Therapeutic Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Models of Pulmonary and Extrapulmonary Acute Lung Injury

Abstract

Bone marrow-derived mesenchymal stem cells (MSCs) offer a promising therapy for acute lung injury (ALI). However, whether the same MSC treatments possess similar potential for different ALI models is not fully clear. The present study evaluated the distribution and therapeutic effects of intravenous MSC administration for the treatment of intratracheal lipopolysaccharide (LPS)-induced intrapulmonary ALI and intravenous LPS/zymosan-induced extrapulmonary ALI, matched with lung injury severity, at 30 min and 1, 3, and 7 days. We found that MSC transplantation attenuated lung injury and inhibited lung inflammation in both ALI models. The benefits of MSCs were more significant in the intrapulmonary ALI mice. In vivo and ex vivo fluorescence imaging showed that MSCs primarily homed into the lung. However, more MSCs were recruited into the lungs of the intrapulmonary ALI mice than those of the extrapulmonary ALI mice over the time course. A few MSCs were also detected in the liver and spleen at days 3 and 7. In addition, the two ALI models showed different extrapulmonary organ dysfunction. A lower percentage of cell apoptosis and SDF-1α levels was found in the liver and spleen of the intrapulmonary ALI mice than in those of the extrapulmonary ALI mice. These results suggested that the two ALI models were accompanied with different degrees of extrapulmonary organ damage, which resulted in differences in the trafficking and accumulation of MSCs to the injured lung and consequently accounted for different therapeutic effects of MSCs for lung repair in the two ALI models. These data suggest that intravenous administration of MSCs has a greater potential for the treatment of intrapulmonary ALI than extrapulmonary ALI matched with lung injury severity; these differences were due to more recruitment of MSCs in the lungs of intrapulmonary ALI mice than those of extrapulmonary ALI mice. This finding may contribute to the clinical use of MSCs for the treatment of ALI.

Keywords

Mesenchymal stem cells (MSCs)Acute lung injury (ALI)Lung repair Homing Retention

Introduction

Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are significant causes of morbidity and mortality in critically ill patients (12,30,37). Despite decades of research, few therapeutic strategies for treating clinical ARDS have emerged. Current treatments are supportive, and the only effective therapies act by limiting the iatrogenic injury that is associated with positive fluid balance or mechanical ventilation (1,27). However, prolonged mechanical ventilation can further contribute to ARDS-related morbidity (5). Currently, poor specific pharmacological therapies for the attenuation of acute lung injury and the promotion of lung repair in ALI/ARDS have been identified (2,5,7,28).

Marrow-derived mesenchymal stem cells (MSCs) are currently proposed as a promising cell therapy for ALI/ARDS. Current available evidence shows that the intravenous administration of MSCs, which has been used by the majority of investigators with great potential clinical applications (44), could potentially be used for the attenuation of lung injury and the augmentation of repair. MSCs exert their therapeutic effects through several mechanisms, including localization to the injured lung, differentiation into mature lung epithelial and endothelial cells, and paracrine secretion of mediators such as growth factors and cytokines to modulate localized inflammation and promote local endothelium and epithelium repair and ultimately reduce the mortality of ALI mice (6,13).

To date, a number of factors could initiate and cause the development of ALI/ARDS, including sepsis, pneumonia, trauma, or aspiration of gastric contents (37). Based on these etiological factors, clinicians categorize ALI/ARDS into intra- and extrapulmonary ALI/ARDS. Sepsis-induced extrapulmonary ALI/ARDS and pneumonia-induced intrapulmonary ALI/ARDS are the leading types of ALI/ARDS in clinics (11,12,37). Excessive inflammatory responses in lung and noncardiogenic pulmonary edema are common pathological changes in both intra- and extrapulmonary ALI/ARDS (38,39). However, sepsis-induced ALI is always accompanied by systemic endothelial cell injury and multiple organ dysfunction. The different extrapulmonary organ dysfunctions between the two types of ALI/ARDS might result in different distributions of systemically infused MSCs and ultimately result in different responses to the same MSC-based therapies.

The currently available data demonstrate that the intravenous administration of MSCs has beneficial effects in intrapulmonary ARDS/ALI induced by intratracheal bleomycin (26), endotoxin (10), and ventilator (9), and extrapulmonary ARDS/ALI, which is induced by intraperitoneal or intravenous endotoxin (17,41), cecal ligation, and puncture (25). However, these studies used different ALI models, different doses and times of MSC delivery, and they also varied with different measured time points and the severity of lung injury. The therapeutic effects of the same systemically infused MSC treatment strategies in both intra- and extrapulmonary ARDS/ALI with comparable lung injury are not fully clear.

The present study evaluated the distribution and therapeutic effects of the intravenous administration of MSCs for the treatment of intra- and extrapulmonary ALI, which matched the severity of lung injury.

Materials and Methods

Ethics Statement

Eight- to 10-week-old male C57BL/6 mice (Laboratory Animal Center, Academy of Military Medical Sciences, Beijing, China) were housed in a specific pathogen-free facility and received humane care. The Committee of Animal Care and Use of Southeast University approved the study.

