http://informahealthcare.com/rst ISSN: 1079-9893 (print), 1532-4281 (electronic) J Recept Signal Transduct Res, 2014; 34(4): 299–306 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2014.896380
RESEARCH ARTICLE
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p38 Mitogen-activated protein kinase accelerates emphysema in mouse model of chronic obstructive pulmonary disease Hiroyuki Amano1,2, Kazuya Murata3, Hirofumi Matsunaga1,4, Kensuke Tanaka1,2, Kento Yoshioka1, Takeshi Kobayashi1,2, Junji Ishida3, Akiyoshi Fukamizu3, Fumihiro Sugiyama5, Tatsuhiko Sudo6, Sadao Kimura1, Koichiro Tatsumi2, and Yoshitoshi Kasuya1 1
Department of Biochemistry and Molecular Pharmacology, 2Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan, 3Life Science Center, Tsukuba Advanced Research Alliance, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennoudai, Ibaraki, Japan, 4Pharmaceuticals Research Laboratory, Ube Industries, Ltd., Ube, Japan, 5Laboratory Animal Resource Center, University of Tsukuba, Tennoudai, Ibaraki, Japan, and 6Chemical Biology Core Facility and Antibiotics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan Abstract
Keywords
Context: There are few short-term mouse models of chronic obstructive pulmonary disease (COPD) mimicking the human disease. In addition, p38 is recently recognized as a target for the treatment of COPD. However, the precise mechanism how p38 contributes to the pathogenesis of COPD is still unknown. Objective: We attempted to create a new mouse model for COPD by intra-tracheal administration of a mixture of lipopolysaccharide (LPS) and cigarette smoke solution (CSS), and investigated the importance of the p38 mitogen-activated protein kinase (p38) pathway in the pathogenesis of COPD. Methods: Mice were administered LPS + CSS once a day on days 0–4 and 7–11. Thereafter, CSS alone was administered to mice once a day on days 14–18. On day 28, histopathological changes of the lung were evaluated, and bronchoalveolar lavage fluid (BALF) was subjected to western blot array for cytokines. Transgenic (TG) mice expressing a constitutive-active form of MKK6, a p38-specific activator in the lung, were subjected to our experimental protocol of COPD model. Results: LPS + CSS administration induced enlargement of alveolar air spaces and destruction of lung parenchyma. BALF analyses of the LPS + CSS group revealed an increase in expression levels of several cytokines involved in the pathogenesis of human COPD. These results suggest that our experimental protocol can induce COPD in mice. Likewise, histopathological findings of the lung and induction of cytokines in BALF from MKK6 c.a.-TG mice were more marked than those in WT mice. Conclusion: In a new experimental COPD mouse model, p38 accelerates the development of emphysema.
Cigarette smoke, COPD mouse model, lipopolysaccharide, MKK6 c.a., transgenic mouse
Introduction Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease characterized by airflow limitation that is not fully reversible. Cigarette smoking has been generally accepted as the most important risk factor for the development of COPD. However, only 15–20% of heavy smokers develop clinically significant airflow obstruction (1), which suggests that a genetic susceptibility to the effects of cigarette smoking. Several pathophysiologic processes interact on a complex background of genetic and environmental factors, but the detailed course of the disease is not fully elucidated.
Address for correspondence: Yoshitoshi Kasuya, Ph.D., Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Tel: +81-43-226-2193. Fax: +81-43-226-2196. E-mail: kasuya@ faculty.chiba-u.jp
History Received 23 November 2013 Revised 30 January 2014 Accepted 17 February 2014 Published online 5 March 2014
Based on prevalence surveys, it is estimated that 10% of adults aged 40 years and older have COPD (2). COPD was ranked sixth in the causes of death in 1990, and will become the third leading cause of death by 2030 (3). These data suggest that COPD will become a major health problem in the next few decades. The mainstay of current therapy for COPD is inhaled bronchodilators. There is a pressing need to develop new treatments for COPD, because most studies have indicated that the existing medications for COPD do not modify the long-term decline in lung function (4). The use of appropriate experimental COPD animal models is indispensable for the development of new therapy and for better understanding of the disease. A variety of different approaches have been used to attempt to model COPD in animals (5). Administration of proteinases, chemicals and particulates, cigarette smoke exposure and genetic manipulation have successfully produced features
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characteristic of human COPD. Although the cause of the disease tempts us to consider a smoke exposure model as the most reasonable model, it takes more than 3 months to induce emphysema (6). In order to shorten the period, Mizutani et al. (7) employed intra-tracheal administration of cigarette smoke solution (CSS) and lipopolysaccharide (LPS) and created a guinea pig COPD model that requires only 19 days, indicating the possibility that this protocol could enable us to investigate the pathogenesis of COPD more easily. The p38 mitogen-activated protein kinase (p38) signaling pathway is activated by a variety of extracellular stimuli including cellular stress, inflammatory cytokines and cell surface receptors, and plays a role in a wide range of cellular responses, such as proliferation, differentiation and apoptosis (8). In particular, the notion that p38 functions as a central player in inflammatory diseases has been supported by various reports including our studies (9,10). Also, in lung inflammatory diseases, p38 is recognized as a potential therapeutic target. Indeed, pirfenidone, a p38g inhibitor, is used for the treatment of patients with idiopathic pulmonary fibrosis (11). Likewise, it has been reported that p38 is activated in the alveolar macrophages of COPD patients compared to healthy controls (12), suggesting that p38 might be a plausible target for the treatment of COPD. Here, we first investigated whether the protocol for the guinea pig COPD model could be applied to mice. Then, we established a new experimental COPD mouse model. We next investigated the participation of p38 in the pathogenesis of COPD, using MKK6 c.a.-TG mice in which the MKK6 constitutive-active gene is introduced under control of the human surfactant protein C (SP-C) gene promoter.
