American Journal of Respiratory Cell and Molecular Biology

We recently reported that IL-17A plays a critical role in the development of porcine pancreatic elastase (PPE)–induced emphysema. The proliferation of T-helper type 17 (Th17) cells was induced by IL-23. To determine the contribution of IL-23 to the development of pulmonary emphysema, a mouse model of PPE-induced emphysema was used in which responses of IL-23p19–deficient (IL-23−/−) and wild-type (WT) mice were compared. Intratracheal instillation of PPE induced emphysematous changes in the lungs and was associated with increased levels of IL-23 in lung homogenates. Compared with WT mice, IL-23−/− mice developed significantly lower static compliance values and markedly reduced emphysematous changes on histological analyses after PPE instillation. These changes were associated with lower levels of IL-17A and fewer Th17 cells in the lung. The neutrophilia seen in bronchoalveolar lavage fluid of WT mice was attenuated in IL-23−/− mice, and the reduction was associated with decreased levels of keratinocyte-derived cytokine and macrophage inflammatory protein-2 in bronchoalveolar lavage fluid. Treatment with anti–IL-23p40 monoclonal antibody significantly attenuated PPE-induced emphysematous changes in the lungs of WT mice. These data identify the important contributions of IL-23 to the development of elastase-induced pulmonary inflammation and emphysema, mediated through an IL-23/IL-17 pathway. Targeting IL-23 in emphysema is a potential therapeutic strategy for delaying disease progression.

IL-23 has previously been linked to autoimmune inflammation, but its role in the development of emphysema is uncertain. IL-23 induces elastase-induced airway inflammation and emphysematous changes in the lung.

The major features of chronic obstructive pulmonary disease (COPD) are progressive alveolar destruction and inflammation of the airways, mainly induced by exposure to cigarette smoke (1). COPD is estimated to become the third most common cause of death by 2020 (2). Macrophages, neutrophils, and T lymphocytes play important roles in the development and progression of COPD (3), and current antiinflammatory therapies are weakly effective in maintaining lung function and controlling symptoms in COPD (4). New antiinflammatory therapeutic strategies are urgently needed.

IL-17 has been associated with the development of lung inflammatory diseases, such as COPD (5). Production of IL-17A was increased in the bronchial submucosa and subepithelium of patients with COPD (6, 7). We reported that IL-17A–deficient mice showed impaired porcine pancreatic elastase (PPE)–induced airway inflammation and emphysema, suggesting that IL-17A contributes to development of emphysema (8).

IL-23 is a novel member of an emerging heterodimeric cytokine family (9), is composed of p19 and p40 subunits, shares a common p40 subunit with IL-12, and induces the differentiation of naive CD4+ T cells into Th17 cells (10). Recently, IL-23 was reported to have effects on the generation of T cytotoxic 17 (Tc17) cells (11). The IL-23/IL-17 pathway is an important area in immunology research, because of its role in the development of chronic inflammation and in host defense against bacterial infection (12). In animal studies, IL-23p19–deficient (IL-23−/−) mice are reported to be resistant to experimental autoimmune encephalomyelitis. In humans, the anti–IL-23p40 antibody, ustekinumab, has demonstrated efficacy in the treatment of psoriasis (13, 14). Levels of IL-23 were increased in the lung tissue of patients with COPD (7); however, the role of IL-23 in the development of emphysema remains unclear.

In the present study, we investigated the susceptibility of IL-23−/− mice to PPE-induced emphysema to define the role of IL-23 in the progression of emphysema. Instilled PPE in the trachea first induces lung inflammation and subsequently results in alveolar wall destruction (15, 16). These pathological changes closely mimic those seen in human emphysema. We also assessed the effects of treatment with anti–IL-23p40 monoclonal antibody (mAb; anti–IL-23p40) and showed that both approaches attenuated development of emphysematous changes after PPE instillation.

Part of the content of this article was presented at the 2015 ATS Conference and published in the conference abstracts (17).

An expanded description of the methods can be found in the online supplement.

Animals and Treatment

C57BL/6 (wild-type [WT]) mice were obtained from Charles River Japan (Yokohama, Japan). IL-23−/− mice were generated as described by Cua and colleagues (18). Female WT and IL-23−/− mice (10 wk old) received 3.75 U of PPE by intratracheal instillation or saline alone on Day 0 (19). To evaluate emphysematous changes in the lungs, lung function was measured, and lung histology and morphometric measurements of air space size were assessed 7, 14, and 21 days after PPE instillation. Numbers of mice in each group ranged from 4 to 10, and experiments were repeated at least twice.