Production of Enhanced Green Fluorescent Protein (eGFP)-Expressing MSCs with Lentiviral Vectors

EGFP-expressing MSC lines were constructed in our laboratory, and the methods are described in an unpublished paper. Briefly, MSCs from the bone marrow of male C57BL/6 mice and 293FT cells were purchased from Cyagen Biosciences, Inc. (Guangzhou, China). MSCs in passages 4-7 were used for transduction. The third-generation self-inactivating HIV-based vectors (HIV-EF1α-eGFP) were produced in 293FT cells using four plasmids: lentiviral expression vector pLV.EX3d.P/neo, which contains the eGFP gene, and three packaging plasmids of pLV/helper-SL3, pLV/helper-SL4, and pLV/helper-SL5 (Cyagen Biosciences, Inc., Guangzhou, China). A high titer of recombinant lentiviral vectors were obtained and transducted into MSCs. MSCs carrying eGFP (MSC-GFPs) were harvested after selection with G418 (0.5 mg/ml; Amresco, Inc., Solon, OH, USA) for 7-14 days. The MSC-GFPs were cultured in normal culture medium, which were composed of a 1:1 mix of Dulbecco's modified Eagle media/nutrient mixture F-12 (DMEM/F12; Wisent, Inc., St-Bruno, Quebec, Canada) containing 10% FBS (Wisent, Inc.) and 1% antibiotics (streptomycin and penicillin; Wisent, Inc.) for 30 passages after transduction to evaluate their long-term transduction efficiency by detection of eGFP expression using an FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The transduction efficiency of MSCs mediated by lentiviral vectors was as high as 86-94%, even after transduced MSCs were cultured for 30 generations. Cells in passages 7-10 were used for in vivo experiments.

Murine Models of ALI

Intra- and extrapulmonary ALI models, matched for lung injury severity, were produced in this study. The anesthetized mice in the extrapulmonary ALI models received 3 mg/kg LPS (Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO, USA) intravenously (IV) and 10 mg/kg zymosan A (Saccharomyces cerevisiae; Sigma-Aldrich) IV 2 h later (22–24). The anesthetized mice in the intrapulmonary ALI model received intratracheal (IT) LPS at 0, 1, 2, 3, 4, or 5 mg/kg. The mice were sacrificed 4 h after the LPS/zymosan IV or LPS IT challenge, and lung tissues were collected for analyses. Lung histopathology was evaluated with hematoxylin and eosin (Beyotime Institute of Biotechnology, Haimen, China) staining, and lung edema was measured using the lung wet weight/body weight ratio (LWW/BW) to match the severity of the lung injury.

Labeling of MSC-GFPs and Cell Transplantation

MSC-GFPs were labeled with CellVue NIR815 dye (eBioscience, Inc., San Diego, CA, USA) according to the manufacturer's instruction. Four hours after the LPS 3 mg/kg IT or LPS/zymosan IV challenge, labeled MSC-GFPs [5 × 10⁵ cells resuspended in 100 μl phosphate-buffered saline (PBS; Wisent, Inc.)] (41) were transplanted into the ALI mice via tail vein injection [ALI(IT) + MSC-GFP; ALI(IV) + MSC-GFP]. ALI mice that received LPS IT 3 mg/kg IT and the same amount of PBS IV 4 h later served as the treatment control [ALI(IT)+PBS(IV)]. The mice received an initial aliquot of PBS IT, and the same amount of PBS IV 4 h later served as the negative control [PBS(IT) + PBS(IV)]. The mice in the PBS(IT) + MSC-GFP(IV) group received an initial aliquot of PBS IT and the same amount of MSC-GFPs IV 4 h later [PBS(IT) + MSC-GFP]; we set this group to evaluate the effects of transplanted MSC-GFPs on the normal lung of mice. Three groups of mice [PBS(IT) + MSC-GFP; ALI(IT) + MSC-GFP; ALI(IV) + MSC-GFP] were subjected to in vivo and ex vivo optical imaging and sacrificed. Tissues were collected for analysis at 30 min, 1 day, 3 days, and 7 days after MSC-GFPs treatment.

In Vivo and Ex Vivo Optical Imaging

In vivo and ex vivo optical imaging were performed to track the transplanted MSC-GFPs in mice. At 30 min, 1 day, 3 days, and 7 days post-MSC-GFP injection, three mice at each time point were subjected to optical imaging under a Maestro In Vivo Optical Imaging System (CRi, Woburn, MA, USA) (43). A filter set (excitation = 786 nm; emission = 814 nm; exposition time = 4,000 ms) was used to detect target fluorescence (from NIR815 dye) and autofluorescence (from the skin of the mice). Images from decubitus were obtained and analyzed using Maestro 2.10.0 software (CRi). The mice were sacrificed, and the right ventricle of the heart was perfused with cold PBS to flush the organs and avoid potential contamination of MSC-GFPs from the blood stream. Ex vivo imaging of the excised organs, including the lung, heart, kidney, spleen, pancreas, small intestine, and liver, was performed. Fluorescence signals were separated using multispectral imaging technology. The fluorescence intensity of each organ was measured by placing ROIs on the organ and determined by a person who was blinded to the experimental groups. The average signals were normalized to the exposure time and the ROI area (scaled counts/s) (35,43).

