Interleukin 6 Mediates the Therapeutic Effects of Adipose-Derived Stromal/Stem Cells in Lipopolysaccharide-Induced Acute Lung Injury

Authors

  • Shijia Zhang,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    2. Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Svitlana D. Danchuk,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Ryan W. Bonvillain,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Beibei Xu,

    1. Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Brittni A. Scruggs,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    2. Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Amy L. Strong,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Julie A. Semon,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Jeffrey M. Gimble,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    2. Stem Cell Biology Laboratory, Pennington Biomedical Research Center, Louisiana State University System, Louisiana, USA
    Search for more papers by this author
  • Aline M. Betancourt,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Deborah E. Sullivan,

    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    2. Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
    Search for more papers by this author
  • Bruce A. Bunnell

    Corresponding author
    1. Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA
    2. Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA
    3. Division of Regenerative Medicine, Tulane National Primate Research Center, Covington, Louisiana, USA
    • Correspondence: Bruce A. Bunnell, Ph.D., Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, SL-99, New Orleans, Louisiana 70112, USA. Telephone: +1-504-988-7711; Fax: +1-504-988-7710; e-mail: bbunnell@tulane.edu

    Search for more papers by this author

Abstract

Adipose-derived stromal/stem cells (ASCs) have anti-inflammatory as well as immunosuppressive activities and are currently the focus of clinical trials for a number of inflammatory diseases. Acute lung injury (ALI) is an inflammatory condition of the lung for which standard treatment is mainly supportive due to lack of effective therapies. Our recent studies have demonstrated the ability of both human ASCs (hASCs) and mouse ASCs (mASCs) to attenuate lung damage and inflammation in a rodent model of lipopolysaccharide-induced ALI, suggesting that ASCs may also be beneficial in treating ALI. To better understand how ASCs may act in ALI and to elucidate the mechanism(s) involved in ASC modulation of lung inflammation, gene expression analysis was performed in ASC-treated (hASCs or mASCs) and control sham-treated lungs. The results revealed a dramatic difference between the expression of anti-inflammatory molecules by hASCs and mASCs. These data show that the beneficial effects of hASCs and mASCs in ALI may result from the production of different paracrine factors. Interleukin 6 (IL-6) expression in the mASC-treated lungs was significantly elevated as compared to sham-treated controls 20 hours after delivery of the cells by oropharyngeal aspiration. Knockdown of IL-6 expression in mASCs by RNA interference abrogated most of their therapeutic effects, suggesting that the anti-inflammatory properties of mASCs in ALI are explained, at least in part, by activation of IL-6 secretion. Stem Cells 2014;32:1616–1628

Introduction

Acute lung injury (ALI) is a common clinical complication that involves severe pulmonary inflammation and results from a number of localized and systemic pathological conditions. ALI and its more severe form, acute respiratory distress syndrome (ARDS), are significant causes of morbidity and mortality in critically ill patients. Diffuse alveolar damage, overwhelming pulmonary inflammation, and bilateral pulmonary infiltrates are the major pathophysiological features of ALI and ARDS [1-3].

The disease process in ALI/ARDS is initially incited by local or distant tissue injury leading to release of inflammatory mediators (e.g., cytokines, chemokines, proteases, and reactive oxygen species). These inflammatory mediators cause diffuse alveolar epithelium damage, increased alveolar capillary permeability, and leakage of proteinaceous fluid into the alveoli. Clinically, ALI/ARDS patients develop acute onset of tachypnea, fever, tachycardia, and dyspnea. In extreme cases, ALI/ARDS can lead to death from intractable respiratory failure, multiorgan failure, or complications with underlying diseases. Current treatment options are mainly limited to supportive care and ventilatory strategies; however, morbidity and mortality in ALI/ARDS patients remain high [1-3].

Recent studies have demonstrated that cell-based therapy for ALI using bone marrow-derived multipotent stromal cells (BMSCs) produced promising results in rodent models and isolated human lungs [4-6]. These therapeutic effects are believed to be due to the potent immunomodulatory and immunosuppressive properties of BMSCs. Adipose-derived MSCs, also known as adipose-derived stromal/stem cells (ASCs), are considered an attractive alternative to BMSCs as cell-based therapeutics for inflammatory diseases because ASCs are easier to isolate with high yields using less invasive procedures and have similar or greater anti-inflammatory activities than BMSCs [7-9]. Our group recently reported that administration of both human and mouse ASCs (hASCs and mASCs, respectively) via oropharyngeal aspiration (OA) significantly attenuated lipopolysaccharide (LPS)-induced ALI in mice [10].

To advance ASC-based therapy for ALI/ARDS into human clinical trials, it is essential that the molecular mechanisms of their therapeutic effect be understood. Although we previously proposed that the protective effects of ASCs in ALI might be due to increased interleukin 10 (IL-10) levels in the treated lungs [10], the detailed mechanism(s) of action involved are yet to be determined. Multiple studies have demonstrated that the beneficial effects of BMSCs in ALI/ARDS are largely due to their capacity to secrete paracrine soluble factors that could modulate immune responses and alter the responses of epithelium or endothelium to injury (discussed in [11]). A review of recent studies revealed a myriad of molecules that might be beneficial in the treatment of inflammatory diseases, such as tissue factor pathway inhibitor 2 (TFPI-2) [12], IL-6 [13, 14], transforming growth factor beta (TGF-β) [15], stanniocalcin 1 (STC-1) [16, 17], nitric oxide (NO) [18], leukemia inhibitory factor (LIF) [19, 20], interleukin 1 receptor antagonist (IL1RN) [21], keratinocyte growth factor (KGF) [5], angiopoietin 1 (ANGPT-1) [22], indoleamine 2,3-dioxygenase (IDO) [23], and tumor necrosis factor alpha-induced protein 6 (TSG-6) [6]. Among these factors, IL1RN, KGF, ANGPT-1, and TSG-6 have been shown to mediate some of the protective effects of BMSCs in different models of ALI. Whether hASCs or mASCs use the same or similar mechanisms in the LPS-induced murine ALI model is yet to be elucidated.