All of the experimental animals used in the present study were housed under a protocol approved by the Institutional Animal Care and Use Committee of Okayama University Medical School (Okayama, Japan).

Determination of Static Lung Compliance

Static lung compliance (Cst) was measured 7, 14, and 21 days after PPE administration in tracheostomized mice using a computer-controlled small-animal ventilator (flexiVent; SCIREQ, Montreal, PQ, Canada) (2022).

Bronchoalveolar Lavage

On Days 4, 7, 14, and 21 after PPE administration, lungs were lavaged, and numbers of cells in the bronchoalveolar lavage (BAL) fluid were counted as previously described (23).

Lung Histology and Morphometric Measurements of Air Space Size

The right lung was inflated and fixed by intratracheal instillation of formalin (24). The tissue sections were then stained with hematoxylin-eosin. Air space enlargement was quantified by the mean linear intercept (Lm) as previously described (25, 26).

Lung Homogenates

Lung tissue was frozen, and lung homogenates were prepared as previously described (8).

Measurement of Cytokines and Chemokines

Cytokine levels in the BAL fluid and homogenized lung were measured by ELISA as previously described (27).

Administration of the Anti–IL-23p40 mAb

The C57BL/6 mice received intraperitoneal injections of 375 μg of anti–IL-23p40 mAb (provided by Dr. Akihiko Yoshimura, Keio University School of Medicine, Tokyo, Japan) on Days −1, 4, 9, and 14. The control mice were administered rat IgG (control IgG). The mice received PPE or saline by intratracheal instillation on Day 0 (see Figure E1A in the online supplement). To assess the effect of IL-23 blockade in mice with established emphysematous changes, the mice received anti–IL-23p40 or control IgG on Days 7, 12, and 17 after PPE instillation (see Figure E1B).

Flow Cytometry

Lung cells were isolated as previously described after collagenase digestion (27). Intracytoplasmic cytokine staining was performed as previously described (28, 29). Cells were stained for cell surface markers with adenomatous polyposis coli (APC)-conjugated anti-CD4 or APC-conjugated anti-CD8, and peridinin chlorophyll protein complex (PerCP)-conjugated anti-CD3. Cells were stained with phycoerythrin (PE)-conjugated anti–IL-17A or fluorescein isothiocyanate (FITC)-conjugated anti–IFN-γ, and analyzed on MACS Quant flow cytometer (Miltenyi Biotec, Auburn, CA) with FlowJo software (TreeStar, Ashland, OR). Cells were also stained for cell surface markers with APC-conjugated anti-CD11b and FITC-conjugated anti-CD11c, and then stained with PE-conjugated anti–IL-23.

Statistical Analysis

All results are expressed as means (±SEM). ANOVA was used to determine the levels of difference between all groups. The Tukey–Kramer test was used for comparisons between multiple groups. Pairs of groups of samples distributed parametrically were compared by unpaired, two-tailed Student’s t test. As measured values may not be normally distributed due to the small sample sizes, nonparametric analysis, Mann–Whitney U test was also used to confirm that the statistical differences remained significant. Significance was assumed at P values of less than 0.05.

Development of Emphysema Is Reduced in IL-23−/− mice

We measured lung function after PPE instillation to clarify the role of IL-23 in the development of emphysema. Cst values were higher in PPE-instilled WT mice than the values in saline-instilled WT mice or saline-instilled IL-23−/− mice on Days 14 and 21. The Cst values in PPE-instilled IL-23−/− mice were similar to the values in saline-instilled WT mice, and the values were significantly lower than the values in PPE-instilled WT mice (Figure 1A). There were no pathological differences in WT and IL-23−/− mice after saline instillation (Figure 1B). Instillation of PPE into WT mice resulted in air space enlargements and alveolar wall destruction. These pathological changes were markedly reduced in PPE-instilled IL-23−/− mice (Figure 1B).

To further assess emphysema development, we monitored airspace enlargement by measuring Lm from hematoxylin-eosin–stained tissue sections. Lm values in WT mice after PPE were significantly increased compared with saline-instilled mice; these changes were significantly reduced in IL-23−/− mice on Days 7, 14, and 21 after instillation of PPE (Figure 1C). These histological and physiological assays demonstrated that IL-23−/− mice are less susceptible to PPE-induced emphysema.