Fluorescence Microscopy

Fluorescence microscopy of a lung section from mice was also performed to detect the expression of eGFP and identify the recruitment of MSC-GFPs into the lung. After ex vivo imaging studies, the right upper lobes of the mice (n = 3 per group at each time point) were collected and fixed in 4% paraformaldehyde (Shanghai Ling Feng Chemical Reagent Co., Ltd., Shanghai, China) overnight at 4°C. Tissues were embedded in OCT compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and cut into 5-μm thick sections. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and eGFP fluorescence was detected using an Olympus IX71 microscope (Olympus Co., Tokyo, Japan) equipped with a CCD camera.

Lung Histopathology Analysis

The lower lobe of the right lung of the mice from the PBS(IT) + PBS(IV), PBS(IT) + MSC-GFP, ALI(IT) + PBS(IV), ALI(IT) + MSC-GFP, and ALI(IV) + MSC-GFP groups (n=6 for each group) was removed immediately after the mice were euthanized, fixed in 10% PBS-buffered formalin (Shanghai Ling Feng), embedded in paraffin (Nanjing WanQing Chemical Glassware Instrument Co., Ltd., Nanjing, Jiangsu, China), and cut into 5-μm-thick sections. Sections were stained with a hematoxylin and eosin staining kit (Beyotime Institute of Biotechnology). The severity of lung injury was quantified blindly by a pathologist based on images in 10 randomly selected high-power fields (400x) for each section following a previously published scoring system (31). The scoring items included six criteria: edema, alveolar and interstitial inflammation, alveolar and interstitial hemorrhage, atelectasis, necrosis, and hyaline membrane formation. Each criterion was graded according to a 0- to 4-point scale, and the mean sum of each field score was compared between groups.

Apoptosis Assessment

Apoptosis in organ tissues after injury indicated the severity of damage, which may influence the recruitment of MSC-GFPs. We observed that fluorescence signals were detected mainly in the liver, spleen, and lung. Therefore, the percentage of apoptotic cells in these organs (n=3 for each group) was assessed using an Annexin V-FITC assay kit (Sigma-Aldrich). After the mice were euthanized, the left upper lobe of the lung, left lobe of the liver, and half of the spleen were collected and collagenase (1 mg/ml; Sigma-Aldrich) digested into single-cell suspensions, as previously described with minor modifications that we used DMEM/F12 (Wisent, Inc.) containing 10% FBS (Wisent, Inc.) and 1% antibiotics (streptomycin and penicillin; Wisent, Inc.) as the tissue culture medium in this study (34). Red blood cells were lysed, and the cells were suspended in a 1x binding buffer at a concentration of ~1 × 10⁶ cells/ml. A total of 5 μl annexin V FITC conjugate (annexin V) and 10 μl of a propidium iodide solution (PI) were added to each cell suspension. The cells were stained for 10 min in the dark at room temperature and analyzed using a flow cytometer (BD Biosciences, Mountain View, CA, USA) within 1 h after staining. Live cells were not stained with PI or annexin V. Cells undergoing the apoptotic process were stained with annexin V.

Measurement of Chemokine and Cytokine

The left lower lobe of the lung, right lobe of the liver, and half of the spleen were snap-frozen and later processed as homogenates. The levels of SDF-1α in the liver, spleen, and lung were measured using an ELISA kit (Cusabio Biotech Co., Ltd., Wuhan, China). IL-1β, IL-6, and IFN-γ concentrations in the lungs were assessed using an ELISA kit (ExCellBio, Shanghai, China). All of the ELISAs were performed strictly according to the instructions of the manufacturer.

Statistical Analysis

Data are expressed as the means ± standard deviations (SD). For multiple group comparisons, one-way ANOVA followed by Tukey's post hoc test was performed using statistical analysis software SPSS 16.0. Values of p < 0.05 were considered statistically significant.

Results

The Severity of Lung Injury of LPS 3 mg/kg IT-Induced Intrapulmonary ALI Matched with LPS/Zymosan IV-Induced Extrapulmonary ALI

Lung sections of the ALI(IV) mice stimulated with LPS/zymosan IV displayed interalveolar septal thickening, patches of inflammatory infiltrates, and interstitial and alveolar edema. The lung injury score was approximately 6.6, which was significantly increased compared to the negative control group. Treatment with LPS 0, 1, 2, 3, 4, or 5 mg/kg IT increased the severity of lung injury in a dose-dependent manner. Lung histopathology appearance of LPS 3 mg/kg IT with a lung injury score of 6.4 was identical to LPS/zymosan IV with a lung injury score of 6.6 (Fig. 1A, B). The LWW/BW of LPS 3 mg/kg IT and LPS/zymosan IV also matched (6.03 ± 0.15 vs. 6.03 ± 0.4, p > 0.05) (Fig. 1C). Therefore, we used LPS 3 mg/kg IT to produce the intrapulmonary ALI model in our subsequent experiments.

