSC infusion markedly reduced intragraft inflammatory cell infiltration. b Decreased expression of TNF-a and IL-6 was detected in lung grafts of MSC-treated mice as compared with those of untreated controls. Data were a representative of six determinants. Magnification 9200
that of MSC-treated animals (Fig. 4). Similar changes were also found in lung grafts at both 4 and 24 h post-transplantation (data not shown). These results suggested that MSC delivery effectively prevented cell apoptosis postreperfusion, which was beneficial to the gaseous exchange of lung grafts. MSC Therapy Suppresses Intragraft Inflammation The infiltrating cells comprising macrophages, neutrophils, CD4?, and CD8? T cells were examined by immunohistochemistry staining 72 h post-reperfusion. As shown in Fig. 5a, much more positively staining of CD68 (macrophages), MPO (neutrophils), and CD8 (CD8? T cells) was seen in lung grafts from untreated animals as compared to fewer of these infiltrates in MSC-treated grafts (Fig. 5a). While, only a few CD4? T cells were observed in lung grafts (data not shown). These data indicate that MSC infusion could effectively attenuate inflammatory immune response to the transplanted lungs after cold IRI in this model. In consistence with the levels of inflammatory cell infiltration, a high level of intragraft expressions of
MSC-treated
Fig. 4 Cell apoptosis in lung grafts at 72 h post-transplantation. Intragraft apoptotic cell death was found in untreated group. In contrast, MSC administration prevented cell apoptosis in lung grafts. Data were a representative of six determinants
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Macrophages
Neutrophils
CD8+ cells
MSC-treated
Untreated
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MSC-treated
TNF-α
IL-6
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Lung Fig. 6 MSCs reduce intragraft inflammatory cytokines and TLR Expression, but increase TSG-6 expression at 72 h following Lung transplantation. a The level of TLR-4 expression in lung grafts of untreated animals was much higher than that of MSC-treated animals. b MSC infusion markedly increased TSG-6 level in lung grafts as compared to untreated controls. Data were a representative of six determinants. Magnification 9200
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Untreated
MSC-treated
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proinflammatory cytokine TNF-a and IL-6 was also detected in untreated controls (Fig. 5b) 72 h following transplantation, whereas MSC infusion markedly decreased the cytokine expression at the same time point (Fig. 5b), implying that the suppression of intragraft inflammation achieved by MSCs could contribute to the improvement of survival and function of lung grafts.
MSC Treatment Reduces TLR-4 but Enhances TSG-6 Expression in Lung Transplants Toll-like receptors (TLRs), TLR-4 in particular, play a pivotal role in the inflammatory process of pulmonary IRI, and are an important functional biomarker for the local inflammation [19, 20]. We examined TLR-4 protein in lung
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grafts after 72 h of reperfusion, and found that MSC infusion greatly suppressed intragraft TLR-4 expression compared with untreated group (Fig. 6a), which was in line with its suppression of inflammatory factors as presented in Fig. 5. Tumor necrosis factor-a stimulated gene/protein (TSG6) secreted by MSCs has been reported to have multipotency in anti-inflammation, and plays important roles upon tissue injury [21, 22]. In the present study, TSG-6 expression was assessed in lung grafts 72 h post-transplantation. The results showed that MSC treatment was obviously associated with promoted intragraft TSG-6 expression as compared with untreated controls (Fig. 6b), indicating that TSG-6 production could be one of the inherent mechanisms for anti-inflammatory efficacy of MSCs in the present study.
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Discussion The present study was designed to investigate the efficacy of autologous bone marrow-derived MSCs in the prevention of pulmonary cold IRI post-lung transplantation using a murine model of syngeneic lung transplantation. The lung is vulnerable to pathological damage, and any condition impairing the alveolar oxygenation or pulmonary blood flow will finally result in lung ischemia [3]. The rigorous anoxic ischemia due to the interruption of both pulmonary bloodstream and ventilation for up to several hours are inevitable in lung transplantation. The subsequent reperfusion of grafted lungs usually leads to alveolar damage, pulmonary edema, and hypoxemia within 72 h, which is the common risk factor for graft dysfunction and chronic function loss following lung transplantation. Animal studies have prompted the development of pulmonary IRI therapy post-lung transplantation. In the recent years, investigations have been focusing on the benefits of MSCs in the prevention of lung IRI; however, most of these studies used a warm ischemia model with pulmonary hilum clamped by a clip [5, 7, 18, 23]. Actually in clinic, IRI following lung transplantation is inevitable during the surgical process. The applications of rat and other large animal implant models based on cuff technique greatly contribute to the understanding of pathogenesis of lung IRI [24]. Although the mouse model of orthotopic lung transplantation subjects to high microsurgical demands [25, 26], mice models offer a broader research scope because of knockout and transgenic backgrounds. In the present study, we developed the orthotopic, single-lung transplantation model in mice by anastomosis of pulmonary vein and bronchus using appropriate cuffs and pulmonary artery sutured with 11-0 silk suture, which could effectively prevent thrombosis. With 6-hour lung cold ischemia, we tested the role of intravenously infused MSCs on the prevention of lung cold I/R injury at 4, 24, and 72 h following transplantation. Data presented here indicated that grafted lungs in MSC-treated mice showed near normal appearance without any visible edemas or collapses at 24 h. The gas exchange assay demonstrated that infused MSCs significantly improved the blood oxygenation (PaO2/FiO2) of lung grafts at as early as 4 h after reperfusion, with further amelioration that approached normal values by 72 h post-transplantation. By contrast, the gas exchange of mouse recipients received saline (as untreated controls) never reached to the level that was achieved by MSC treatment, indicating that intravenously delivered MSCs effectively improved the function of lung grafts. It has been widely accepted that MSC-based cell therapy has been a promising strategy for the treatment of degenerating and immunological disorders, and animal studies