Materials and methods Animals Male C57BL/6J mice at 10 weeks of age were purchased from Clea Japan (Tokyo, Japan). To generate MKK6 constitutiveactive (c.a., dual mutations in wild human MKK6: Ser207 to Asp; Tyr211 to Asp) TG mice, the linear 5.3 k-bp hSPC3HA-tag-hMKK6 c.a. NarI/NotI-fragment (Supplemental Figure S1A) was microinjected in eggs from C57BL/6J mice, which were subsequently transplanted into the uteli of pseudopregnant female ICR mice. For construction of the transgene, the 3.7SPC/SV40 vector was provided by Dr. Jeffrey A. Whitsett, Children’s Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio. Although we obtained five lines of TG F1 mice, the four TG lines with high expression level of transgene-derived MKK6 c.a. showed low fertility rate. As a result, only one line of MKK6 c.a.-TG mice could be maintained. In the lung of MKK6 c.a.-TG mice, the expression of HA-tag+/41-KDa protein corresponding to the predicted size of 3HA-tag-MKK6 c.a. was confirmed by western blot (WB) analysis, and immunofluorescence study revealed the co-localization of HA-tag-like immunoreactivity (LI) and proSP-C-LI (Supplemental Figure S1B and C). These indicate that the transgene-derived MKK6 c.a. is successfully expressed at least in type II alveolar epithelial cells. Male heterozygous TG mice and WT littermates at 10 weeks of age were used for experiments (Supplemental Figure 1D). Animals were housed in standard laboratory
J Recept Signal Transduct Res, 2014; 34(4): 299–306
cages and allowed food and water throughout the experiments. The studies were performed according to a protocol approved by the Committee of Animal Welfare of Chiba University. Reagents LPS (Escherichia coli, serotype) was purchased from SigmaAldrich Co. Cigarette smoke solution (CSS) was purchased from Sugi Institute of Biological Science Co. (Yamanashi, Japan). Experimental protocol of intra-tracheal administration The two administration protocols employed in this study are shown in Figure 1(A) (Protocol A) and Figure 2(A) (Protocol B). In Protocol A, CSS (diluted 4-fold with PBS, 50 ml/body) was intra-tracheally administered to mice once a day on days 0–3, 5–8, 10–13 and 15–18, and LPS (200 mg/ml, 50 ml/body) was administered once a day on days 4, 9 and 14. Mice were sacrificed on day 19. In Protocol B, a mixture of CSS (diluted 4-fold with PBS, 25 ml/body) and LPS (25 mg/ml, 25 ml/body) was intra-tracheally administered to mice once a day on days 0–4 and 7–11, and then CSS was administered to mice once a day on days 14–18. Mice were sacrificed on day 28 after a 10-day period of no treatment. As a negative control, PBS was administered with the same schedule. Intra-tracheal administration was performed using a Micro SprayerÕ aerosolizer (IA-1C; Penn-Century Inc., Philadelphia, PA) attached to a high-pressure syringe (FMJ250; Penn-Century). Mice were anesthetized by intraperitoneal injection of tribromoethanol, and 50 ml of reagent was instilled with the sprayer. After intra-tracheal treatment, mice were kept in an upright position for 1 min to allow sufficient spreading of the fluid throughout the lungs. Histological analyses The trachea was cannulated and the lungs were perfused with paraformaldehyde at a constant pressure of 20 cmH2O. The trachea was clamped and the lungs were removed and fixed overnight at 4 C in 4% paraformaldehyde. After paraffin embedding, sections (4 mm thickness) were subjected to hematoxylin and eosin (HE) staining. A standard morphometric technique was used to determine the presence of emphysematous changes in the lungs. To quantify mean linear intercept (MLI) and destructive index (DI), 10 randomly selected fields in each section were evaluated by microscopic observation. MLI was obtained by dividing the total length of a line drawn across the lung section by the total number of intercepts encountered. DI was measured by manual point counting and calculated with the formula: DI ¼ D/(D + N) 100 (%), where D indicates ‘‘destroyed’’ and N indicates ‘‘normal’’ points according to methods previously described (13). Western blot array analysis Mice with instillation of CSS + LPS according to Protocol B were anesthetized and sacrificed on day 28. Then, the trachea of each mouse was exposed and lavaged with 1 ml ice-cold PBS twice (2 ml total) using a 20-gauge catheter.
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Figure 1. CSS and LPS-induced histopathological changes in the lung. (A) Time schedule of CSS and LPS administration (protocol A). (B) Histopathological findings in the lung in response to CSS–LPS. Arrows and arrowheads represent infiltrated macrophages and neutrophils, respectively.
Collected bronchoalveolar lavage fluid (BALF) from three mice was mixed and concentrated with an Amicon Ultra Centrifugal Filter Device (Millipore, Billerico, MA) to a final volume of 2 ml. The resulting sample was subjected to RayBioÕ Biotin Label-based Mouse Antibody Array 1 (RayBiotech, Inc., Norcross, GA), and changes in expression levels of 308 inflammation-related proteins in the BALF sample were evaluated. The array was performed according to the manufacturer’s instructions. Using a densitometer, each signal was normalized to the positive internal control included in the array membrane and expressed as arbitrary units. Statistical analyses Data are shown as mean ± SE. Statistical analysis of differences between the two groups was performed using Student’s t-test. A probability value of P50.05 was considered to indicate statistical significance.