Figure 1

Production of intra- and extrapulmonary ALI animal models matched in the severity of lung injury. (A) Representative images of histopathology of LPS/zymosan IV or different doses of LPS IT-induced lung injury (200x). (B) Lung injury scores of LPS/zymosan IV or different doses of LPS IT-induced lung injury. (C) Comparison of lung wet weight/body weight (LWW/BW) of LPS/ zymosan IV or different doses of LPS IT-induced lung injury. The lung histopathology appearance of LPS 3 mg/kg IT with a lung injury score of 6.4 was identical to LPS/zymosan IV with a lung injury score of 6.6. The LWW/BW of LPS 3 mg/kg IT and LPS/zymosan IV also matched. Therefore, we used LPS 3 mg/kg IT to produce the intrapulmonary ALI model in our subsequent experiments (n = 5 per group; *p < 0.05 vs. IT-0 mg/kg LPS group l; #p < 0.05 vs. IV- LPS/zymosan group).

Intravenous MSC-GFP Transplantation Showed Greater Improvements in Lung Histopathology of Intrapulmonary ALI Than Extrapulmonary ALI

We used lung histopathology to evaluate the severity of lung injury in the mice. There were minimal pathological changes in the lung tissues and a low lung injury score in the PBS(IT) + PBS(IV) and PBS(IT) + MSC-GFP groups, which suggested that MSC-GFP transplantation had no detrimental effect in mouse lungs. However, lung sections showed severe lung injury in the ALI(IT) + PBS(IV) group with marked interalveolar septal thickening, diffuse inflammatory infiltrates, interstitial and alveolar edema, and interstitial hemorrhage that was aggravated gradually until day 3 with a mean maximum lung injury score of 12.1. The injury score attenuated slightly over time, but it remained serious at day 7 with a lung injury score of 9.6. Lung sections from the ALI(IT) + MSC-GFP and ALI(IV) + MSC-GFP mice showed similar severities of lung injury with those of the ALI(IT) + PBS(IV) group at 30 min and day 1, but the injuries attenuated significantly at 3 and 7 days. Lung injury scores decreased in the two ALI groups treated with MSC-GFPs over time, but the beneficial effects of MSC-GFPs were more pronounced in the ALI(IT) group at day 7, in which the lung injury score was only 1.6, which is an almost normal level. The lung injury score was 3.4 in the ALI(IV) + MSC-GFP group. These results suggested that the administration of MSC-GFPs improved the lung histopathology of the two ALI models to different degrees over our time course, and the therapeutic effects in the ALI(IT)+MSC-GFP group were more significant than those in the ALI(IV)+MSC-GFP group (Fig. 2A, B).

Figure 2

Therapeutic potential of intravenous MSC transplantation in intra- and extrapulmonary ALI animal models. (A) Histological evaluation of the therapeutic potential of MSCs on LPS 3 mg/kg IT- and LPS/zymosan IV-induced lung injury in mice at 30 min, 1 day, 3 days, and 7 days (200x). (B) Lung injury scores of the two ALI mouse models at different time points to evaluate the severity of lung injury. (C) Levels of IL-1β in lungs of the two models of ALI mice at 30 min, day 1, day 3, and day 7. (D) Levels of IL-6 in lungs of the two models of ALI mice at 30 min, day 1, day 3, and day 7. (E) Levels of IFN-γ in lungs of the two models of ALI mice at 30 min, day 1, day 3, and day 7. The results showed that intravenous MSC-GFP transplantation improved lung histopathology and inhibited lung inflammation more significantly in the intrapulmonary ALI mice than in the extrapulmonary ALI mice [n = 6 per group; *p < 0.05 vs. 30 min; #p < 0.05 vs. PBS(IT) + PBS(IV) group; *p < 0.05 vs. PBS(IT) + MSC-GFP group; %p< 0.05 vs. ALI(IT) + PBS(IV) group; &p < 0.05 vs. ALI(IT) + MSC-GFP group].

Intravenous MSC-GFP Transplantation Inhibited Lung Inflammation More Significantly in Intrapulmonary ALI Mice Than Extrapulmonary ALI Mice

The levels of proinflammatory cytokines IL-1β, IL-6, and IFN-γ were measured in lung homogenates at 30 min, 1 day, 3 day, and 7 days. IL-1β, IL-6, and IFN-γ levels in the three ALI groups in response to LPS challenge were elevated significantly at 30 min compared to the PBS(IT) + PBS(IV) and PBS(IT) + MSC-GFP control groups. The levels of IL-1β and IFN-γ in the ALI group with PBS treatment was persistently increased until day 3, then decreased gradually at day 7, but still higher than the PBS(IT) + PBS(IV) and PBS(IT) + MSC-GFP control groups; IL-6 level peaked at day 1, decreased gradually, and almost reached the level of the control group at day 7. By day 1, most proinflammatory mediators were reduced by MSC-GFP treatment in both ALI groups and almost returned to the normal level at day 7. The decrease in the ALI(IT) + MSC-GFP group was more significant than in the ALI(IV) + MSC-GFP group (Fig. 2C-E).