Elastase-Induced Pulmonary Inflammation Is Reduced in IL-23−/− Mice

Acute-phase lung inflammation is thought to be a major contributor to the development of emphysema (8, 30). The severity of the initial inflammatory response was compared in WT and IL-23−/− mice 4 days after PPE administration. In WT mice, inflammatory cell recruitment into the airways was increased after PPE instillation (Figure 2A). Increased total cell numbers were largely due to increased numbers of neutrophils and macrophages in BAL fluid. The increases in neutrophil numbers were significantly lower in IL-23−/− mice compared with WT mice 4 days after PPE administration (Figure 2A). We also assessed inflammatory cell populations in BAL fluid 21 days after PPE administration when emphysematous changes were established. The numbers of macrophages were similar in PPE-instilled WT mice and IL-23−/− mice, but were significantly increased in both groups compared with the saline-instilled groups (see Figure E2).

Cytokine and Chemokine Levels in BAL Fluid

To determine the basis for the altered inflammatory cellular recruitment in IL-23−/− mice, we measured levels of BAL neutrophil-related chemokines after PPE instillation. Keratinocyte-derived cytokine (KC) and macrophage inflammatory protein-2 (MIP-2) levels were increased in PPE-instilled WT mice compared with the mice that received saline on Day 4. Levels of KC and MIP-2 in BAL fluid of IL-23−/− mice were significantly lower compared with the WT mice (Figure 2B). The levels of KC and MIP-2 were not different among the groups on Days 7, 14, and 21 after PPE instillation (see Figure E3).

IL-17A Levels and the Numbers of IL-17A+ CD4+ T Cells in the Lung Are Lower in IL-23−/− Mice Compared with WT Mice after PPE Administration

IL-23 is known to induce proliferation of Th17 cells, and we have recently shown that IL-17A plays an essential role in PPE-induced emphysema. We assessed IL-17A levels in lung homogenates and the numbers of IL-17A–producing CD4+ T cells in the lung. Th1 cells have been shown to contribute to the pathogenesis of COPD; therefore, we also examined IFN-γ–producing cells (31, 32). To determine the production of IL-17A in the airways after PPE instillation, we measured levels in lung homogenates from WT mice and IL-23−/− mice on Days 4 and 21 after PPE treatment. The levels of IL-17A in the lungs of PPE-instilled WT mice were increased compared with saline-instilled WT mice, and this increase was attenuated in IL-23−/− mice on Days 4 and 21 (Figures 2C and 2D). In contrast, the levels of IFN-γ were not different among the groups.

We next compared the numbers of IL-17A– and IFN-γ–producing cells in the lungs of IL-23−/− and WT mice 4 days after PPE instillation. Figure 3A shows the representative percentages of IL-17A+ and IFN-γ+ CD4+ T cells in the lung after PPE instillation, and Figure 3B shows the total numbers of IL-17A+ and IFN-γ+ CD4+ T cells in the lung. In WT mice, the numbers of IL-17A+ CD4+ T cells were significantly increased after PPE instillation, and the numbers in PPE-instilled IL-23−/− mice were significantly lower (Figures 3A and 3B).

The importance of IL-17A/IFN-γ double-positive CD4+ T cells in the pathogenesis of graft-versus-host disease was recently reported (33). In WT mice, the numbers of IL-17A+ IFN-γ CD4+ T cells were significantly increased after PPE instillation, but the numbers of IL-17A+ IFN-γ+ CD4+ T cells were not affected by PPE instillation (Figure 3C). The numbers of IL-17A+ IFN-γ CD4+ T cells in the lungs of IL-23−/− mice were significantly lower compared with WT mice after PPE instillation. We also assessed the numbers of IFN-γ+ CD4+ T cells, but these numbers were not different between WT and IL-23−/− mice 4 days after PPE instillation (Figure 3B). These data suggest that IL-23 contributes to the accumulation of IL-17A+ IFN-γ CD4+ T cells in the lungs.

Figure 3D shows the numbers of IL-17A+ and IFN-γ+ CD8+ T cells in the lung. At 4 days after PPE instillation, the numbers of IL-17A+ CD8+ T cells tended to be increased in both WT and IL-23−/− mice, but these numbers were not statistically different between the groups.

Kinetics of IL-23 and IL-17A Levels in the Lung

To assess the kinetics of IL-23 production in the airways after PPE instillation, we measured the levels of IL-23 in lung homogenates of WT mice at different time points (Figure 4A). IL-23 levels were transiently increased on Days 1 and 2 after PPE instillation and increased again on Days 9, 12, 14, and 21.