Results and discussion Establishment of mouse COPD model It has been clearly shown that intra-tracheal administration of LPS and CSS can induce emphysema in the guinea pig in an extremely short time compared with animal models of cigarette smoke exposure (6,7). We therefore applied the protocol of the guinea pig COPD model to mice, with adjustment of reagent volume and concentration according to the weight of each animal, and examined the histopathological changes in the lungs of mice. Protocol A followed in this experiment is shown in Figure 1(A). Typical histopathological findings are shown in Figure 1(B). The lung architecture was
normal in the PBS group. However, the lung tissue exposed to CSS and LPS in an alternating sequential manner showed marked accumulation of inflammatory cells and thickening of the alveolar walls. On the other hand, typical pathologic changes of pulmonary emphysema were rarely observed, and hyperplasia of bronchial epithelial cells observed in patches was mild but not severe. Thus, the histological changes in the lung of mice committed to Protocol A were similar to those of the inflammatory phase of acute lung injury (ALI) (14). In preliminary experiments, we confirmed that the manifestation of an ALI-like profile could be dependent on the concentration or administration timing of LPS. Therefore, we evaluated the method with the following modifications: (A) providing a recovery period after final administration; (B) diluting the LPS concentration; and (C) increasing the frequency of LPS administration. Then, we established an efficient protocol to induce COPD in mice, which is shown as Protocol B (Figure 2A). In the CSS + LPSadministered group, alveolar wall destruction associated with alveolar space enlargement was observed. On the other hand, the lung of mice in the CSS + LPS group did not show typical bronchial epithelial cell hyperplasia and the findings were nearly consistent with the histological profile in the PBS group (Figure 2B). To further assess the emphysematous profile in the lung in the CSS + LPS-administered group, MLI and DI were measured. Both MLI and DI were significantly higher in the CSS + LPS group than in the PBS group (Figure 2C and D). These results statistically support the histopathological findings. LPS is a glycolipid component of gram-negative bacterial cell walls and is used because of its pro-inflammatory effect. In an acute model, LPS induces a mixed inflammatory
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Figure 2. CSS + LPS-induced histopathological changes in the lung. (A) Time schedule of CSS + LPS administration (protocol B). (B) Histopathological findings in the lung in response to CSS + LPS. (C) Mean linear intercept (MLI), as a measure of interalveolar wall distance, was determined by light microscopy. Data represent mean ± SE (n ¼ 7). The difference between the two groups was statistically significant (*p50.05) by Student’s t-test for unpaired values. (D) Destructive index (DI) as a measurement of lung parenchymal destruction. Data represent mean ± SE (n ¼ 7). The difference between the two groups was statistically significant (*p50.05) by Student’s t-test for unpaired values.
reaction with an increase in neutrophils and upregulation of inflammatory cytokines (14). However, with chronic administration over several weeks, LPS administration produces emphysema-like changes in the mouse lung that are associated with apoptosis of structural cells (15). In humans, chronic occupational exposure to LPS contained in organic dusts, such as grain dust and swine dust, is known to result in airflow obstruction in agricultural workers (16,17). These reports suggest that, similarly to cigarette smoking, LPS is a potent pathogenic substance for COPD. At least in inducing emphysematous changes in mice, frequent administration of a relatively low dose of LPS in combination with CSS may be crucial. BALF analysis of mouse COPD model To elucidate the change of microenvironment in the lung showing emphysema, BALF was subjected to WB array. As shown in Figure 3(A), WB array analysis showed that a clear tendency toward an increase in various cytokine/ chemokine levels in BALF from the CSS + LPS group compared with that from the PBS group. In BALF from the
PBS group, no signal was detected except ICK (inhibitor of cyclin-dependent kinase) under our experimental condition. Among the CSS + LPS-induced proteins, molecules reported to be related to COPD are shown in Figure 3(B) (18–25). It is generally believed that cigarette smoke-induced emphysema is caused by an imbalance between protease and antiprotease activity, where an excess of proteolytic enzymes overwhelms the local antiproteolytic defenses, leading to matrix destruction and emphysema (2). At least under our experimental condition, antiproteolytic molecules like tissue inhibitor of metalloproteinases (TIMPs) listed in the array as target proteins were not detected in BALF. In contrast, one of the matrix metalloproteinases (MMPs), MMP-12, was increased in BALF from the CSS + LPS group (Figure 3B). MMP-12 is mainly produced by macrophages and degrades extracellular matrix components, among which elastin is the major constituent of the alveolar wall (22). Targeted disruption of the MMP12 (macrophage metalloelastase) gene protects mice against smoke-induced emphysema (23). In humans, enhanced MMP-12 activity was shown in sputum from patients with COPD in comparison with samples from control subjects, and the number of
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smokers (20). IL-27 is an early product of activated antigenpresenting cells and drives T-cell differentiation (26). Sputum and plasma levels of IL-27 were significantly elevated in COPD patients compared with healthy control subjects. Likewise, the concentration of IL-27 in sputum was negatively correlated with forced expiratory volume in 1 s (FEV1) in COPD patients, suggesting a possible relationship between IL-27 and disease severity (19). Taken together, these results indicate that CSS + LPS-induced MMP-12, KC and IL-27 may in concert contribute to the development of emphysema. Furthermore, IL-16, leptin and TCA-3 have been reported as biomarkers in an animal COPD model or in patients with COPD (18,21,25). Their upregulation in BALF from the CSS + LPS group supports the reliability of our mouse COPD model. Involvement of p38 in pathogenesis of COPD
Figure 3. WB array analysis of BALF from mice treated with CSS + LPS. (A) Typical profiles of protein array for 308 molecules. CSS + LPS-induced expression levels of cytokines/chemokines in BALF. (B) CSS + LPS-upregulated molecules related to progression of COPD. Six molecules were selected, and their signals from two experiments were shown. (C) Densitometric analysis of the molecules shown in B. Closed bars represent molecules that participate in COPD proved experimentally. Open bars represent molecules recognized as biomarkers for COPD. Induction ratio is expressed as arbitrary units relative to the positive internal control.