More Labeled MSC-GFPs Were Recruited Into the Lungs of Intrapulmonary ALI Mice After Intravenous MSC-GFP Transplantation

In vivo optical imaging showed enhanced fluorescence intensity in the lung and liver 30 min postinjection in the PBS(IT) + MSC-GFP, ALI(IT) + MSC-GFP, ALI(IV) + MSC-GFP groups, which suggests that NIR815 dye labeled MSC-GFPs that were predominantly recruited within the lung and liver. No differences were observed between the three groups (Fig. 3A). The signal intensity in the lungs of the PBS(IT)+MSC-GFP group decreased rapidly over time, and no signals were detected 3 days postinjection. However, the signal intensity in the lungs of the ALI(IT)+MSC-GFP group peaked at day 1 and then decreased gradually. A signal could be detected until 7 days after transplantation. The signal intensity in the lungs of the ALI(IV)+MSC-GFP group decreased gradually postinjection, but it also persisted until day 7. The signal intensity in the ALI(IV) + MSC-GFP group was weaker than in the ALI(IT)+MSC-GFP group at days 1, 3, and 7 (Fig. 3A, B). Weak signals were also detected in the spleen at day 1 in the PBS(IT)+MSC-GFP group and day 3 in the ALI(IT) + MSC-GFP and ALI(IV) + MSC-GFP groups. The signal intensity in the ALI(IV)+MSC-GFP group seemed stronger than in the ALI(IT)+MSC-GFP group, but there was no significant difference between these groups. The signals almost disappeared in all three groups at day 7 (Fig. 3A, C). The fluorescence intensity in the livers of three groups enhanced gradually, peaking at day 3 and decreasing at day 7. The uptake of the labeled MSC-GFP in the liver of the PBS(IT)+MSC-GFP group at day 1 and day 3 was higher than that of the ALI(IT)+MSC-GFP group, and the signal intensity of PBS(IT)+MSC-GFP group decreased more rapidly at day 7 than in the ALI(IT) + MSC-GFP and ALI(IV) + MSC-GFP groups. The uptake of labeled MSC-GFP in the liver of the ALI(IV) + MSC-GFP group was higher than in the ALI(IT)+MSC-GFP group at day 1, day 3, and day 7 postinjection (Fig. 3A, D).

Figure 3

Changes in fluorescence intensity of mice using optical imaging. (A) Near infrared color-coded fluorescence images of mice in a supine position at 30 min, day 1, day 3, and day 7 post-MSC transplantation. (B) The value of the average fluorescent signal intensities in the lung was measured from 30 min to day 7. (C) The value of the average fluorescent signal intensities in the spleen was measured from 30 min to day 7. (D) The value of the average fluorescent signal intensities in the liver was measured from 30 min to day 7. The results showed that the fluorescent signals were detected mainly in the lung, spleen, and liver after cell delivery. There was even a notable enhancement in the lung in the intrapulmonary ALI group at day 1. The difference between the two ALI groups remained significant even 7 days postinjection. The results also showed that fluorescent signals in the liver and spleen appeared higher in the extrapulmonary ALI group over the time course [n = 3 per group; *p < 0.05 vs. 30 min; #p < 0.05 vs. PBS(IT) + MSC-GFP group; &p < 0.05 vs. ALI(IT) + MSC-GFP group].

Ex vivo imaging was also performed on excised organs to further verify the MSC-GFP homing observed in vivo (Fig. 4A). Labeled MSC-GFPs were mainly recruited to the lung, liver, and spleen over the time course, but the uptake in the lung was higher than in the liver and spleen initially. The fluorescence intensity in the liver and spleen increased gradually over time, especially in the livers, and the intensities in these organs surpassed those in the lungs at day 7 postinjection. Signal intensities in the lung, spleen, and liver exhibited nearly identical changes as those seen in the in vivo optical imaging (Fig. 4A–D).

Figure 4

Ex vivo near infrared imaging of organs. (A) Near infrared color-coded fluorescent images of organs of a mouse at 30 min, 1 day, 3 days, and 7 days post-MSC transplantation. (B) The value of the average fluorescent signal intensity in the lung was measured from 30 min to day 7. (C) The value of the average fluorescent signal intensity in the spleen was measured from 30 min to day 7. (D) The value of the average fluorescent signal intensity in the liver was measured from 30 min to day 7. These results are consistent with in vivo optical imaging and suggest that a greater number of labeled MSC-GFP were recruited to the lungs of the intrapulmonary ALI mice than the extrapulmonary ALI mice after intravenous MSC-GFP transplantation [n = 3 per group; *p<0.05 vs. 30 min; #p<0.05 vs. PBS(IT)+MSC-GFP group; &p<0.05 vs. ALI(IT) + MSC-GFP group].