This study aims to identify the molecular mechanism(s) behind the observed therapeutic effects of ASC treatment in ALI. To accomplish this goal, gene expression analysis was used to screen for potential anti-inflammatory factors expressed by hASCs or mASCs in a mouse model of LPS-induced ALI. Since mASCs are more potent than hASCs in mediating the therapeutic effects following LPS-induced ALI in mice [10], the expression of anti-inflammatory mediators by hASCs and mASCs was compared. The results revealed a dramatic difference between the expression of anti-inflammatory molecules by hASCs and mASCs. Very high levels of IL-6 were detected in the mASC-treated lungs relative to sham-treated controls 20 hours after OA delivery to the LPS-treated mice. Knockdown of IL-6 expression in mASCs by RNA interference abrogated most of their therapeutic effects in this model, suggesting that the anti-inflammatory properties of mASCs in ALI are explained, at least in part, by activation of mASCs to secrete IL-6.

Materials and Methods

Human and Mouse ASCs

hASCs and mASCs were isolated and characterized using standard procedures developed at the Pennington Biomedical Research Center, Louisiana State University System [24, 25]. All protocols were approved by the Pennington Biomedical Research Center Institutional Review Board (IRB), and all human participants provided written informed consent (PBRC #23040). The mASCs were obtained from eGFP transgenic mice (C57Bl/6-Tg (UBC-GFP) 30Scha/J strain; Jackson Laboratory, Bar Harbor, ME, http://www.jax.org). Both hASCs and mASCs (passages 3–4) were grown and expanded in complete culture medium (CCM) at 37°C with humidified air containing 5% CO2. CCM consisted of Dulbecco's modified Eagle's medium:Nutrient Mixture F-12 (DMEM/F-12, GIBCO, Grand Island, NY, http://www.invitrogen.com), 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA, http://www.atlantabio.com/), and 1% Anti-Anti (antibiotic-antimycotic, GIBCO) as previously reported [10]. The culture medium was changed to antibiotic-antimycotic-free CCM 1 day before the cells were to be delivered to mice. Harvested ASCs were washed with phosphate buffered saline (PBS, GIBCO), counted, resuspended in Hank's balanced salt solution (HBSS, GIBCO), and administered to different groups of mice via OA as described below. HBSS alone was used as a vehicle-only (sham-treated) control.

Mouse Model of ALI

Eight- to ten-week-old female C57Bl/6 mice (National Cancer Institute-Frederick, Frederick, MD, http://ncifrederick.cancer.gov/) were used for ALI induction via OA delivery of LPS as previously described [6, 10, 26]. Briefly, anesthetized mice (2% isoflurane, VetOne, Meridian, ID, http://www.vetone.net/) were suspended by the cranial incisors, and LPS from Escherichia coli 055:B5 (15 mg/kg, Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) was pipetted into the back of the throat. The tongue was extracted to full extension to prevent the swallowing reflex, and the nares were pinched shut to force breathing through the mouth and subsequent LPS aspiration. Four hours after LPS exposure, hASCs or mASCs (3.75 × 105/75 μL HBSS) were delivered similarly by OA, and 30 minutes later a second dose of an equal number of cells was administered for a total of 7.5 × 105 cells. For the control, two doses of HBSS (75 µL each) were delivered. The animals were sacrificed 24 or 72 hours after ALI induction by anesthesia with 80 mg/kg ketamine plus 8 mg/kg xylazine followed by laparotomy and exsanguination via laceration of the inferior vena cava. The lungs were processed for histology, bronchoalveolar lavage collection, and RNA or protein isolation as described below. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Tulane University and conformed to the requirements of the Animal Welfare Act (AWA).

Bronchoalveolar Lavage

The lungs were cannulated with 20-gauge intravenous (IV) catheters (Exel International Medical Products, St. Petersburg, FL, http://www.exelint.com/) immediately after exsanguination and gently washed five times with 530 μL (right lung) or 1 mL (whole lung) lavage buffer. The lavage buffer consisted of PBS supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, http://www.roche-applied-science.com) and 0.4 mM EDTA (GIBCO), except that EDTA was not added when the lavage fluid was to be used in the in vitro mASC stimulation assay. The lavage fluid was spun at 1,500g for 5 minutes at 4°C to pellet the cells. Cells from all five lavage collections were pooled for total cell counting while the supernatant from the first lavage was (a) used to stimulate mASCs in vitro or (b) stored at −80°C for biochemical analysis. The protein concentration in the bronchoalveolar lavage fluid (BALF) was measured using the micro bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL, http://www.piercenet.com). For differential cell counts, the cells were spun onto glass slides by cytospin (Thermo-Shandon, Wilmington, DE, http://www.thermoscientific.com) and stained with a modified Wright-Giemsa stain (Diff-Quik, Fisher Scientific, Pittsburgh, PA, http://www.fishersci.com). The numbers of neutrophils, macrophages, eosinophils, basophils, and lymphocytes were determined up to a total of 100 cells in three random fields per sample.

Histological Analysis

Lungs were perfused with 10% neutral buffered formalin (Sigma-Aldrich) at a pressure of 25 cm H2O for 20 minutes, removed from the mice, and placed in fresh 10% neutral buffered formalin for 20 hours at 4°C prior to processing and embedding. Sections (6 μm) from each sample were stained with hematoxylin and eosin (H&E), and images were captured on an Aperio ScanScope (Aperio Technologies, Vista, CA, http://www.aperio.com/). Histopathological evaluation was performed by two independent investigators blinded to the treatment. The degree of inflammation and hemorrhage was scored in six random fields using the following criteria: no injury, 0; injury to 25% of the field, 1; injury to 50% of the field, 2; injury to 75% of the field, 3; injury throughout the field, 4 [4, 27].

