Pulmonary fibrosis, characterized by progressive accumulation of extracellular matrix protein, compromises lung function (1, 2), manifests clinically during acute respiratory distress syndrome, which has a mortality rate of up to 40%. It is also an extremely complex biologic process requiring the coordinated proliferation and migration of multiple cell types, including leukocytes, macrophages, endothelial cells, epithelial cells, and fibroblasts (3). Bleomycin has been used to model fibrotic lung injury in animal studies because the characteristics of bleomycin-induced lung damage in animals include acute inflammatory injury of the alveolar epithelium, followed by reversible fibrosis, which overlaps with the symptoms of acute respiratory distress syndrome and idiopathic pulmonary fibrosis (4, 5). Previous studies have suggested that the early reduction of inflammation may result in the attenuation of downstream events leading to collagen deposition (6). Therefore, developing an effective strategy for ameliorating pulmonary damage during the early phases of pulmonary fibrosis pathogenesis is therefore a top priority.
Stem cell–based therapies, including the use of embryonic stem cells (ESCs) or adult stem cells, have been considered novel strategies for treating several devastating and incurable lung diseases. Because of the ethical and safety issues of ESCs, most studies have focused on adult stem cell therapy (7). Previous studies using mesenchymal stem cell (MSC)–based therapy in bleomycin-induced pulmonary fibrosis have been reported (8–13). Induced pluripotent stem cells (iPSCs) represent a more accessible stem cell population that can be induced from mouse and human adult somatic cells through reprogramming by transduction with defined transcription factors (14, 15). Induced pluripotent stem cells have the potential for multilineage differentiation and can provide a resource for stem cell–based treatment. Although recent reports have suggested the use and low tumorigenic risk of c-Myc– free iPSCs (16, 17), it remains uncertain whether 3-gene iPSCs are capable of blocking inflammatory cascades and rescuing pulmonary fibrosis.
Cytokines and chemokines have long been implicated in the pathogenesis of pulmonary fibrosis (3). Systemic administration of MSCs after bleomycin exposure reduces inflammation and fibrosis formation through the release of the interleukin 1α (IL-1α) receptor antagonist and suppression of nitric oxide metabolites and proinflammatory angiogenic cytokines (8–13). Recently, we reported that iPSCs produce paracrine mediators that regulate neutrophil activity in response to endotoxin stimulation and reduce the inflammatory response in endotoxin-induced acute lung injury (16). In the current study, we evaluated the protective effects of 3-gene iPSCs and iPSC-conditioned medium (iPSC-CM) in a bleomycin-induced pulmonary fibrosis mouse model. The treatment effects of 3-gene iPSCs/iPSC-CM were simultaneously investigated by measuring pulmonary function, histology, and inflammatory and fibrotic responses to bleomycin challenge. Our findings may provide a novel therapeutic alternative using 3-gene iPSCs/iPSC-CM for the management of pulmonary fibrosis.
MATERIALS AND METHODS
C57BL/6 mice (from the National Laboratory Animal Center, Taipei, Taiwan) that were aged 8 to 10 weeks were maintained on a 12-h light-dark cycle and were provided free access to food and water. The Ethical Committee for the Use of Laboratory Animals of Taipei Veterans General Hospital approved the experimental procedures and protocols, which also complied with the Guide for the Care and Use of Laboratory Animals.
Mouse 3-gene iPSCs generation
Mouse 3-gene iPSCs were established by introducing three genes (Oct4/Sox2/Klf4) not including c-Myc. Briefly, murine iPSCs were generated from mouse embryonic fibroblasts (MEFs) derived from 13.5-day-old embryos of C57BL/6 mice. The iPSCs were reprogrammed via the transduction of pMX-based retroviral vectors encoding three transcription factors, Oct-4, Sox2, and Klf4, according to the protocol described in previous studies with minor modifications (14). Plat-E packing cells were incubated overnight at a density of 3.6 × 106 cells per 100-mm dish. The next day, pMX-based retroviral vectors encoding mouse complementary DNAs were introduced into the Plat-E cells using the Fugene 6 transfection reagent (Roche Applied Science, Indianapolis, Ind). Forty-eight hours after transfection, virus-containing supernatants were collected for target cell infection. In preparation for viral infection, 8 × 105 MEFs were seeded per well into six-well plates 1 day before transduction. The virus-containing supernatants were filtered through a 0.45-μm filter and supplemented with 4 μg/mL polybrene (Sigma-Aldrich, St. Louis, Mo). Equal amounts of supernatants containing each of the three retroviruses were mixed, transferred to the fibroblast dish, and incubated overnight. After infection, the cells were replated in fresh medium. Six days after transduction, the cells were passaged on an SNL feeder layer and cultured using mouse ESC culture medium. Colonies were selected for 2 to 3 weeks.