The retention of MSC-GFPs in the lung was also verified using fluorescence microscopy (Fig. 5). The results are consistent with the results of in vivo and ex vivo optical imaging and showed that the GFP-positive cells (GFP⁺) were observed in all three groups at 30 min postinjection. However, the presence of GFP⁺ reduced rapidly and almost disappeared at day 7 in the PBS(IT)+MSC-GFP group. The numbers of GFP⁺ cells were higher in the injured lung tissues of the ALI(IT) + MSC-GFP and ALI(IV) + MSC-GFP groups than in those of the PBS(IT) + MSC-GFP group over the time course. The numbers of GFP⁺ cells in the ALI(IT) + MSC-GFP group increased greatly at day 1 and then decreased gradually but remained high until 7 days after cell transplantation. The number of GFP⁺ cells in the ALI(IV) + MSC-GFP group decreased faster than in the ALI(IT) + MSC-GFP group but more slowly than in the PBS(IT) + MSC-GFP group. GFP⁺ cells were also detected in the lungs at day 7 (Fig. 5).

Figure 5

Fluorescence microscopy validation in recipient lungs. The retention of MSCs in the lungs was also verified using fluorescence microscopy (400x). The results showed that GFP⁺ cells were observed in all three groups at 30 min postinjection, but the numbers reduced rapidly and almost disappeared at day 7 in the PBS(IT) + MSC-GFP group. The numbers of GFP⁺ cells were higher in the injured lung tissues of the ALI(IT) + MSC-GFP and ALI(IV) + MSC-GFP groups than in those of the PBS(IT) + MSC-GFP group during the time course. Especially in the ALI(IT) + MSC-GFP group, the numbers of GFP⁺ cells increased greatly at day 1 and decreased gradually but remained high until 7 days after cell transplantation. The numbers of GFP⁺ cells in this group decreased faster than in the ALI(IT) + MSC-GFP group and more slowly than in the PBS(IT) + MSC-GFP group, but the GFP⁺ cells were also detected in the lungs at day 7.

Greater Apoptosis Was Detected in the Lungs and Extrapulmonary Organs of Extrapulmonary ALI Mice

Cell apoptosis in the lungs, spleens, and livers was evaluated to find the association between the distribution of MSC-GFPs in these organs and the severity of organ damage. The percentage of apoptotic cells in the lung, spleen, and liver of the PBS(IT)+MSC-GFP group did not change significantly at any time point. However, the percentage of apoptotic cells in the lungs in the two ALI groups increased and peaked at day 1 and then decreased at day 3 and day 7. The percentage of apoptotic cells in the lungs of the ALI(IT)+MSC-GFP group decreased more rapidly than in the ALI(IV) + MSC-GFP group, and this difference was significant at day 3. Cell apoptosis in the ALI(IT) + MSC-GFP group decreased to near normal levels (Fig. 6A). The percentage of apoptotic cells in the spleens and livers of the two ALI groups also increased gradually to reach a maximum at day 3 and then dropped at day 7. Cell apoptosis in the spleen and liver seemed more significantly increased in the ALI(IV) + MSC-GFP group. This increase did not reach statistical significance in the spleen, but the difference between these groups was significant in the liver at day 7 (Fig. 6B, C).

Figure 6

Organ damage in the two ALI models after MSC-GFP transplantation. (A–C) Apoptosis in the lung, spleen, and liver of the two ALI models after MSC-GFP transplantation showed that more apoptotic cells in the lung and extrapulmonary organs, including the spleen and liver, were detected in extrapulmonary ALI mice. (D–F) Expression of SDF-1α in lung, spleen, and liver of two ALI models after MSC-GFP transplantation. The result showed that higher level of SDF-1α in the lung and extrapulmonary organs, including the spleen and liver, of the extrapulmonary ALI mice than in those of the intrapulmonary ALI mice [n = 6 per group; *p < 0.05 vs. 30 min; #p < 0.05 vs. PBS(IT) + MSC-GFP group; &p < 0.05 vs. ALI(IT) + MSC-GFP group].

Higher Levels of SDF-1α in Extrapulmonary Organs of Extrapulmonary ALI Mice

Chemokines result in the homing of MSC-GFPs to the injury site. Therefore, we examined the expression of SDF-1α in the lung, spleen, and liver within 3 days of ALI. SDF-1α levels in the lung increased significantly in the two ALI groups compared to the PBS(IT) + MSC-GFP group. These levels peaked at day 1 and decreased at day 3. The SDF-1α level in the lung in the ALI(IV)+MSC-GFP group was higher than that in the ALI(IT) + MSC-GFP group at day 3 (Fig. 6D). The concentration of SDF-1α in the liver and spleen in the two ALI groups was also elevated significantly compared to that of the PBS(IT)+MSC-GFP group, and this level was gradually upregulated over the time course. The increase in SDF-1α in the liver and spleen in the ALI(IV)+MSC-GFP group was more significant than the corresponding increase in the ALI(IT)+MSC-GFP group (Fig. 6E, F).