IL-6 Knockdown by siRNA Transfection

mASCs for transfection were cultured in CCM, harvested, and washed twice with PBS. The cells were then resuspended in resuspension buffer R (1 × 107 cells per mL; Neon Transfection System, Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with 600 nM Silencer Select Pre-Designed & Validated small interfering RNA (siRNA) for mouse IL-6 (s68290; Ambion, Life Technologies, Carlsbad, CA, http://www.lifetech.com) or Silencer Select Negative Control No. 1 siRNA (NC siRNA, Ambion, Life Technologies). The final siRNA working concentration was 5 nM. For each sample, 1 × 106 mASCs were transfected in a total cell suspension of 100 μL using the Neon Transfection System (Invitrogen) using two pulses (1,400 V input pulse voltage/20 ms input pulse width). Transfected mASCs were immediately plated on 10-cm tissue culture dishes in 12 mL of antibiotic-free CCM. The transfected cells were cultured for 30 hours, harvested, washed, and then delivered to LPS-challenged mice.

To determine the knockdown efficiency, mASCs transfected with IL-6 siRNA were plated in six-well plates in antibiotic-antimycotic-free CCM for 30 hours, and the medium was then changed to CCM with antibiotics and antimycotics followed by addition of LPS (30 ng/mL) to activate the cells. Twenty hours later, the cells were harvested for RNA extraction using RNeasy mini kit (Qiagen, Valencia, CA, http://www1.qiagen.com), and real-time reverse transcriptase polymerase chain reaction (RT-PCR) was conducted to quantify the knockdown efficiency. Untransfected cells in the presence or absence of LPS were used as controls.

In Vitro ASC Stimulation

For in vitro mASC stimulation experiments, BALF from LPS-challenged mice was diluted 1:1 with DMEM/F-12 (containing 4% FBS, 200 IU/mL penicillin, and 200 mg/mL streptomycin) and, after removal of the original CCM, added to mASCs in 10-cm dishes (106 cells per 3 mL final concentration). After 6 hours and 20 hours, cells were harvested for RNA isolation followed by gene expression analysis. Cells stimulated with lavage buffer were used as the control.

Lung Homogenization, RNA Isolation, and cDNA Synthesis

Excised lung tissue was weighed and homogenized using a Bio-Plex cell lysis kit (Bio-Rad, Hercules, CA, http://www.bio-rad.com) as per the manufacturer's instruction. The homogenates were centrifuged at 10,000g for 15 minutes, and the supernatant was aliquoted and stored at −80°C until analyzed. The protein concentration in the supernatant was measured by BCA assay.

Total RNA from cultured cells was isolated using RNeasy mini kit (Qiagen). Total RNA from lung was isolated from homogenized tissue in TriPure Isolation Reagent (Roche) and was purified with the RNeasy mini kit. The RNA was first treated with DNase I (Amplification grade, Invitrogen) and then converted into cDNA using iScript cDNA Synthesis Kit (Bio-Rad) following the manufacturer's instructions in a PTC-200 Peltier Thermal Cycler (MJ Research, Ramsey, MN, www.bio-rad.com).

Real-Time RT-PCR

For gene expression analysis, the real-time PCR reactions were performed as previously described [10]. Briefly, each reaction mixture contained 1 µL of commercially available Applied Biosystems TaqMan Gene Expression Assay primer/probe set, 10 µL TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com), 50 ng cDNA template, and RNase-free water with a total volume of 20 µl. For human tumor necrosis factor (TNF)-α-induced protein 6 (hTNFAIP6/hTSG-6) the following primer/probe set was used instead of Applied Biosystems TaqMan Gene Expression Assay: forward 5'-AAGCACGGTCT GGCAAATACAAGC-3', reverse 5'-ATCCATCCAGCAGCACAGACA TGA-3', probe 5'-6FAM-ATTTGAAGGCGGCCATCTCGCAACTT-TA MRA-3'. The reaction was performed at 50°C for 2 minutes, 95°C for 10 minutes followed by a 40-cycle two-step PCR (95°C for 15 seconds and 60°C for 1 minute) using the CFX96 Real-Time System (Bio-Rad). Mouse beta-actin (β-actin) and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as endogenous controls for mouse and human genes, respectively. The final expression of human genes in the lung after hASC treatment was further corrected by parallel real-time RT-PCR assays with primers that amplified both the human and the mouse genes for GAPDH (forward 5′-CAGCGACACCCACTCCTCCACCTT-3′, reverse 5′-CATGAGGTCCAC CACCCTGTTGCT-3′). The TaqMan Gene Expression Assays used in real-time RT-PCR are listed in Supporting Information Table 1.

Myeloperoxidase Activity Assay, ELISA, and Multiplex Immunoassay

The BALF myeloperoxidase (MPO) activity assay was performed as previously described [6]. Briefly, BALF was added to a reaction buffer containing 50 mM potassium phosphate (pH 6.0), 0.0005% (v/v) H2O2, and 0.167 mg/mL o-dianisidine dihydrochloride (Sigma-Aldrich); the absorbance at 460 nm was monitored for 30 minutes using the Synergy HT multidetection microplate reader (Bio-Tek Instruments, Winooski, VT, http://www.biotek.com/). The albumin and IL-6 levels in BALF or lung homogenates were measured using mouse albumin and IL-6 ELISA kits, respectively, which were purchased from Bethyl Laboratories (Montgomery, TX, http://www.bethyl.com/). Measurement of cytokines and chemokines in lung homogenates was performed by multiplex immunoassay using a Millipore mouse cytokine/chemokine 32-plex kit (Millipore, Billerica, MA, http://www.millipore.com).

Statistical Analysis

Data were summarized as mean ± SEM. The statistical differences among three or more groups were determined by One-way ANOVA, followed by Bonferroni multiple comparison post hoc tests. The statistical differences between two groups were performed by Student's t test. The statistical significance value was set at p < .05.