confirm the long-term regenerative and immunomodulatory function of MSCs, while the fate and distribution of MSCs in vivo post infusion remain to be elucidated. Undoubtedly, intravenous administration is the most suitable route for MSC delivery in potential clinical application. Although MSCs have the properties of migrating and homing to injured sites, some reports concerning cell tracking have indicated that the majority of MSCs localizes to the lungs post intravenous infusion with only a small portion homing to the target organs along with circulation [13, 14]. In fact, this provides a real benefit to the treatment of pulmonary injury. In the current study, we used eGFP-transgenic MSCs for IRI treatment and tracked the migration in a short term. As expected, infused MSCs mainly homed to grafted lungs rather than other native organs of recipient mice, providing important preconditions for their therapeutic effect. Histological assessment indicated that MSC infusion effectively protected the alveolar network and reduced interstitial hemorrhage, edema, alveolar congestion and collapse 24 h post-transplantation. The injury score analysis based on the pathological features demonstrated that MSC treatment significantly decreased the injury score compared with controls at each time point. In addition, we did wet/dry ratio assay and found less fluid inside lung grafts of MSC-treated mice as compared to those of untreated recipients (data not shown). Apoptosis is the main mechanism of cell death observed in lung transplantation-triggered IRI. Donor lungs are unavoidably confronted parenchyma cell apoptotic death during the preservation period ex vivo, and cell apoptosis in lung grafts will be worsened following blood reperfusion [2, 27]. TUNEL detection indeed showed that the amount of apoptotic cells located in grafted lungs of untreated mice 72 h post-transplantation, which got markedly reduced in that of MSC-treated mice. We believe that this reduction could contribute to the protective role of MSCs in pulmonary function. Experimental studies regarding the mechanism of IRI highlight that it is a pathologically inflammatory course involves a neutrophil predominating immunocyte infiltration and proinflammatory cytokine extravasation [3]. We also checked the intragraft cellular infiltration and inflammation-related molecular expression by 72 h. The results showed that much more macrophages, neutrophils, and CD8? T cells located in lung grafts of untreated recipients, while MSC administration greatly reduced intragraft inflammatory cell recruitment. It is worth mentioning that we did not find infiltrating CD4? T cells in both MSCtreated and untreated control groups at 72 h, which was different from some earlier reports [7, 25], possibly due to the different detection time points and the different transplant models. In addition, cytokine examination indicated
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that both IL-6 and TNF-a expressions were effectively attenuated in lung grafts of MSC-treated mice compared with controls. The marked decrease of inflammatory cells and cytokines suggested the diminished intragraft inflammation achieved by MSC infusion, which benefited to the protection of lung grafts and restored the pulmonary function. As the first line of defense, the innate immune system is activated with the initial reperfusion in minutes posttransplantation. TLR signaling, a key innate immune pathway, has been demonstrated to contribute to pulmonary IRI-associated inflammatory response [28, 29]. In particular, TLR4 activation plays critical roles in initial edema formation and onset of downstream signal pathways concerning inflammation process of lung IRI in pulmonary hilum clamping model [19, 20, 30]. Thus, in current study, we evaluated the TLR4 level 72 h post-transplantation and found that the intragraft TLR4 expression in the untreated control group was much higher than that of MSC-treated mice. In other words, MSC infusion effectively inhibited TLR4 signaling pathway, which might make important contributions to the low proinflammatory cytokine production, since IL-6 and TNF-a are both downstream molecules in TLR4 pathway [31]. Considerable efforts have been made to explain the therapeutic potentials of MSCs in many disorder treatments. The damage repair and function improvement observed in subsequent investigations showed much more close connection with the paracrine secretions or cell-tocell contacts [32, 33]. It is now believed that MSCs cultured in vitro secrets a large number of cytokines and expresses high levels of additional beneficial factors after engrafting into injured sites [32]. Among these factors, TSG-6 is a multipotent anti-inflammatory protein and makes contributions to the immuno-modulatory function of MSCs [13, 22]. Therefore, we examined intragraft level of TSG-6 and did find TSG-6 expression in lung grafts of MSC-treated recipients, but hardly found this protein in lung grafts of untreated mice. The tissue repair and function restore achieved by MSC infusion could be partly ascribed to the TLR-4 attenuation together with TSG-6 secretion.
Conclusion Owing to the important role of MSCs in the suppression of inflammatory response and improvement of pulmonary function, our data suggest that MSC infusion could be used as a potential strategy for preventing cold IRI following lung transplantation. In addition, the modified mouse orthotopic lung transplantation model used in this study would provide a useful tool to the further mechanism and
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efficacy studies in achieving long-term lung transplant acceptance. Acknowledgments We are grateful to Dr. Bertha Garcia at Western University, London, Canada for her critical review of the histological slides, and to Drs. Shuhua Luo and Weihua Liu for their technical assistance. This work was supported by Grant to H.W. from National Natural Science Foundation of China (No. 81273257), and National High Technology Research and Development Program 863 (2012AA021003). C.D. was supported by grants from the Kidney Foundation of Canada and the Canadian Institutes of Health Research. Conflict of interest disclose.
The authors have no conflicts of interest to
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