Discussion

Several preclinical studies have reported that MSCs reduce lung injury caused by systemic (17,41) or intratracheal (10,13) LPS challenge. Recently, one study also demonstrated that human MSCs attenuated LPS-induced injury in explanted human lungs (16). Therefore, the clinical potential of MSCs for the treatment of ALI has been considerably enhanced, and MSC-based therapies are currently being tested in clinical trials to provide novel treatments for ALI/ARDS. The pathophysiology of ALI is very complex, and numerous factors can induce the development of ALI directly or indirectly. Therefore, no unified MSC-based treatment of ALI has been identified until now, and whether the same treatment strategy using MSCs has similar distribution and therapeutic effects in different ALI animal models is not fully elucidated.

The present study found that the infusion of MSCs had protective effects on attenuating lung injury and inhibiting the lung inflammation in intra- and extrapulmonary ALI mice, the results were in accordance with previous studies that MSC administration attenuated lung inflammation in both pulmonary and extrapulmonary ALI groups, no matter if the cells were delivered within 6 h or 24 h of ALI (3,18). However, the benefits were more significant in intrapulmonary ALI mice than extrapulmonary ALI mice. On the contrary, Araújo and colleagues’ study (3) found that MSC delivery showed better therapeutic effects on the histology of extrapulmonary ALI mice than that of pulmonary ALI mice. This may be explained by different animal models and different cell types being used in the two studies, which resulted in different recruitment of MSCs in the injured lungs (our study showed more MSCs being recruited in the lung of pulmonary ALI mice over the time course, while Araújo and colleagues’ study showed more bone marrow-derived mononuclear cells recruited in the lung of extrapulmonary ALI mice at day 7), and finally leading to different benefits in two ALI models of the two studies. This study showed the different fates and therapeutic effects of the same MSC treatment strategies in intra- and extrapulmonary ALI models. These results indicated that the short-term retention of MSCs in the lung was very important for MSCs to promote lung repair, and the treatment of MSCs clinically should be based on the type of ALI being treated.

We first needed to match the severity of lung injuries of different ALI models to evaluate the treatment effects of MSCs on different ALI models. ALI is characterized by uncontrolled inflammation and damage to endothelial and epithelial barriers of the lung. Severe bacterial infections systemically or locally in the lung are the primary causes of ALI development, and many toxic agents, especially LPS, are produced (39). LPS is a major component of the cell wall of Gram-negative bacteria, and it has the ability to induce a persistent and severe inflammatory response that damages tissue. Experimental LPS administration, systemically and intratracheally, induces ALI in animal models (4,19,29). However, systemic LPS administration causes only a transient systemic inflammatory response and transient lung injury and dysfunction (29). However, lung injury increases markedly when LPS is followed by treatment with zymosan A, a component extracted from yeast cell walls (21). Therefore, we infused 3 mg/kg LPS followed with 10 mg/kg zymosan A 2 h later to establish the extrapulmonary ALI murine model, as previously reported (23). Four hours after the LPS/zymosan challenge, histological examination, which is widely considered a gold standard methodology, showed significant inflammatory infiltrates and interstitial edema, and the lung injury score was higher than in the control group. These observations suggest that LPS/zymosan infusion resulted in the development of ALI because the severity of lung injury is comparable with a previous study (23). Previous studies show that intratracheal LPS administration produces stable ALI models and induces different degrees of lung injury in C57BL/6 mice from doses of 0.3 mg/kg to 5 mg/kg LPS (E. coli 0111:B4) (8,15,33). The intrapulmonary ALI mice group received LPS from doses of 0 mg/kg to 5 mg/kg to match the severity of lung injury with extrapulmonary ALI mice, and we found that intratracheal administration of 3 mg/kg LPS produced a coordinated lung injury and lung edema in mice challenged with LPS/zymosan.

First, our study found that the same treatment strategy of intravenous MSC administration showed differential benefits in improving lung repair of intra- and extrapulmonary ALI mice. The histopathology showed that the effects of unified MSC treatment on lung repair were more significant in the intrapulmonary ALI mice, in which lung injury was attenuated at day 3 and almost normalized at day 7 postinjection. Lung sections from extrapulmonary ALI mice at day 7 still showed inflammatory cell infiltration and interstitial edema. Anti-inflammatory responses of MSCs play an important role in the promotion of the lung repair process. Therefore, we also measured the levels of the proinflammatory cytokines IL-1β, IL-6, and IFN-γ in the lungs over the time course of ALI. The results showed that most of the inflammatory cytokines were decreased at days 1, 3, and 7 after MSC transplantation in the two ALI groups. However, the decrease in inflammatory cytokines was more significant in the intrapulmonary ALI mice, which suggests that the inhibition of inflammation in lung tissues by MSC transplantation was more notable in the intrapulmonary ALI mice than in the extrapulmonary ALI mice.