Mouse 3-gene iPSCs culture
Undifferentiated 3-gene iPSCs were routinely cultured and expanded on mitotically inactivated MEFs (50,000 cells/cm2) in six-well culture plates (BD Technology, Franklin Lakes, NJ) in the presence of 0.3% leukemia inhibitory factor in an iPSC medium consisting of Dulbecco modified Eagle medium (Sigma-Aldrich) supplemented with 15% fetal bovine serum (Invitrogen, Carlsbad, Calif), 100 mM minimal essential medium nonessential amino acids (Sigma-Aldrich), 0.55 mM 2-mercaptoethanol (Gibco, Gaithersburg, Md), and antibiotics (Invitrogen). Every 3 to 4 days, colonies were detached with 0.2% collagenase IV (Invitrogen), dissociated into single cells with 0.025% trypsin (Sigma-Aldrich) and 0.1% chicken serum (Invitrogen) in phosphate-buffered saline (PBS), and replated onto MEFs.
Preparation of the conditioned medium
Mouse 3-gene iPSCs were placed at 20,000 cells/cm2 and incubated in a 10-mL volume of serum-free basal medium (Dulbecco modified Eagle medium, high glucose [Gibco], with a 100-mM concentration of nonessential amino acids [Gibco], 0.3% leukemia inhibitory factor, and 1% penicillin-streptomycin) in a 10-cm dish (Corning Incorporated) for 48 h. The trypsinized iPSCs in whole culture medium were then collected and centrifuged for 10 min at 1,500 rpm to obtain the supernatant to be used as the conditioned medium in subsequent in vivo experiments.
C57BL/6 mice were intratracheally injected with 1.5 U/kg bleomycin sulfate (Merck, Darmstadt, Germany) in 50 μL PBS under light anesthesia to induce pulmonary fibrosis. In the designated experiments, mice received either 3-gene iPSCs (2 × 106 cells in 200 μL of PBS; iPSC-treated mice) or PBS 200 μL (PBS-treated mice) via tail vein injection 24 h after the induction of lung injury. Cell administration 24 h after lung injury was chosen to optimize cell incorporation into the lungs during early inflammation (Results and Fig. 1D). Cell engraftment was routinely examined at another 24 h after 3-gene iPSC transplantation. Further experiments were performed in which nonreprogrammed MEFs (2 × 106 cells in 200 μL of PBS; MEF-treated mice) and the conditioned medium from iPSCs (200 μL of iPSC-CM) were injected into the tail vein 24 h after the induction of bleomycin-induced lung injury as control cell therapy and paracrine therapy, respectively. The animals were sacrificed after 3, 7, 14, or 21 days after bleomycin injection because these time points represent the phases of maximal inflammation (3 and 7 days) and fibrosis (14 and 21 days). Samples were collected from each mouse for the assessment of lung injury, which was performed according to the hydroxyproline assay, pulmonary physiology, histology, immunohistochemistry, and cytokine and myeloperoxidase (MPO) analysis. Other methods and procedures are described in Methods, Supplemental Digital Content 1, at https://links.lww.com/SHK/A154.
Results are reported as mean ± SD. Statistical analysis was performed using Student t test or a one-way or two-way analysis of variance followed by Turkey test, as appropriate. The survival rate analysis was performed using log-rank test. Results were considered statistically significant at P < 0.05.