Results

Histology

Histological analysis was performed to evaluate the effects of ASC infusion on the lung injury. H&E staining of lung sections from both hASC- and mASC-treated mice had significantly less injury compared with mice given HBSS (Fig. 1A). Lung injury scores showed that mice treated with hASCs or mASCs had a significant improvement in the degree of inflammation (p < .01) and hemorrhage (p < .05 and p < .01, respectively) compared to HBSS sham treatment (Fig. 1B).

Figure 1.

Lung histology and expression of anti-inflammatory/trophic factors of human origin in the lungs of acute lung injury (ALI) mice treated with hASCs. Both hASC and mASC treatments improved lung injury as assessed by histological methods. (A): H&E staining of lung sections demonstrated attenuated lung injury in the mice treated with hASCs and mASCs at 72 hours after ALI induction. (B): Quantification of lung injury showed a significant reduction in the degree of inflammation and hemorrhage in the mice receiving hASCs and mASCs compared to mice receiving HBSS. N = 4 for each group. Significance was defined as * and ** for p < .05 and p < .01, respectively, compared to HBSS sham treatment. Steady-state mRNA levels of (C) hTFPI-2, (D) hTGF-β, (E) hSTC-1, (F) hANGPT-1, (G) hKGF, (H) hIL1RN, (I) hTSG-6, (J) hLIF, (K) hiNOS, (L) hIL-6, and (M) hIDO are shown for PBS- or LPS- challenged mice injected with hASCs. All levels were normalized to GAPDH and reported as fold changes compared to in vitro control. N = 4 for both PBS+hASC and LPS+hASC groups. Significance was defined as *, **, and *** for p < .05, p < .01, and p < .001, respectively, compared to in vitro control; significant differences between PBS+hASC and LPS+hASC groups were denoted by #, ##, and ### for p < .05, p < .01, and p < .001, respectively. Abbreviations: hANGPT-1, human angiopoietin 1; HBSS, Hank's balanced salt solution; hASC, human adipose-derived stem cell; hIDO, human indoleamine 2,3-dioxygenase; hIL-6, human interleukin; hIL1RN, human interleukin 1 receptor antagonist; hLIF, human leukemia inhibitory factor; hKGF, human keratinocyte growth factor; hTFPI-2, human tissue factor pathway inhibitor 2; hTGF-β, human transforming growth factor beta; hTSG-6, human tumor necrosis factor alpha-induced protein 6; hSTC-1, human stanniocalcin 1; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; mASC, mouse adipose-derived stem cells; PBS, phosphate buffered saline.

Screening for Anti-Inflammatory/Trophic Factors Derived from ASCs

To screen for anti-inflammatory/trophic factors derived from ASCs, real-time RT-PCR using human and mouse specific primers and probes was performed to quantify the expression levels of the abovementioned potential mediators in the lungs after hASC or mASC treatments. To establish baseline levels of gene expression in hASCs prior to delivery to the lung, 7.5 × 105 hASCs were added to intact mouse lung immediately prior to homogenization (in vitro control). Administration of hASCs (7.5 × 105) to LPS-challenged mice significantly increased the steady-state mRNA levels of human TFPI-2 (hTFPI-2, 62.7-fold, p < .01), hTGF-β (2.9-fold, p < .01), hSTC-1 (54.4-fold, p < .01), hKGF (3.5-fold, p < .05), hIL1RN (110.4-fold, p < .01), hTSG-6 (424.8-fold, p < .01), hLIF (26.8-fold, p < .01), hIL-6 (14.4-fold, p < .01), and hIDO (533.1-fold, p < .01) relative to the in vitro control. The expression levels of hTFPI-2 (45.5-fold, p < .01), hTGF-β (3.5-fold, p < .01), hSTC-1 (17.9-fold, p < .01), hKGF (1.6-fold, p < .01), hIL1RN (60.5-fold, p < .01), and hTSG-6 (209.4-fold, p < .01) were also upregulated in the PBS-challenged mice treated with hASCs as compared to the in vitro control (Fig. 1C–1M). Since the primers and probes used were specific for human genes, hASCs were assumed to be the sources of these molecules.

When mouse-specific primers and probes were used to detect the expression of similar molecules in the treated lungs, a different pattern was observed. It is noted that LPS challenge resulted in downregulation of mSTC-1, mANGPT-1, and mKGF and upregulation of mIL1RN, mLIF, mouse inducible nitric oxide synthase (miNOS, responsible for nitric oxide release), and mIL-6 compared to unchallenged and untreated controls (intact). In human ASC-treated mice only the expression of mTFPI-2 (2.8-fold vs. 1.2-fold, p < .001) was increased, and the expression of mIL1RN (67.6-fold vs. 93.4-fold, p < .05) was decreased compared to HBSS sham treatment, without significantly affecting other mouse genes. Treatment with mASCs resulted in either no change or decrease in the expression of mTFPI-2 (1.4-fold vs. 1.2-fold, p > .05), mTGF-β (0.54-fold vs. 0.85, p < .001), mSTC-1 (0.16-fold vs. 0.12-fold, p > .05), mANGPT-1 (0.11-fold vs. 0.16-fold, p > .05), mIL1RN (40.5-fold vs. 93.4-fold, p < .001), and mTSG-6 (0.98-fold vs. 0.85-fold, p > .05) in the lung compared to HBSS sham treatment. On the other hand, administration of mASCs significantly increased the steady-state mRNA levels of mKGF (0.52-fold vs. 0.23-fold, p < .001), mLIF (19.5-fold vs. 5.6-fold, p < .001), miNOS (12.8-fold vs. 6.5-fold, p < .001), and mIL-6 (721.4-fold vs. 81.0-fold, p < .001) in the lung compared to HBSS sham treatment group, with the latter three genes having high levels of induction (Fig. 2).