MMP-12-expressing-macrophages was higher in BAL samples from COPD patients than in those from control subjects (24). These findings strongly suggest that MMP-12 has a crucial role in the structural changes in the lungs of COPD patients, and support that MMP-12 plays a pathophysiological role also in our mouse COPD model. In addition to MMP-12, molecules such as KC and IL-27 that were increased in BALF from the CSS + LPS group (Figure 3B) were reported to be associated with the pathogenesis of COPD (19,20). KC is a mouse homolog of human chemokine CXCL1 and plays an important role in the recruitment and activation of inflammatory cells. In humans, CXCL1 level was significantly increased in sputum from patients with COPD compared with non-smokers and healthy
We established a mouse emphysema model. We next elucidated whether p38 activation in alveoli could contribute to the pathogenesis of COPD. To precisely assess this possibility, MKK6 c.a.-transgenic (TG) mice and WT littermates were used to create a mouse COPD model. As shown in Figure 4(A), the resting lung histological profiles (PBS groups) were consistent between MKK6 c.a.-TG mice and WT littermates, indicating that the SP-C-3HA-tag-MKK6 transgene itself did not induce histological changes in the lung parenchyma. We have confirmed by real-time PCR that MKK6 c.a.-TG mice have two copies of the transgene (Supplemental Figure S2), whose effect is modest and may upregulate the intrinsic activity of p38 but not lead to sufficient p38 activation to drive an inflammatory response. This notion was supported by the facts that detectable probability but not intensity of phospho-p38-LI in type II alveolar epithelial cells increased in MKK6 c.a.-TG mice compared with WT littermates (Supplemental Figure 1E). On the other hand, alveolar wall destruction and space enlargement induced by CSS + LPS were observed in both MKK6 c.a.-TG mice and WT littermates, the severity of which was advanced in MKK6 c.a.-TG mice compared with WT littermates (Figure 4A). The hyperplasic changes in bronchial epithelial cells were not typical and were consistent between the two genotypes (data not shown). In accordance with the histopathological findings, both MLI and DI were significantly higher in MKK6 c.a.-TG mice than in WT littermates (Figure 4B and C). These results clearly suggest that p38 activation in the alveoli can cause deterioration of emphysema induced by CSS + LPS. To further examine whether some microenvironmental changes in the alveoli are related to the deterioration of emphysema in MKK6 c.a.-TG mice, WB array analysis was performed. BALF showed an increase in 72/308 and 15/308 proteins in MKK6 c.a.-TG mice and WT littermates, respectively, indicating that MKK6 c.a.-TG mice show high sensitivity to CSS + LPS application compared with WT littermates. Molecules reported to be related to COPD and induction of its indices are shown in Table 1 (18–25, 27–31). An increase in COPD-related molecules such as IL-16, IL-27, KC, leptin, MMP-12 and TCA-3 was observed in both MKK6 c.a.-TG mice and WT littermates. Most of them were
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Figure 4. Enhancement of emphysematous changes induced by CSS + LPS in MKK6 c.a.-TG mice. (A) Histopathological findings in the lung in response to CSS + LPS in MKK6 c.a.-TG mice and WT littermates. (B) MLI, an index of enlargement of alveolar air spaces. Data represent mean ± SE (n ¼ 7). The difference between the two groups was statistically significant (*p50.05) by Student’s t-test for unpaired values. (C) DI, a measurement of lung parenchymal destruction. Data represent mean ± SE (n ¼ 7). The difference between the two groups was statistically significant (*p50.05) by Student’s t-test for unpaired values. Table 1. Increase of COPD-related molecules in BALF from two genotypes of mouse. Induction index (Arbitrary unit)
Category
Protein name
WT
MKK6 c.a.
Function
IL-16 IL-27 KC Leptin MMP-12 TCA-3 Adiponectin CRP I-TAC Prolactin uPAR
6 9 6 4 4 7 () () () () ()
9.5 9 9 6 19 10 4 3 8 6 3
Cytokine Cytokine Chemokine Adipokine Protease Chemokine Adipokine Acute phase protein Chemokine Hormone Receptor
A
B *
* * * * * * * * * *
Category A: participation in COPD has been proved experimentally. Category B: biomarker for COPD (recognized in animal models and patients). *, means that each protein satisfies alternative category.
further upregulated in MKK6 c.a.-TG mice compared with WT littermates. In addition, an increase in expression of adiponectin, CRP (C-reactive protein), I-TAC (interferoninducible T cell a chemoattractant), prolactin and uPAR (urokinase-type plasminogen activator receptor) was observed specifically in BALF from MKK6 c.a.-TG mice. Adiponectin has been reported to be involved in the pathogenesis of COPD as follows: adiponectin stimulates airway epithelial cells in an autocrine/paracrine manner to produce IL-8 in COPD patients, and plasma adiponectin level is increased in COPD patients (27,28). Although the correlation between p38 activity and expression of adiponectin has never been
shown, our present data suggest that p38 mediates an increase in adiponectin expression. The p38/adiponectin/IL-8 axis is of interest in the pathogenesis of COPD. Previous reports have demonstrated that both human and mouse adiponectin promoter regions involve the cAMP-responsive element (CRE) and that the CRE functions as a positive regulator of the mouse adiponectin gene transcription (32,33). Moreover, we have demonstrated that p38 could activate the CRE in the proximal promoter region of IL17 gene through phosphorylation of CREB and ATF2 (9). These facts tempt us to consider that the CRE may play a crucial role in the p38-induced adiponectin expression. In addition to adiponectin, CRP, I-TAC, prolactin and uPAR are recognized as biomarkers of COPD (29–31). Taken together, these findings indicate that alveolar epithelial cells with high intrinsic p38 activity may be committed to efficiently produce pathophysiological substances leading to emphysema in response to CSS + LPS. Notably, the expression level of MMP-12 in BALF from MKK6 c.a.-TG mice was nearly five times higher than that in WT littermates. In chondrocytes, a possible relationship between p38 signaling and MMP-12 expression is suggested (34). Although a precise mechanism for p38-regulated MMP12 expression in the lung is still unknown, p38 can contribute to the development of emphysema at least through MMP-12 induction. Likewise, expression of TIMPs, antiproteolytic molecules, was not detected in BALF from either MKK6 c.a.TG mice or WT littermates. Thus, protease-antiprotease imbalance may be more enhanced in the bronchoalveolar space of MKK6 c.a.-TG mice with CSS + LPS instillation compared with WT littermates. The p38 signaling pathway is activated by a variety of extracellular stimuli including osmotic shock,
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inflammatory cytokines, and toll like receptor (TLR) agonist. Once activated, p38 phosphorylates a variety of substrates such as other downstream kinases and transcription factors, and plays a central role in inflammatory processes. p38 inhibitors efficiently reduce cytokine production from various types of cells including alveolar macrophages (8,35). By virtue of the anti-inflammatory potency of p38 inhibitors, several p38 inhibitors are in clinical trials for the treatment of COPD (36,37). However, little is known about the precise mechanism by which p38 mediates the pathogenesis of COPD. In this study, we clearly demonstrated that p38 in type II alveolar epithelial cells positively contributes to the pathogenesis of emphysema.
Conclusions We have established a mouse model of COPD that can be produced in a relatively short period of time. Furthermore, p38 in alveolar epithelial cells can play a crucial role as a driving force for emphysematous changes in our mouse COPD model. This mouse model is expected to be a useful tool for elucidating the pathogenesis of COPD and drug development.
Acknowledgements The human SPC/SV40 vector was kindly provided by Dr. Jeffrey A. Whitsett (Children’s Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio). We thank Dr. Wendy Gray for editing our manuscript.