We assessed changes in lung morphology after PPE instillation in WT mice. It was histologically difficult to measure Lm values at an earlier stage because of severe hemorrhage and infiltration of inflammatory cells (data not shown), but they became measurable 7 days after PPE instillation. Lm values remained significantly higher from Day 7 to Day 21 compared with baseline (Day 0) values (Figure E4).

We next assessed kinetics of IL-17A production in the airways of WT and IL-23−/− mice after PPE instillation (Figure 4B). In WT mice, IL-17A levels were increased on Days 2 and 4 compared with baseline, and, similar to IL-23, the levels were transiently decreased, but increased again on Day 21. Thus, increases in IL-17A levels appeared after increases in IL-23. In IL-23−/− mice, IL-17A levels remained significantly lower compared with WT mice on Days 4, 14, and 21. These data suggest that, in the lung, elastase induces IL-23 production followed by IL-17A production, and then promotes development of emphysematous changes.

Treatment with Anti–IL-23p40 Attenuates Emphysema in PPE-Instilled Mice

In parallel to examining responses in IL-23−/− mice, we determined if anti–IL-23p40 similarly altered the responses to PPE instillation (see Figure E1A). After administration of anti–IL-23p40 to mice that received PPE, changes in Cst values were significantly reduced (Figure 5A). In these mice, lung pathology was reduced, with less-severe emphysematous changes observed (Figures 5B and 5C). As seen in Figure 5B, the alveolar walls were less-severely damaged in the lungs of anti–IL-23p40–treated mice compared with control IgG-treated mice after PPE instillation. Lm values of anti–IL-23p40–treated mice were significantly lower compared with control IgG-treated mice after PPE instillation (Figure 5C).

Treatment with Anti–IL-23p40 Reduces Inflammatory Cell Accumulation in the BAL Fluid of PPE-Instilled Mice

As shown in IL-23−/− mice, administration of anti–IL-23p40 to PPE-treated WT mice significantly reduced the numbers of cells in the BAL fluid, including lymphocytes and neutrophils (Figure 6A).

The levels of KC and MIP-2 in the BAL fluid were significantly reduced in anti–IL-23p40–treated mice compared with control IgG-treated mice on Day 4 after PPE instillation (Figure 6B).

Treatment with Anti–IL-23p40 Reduces Both IL-17A Levels in Lung Homogenates and the Numbers of IL-17A–Producing Cells in the Lung

The levels of IL-17A in the lung homogenates on Days 4 and 21 after intratracheal instillation of PPE were significantly reduced after administration of anti–IL-23p40 (Figures 6C and 6D). The levels of IFN-γ showed a similar trend 21 days after PPE instillation, but the levels were not statistically different between the groups.

We quantitated numbers of IL-17A–producing cells in the lungs 4 days after elastase instillation. Figure 7A shows the numbers of IL-17A+ CD4+ T cells in the lung. In control IgG-treated mice, the numbers of IL-17A+ CD4+ T cells, especially IL-17A+ IFN-γ CD4+ T cells, were increased after PPE instillation, whereas these increases were significantly reduced after anti–IL-23p40 administration (Figures E5A and E5B). The numbers of IFN-γ+ CD4+ T cells in the lung were not different between the groups. The numbers of L-17A+ CD8+ T cells and IFN-γ+ CD8+ T cells tended to be increased after PPE instillation, but those numbers were not affected by treatment with anti–IL-23p40 (see Figure E5C).

Figure E5D shows the numbers of CD11b+ cells and CD11c+ cells in the lung 4 days after PPE instillation. The numbers of CD11b+ and CD11c+ cells were increased after PPE instillation, and those numbers were significantly reduced by treatment with anti–IL-23p40. IL-23–producing cells, including IL-23+CD11b+ cells and IL-23+CD11c+ cells, in the lung were also assessed 4 days after PPE instillation (Figure E5E). The numbers of IL-23+CD11b+ cells in the control IgG-treated mice were increased after PPE instillation, and this increase was reduced by treatment with anti–IL-23p40. These data suggest that treatment with anti–IL-23p40 not only abrogated IL-23 activity in vivo, but also suppressed accumulation of IL-23–producing macrophages and dendritic cells (DCs) in the airway, and consequently suppressed generation of Th17 cells and the development of emphysema.