Figure 2.

Expression of anti-inflammatory/trophic factors of mouse origin in the lungs of acute lung injury (ALI) mice treated with hASCs and mASCs. Steady-state mRNA levels of (A) mTFPI-2, (B) mTGF-β, (C) mSTC-1, (D) mANGPT-1, (E) mKGF, (F) mIL1RN, (G) mTSG-6, (H) mLIF, (I) miNOS, and (J) mIL-6 are shown for PBS- or LPS-challenged mice injected with HBSS, hASCs, or mASCs. All levels were normalized to beta actin and reported as fold changes compared to unchallenged and untreated controls (Intact). N = 2, 4, 4, 5, 4, and 5 for Intact, PBS+hASC, PBS+mASC, LPS+HBSS, LPS+hASC, and LPS+mASC, respectively. Significance was defined as *, **, and *** for p < .05, p < .01, and p < .001, respectively, compared to control groups; significant differences between the LPS-challenged cell-treatment groups (LPS+hASC and LPS+mASC) were denoted by #, ##, and ### for p < .05, p < .01, and p < .001, respectively. Abbreviations: hASC, human adipose-derived stem cells; HBSS, Hanks' balanced salt solution; LPS, lipopolysaccharide; mASCs, mouse adipose-derived stem cells; mTFPI, mouse tissue factor pathway inhibitor 2; mTGF-β, mouse transforming growth factor beta; mSTC-1, mouse stanniocalcin 1; mANGPT-1, mouse angiopoietin 1; mKGF, mouse keratinocyte growth factor; mILRN, mouse interleukin 1 receptor antagonist; mTSG-6, mouse tumor necrosis factor, alpha-induced protein 6; mILF, mouse leukemia inhibitory factor; miNOS, mouse inducible nitric oxide synthase; mIL-6, mouse interleukin 6; PBS, phosphate buffered saline.

The protein levels of mLIF and mIL-6 in BALF and lung lysates were determined by mouse Bio-Plex immunoassay. Similar to the real-time RT-PCR results, administration of mASCs significantly increased mLIF (0.86 vs. 0.03 ng/mL, p < .001 and 29.6 vs. 3.9 ng/mL per mg, p < .001, respectively) and mIL-6 (12.8 vs. 0.7 ng/mL, p < .001 and 1270.0 vs. 43.8 ng/mL per mg, p < .001, respectively) levels in both BALF and lung lysate compared to HBSS treatment group, whereas hASCs did not affect mLIF (0.02 vs. 0.03 ng/mL, p > .05 and 3.3 vs. 3.9 ng/mL per mg, p > .05, respectively) and mIL-6 (0.65 vs. 0.69 ng/mL, p > .05 and 32.8 vs. 43.8 ng/mL per mg, p > .05, respectively) protein levels (Fig. 3).

Figure 3.

LIF and IL-6 in BALF and lungs from mASC- and hASC-treated acute lung injury (ALI) mice. Protein levels of LIF and IL-6 in BALF (A, C) and lung lysates (B, D) were quantified using a mouse Bio-Plex immunoassay 24 hours after PBS or LPS challenge. Values for LIF and IL-6 in lung lysates were normalized to total lung lysate protein. N = 4, 4, 5, 4, and 5 for PBS+hASC, PBS+mASC, LPS+HBSS, LPS+hASC, and LPS+mASC, respectively. Significance was defined as *** for p < .001 compared to LPS+HBSS; significant differences between LPS+hASC and LPS+mASC groups were denoted by ### for p < .001. Abbreviations: BALF, bronchoalveolar lavage fluid; HBSS, Hanks' balanced salt solution; hASCs, human adipose-derived stem cells; LPS, lipopolysaccharide; PBS, phosphate buffered saline; mIL-6, mouse interleukin-6; mLIF, mouse leukemia inhibitory factor; mASCs, mouse adipose-derived stem cells.

Stimulation of mASCs with BALF

Since the detected steady-state mRNA levels of mouse molecules could be derived from either mouse lung tissue or administered mASCs, it was necessary to determine whether mASCs could actually express these genes. Thus, in vitro mASCs stimulation experiments were performed using BALF from LPS-challenged mice, and gene expression assays were conducted to examine the expression of some of the possible anti-inflammatory/trophic factors in mASCs. BALF stimulation for 6 or 20 hour significantly increased the expression of mTFPI-2 (2.1-fold, p < .001 and 3.8-fold, p < .001, respectively), mTGF-β (3.0-fold, p < .001 and 2.3-fold, p < .001, respectively), mTSG-6 (1.4-fold, p < .001 and 1.4-fold, p < .01, respectively), mLIF (178.6-fold, p < .01 and 53.2-fold, p < .05, respectively), miNOS (82.5-fold, p < .001 and 286.7-fold, p < .001, respectively), and mIL-6 (98.8-fold, p < .001 and 137.1-fold, p < .001, respectively) by mASCs compared to the lavage buffer stimulation control. Similar to the in vivo results, mLIF, miNOS, and mIL-6 had high levels of induction. The expression of mSTC-1 (1.0-fold, p > .05 and 0.6-fold, p > .05, respectively) and mANGPT-1 (0.5-fold, p < .01 and 2.1-fold, p > .05, respectively) either did not change or decreased upon BALF stimulation. The effects of BALF stimulation on the expression of mKGF (1.3-fold, p > .05 and 1.2-fold, p < .01, respectively) and mIL1RN (0.3-fold, p < .001 and 3.6-fold, p < .01, respectively) by mASCs were variable at different time points (Fig. 4).

Figure 4.