Characterization of 3-gene iPSCs
In the present study, we generated mouse iPSCs by introducing three genes (Oct4/Sox2/Klf4) not including c-Myc. Undifferentiated 3-gene iPSCs were cultured on inactivated MEFs (Fig. 1A) and formed colonies very similar to ESCs. These 3-gene iPSCs clones were positive immunofluorescent staining for stage-specific embryonic antigen 1 (Fig. 1A) and alkaline phospatase (Fig. 1B). The reverse transcription polymerase chain reaction (PCR) showed that 3-gene iPSCs expressed a gene signature similar to that of mouse ESCs, including Oct4, Sox2, Nanog, Klf-4, Fbx5, Eras, Dppa5a, and Rex1 (data not shown). We investigated the pluripotency of 3-gene iPSCs using embryonic body formation (Fig. 1B) and various differentiation protocols of three dermal lineages. These 3-gene iPSCs could be differentiated into hepatocyte-like (endodermal-lineage) cells (Fig. 1B), osteocyte-like (mesodermal-lineage) cells (data not shown), and astrocyte-like (neuroectodermal-lineage) cells (Fig. 1B).
Effects of 3-gene iPSCs and iPSC-CM on the histopathology of bleomycin-induced lung injury
Intratracheal injection of bleomycin resulted in the previously documented sequence of events leading to lung injury (Fig. 2A), which exhibited neutrophilic alveolitis during early phase and was characterized by patchy areas during late phase. Intravenously delivered 3-gene iPSCs were engrafted to lungs in response to bleomycin challenge (Fig. 1C). Delivery of 3-gene iPSCs 24 h after bleomycin challenge led to optimal engraftment to the injury areas (Fig. 1D). Using 3-gene iPSCs as cell resource, no teratoma formation was observed during a 2-month extended follow-up study after transplantation (see Methods, Supplemental Digital Content 1, at https://links.lww.com/SHK/A154), as previously described (17). Intravenous delivery of 3-gene iPSCs or iPSC-CM, but not MEFs, reduced infiltrative neutrophils and injury areas observed in lungs at day 7 after bleomycin treatment. In addition, bleomycin-induced interstitial thickening, inflammation, and distortion of lung architecture were attenuated after the administration of 3-gene iPSCs or iPSC-CM at days 14 and 21 after bleomycin treatment (Fig. 2A). The bleomycin-treated recipients of 3-gene iPSCs also exhibited lower Ashcroft lung fibrosis scores than those of MEFs or PBS alone (Fig. 2B). Restoration of bleomycin-impaired lung structure after treatment with 3-gene iPSCs was also observed according to measurements of the destructive index (Fig. 2C). The restorative effect of iPSC-CM on Ashcroft lung fibrosis scores and the destructive index was slightly less than that of 3-gene iPSCs. The alveolar mean linear intercept (Lm), a morphometric parameter of average alveolus size, also recovered to near-normal levels after the delivery of 3-gene iPSCs or iPSC-CM (Fig. 2D).
Both 3-gene iPSCs and iPSC-CM attenuate the intensity of bleomycin-induced pulmonary fibrosis
Pulmonary fibrosis is characterized by the accumulation of fibrillar collagens like type 1 collagen. Immunostaining for collagen-1 demonstrated thick bands of fibrosis in PBS-treated mice, whereas the lungs of bleomycin-challenged recipients treated with either 3-gene iPSCs or iPSC-CM were significantly protected from bleomycin-induced fibrosis (Fig. 3, A and C). The accumulation of myofibroblasts, serving as fibrogenic effector cells, was also examined (18). As indicated by the staining of lung sections for α-smooth muscle actin (α-SMA) at 14 and 21 days after bleomycin treatment, recipients of 3-gene iPSCs or iPSC-CM mice exhibited a significant decrease in myofibroblast accumulation in comparison with that of PBS-treated mice (Fig. 3, B and D). Lung collagen content was further assessed by measuring hydroxyproline content. Values in the iPSC-treated group and iPSC-CM–treated group were significantly lower than those in the PBS-treated group at 14 days and 21 days, indicating that bleomycin-induced collagen synthesis was significantly suppressed in mice treated with 3-gene iPSCs or iPSC-CM (Fig. 3E).