Treatment with Anti–IL-23p40 Attenuates Progression of Emphysematous Changes Induced by PPE

After PPE instillation, emphysematous changes in the lung were observed as early as 7 days (Figures 1C and 5C). To assess the effect of IL-23 blockade in mice with established emphysematous changes, administration of anti–IL-23p40 was initiated 7 days after PPE instillation (see Figure E1B). As shown in Figure 7A, treatment with anti–IL-23p40 significantly reversed the changes in Cst; mice which received anti–IL-23p40 showed less severe emphysematous changes compared with control IgG-treated mice (Figure 7B). Lm values of anti–IL-23p40–treated mice were significantly lower compared with control IgG-treated mice after PPE instillation (Figure 7C). These data suggest that treatment with anti–IL-23p40 was effective in attenuating the progression of lung damage and emphysematous changes induced by the earlier administration of PPE.

There is increasing evidence demonstrating critical roles for an IL-23/IL-17 axis in autoimmune disease, highlighting the importance of understanding the mechanisms leading to development of IL-17 effector cells (10, 34). It has been reported that IL-23−/− mice were completely resistant to the development of collagen-induced arthritis, and that IL-23 was required for the induction of inflammatory mediators, including IL-17 (35). These findings suggest that IL-23 has a pivotal role in the establishment and maintenance of inflammatory autoimmune diseases; however, a role in the development of emphysema remains unclear.

In the present study, we demonstrated that both the PPE-induced acute-phase inflammatory response and late-phase emphysematous changes seen in WT mice were attenuated in IL-23−/− mice. Increased levels of neutrophil-related chemokines, such as KC and MIP-2 seen in the BAL fluid of WT mice, were significantly reduced in IL-23−/− mice after PPE instillation, associated with decreased numbers of IL-17A+ CD4+ T cells in the lung. Administration of anti–IL-23p40 resulted in similar reductions in the inflammatory responses and pathological changes, and was effective in reducing the progression of pathological and physiological changes once established. Taken together, the data indicate that IL-23 plays a critical role in the development of elastase-induced emphysema IL-23/IL-17 pathway, and that targeting IL-23 suppresses the development of elastase-induced emphysema.

IL-17A is a proinflammatory cytokine produced mainly by Th17 cells and neutrophils (36). Levels of IL-17A in the sputum of patients with COPD were reportedly higher compared with those with asthma (6), and the numbers of Th17 cells in the bronchial submucosa were increased in COPD compared with smokers without COPD and nonsmokers (6). Using a mouse model of PPE-induced emphysema, we recently showed that IL-17A plays a critical role in the development of emphysema (8). During preparation of this article, the importance of IL-17A in acute exacerbations and lymphoid neogenesis in COPD was reported (37, 38). The generation and development of Th17 cells were thought to be dependent on IL-23, IL-6, and transforming growth factor-β (39). In the present study, the levels of IL-17A in the lungs were increased in WT mice after PPE instillation, associated with increased numbers of IL-17A+ CD4+ T cells. This increase was reduced in IL-23−/− mice after PPE instillation, suggesting that IL-23 induces IL-17A production, especially from CD4+ T cells, and contributes to development of PPE-induced emphysema in mice. When the kinetics of IL-23 and IL-17 production in the lungs of WT mice after elastase instillation were monitored, increases in IL-23 were followed by increases in IL-17, which support the importance of the IL-23/IL-17 pathway in the pathology induced by elastase. Interestingly, increased IL-23 levels during the early phase transiently decreased, but increased again at the later stages. Although the mechanism leading to the later increases in IL-23 levels is unclear, increased IL-23 levels in both early and late phases appeared to contribute to the increases in IL-17A and the development of emphysematous changes.

Anti–IL-23p40 treatment is recognized as efficacious in the treatment of inflammatory autoimmune disease models. In animal models, therapeutic targeting of IL-23 effectively inhibited multiple inflammatory pathways that are critical for driving autoimmune inflammation of the central nervous system (40). Ustekinumab, a human anti–IL-23p40 mAb, inhibited the bioactivity of IL-12 and IL-23, and effectively neutralized both IL-12 (Th1)–mediated and IL-23 (Th17)–mediated cellular responses (41). Clinical efficacy has been shown in moderate to severe psoriasis (13, 14), and a positive benefit/risk profile in early clinical trials in psoriatic arthritis (42) and Crohn’s disease (43, 44) has been demonstrated. In the present study, anti–IL-23p40–treated mice showed reduced airway inflammation and emphysematous changes compared with control IgG-treated mice. Increased neutrophil-related chemokine levels, as well as IL-17A levels and Th17 cell numbers, in the airway were significantly reduced. In contrast, IFN-γ levels or the numbers of IFN-γ–producing cells in the lung were not affected by treatment with anti–IL-23p40. Although anti–IL-23p40 may suppress both Th17 signaling through p19 and Th1 signaling through p40, these data suggest that the blockade of IL-23 by anti–IL-23p40 mainly inhibited Th17 signaling rather than Th1 signaling, and attenuated airway inflammation and development of emphysema. However, the role of IL-12 in elastase-induced emphysema has not been investigated, and remains to be defined.