Assessing gene expression by mouse adipose-derived stem cells (mASCs) upon stimulation with BALF. The expression of various genes by mASCs was quantified using real-time reverse transcription polymerase chain reaction. Steady-state mRNA levels of (A) mTFPI-2, (B) mTGF-β, (C) mSTC-1, (D) mANGPT-1, (E) mKGF, (F) mIL1RN, (G) mTSG-6, (H) mLIF, (I) miNOS, and (J) mIL-6 are shown for mASCs stimulated by BALF or lavage buffer alone at 6 hours and 20 hours poststimulation. All expression levels were normalized to beta actin expression and reported as fold changes compared to lavage buffer stimulated controls (Ctrl). N = 6 for each group. Significance was defined as *** for p < .001, compared to control groups. Abbreviations: BALF, bronchoalveolar lavage fluid; mTFPI, mouse tissue factor pathway inhibitor 2; mTGF-β, mouse transforming growth factor beta; mSTC-1, mouse stanniocalcin 1; mANGPT-1, mouse angiopoietin 1; mKGF, mouse keratinocyte growth factor; mILRN, mouse interleukin 1 receptor antagonist; mTSG-6, mouse tumor necrosis factor, alpha-induced protein 6; mILF, mouse leukemia inhibitory factor; miNOS, mouse inducible nitric oxide synthase; mIL-6, mouse interleukin 6.

Effects of IL-6 Knockdown mASCs on ALI

Among the few candidate anti-inflammatory/trophic factors which had high levels of induction in the lung after mASC therapy, IL-6 was chosen for further investigation as the potential mechanism through which mASCs attenuate LPS-induced ALI because IL-6 had the highest fold-induction in mASC-treated ALI mouse lung. To test this hypothesis, an IL-6 RNA interference study was performed, and the therapeutic effects of control mASCs and IL-6 knockdown mASCs were compared in the LPS-induced ALI model in mice.

mASCs were transfected with IL-6 siRNA by electroporation and stimulated with LPS. Real-time RT-PCR results demonstrated that the knockdown efficiency was 85%–90% in mASCs transfected with IL-6 siRNA compared to mASCs transfected with nonsilencing negative control (NC) siRNA (Fig. 5A). These cells were then delivered to LPS-challenged mice by OA, and 20 hours later real-time RT-PCR analysis showed that lungs receiving mASCs transfected with IL-6 siRNA had significantly lower level (29.1-fold, p < .001) of IL-6 mRNA compared to lungs that received NC siRNA (117.7-fold) (Fig. 5B). The protein levels of mIL-6 in BALF and lung lysates were also determined by ELISA. Similar to the real-time RT-PCR results, mice receiving mASCs transfected with IL-6 siRNA had significantly lower levels of IL-6 in BALF (1.4 vs. 15.7 ng/mL, p < .001) and lung lysates (128.2 vs. 1211.0 ng/mL per mg, p < .001, respectively) compared to mice that received mASCs transfected with NC siRNA (Fig. 5C, 5D).

Figure 5.

Evaluation of IL-6 knockdown efficiency in mASCs. The IL-6 knockdown efficiency in mASCs was quantified using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA. (A): Steady-state mRNA levels of IL-6 are shown for mASCs with or without siRNA transfection followed by LPS stimulation for 20 hours. All expression levels were normalized to beta actin expression and reported as fold changes compared to mASCs without LPS activation (Control). N = 3 for each group. Significance was defined as *** for p < .001, compared to control group. (B): The in vivo lung mRNA levels of IL-6 24 hours after LPS challenge were quantified using real-time RT-PCR. Steady-state mRNA levels of IL-6 are shown for LPS-challenged mice injected with HBSS, mASCs transfected with Silencer Select Negative Control No. 1 (NC) siRNA, or mASCs transfected with IL-6 siRNA. All expression levels were normalized to beta actin expression and reported as fold changes compared to unchallenged and untreated controls (Intact). N = 2, 6, 5, and 4 for Intact, LPS+HBSS, LPS+mASC/NC siRNA, and LPS+mASC/IL-6 siRNA, respectively. The protein levels of IL-6 in BALF (C) and lung lysates (D) 24 hours after LPS challenge were quantified by ELISA. IL-6 levels are shown for intact mice and LPS-challenged mice injected with HBSS, mASCs transfected with NC siRNA, or mASCs transfected with IL-6 siRNA. Values for IL-6 in lung lysates were normalized to total lung lysate protein. N = 3, 6, 5, and 4 for Intact, LPS+HBSS, LPS+mASC/NC siRNA, and LPS+mASC/IL-6 siRNA, respectively. Significance was defined as *** for p < .001, compared to LPS+HBSS group; significant differences between LPS+mASC/NC siRNA and LPS+mASC/IL-6 siRNA groups were denoted by ### for p < .001. Abbreviations: BALF, bronchoalveolar lavage fluid; HBSS, Hanks' balanced salt solution; mASCs, mouse adipose-derived stem cells; LPS, lipopolysaccharide; NC, negative control; mIL-6, mouse interleukin-6; siRNA, small interfering RNA.

To determine if IL-6 mediates the therapeutic benefits of mASCs, equal numbers (7.5 × 105) of mASCs transfected with IL-6 siRNA or NC siRNA were administered to mice 4 hours after LPS exposure. Lungs were either lavaged or processed for RNA extraction 24 hours after LPS exposure as described above. LPS-induced increases in total protein and albumin in BALF were both significantly decreased by delivery of NC siRNA transfected mASCs (2.6-fold vs. 1.9-fold, p < .001 and 3.9-fold vs. 2.6-fold, p < .01, respectively) but not IL-6 siRNA transfected mASCs (2.6-fold vs. 2.2-fold, p > .05 and 3.9-fold vs. 3.2-fold, p > .05, respectively) (Fig. 6A, 6B).

Figure 6.