Declaration of interest The authors declare no conflict of interest. This work was supported in part by Grants-in-Aid for Scientific Research [(B), 24390137 to Y.K.] and for Challenging Exploratory Research (25670256 to Y.K.), by the Hamaguchi Foundation for the Advancement of Biochemistry (to Y.K.), and by the Takeda Science Foundation for Visionary Research (to Y.K.).
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11. Okuda R, Hagiwara E, Baba T, et al. Safety and efficacy of pirfenidone in idiopathic pulmonary fibrosis in clinical practice. Respir Med 2013;107:1431–7. 12. Renda T, Baraldo S, Pelaia G, et al. Increased activation of p38 MAPK in COPD. Eur Respir J 2008;31:62–9. 13. Saetta M, Shiner RJ, Angus GE, et al. Destructive index: a measurement of lung parenchymal destruction in smokers. Am Rev Respir Dis 1985;131:764–9. 14. Ishizaki S, Kasuya Y, Kuroda F, et al. Role of CD69 in acute lung injury. Life Sci 2012;90:657–65. 15. Brass DM, Hollingsworth JW, Cinque M, et al. Chronic LPS inhalation causes emphysema-like changes in mouse lung that are associated with apoptosis. Am J Respir Cell Mol Biol 2008;39: 584–90. 16. Schwartz DA, Thorne PS, Yagla SJ, et al. The role of endotoxin in grain dust-induced lung disease. Am J Respir Crit Care Med 1995;152:603–8. 17. Vohlonen I, Tupi K, Terho EO, et al. Prevalence and incidence of chronic bronchitis and farmer’s lung with respect to the geographical location of the farm and to the work of farmers. Eur J Respir Dis Suppl 1987;152:37–46. 18. Andersson A, Qvarfordt I, Laan M, et al. Impact of tobacco smoke on interleukin-16 protein in human airways, lymphoid tissue and T lymphocytes. Clin Exp Immunol 2004; 138:75–82. 19. Cao J, Zhang L, Li D, et al. IL-27 is elevated in patients with COPD and patients with pulmonary TB and induces human bronchial epithelial cells to produce CXCL10. Chest 2012;141: 121–30. 20. Traves SL. Increased levels of the chemokines GROalpha and MCP-1 in sputum samples from patients with COPD. Thorax 2002; 57:590–5. 21. Broekhuizen R, Vernooy JHJ, Schols AMWJ, et al. Leptin as local inflammatory marker in COPD. Respir Med 2005;99: 70–4. 22. Hunninghake GM, Cho MH, Tesfaigzi Y, et al. MMP12, lung function, and COPD in high-risk populations. N Engl J Med 2009; 361:2599–608. 23. Hautamaki RD. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997;277: 2002–4. 24. Demedts IK, Morel-Montero A, Lebecque S, et al. Elevated MMP12 protein levels in induced sputum from patients with COPD. Thorax 2006;61:196–201. 25. Ying S, O’Connor B, Ratoff J, et al. Expression and cellular provenance of thymic stromal lymphopoietin and chemokines in patients with severe asthma and chronic obstructive pulmonary disease. J Immunol 2008;181:2790–8. 26. Villarino AV, Huang E, Hunter CA. Understanding the pro- and anti-inflammatory properties of IL-27. J Immunol 2004;173: 715–20. 27. Miller M, Cho JY, Pham A, et al. Adiponectin and functional adiponectin receptor 1 are expressed by airway epithelial cells in chronic obstructive pulmonary disease. J Immunol 2009;182: 684–91. 28. Tomoda K, Yoshikawa M, Itoh T, et al. Elevated circulating plasma adiponectin in underweight patients with COPD. Chest 2007;132: 135–40. 29. Dahl M, Vestbo J, Lange P, et al. C-reactive protein as a predictor of prognosis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2007;175:250–5. 30. Pinto-Plata V, Toso J, Lee K, et al. Profiling serum biomarkers in patients with COPD: associations with clinical parameters. Thorax 2007;62:595–601. 31. Xiao W, Hsu Y-P, Ishizaka A, et al. Sputum cathelicidin, urokinase plasminogen activation system components, and cytokines discriminate cystic fibrosis, COPD, and asthma inflammation. Chest 2005; 128:2316–26. 32. Laumen H, Saningong AD, Heid IM, et al. Functional characterization of promoter variants of the adiponectin gene complemented by epidemiological data. Diabetes 2009;58:984–91. 33. Kim HB, Kim WH, Han KL, et al. cAMP-response element binding protein (CREB) positively regulates mouse adiponectin gene expression in 3T3-L1 adipocytes. Biochem Biophys Res Commun 2010;391:634–9.
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36. Singh D, Smyth L, Borrill Z, et al. A randomized, placebocontrolled study of the effects of the p38 MAPK inhibitor SB-681323 on blood biomarkers of inflammation in COPD patients. J Clin Pharmacol 2010;50:94–100. 37. Singh D. p38 inhibition in COPD; cautious optimism. Thorax 2013; 68:705–6.