3-gene iPSCs/iPSC-CM treatment rescues physiologic impairments, diminished inflammatory cell accumulation, and reduced MPO activity
To characterize the physiologic processes that occur as a result of bleomycin treatment, various measurements of pulmonary function were assessed, including lung tidal volume, respiratory frequency, and minute ventilation. Decreased lung tidal volume, respiratory frequency, and minute ventilation were noted in mice with bleomycin-induced pulmonary fibrosis as compared with those of normal control mice (Fig. 4, A – C). Lung tidal volume during spontaneous respiration in mice treated with 3-gene iPSCs recovered to a near-normal level at 21 days after bleomycin injection (Fig. 4A). Respiratory frequency and minute ventilation showed similar trends. Mice treated with PBS exhibited decreased respiratory frequency and minute ventilation at day 7 after bleomycin exposure compared with normal control mice. Notably, 3-gene iPSCs treatment resulted in a minor reduction in respiratory frequency and minute ventilation, which then gradually increased to within basal levels by day 21 after treatment (. Fig. 4, B and C). Treatment with control MEFs had no effect on physiologic functions, and iPSC-CM treatment had slightly less pronounced effects compared with those observed in mice treated with 3-gene iPSCs. The mice used in this study had a baseline body weight of 23.4 ± 0.3 g but developed early weight loss after the intratracheal injection of bleomycin. Mice treated with iPSCs showed less decline in body weight and regained normal body weight at 14 days after bleomycin exposure (Fig. 4D). Moreover, iPSC-CM–treated mice also regained normal body weight at 21 days after bleomycin exposure (Fig. 4D).
Histologically, treatment with either 3-gene iPSCs or iPSC-CM reduced the exudative infiltration observed in the lungs at 7 days after bleomycin injection, suggesting that early reduction of inflammation may result in the attenuation of the downstream events leading to collagen deposition. In addition, intratracheal bleomycin treatment resulted in a significant increase in the accumulation of inflammatory cells in the lungs, as shown by Ly6C staining (positive staining indicates neutrophils and monocytes) (Fig. 5A). In contrast, i.v. administration of 3-gene iPSCs or iPSC-CM was associated with a significant decrease in positive Ly6C staining, and the quantification of Ly6C staining confirmed that both 3-gene iPSCs and iPSC-CM treatment reduced the degree of inflammatory cell accumulation in the lungs (Fig. 5B). Moreover, 3-gene iPSC treatment decreased the neutrophilic burden, as reflected by reduced MPO activity in the lungs (Fig. 5C). In contrast, MEF administration had only a minor diminishing effect on the bleomycin-induced elevation of MPO activity.
3-gene iPSCs/iPSC-CM decreased levels of cytokines and chemokines that promote inflammation and fibrosis and increased production of antifibrotic chemokine IP-10 in injured lungs
Because we observed reduced inflammation and fibrosis after bleomycin-induced lung injury as a result of treatment with 3-gene iPSCs and iPSC-CM, we next sought to investigate the potential mechanisms responsible for this effect. As the transition from inflammation to fibrosis occurs at an early phase during pathogenesis, we hypothesized that cytokine profiling would reflect the influence of cytokines on subsequent collagen deposition. The effect of 3-gene iPSCs and iPSC-CM on lung cytokine and chemokine levels at 3 days after bleomycin treatment was evaluated using a cytokine array assay (Fig. 6A). After treatment with 3-gene iPSCs or iPSC-CM, there was a reduction in the expression of cytokines and chemokines known to mediate inflammation (i.e., IL-1, IL-2, IL-10, tumor necrosis factor-α, and monocyte chemotactic protein 1 [MCP-1]) and fibrosis (i.e., interferon-γ [IFN-γ] and MCP-5).