Effects of IL-6 knockdown mASCs on BALF protein and albumin levels, inflammatory cell infiltration, and MPO activity in the lungs after LPS injury. Levels of total protein (A) and albumin (B) in the BAL fluid were used to assess the extent of vascular leakage after treatment with mASCs transfected with either NC siRNA or IL-6 siRNA. Intact or HBSS-treated mice were used as controls. Values were presented as fold changes relative to unchallenged and untreated controls (Intact). Administration of NC siRNA-transfected mASCs, but not IL-6 siRNA-transfected mASCs, significantly decreased the (C) total infiltrating cells, (D) neutrophils, and (E) MPO activity in BALF 24 hours after LPS exposure when compared to HBSS sham treatment controls. N = 3, 6, 5, and 4 for intact, LPS+HBSS, LPS+mASC/NC siRNA, and LPS+mASC/IL-6 siRNA groups, respectively. Significance was defined as ** and *** for p < .01 and p < .001, respectively, as compared to the HBSS-treated LPS-challenged mice; significant differences between LPS+mASC/NC siRNA and LPS+mASC/IL-6 siRNA groups were denoted by # for p < .05. Abbreviations: BAL, bronchoalveolar lavage; HBSS, Hanks' balanced salt solution; IL-6, interleukin-6; LPS, lipopolysaccharide; MPO, myeloperoxidase; mASCs, mouse adipose-derived stem cells; NC, negative control; siRNA, small interfering RNA.

Administration of NC siRNA transfected mASCs dramatically decreased inflammatory cell infiltration in the lung, as shown by the reduced total cell (0.5 × 106 vs. 2.0 × 106, p < .001) (Fig. 6C) and neutrophil (0.4 × 106 vs. 1.9 × 106, p < .001) (Fig. 6D) numbers and MPO activity (1.8 vs. 4.7 mU/minute per mL, p < .01) (Fig. 6E) in BALF compared to HBSS sham treatment. However, IL-6 mRNA knockdown abrogated most, but not all, effects of mASCs in LPS-exposed lungs (Fig. 6C–6E).

Treatment with mASCs transfected with NC siRNA significantly downregulated the expression of various proinflammatory cytokines/chemokines, such as macrophage inflammatory protein-2 (MIP-2, 228.1-fold vs. 649.0-fold, p < .001), IL-1α (15.4-fold vs. 38.9-fold, p < .001), IL-1β (20.3-fold vs. 74.0-fold, p < .001), MIP-1α (15.2-fold vs. 107.7-fold, p < .001), and TNF-α (13.9-fold vs. 50.6-fold, p < .001) in LPS-exposed lungs compared to HBSS sham treatment. IL-6 mRNA knockdown partially reversed the suppressive effects of mASCs on the expression of MIP-2, IL-1α, IL-1β, MIP-1α, and TNF-α in LPS-exposed lungs (Fig. 7A–7E). Interestingly, NC siRNA-transfected mASCs significantly increased the expression of the anti-inflammatory cytokine IL-10 compared to HBSS sham treatment (102.4-fold vs. 52.3-fold, p < .05), but IL-6 siRNA-transfected mASCs had no effect on the upregulation of IL-10 (Fig. 7F).

Figure 7.

Effects of IL-6 knockdown mASCs on inflammatory responses in LPS-treated lung. The lung mRNA levels of various cytokines and chemokines produced in response to LPS challenge were quantified 24 hours after exposure using real-time reverse transcriptase polymerase chain reaction. Steady-state mRNA levels of (A) MIP-2, (B) IL-1α, (C) IL-1β, (D) MIP-1α, (E) TNF-α, and (F) IL-10 are shown for each group. All expression levels were normalized to beta actin expression and reported as fold changes compared to unchallenged and untreated mouse levels. N = 3, 6, 5, and 4 for Intact, LPS+HBSS, LPS+mASC/NC siRNA, and LPS+mASC/IL-6 siRNA, respectively. Significance was defined as * and *** for p < .05 and p < .001, respectively, as compared to the HBSS-treated LPS-challenged mice; significant differences between LPS+mASC/NC siRNA and LPS+mASC/IL-6 siRNA groups were denoted by # and ## for p < .05 and p < .01, respectively. Abbreviations: HBSS, Hanks' balanced salt solution; IL-6, interleukin-6; LPS, lipopolysaccharide; mSACs, mouse adipose-derived stem cells; mMIP-2, mouse macrophage inflammatory protein-2; mIL-1α, mouse interleukin 1α; mTNF-α, mouse tumour necrosis factor α; siRNA, small interfering RNA.

Discussion

Due to the severe complications and high mortality of ALI/ARDS and the limited effectiveness of current therapies, a novel and more effective therapeutic modality is needed. ASCs have been shown to be able to regulate inflammatory/immune responses in many disease models, including experimental inflammatory colitis [7, 8], globoid cell leukodystrophy [28, 29], and experimental autoimmune encephalomyelitis model for multiple sclerosis [30, 31]. As adult stem cells, ASCs have a limited risk of tumor formation and are not associated with the ethical controversy that surrounds embryonic stem cell research. Our previous study demonstrated the ability of ASCs to attenuate lung damage and inflammation in a rodent model of LPS-induced ALI, and the syngeneic mASCs are more potent that the xenogeneic hASCs [10]. These studies have shown that ASCs are attractive candidates for cell-based therapies for diseases that involve excessive inflammation; however, the mechanisms of action are poorly understood. Before being implemented in clinical trials, it is critical to understand the mechanisms integral to the beneficial effects of ASCs in ALI. The goal of this study was to investigate the mechanisms by which ASCs modulate lung inflammation and repair.

Several studies have shown that the immunomodulatory and immunosuppressive effects of MSCs are largely mediated through production of paracrine factors [11, 32, 33]. However, most of what is known about the mechanisms of MSC-mediated immune regulation comes from studies that used BMSCs. Our previous study suggested that increased IL-10 levels in the ASC-treated lungs might contribute to the protective effects in ALI [10]. An increasing number of studies support the idea that it is not likely that one single mechanism accounts for all the immune regulatory effects of MSCs.