RESEARCH ARTICLE
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p38 Mitogen-activated protein kinase accelerates emphysema in mouse model of chronic obstructive pulmonary disease Hiroyuki Amano1,2, Kazuya Murata3, Hirofumi Matsunaga1,4, Kensuke Tanaka1,2, Kento Yoshioka1, Takeshi Kobayashi1,2, Junji Ishida3, Akiyoshi Fukamizu3, Fumihiro Sugiyama5, Tatsuhiko Sudo6, Sadao Kimura1, Koichiro Tatsumi2, and Yoshitoshi Kasuya1 1
Department of Biochemistry and Molecular Pharmacology, 2Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan, 3Life Science Center, Tsukuba Advanced Research Alliance, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennoudai, Ibaraki, Japan, 4Pharmaceuticals Research Laboratory, Ube Industries, Ltd., Ube, Japan, 5Laboratory Animal Resource Center, University of Tsukuba, Tennoudai, Ibaraki, Japan, and 6Chemical Biology Core Facility and Antibiotics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan Abstract
Keywords
Context: There are few short-term mouse models of chronic obstructive pulmonary disease (COPD) mimicking the human disease. In addition, p38 is recently recognized as a target for the treatment of COPD. However, the precise mechanism how p38 contributes to the pathogenesis of COPD is still unknown. Objective: We attempted to create a new mouse model for COPD by intra-tracheal administration of a mixture of lipopolysaccharide (LPS) and cigarette smoke solution (CSS), and investigated the importance of the p38 mitogen-activated protein kinase (p38) pathway in the pathogenesis of COPD. Methods: Mice were administered LPS + CSS once a day on days 0–4 and 7–11. Thereafter, CSS alone was administered to mice once a day on days 14–18. On day 28, histopathological changes of the lung were evaluated, and bronchoalveolar lavage fluid (BALF) was subjected to western blot array for cytokines. Transgenic (TG) mice expressing a constitutive-active form of MKK6, a p38-specific activator in the lung, were subjected to our experimental protocol of COPD model. Results: LPS + CSS administration induced enlargement of alveolar air spaces and destruction of lung parenchyma. BALF analyses of the LPS + CSS group revealed an increase in expression levels of several cytokines involved in the pathogenesis of human COPD. These results suggest that our experimental protocol can induce COPD in mice. Likewise, histopathological findings of the lung and induction of cytokines in BALF from MKK6 c.a.-TG mice were more marked than those in WT mice. Conclusion: In a new experimental COPD mouse model, p38 accelerates the development of emphysema.
Cigarette smoke, COPD mouse model, lipopolysaccharide, MKK6 c.a., transgenic mouse
Introduction Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease characterized by airflow limitation that is not fully reversible. Cigarette smoking has been generally accepted as the most important risk factor for the development of COPD. However, only 15–20% of heavy smokers develop clinically significant airflow obstruction (1), which suggests that a genetic susceptibility to the effects of cigarette smoking. Several pathophysiologic processes interact on a complex background of genetic and environmental factors, but the detailed course of the disease is not fully elucidated.
Address for correspondence: Yoshitoshi Kasuya, Ph.D., Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Tel: +81-43-226-2193. Fax: +81-43-226-2196. E-mail: kasuya@ faculty.chiba-u.jp
History Received 23 November 2013 Revised 30 January 2014 Accepted 17 February 2014 Published online 5 March 2014
Based on prevalence surveys, it is estimated that 10% of adults aged 40 years and older have COPD (2). COPD was ranked sixth in the causes of death in 1990, and will become the third leading cause of death by 2030 (3). These data suggest that COPD will become a major health problem in the next few decades. The mainstay of current therapy for COPD is inhaled bronchodilators. There is a pressing need to develop new treatments for COPD, because most studies have indicated that the existing medications for COPD do not modify the long-term decline in lung function (4). The use of appropriate experimental COPD animal models is indispensable for the development of new therapy and for better understanding of the disease. A variety of different approaches have been used to attempt to model COPD in animals (5). Administration of proteinases, chemicals and particulates, cigarette smoke exposure and genetic manipulation have successfully produced features
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characteristic of human COPD. Although the cause of the disease tempts us to consider a smoke exposure model as the most reasonable model, it takes more than 3 months to induce emphysema (6). In order to shorten the period, Mizutani et al. (7) employed intra-tracheal administration of cigarette smoke solution (CSS) and lipopolysaccharide (LPS) and created a guinea pig COPD model that requires only 19 days, indicating the possibility that this protocol could enable us to investigate the pathogenesis of COPD more easily. The p38 mitogen-activated protein kinase (p38) signaling pathway is activated by a variety of extracellular stimuli including cellular stress, inflammatory cytokines and cell surface receptors, and plays a role in a wide range of cellular responses, such as proliferation, differentiation and apoptosis (8). In particular, the notion that p38 functions as a central player in inflammatory diseases has been supported by various reports including our studies (9,10). Also, in lung inflammatory diseases, p38 is recognized as a potential therapeutic target. Indeed, pirfenidone, a p38g inhibitor, is used for the treatment of patients with idiopathic pulmonary fibrosis (11). Likewise, it has been reported that p38 is activated in the alveolar macrophages of COPD patients compared to healthy controls (12), suggesting that p38 might be a plausible target for the treatment of COPD. Here, we first investigated whether the protocol for the guinea pig COPD model could be applied to mice. Then, we established a new experimental COPD mouse model. We next investigated the participation of p38 in the pathogenesis of COPD, using MKK6 c.a.-TG mice in which the MKK6 constitutive-active gene is introduced under control of the human surfactant protein C (SP-C) gene promoter.