The different therapeutic effects of MSCs in different ALI animal models may be attributed to differences in MSC homing in mice. Multiple studies demonstrated that the management of ALI/ARDS with MSCs involved two different mechanisms, a cell engraftment mechanism and the paracrine/endocrine mechanism (36). Currently, the therapeutic efficacy of MSCs is greatly dependent on their ability to produce paracrine factors that enhance regeneration, but the migration of MSCs to diseased organ or tissue is required (32). Our study found that a large proportion of cells migrated into normal and injured lungs 30 min after cell transplantation, but the cells started to recruit in injured lungs at day 1 and persisted to day 7. These results suggest that MSCs possess the ability to home into injured tissues, which is consistent with a previous report that bone marrow-derived progenitor cells were attracted to inflammatory sites of lungs and lasted for 1 week following injury (42). The result of our study also showed that the retention of MSCs in injured lungs was more significant in the intrapulmonary ALI group than in the extrapulmonary ALI group at the same time. There was even a notable recruitment in the lungs of the intrapulmonary ALI group at day 1. The difference between the two ALI groups remained significant even 7 days postinjection. Notably, we also found that MSC recruitment to the liver and spleen appeared higher in the extrapulmonary ALI group, which suggested that a smaller proportion of MSCs accumulated in the injured lungs in the extrapulmonary ALI mice than in the intrapulmonary ALI mice with the same MSC treatment strategy. This difference may be attributed to the different therapeutic effects of MSCs in the two ALI models. We also found that the majority of cells were lost from the lungs within 7 days in both ALI groups, which is consistent with previous reports that the lung retention of MSCs was very low (13,20,40). However, the discrepancy of benefits of MSCs between the two ALI groups also indicated that the short-term persistence of transplanted MSCs in the lung played a vital role in promoting lung repair.

Different distributions of transplanted MSCs in the intra- and extrapulmonary ALI mice were associated with organ damage. MSCs can home to injured sites, apoptosis can be activated under pathophysiological conditions, and it contributes to the pathogenesis of multiple organ dysfunction syndrome. Therefore, cell apoptosis could reflect organ injury (14). This study detected the percentage of apoptotic cells in the liver, spleen, and lung at different time points as indications of the degree of organ injury. The results showed that cell apoptosis was more significant in lungs and liver, and the percentage of apoptotic cells increased greatly in the lungs at day 1 and then decreased gradually. The percentage of apoptotic cells in the intrapulmonary ALI group was lower than in the extrapulmonary ALI group at day 3 and day 7, but the retention of MSCs in the lung in the intrapulmonary ALI group was greater than in the extrapulmonary ALI group. This phenomenon may be explained as a greater recruitment of MSCs into the lung resulting in better therapeutic effects in the promotion of lung repair. The percentage of apoptotic cells in the liver and spleen increased gradually, reached a maximum at day 3, and decreased at day 7. Changes in cell apoptosis in these two organs are consistent with changes in the distribution of transplanted MSCs in the liver and spleen. Our data suggest that the two injury methods induced different organ damages despite presenting similar lung injuries, which might result in a differential distribution of MSCs in ALI mice. Therefore, the effects of the same MSC treatment strategy in the two ALI models were greatly distinctive.

The underlying mechanism of MSC migration and homing to injured tissues has not been clarified completely. Chemokines and their receptors and adhesion molecules may play important roles in the tissue-specific homing of transplanted MSCs. SDF-1α, an important chemokine released from the injury site, is involved in the process of chemotaxis of MSCs through its receptor, CXCR4, in many diseases, such as myocardial infarction and ALI. We measured the expression of SDF-1α in lung, liver, and spleen within 3 days, and we found that the changing trends of SDF-1α were nearly consistent with the changes in cell apoptosis in the lung, liver, and spleen and the trends of MSC retention in these three organs. These results suggested that SDF-1α participated in the process of MSC distribution after transplantation in the two ALI animal models.

Conclusions

In conclusion, intravenous administration of MSCs promoted lung repair after intratracheal LPS-induced intrapulmonary ALI and intravenous LPS/zymosan-induced extrapulmonary ALI. However, this MSC-based treatment strategy showed better therapeutic effects in the treatment of intrapulmonary ALI than extrapulmonary ALI, which were matched in lung injury severity. These differences in the benefits of MSCs between the two ALI models might be due to more MSCs recruited into the lungs of the intrapulmonary ALI mice than those of the extrapulmonary ALI mice over the time course. This finding may contribute to the clinical use of MSCs in ALI/ARDS.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China under the contract grant Nos. 81000828, 81170057, 81201489, and 81372093, and Natural Science Foundation of Jiangsu Province under the contract grant Nos. BK20131302 and BK20141344. This work was also supported by the Graduate Innovation Project of Jiangsu Province under the contract grant No. CXLX_0151. The authors declare no conflicts of interest.

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