We, and others, have routinely and consistently used intrapulmonary delivery of LPS to induce ALI in immunocompetent mice [4, 6, 10, 22], which is a well-characterized and widely used animal model of direct lung injury. While no single ALI/ARDS animal model can completely reproduce all the pathophysiological features of human ALI/ARDS, this model recapitulates many of the features of ALI/ARDS in humans, including patchy intra-alveolar neutrophil infiltrates and changes in epithelial permeability [6, 10]. Here, both hASCs and mASCs were delivered to LPS-challenged mice through OA. Real-time RT-PCR was used to screen the possible anti-inflammatory/trophic factors of both human and mouse origins. Our data suggest that the immunosuppressive effects of hASCs and mASCs in the lung might be mediated by different mechanisms. For example, the high-level expression of TFPI-2, TGF-β, STC-1, KGF, IL1RN, and TSG-6 was only seen in hASC-treated lungs but not mASC-treated lungs. These variations indicate that the mechanisms mediating these outcomes may be unique to each cell type. Similarly, it is not clear how closely ASCs resemble BMSCs regarding the therapeutic effects in an ALI model and their respective mechanisms of action. Data have indicated that the beneficial effects of mouse BMSCs in ALI were attributed to the production of IL1RN [21] and ANGPT-1 [22]. On the contrary, our study showed decreased levels of IL1RN and ANGPT-1 in the lungs treated by mouse ASCs. The discrepancies may be due to differences in the animal models or the distinctive biological characteristics of BMSCs and ASCs, which remain to be determined.

Interestingly, a very high level of IL-6 production in the injured lungs following mASC treatment was detected in our ALI model. Our in vitro study showed that both LPS and BALF from LPS-injured mice stimulated mASCs to express enhanced level of IL-6 relative to mASCs without stimulation. IL-6 has multiple biological activities and has been shown to have both pro- and anti-inflammatory effects [13, 34]. Therefore, it was hypothesized that IL-6 secreted by mASCs might play an important role in the attenuation of ALI following mASC treatment. It was found that knockdown of IL-6 in mASCs partially reversed their anti-inflammatory effects in the lung, including reduced alveolar capillary permeability and leakage, decreased inflammatory cell infiltration, and downregulated proinflammatory cytokines. This suggests that the beneficial effects of mASCs in the murine model of LPS-induced ALI are mediated, at least partially, by secreting high level of IL-6.

To the best of our knowledge, this is the first report to demonstrate that the beneficial effects of mASCs for the treatment of LPS-induced ALI are mediated by IL-6. This novel mechanism of action through which ASCs suppress inflammatory responses is consistent with a recent report by Ivanova-Todorova et al., who proposed that IL-6 secreted by ASCs induces CD4+FOXP3+ cells in vitro. These cells in turn increase IL-10 secretion, which mediates the anti-inflammatory effects [14]. Moreover, IL-6 mRNA knockdown abrogated the mASCs-induced IL-10 expression in the treated lungs in our study, further supporting the theory proposed by Ivanova-Todorova et al.

Samavedam et al. [35] demonstrated that the protective effects of IL-6 in the treatment of epidermolysis bullosa acquisita in mice were mediated by IL-6-dependent IL1RN induction. However, in our study decreased levels of mIL1RN expression were detected in the LPS-injured lung after hASC and mASC treatments compared to HBSS vehicle-only controls. This suggests that mIL1RN induction may not contribute to the beneficial effects of mASCs in the murine model of LPS-induced ALI.

Since IL-6 knockdown only partially reversed the therapeutic effects of mASCs in LPS-induced ALI, it is unlikely that IL-6 is the sole contributing factor. High levels of miNOS and mLIF were also detected in the mASC-treated lungs; NO and LIF have been shown to mediate the anti-inflammatory effects of MSCs [18-20]. Future studies will involve the knockdown of iNOS or LIF in mASCs and evaluation of the effects of these mASCs on LPS-induced ALI. Further studies can be directed at silencing combinations of these factors (IL-6, iNOS, and LIF) in mASCs to examine whether or not they work synergistically.

This study strongly suggests that mASCs and hASCs use different mechanisms to suppress inflammation at least in this mouse model of LPS-induced ALI. However, many preclinical studies are currently being done with mouse cells in mouse models. To advance the use of human stem cell therapy in the treatment of ALI/ARDS, it is essential that human cells be used in preclinical studies especially when trying to elucidate the mechanisms behind the therapeutic effects.

Conclusion

As a potential novel therapy for ALI, transplantation of ASCs requires extensive investigation in animal models before proceeding to clinical trials. Understanding the mechanism(s) of action will help predict the physiological responses of the patients to the therapies. Our data indicated that the beneficial effects of hASCs and mASCs might be mediated by different mechanisms. IL-6 was identified for the first time as a major mediator of the therapeutic effects of mASCs in LPS-induced ALI. This study provides important preclinical data for eventual clinical trials using ASCs for treatment of ALI.

Acknowledgments

We thank Margie McCants for her technical assistance with cell culture. The research was supported by funds from Tulane University.

Author Contributions

S.Z.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; S.D.D.: conception and design, collection and/or assembly of data, and data analysis and interpretation; R.W.B.: data analysis and interpretation and manuscript writing; B.X., B.A.S., and A.L.S.: collection and/or assembly of data, data analysis and interpretation; J.A.S., J.M.G., and A.M.B.: data analysis and interpretation; D.E.S.: conception and design and data analysis and interpretation; B.A.B.: conception and design, financial support, administrative support, data analysis and interpretation, and final approval of manuscript.

Disclosure of Potential Conflicts of Interest

J.M.G. is Co-Founder and Chief Scientific Officer for LaCell LLC, and A.M.B. is Founder and Chief Scientific Officer for Wibi+Works, LLC (www.wibiworks.com). The other authors declare that they do not have any conflict of interests.

Ancillary