Interferon-γ–inducible protein 10 (IP-10) is an antifibrotic chemokine responsible for tissue repair and remodeling. Three chemokines, including IP-10, a monokine induced by IFN-γ (MIG), and IFN-γ–inducible T-cell chemoattractant (iTAC), can bind to the common receptor CXCR3 and can be induced by IFN-γ. Among these chemokines, IP-10 has exhibited protective effects against hepatitis (19), pulmonary fibrosis (20, 21), and myocardial infarction (22) and has been shown to be involved in tissue repair and remodeling (22, 23). In addition to demonstrating reduced levels of several cytokines/chemokines capable of mediating inflammation and fibrosis, the enzyme-linked immunosorbent assay data also showed that both 3-gene iPSCs and iPSC-CM treatment stimulated the production of IP-10 (Fig. 6A). Real-time PCR further indicated that administration of 3-gene iPSCs or iPSC-CM significantly increased the mRNA expression of IP-10 at 3 days after bleomycin injury (Fig. 6B). In addition, these treatments reduced INF-γ mRNA expression but showed no effect on MIG, iTAC, or CXCR3 mRNA expression (Fig. 6, C – E).
3-gene iPSCs and iPSC-CM rescued the survival of bleomycin-treated recipient in a cell dose–dependent manner
To verify the protective effect of 3-gene iPSCs and iPSC-CM against bleomycin-induced pulmonary fibrosis, we also evaluated and compared the treatment efficacy of 3-gene iPSCs with that of iPSC-CM regarding recipient survival in response to bleomycin challenge. As shown in Figure 7A, mice were first intratracheally injected with 3.0 U/kg bleomycin sulfate, a dose sufficient to induce recipient lethality. Notably, i.v. delivery of 3-gene iPSCs led to a dose-dependent improvement in recipient survival, and the maximal protective effect of 3-gene iPSCs was observed at a dose of 2 × 106 cells. Treatment with iPSC-CM (collected from iPSC culture at the indicated cell dosages) similarly improved recipient survival in a cell dose–dependent manner (Fig. 7B). Collectively and based on the observed protective effects of 3-gene iPSCs and iPSC-CM on pulmonary fibrosis and recipient survival, these results suggest that 3-gene iPSCs exerted their protective effect in a predominantly paracrine manner.
IP-10 as the major contributor to the iPSC-CM–mediated reparative response
To elucidate whether IP-10 plays a crucial role in the reparative effect of iPSC-CM treatment, we evaluated the effect of neutralizing IP-10 by administration of IP-10–neutralizing antibody ([IP-10 nAb] Abcam, ab9938, Cambridge, UK). For the neutralizing antibody study, mice were given two distinct doses (i.e., 0.5 μg/dose and 0.3 μg/dose, intraperitoneally) of IP-10 nAb at 4 h before and 4 h after injury. In a cell dose–dependent manner, IP-10 neutralization reduced the survival rate of bleomycin-injected mice that received iPSC-CM treatment (Fig. 8A) or 3-gene iPSC transplantation (data not shown). These data further demonstrated that IP-10 nAb administration largely blocked the effects of iPSC-CM on collagen deposition (positive staining for collagen-1; Fig. 8B), myofibroblast accumulation (positive staining for α-SMA; Fig. 8C), inflammatory cell accumulation (positive staining for Ly6C; Fig. 8D), and pulmonary fibrosis (Ashcroft score; Fig. 8E). Administration of IP-10 nAb also deteriorated the reparative effect of 3-gene iPSCs, identical to the observations in that of iPSC-CM (data not shown). Taken together, these findings demonstrate that IP-10 is the major contributor to the 3-gene iPSCs and iPSC-CM–mediated inhibition of collagen deposition and inflammation in bleomycin-induced pulmonary fibrosis.
Chronic lung diseases cause serious morbidity and mortality but currently offer no effective treatments. Previous studies have reported using MSC-based therapy in bleomycin-induced pulmonary fibrosis (9, 10, 24, 25), highlighting the use of cell-based therapy for chronic lung diseases. Although recent reports have suggested the use and low tumorigenic risk of c-Myc–free iPSCs (16, 17), their therapeutic effects on chronic lung disease had not been investigated. In the present study, administration of 3-gene iPSCs or iPSC-CM dramatically improved the histopathologic changes, lung collagen content, and pulmonary function, which was deteriorated by bleomycin. In addition, both 3-gene iPSCs and iPSC-CM diminished neutrophil accumulation and MPO activity in the lungs at day 7 after bleomycin exposure. Moreover, these treatments also reduced the expression of inflammatory and fibrotic cytokines/chemokines 3 days after bleomycin exposure. Remarkably, treatment with 3-gene iPSCs/iPSC-CM also increased the production of the IP-10 in injured lungs. Importantly, iPSC treatment has been associated with the risk of teratoma formation (26), whereas we conducted a 2-month follow-up study but did not observe tumorigenesis after the transplantation of 3-gene iPSCs. Administration of iPSC-CM also did not lead to tumorigenesis, and its reparative effect was partially caused by early reduction of inflammation and the levels of inflammatory and fibrotic cytokines/chemokines and elevation of antifibrotic chemokine IP-10 production in the injured lungs.