Materials and methods Animals Male C57BL/6J mice at 10 weeks of age were purchased from Clea Japan (Tokyo, Japan). To generate MKK6 constitutiveactive (c.a., dual mutations in wild human MKK6: Ser207 to Asp; Tyr211 to Asp) TG mice, the linear 5.3 k-bp hSPC3HA-tag-hMKK6 c.a. NarI/NotI-fragment (Supplemental Figure S1A) was microinjected in eggs from C57BL/6J mice, which were subsequently transplanted into the uteli of pseudopregnant female ICR mice. For construction of the transgene, the 3.7SPC/SV40 vector was provided by Dr. Jeffrey A. Whitsett, Children’s Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio. Although we obtained five lines of TG F1 mice, the four TG lines with high expression level of transgene-derived MKK6 c.a. showed low fertility rate. As a result, only one line of MKK6 c.a.-TG mice could be maintained. In the lung of MKK6 c.a.-TG mice, the expression of HA-tag+/41-KDa protein corresponding to the predicted size of 3HA-tag-MKK6 c.a. was confirmed by western blot (WB) analysis, and immunofluorescence study revealed the co-localization of HA-tag-like immunoreactivity (LI) and proSP-C-LI (Supplemental Figure S1B and C). These indicate that the transgene-derived MKK6 c.a. is successfully expressed at least in type II alveolar epithelial cells. Male heterozygous TG mice and WT littermates at 10 weeks of age were used for experiments (Supplemental Figure 1D). Animals were housed in standard laboratory
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cages and allowed food and water throughout the experiments. The studies were performed according to a protocol approved by the Committee of Animal Welfare of Chiba University. Reagents LPS (Escherichia coli, serotype) was purchased from SigmaAldrich Co. Cigarette smoke solution (CSS) was purchased from Sugi Institute of Biological Science Co. (Yamanashi, Japan). Experimental protocol of intra-tracheal administration The two administration protocols employed in this study are shown in Figure 1(A) (Protocol A) and Figure 2(A) (Protocol B). In Protocol A, CSS (diluted 4-fold with PBS, 50 ml/body) was intra-tracheally administered to mice once a day on days 0–3, 5–8, 10–13 and 15–18, and LPS (200 mg/ml, 50 ml/body) was administered once a day on days 4, 9 and 14. Mice were sacrificed on day 19. In Protocol B, a mixture of CSS (diluted 4-fold with PBS, 25 ml/body) and LPS (25 mg/ml, 25 ml/body) was intra-tracheally administered to mice once a day on days 0–4 and 7–11, and then CSS was administered to mice once a day on days 14–18. Mice were sacrificed on day 28 after a 10-day period of no treatment. As a negative control, PBS was administered with the same schedule. Intra-tracheal administration was performed using a Micro SprayerÕ aerosolizer (IA-1C; Penn-Century Inc., Philadelphia, PA) attached to a high-pressure syringe (FMJ250; Penn-Century). Mice were anesthetized by intraperitoneal injection of tribromoethanol, and 50 ml of reagent was instilled with the sprayer. After intra-tracheal treatment, mice were kept in an upright position for 1 min to allow sufficient spreading of the fluid throughout the lungs. Histological analyses The trachea was cannulated and the lungs were perfused with paraformaldehyde at a constant pressure of 20 cmH2O. The trachea was clamped and the lungs were removed and fixed overnight at 4 C in 4% paraformaldehyde. After paraffin embedding, sections (4 mm thickness) were subjected to hematoxylin and eosin (HE) staining. A standard morphometric technique was used to determine the presence of emphysematous changes in the lungs. To quantify mean linear intercept (MLI) and destructive index (DI), 10 randomly selected fields in each section were evaluated by microscopic observation. MLI was obtained by dividing the total length of a line drawn across the lung section by the total number of intercepts encountered. DI was measured by manual point counting and calculated with the formula: DI ¼ D/(D + N) 100 (%), where D indicates ‘‘destroyed’’ and N indicates ‘‘normal’’ points according to methods previously described (13). Western blot array analysis Mice with instillation of CSS + LPS according to Protocol B were anesthetized and sacrificed on day 28. Then, the trachea of each mouse was exposed and lavaged with 1 ml ice-cold PBS twice (2 ml total) using a 20-gauge catheter.
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Figure 1. CSS and LPS-induced histopathological changes in the lung. (A) Time schedule of CSS and LPS administration (protocol A). (B) Histopathological findings in the lung in response to CSS–LPS. Arrows and arrowheads represent infiltrated macrophages and neutrophils, respectively.
Collected bronchoalveolar lavage fluid (BALF) from three mice was mixed and concentrated with an Amicon Ultra Centrifugal Filter Device (Millipore, Billerico, MA) to a final volume of 2 ml. The resulting sample was subjected to RayBioÕ Biotin Label-based Mouse Antibody Array 1 (RayBiotech, Inc., Norcross, GA), and changes in expression levels of 308 inflammation-related proteins in the BALF sample were evaluated. The array was performed according to the manufacturer’s instructions. Using a densitometer, each signal was normalized to the positive internal control included in the array membrane and expressed as arbitrary units. Statistical analyses Data are shown as mean ± SE. Statistical analysis of differences between the two groups was performed using Student’s t-test. A probability value of P50.05 was considered to indicate statistical significance.
Results and discussion Establishment of mouse COPD model It has been clearly shown that intra-tracheal administration of LPS and CSS can induce emphysema in the guinea pig in an extremely short time compared with animal models of cigarette smoke exposure (6,7). We therefore applied the protocol of the guinea pig COPD model to mice, with adjustment of reagent volume and concentration according to the weight of each animal, and examined the histopathological changes in the lungs of mice. Protocol A followed in this experiment is shown in Figure 1(A). Typical histopathological findings are shown in Figure 1(B). The lung architecture was
normal in the PBS group. However, the lung tissue exposed to CSS and LPS in an alternating sequential manner showed marked accumulation of inflammatory cells and thickening of the alveolar walls. On the other hand, typical pathologic changes of pulmonary emphysema were rarely observed, and hyperplasia of bronchial epithelial cells observed in patches was mild but not severe. Thus, the histological changes in the lung of mice committed to Protocol A were similar to those of the inflammatory phase of acute lung injury (ALI) (14). In preliminary experiments, we confirmed that the manifestation of an ALI-like profile could be dependent on the concentration or administration timing of LPS. Therefore, we evaluated the method with the following modifications: (A) providing a recovery period after final administration; (B) diluting the LPS concentration; and (C) increasing the frequency of LPS administration. Then, we established an efficient protocol to induce COPD in mice, which is shown as Protocol B (Figure 2A). In the CSS + LPSadministered group, alveolar wall destruction associated with alveolar space enlargement was observed. On the other hand, the lung of mice in the CSS + LPS group did not show typical bronchial epithelial cell hyperplasia and the findings were nearly consistent with the histological profile in the PBS group (Figure 2B). To further assess the emphysematous profile in the lung in the CSS + LPS-administered group, MLI and DI were measured. Both MLI and DI were significantly higher in the CSS + LPS group than in the PBS group (Figure 2C and D). These results statistically support the histopathological findings. LPS is a glycolipid component of gram-negative bacterial cell walls and is used because of its pro-inflammatory effect. In an acute model, LPS induces a mixed inflammatory
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Figure 2. CSS + LPS-induced histopathological changes in the lung. (A) Time schedule of CSS + LPS administration (protocol B). (B) Histopathological findings in the lung in response to CSS + LPS. (C) Mean linear intercept (MLI), as a measure of interalveolar wall distance, was determined by light microscopy. Data represent mean ± SE (n ¼ 7). The difference between the two groups was statistically significant (*p50.05) by Student’s t-test for unpaired values. (D) Destructive index (DI) as a measurement of lung parenchymal destruction. Data represent mean ± SE (n ¼ 7). The difference between the two groups was statistically significant (*p50.05) by Student’s t-test for unpaired values.