Of note, IL-17/IFN-γ double-positive CD4+ T cells may play a role in Th-17–related disease pathology, as shown in an experimental model graft-versus-host disease (32). In the present study, the numbers of IL-17A+ IFN-γ CD4+ T cells, but not IL-17/IFN-γ double-positive CD4+ T cells, in the lung were increased after PPE instillation and were reduced by treatment with anti–IL-23p40. In addition, IL-23−/− mice showed fewer IL-17A+ IFN-γ CD4+ T cells compared with WT mice after PPE instillation. Therefore, IL-23 may contribute to the development of emphysema primarily through IL-17A single-positive CD4+ T cells.

IL-17A is also produced by Tc17 cells. It has been shown that the numbers of Tc17 cells in the lung were increased in patients with COPD (11). Zhou and colleagues (45) reported that long-term cigarette smoke exposure induced Tc17 cell expansion. In the present study, IL-17A+ CD8+ T cells in the lungs of control IgG-treated mice tended to be increased after PPE instillation, but treatment with anti–IL-23p40 did not affect the numbers of these T cells. Thus, at least in this model, Th17 rather than Tc17 may be involved in IL-23–dependent development of emphysema.

IL-23 secretion by macrophages and DCs has been shown (9). Through interaction with the IL-23 receptor, IL-23 plays a central role in inflammation, including the induction of Th17 cells (34, 46, 47). In the present study, the numbers of CD11b+ and CD11c+ cells, and especially the numbers of IL-23+ CD11b+ cells, in the lung were increased after PPE instillation, and treatment with anti–IL-23p40 significantly reduced the numbers of these cells. In human rheumatoid arthritis, it has been reported that M1 macrophages produce IL-6 and IL-23, which act to expand the Th1/Th17 cell population, and the Th1/Th17 cells produce granulocyte-macrophage colony–stimulating factor, thereby facilitating differentiation of M1 macrophages (48). IL-23 has also been reported to induce accumulation of CD11b+ DCs at sites of inflammatory bowel diseases (49). Thus, anti–IL-23p40 may not only antagonize the function of IL-23, but also reduce differentiation and accumulation of IL-23–secreting alveolar macrophages and DCs in the airways and affect PPE-induced differentiation of Th17 cells. Together, these activities may underlie the beneficial effects in attenuating the development of emphysema.

In summary, we demonstrated that IL-23 played a critical role in the development of PPE-induced acute-phase neutrophilic inflammation and subsequent emphysematous changes in the lung. These responses were associated with increased levels of IL-17 and increased numbers of Th17 cells in the lung. In a complementary way, antagonizing IL-23 with an IL-23p40 mAb similarly altered development of PPE-induced emphysema, and was effective in reducing the progression of lung pathology even when administered after PPE-induced lung damage had begun. The data support the notion that targeting IL-23 offers promise as a therapeutic strategy for improving clinical outcomes and for delaying emphysema disease progression.

The authors thank Dr. Sachiyo Okamoto, Dr. Yoshinobu Maeda, Misa Ariyoshi, and Kaoru Kawano (Okayama University, Okayama, Japan) for their help in the experiments, and Diana Nabighian (National Jewish Health, Denver, CO) for her assistance in preparing the manuscript.

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Correspondence and requests for reprints should be addressed to Nobuaki Miyahara, M.D., Ph.D., Field of Medical Technology, Okayama University Graduate School of Health Sciences, 2-5-1 Shikata-cho, Okayama, Okayama 700-8558, Japan. E-mail:

This work was supported by the Japan Society for Promotion of Science Kakenhi grant 15K09221 (N.M.).

Author Contributions: Conception and design—N.M. and M.T.; analysis and interpretation—U.F., N.M., A.T., K.W., D.M., E.K., H.K., M.K., E.W.G., D.J.C., A.Y., M.T., and A.K.; drafting the manuscript for important intellectual content—U.F., N.M., E.W.G., M.T., and A.K.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2016-0015OC on June 28, 2016

Author disclosures are available with the text of this article at www.atsjournals.org.

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