Recent studies have shown that systemic administration of MSCs can reduce inflammation, fibrosis formation, and mortality after lipopolysaccharide- or bleomycin-induced acute lung injury (8–13). Our recent study demonstrated that iPSC-CM therapy for endotoxin-induced acute lung injury had effects that were similar to those obtained with iPSC treatment (16). Interferon-γ–induced protein 10 has been shown to attract lymphocytes (27) and to suppress CXCR2-positive neutrophil migration during T-cell priming (19) and to attenuate fibroblast accumulation in bleomycin-induced pulmonary fibrosis by limiting fibroblast migration (20). Moreover, IP-10–deficient mice that genetically overexpress IP-10 or that are given exogenous IP-10 are protected from bleomycin-induced pulmonary fibrosis (20, 28). In this study, we found that treatment with 3-gene iPSCs or iPSC-CM increased the production of IP-10, which we identified as one of secretory factors mediating the protective effect of 3-gene iPSCs and iPSC-CM treatment. Importantly, our in vivo animal study revealed that IP-10 nAb treatment attenuated the protective effects of iPSC-CM administration on recipient survival, collagen deposition, myofibroblast accumulation, inflammatory cell accumulation, and alveolar destruction. These data demonstrate that IP-10 is a significant contributor to the iPSC-CM–mediated reparative response in bleomycin-induced pulmonary fibrosis. This IP-10 upregulation may be directly stimulated by the 3-gene iPSCs or indirectly recruited by IP-10 inducer generated by 3-gene iPSCs infusion. Although IP-10 could be induced by IFN-γ (29), our findings of reduced IFN-γ did not explain the IP-10 upregulation. Actually, IP-10 could be produced by several types of cells, including endothelial cells, Küpffer cells, and immune cells, and the precise mechanism of IP-10 regulation warrants further investigation. Taken together, our data provide preclinical indications suggesting that IP-10 may serve as a prospective diagnostic and therapeutic target.
Because of parenchymal lesions and extracellular matrix remodeling, the lungs of bleomycin-treated animals have reduced compliance, increased elastic recoil, and increased lung resistance (30–32). Consistently, our findings also found that bleomycin-treated recipients exhibited impaired tidal volume, respiratory frequency, and minute ventilation. These changes are likely a consequence of the morphological alterations that occur in the lung after bleomycin exposure. Notably, transplantation of 3-gene iPSCs/iPSC-CM restored the impaired lung tidal volume, respiratory frequency, and minute ventilation 21 days after bleomycin exposure. A previous study suggested that an early blunting of inflammation may result in the attenuation of the downstream events that lead to collagen deposition (6). Recent evidences showed that transplanted cells generated paracrinal effects, including stimulating angiogenesis and modulating local inflammatory responses (7, 33). The present study found that 3-gene iPSCs reduced neutrophil accumulation and MPO activity at 7 days after bleomycin exposure. This early prevention of recruited neutrophil and inflammatory responses by 3-gene iPSCs/iPSC-CM may serve a potential role in mediating the protective effect of 3-gene iPSCs/iPSC-CM against pulmonary fibrosis.
In conclusion, i.v. delivery of 3-gene iPSCs/iPSC-CM attenuated the severity of the histopathologic and physiologic impairments caused by bleomycin-induced lung fibrosis. These protective effects were partially mediated by the early amelioration of inflammation, reduction of inflammatory and fibrotic cytokines/chemokines, and increase of IP-10 production in injured lungs. Thus, 3-gene iPSCs/iPSC-CM may provide a novel therapeutic alternative for the management of pulmonary fibrosis.
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