reaction with an increase in neutrophils and upregulation of inflammatory cytokines (14). However, with chronic administration over several weeks, LPS administration produces emphysema-like changes in the mouse lung that are associated with apoptosis of structural cells (15). In humans, chronic occupational exposure to LPS contained in organic dusts, such as grain dust and swine dust, is known to result in airflow obstruction in agricultural workers (16,17). These reports suggest that, similarly to cigarette smoking, LPS is a potent pathogenic substance for COPD. At least in inducing emphysematous changes in mice, frequent administration of a relatively low dose of LPS in combination with CSS may be crucial. BALF analysis of mouse COPD model To elucidate the change of microenvironment in the lung showing emphysema, BALF was subjected to WB array. As shown in Figure 3(A), WB array analysis showed that a clear tendency toward an increase in various cytokine/ chemokine levels in BALF from the CSS + LPS group compared with that from the PBS group. In BALF from the
PBS group, no signal was detected except ICK (inhibitor of cyclin-dependent kinase) under our experimental condition. Among the CSS + LPS-induced proteins, molecules reported to be related to COPD are shown in Figure 3(B) (18–25). It is generally believed that cigarette smoke-induced emphysema is caused by an imbalance between protease and antiprotease activity, where an excess of proteolytic enzymes overwhelms the local antiproteolytic defenses, leading to matrix destruction and emphysema (2). At least under our experimental condition, antiproteolytic molecules like tissue inhibitor of metalloproteinases (TIMPs) listed in the array as target proteins were not detected in BALF. In contrast, one of the matrix metalloproteinases (MMPs), MMP-12, was increased in BALF from the CSS + LPS group (Figure 3B). MMP-12 is mainly produced by macrophages and degrades extracellular matrix components, among which elastin is the major constituent of the alveolar wall (22). Targeted disruption of the MMP12 (macrophage metalloelastase) gene protects mice against smoke-induced emphysema (23). In humans, enhanced MMP-12 activity was shown in sputum from patients with COPD in comparison with samples from control subjects, and the number of
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smokers (20). IL-27 is an early product of activated antigenpresenting cells and drives T-cell differentiation (26). Sputum and plasma levels of IL-27 were significantly elevated in COPD patients compared with healthy control subjects. Likewise, the concentration of IL-27 in sputum was negatively correlated with forced expiratory volume in 1 s (FEV1) in COPD patients, suggesting a possible relationship between IL-27 and disease severity (19). Taken together, these results indicate that CSS + LPS-induced MMP-12, KC and IL-27 may in concert contribute to the development of emphysema. Furthermore, IL-16, leptin and TCA-3 have been reported as biomarkers in an animal COPD model or in patients with COPD (18,21,25). Their upregulation in BALF from the CSS + LPS group supports the reliability of our mouse COPD model. Involvement of p38 in pathogenesis of COPD
Figure 3. WB array analysis of BALF from mice treated with CSS + LPS. (A) Typical profiles of protein array for 308 molecules. CSS + LPS-induced expression levels of cytokines/chemokines in BALF. (B) CSS + LPS-upregulated molecules related to progression of COPD. Six molecules were selected, and their signals from two experiments were shown. (C) Densitometric analysis of the molecules shown in B. Closed bars represent molecules that participate in COPD proved experimentally. Open bars represent molecules recognized as biomarkers for COPD. Induction ratio is expressed as arbitrary units relative to the positive internal control.
MMP-12-expressing-macrophages was higher in BAL samples from COPD patients than in those from control subjects (24). These findings strongly suggest that MMP-12 has a crucial role in the structural changes in the lungs of COPD patients, and support that MMP-12 plays a pathophysiological role also in our mouse COPD model. In addition to MMP-12, molecules such as KC and IL-27 that were increased in BALF from the CSS + LPS group (Figure 3B) were reported to be associated with the pathogenesis of COPD (19,20). KC is a mouse homolog of human chemokine CXCL1 and plays an important role in the recruitment and activation of inflammatory cells. In humans, CXCL1 level was significantly increased in sputum from patients with COPD compared with non-smokers and healthy
We established a mouse emphysema model. We next elucidated whether p38 activation in alveoli could contribute to the pathogenesis of COPD. To precisely assess this possibility, MKK6 c.a.-transgenic (TG) mice and WT littermates were used to create a mouse COPD model. As shown in Figure 4(A), the resting lung histological profiles (PBS groups) were consistent between MKK6 c.a.-TG mice and WT littermates, indicating that the SP-C-3HA-tag-MKK6 transgene itself did not induce histological changes in the lung parenchyma. We have confirmed by real-time PCR that MKK6 c.a.-TG mice have two copies of the transgene (Supplemental Figure S2), whose effect is modest and may upregulate the intrinsic activity of p38 but not lead to sufficient p38 activation to drive an inflammatory response. This notion was supported by the facts that detectable probability but not intensity of phospho-p38-LI in type II alveolar epithelial cells increased in MKK6 c.a.-TG mice compared with WT littermates (Supplemental Figure 1E). On the other hand, alveolar wall destruction and space enlargement induced by CSS + LPS were observed in both MKK6 c.a.-TG mice and WT littermates, the severity of which was advanced in MKK6 c.a.-TG mice compared with WT littermates (Figure 4A). The hyperplasic changes in bronchial epithelial cells were not typical and were consistent between the two genotypes (data not shown). In accordance with the histopathological findings, both MLI and DI were significantly higher in MKK6 c.a.-TG mice than in WT littermates (Figure 4B and C). These results clearly suggest that p38 activation in the alveoli can cause deterioration of emphysema induced by CSS + LPS. To further examine whether some microenvironmental changes in the alveoli are related to the deterioration of emphysema in MKK6 c.a.-TG mice, WB array analysis was performed. BALF showed an increase in 72/308 and 15/308 proteins in MKK6 c.a.-TG mice and WT littermates, respectively, indicating that MKK6 c.a.-TG mice show high sensitivity to CSS + LPS application compared with WT littermates. Molecules reported to be related to COPD and induction of its indices are shown in Table 1 (18–25, 27–31). An increase in COPD-related molecules such as IL-16, IL-27, KC, leptin, MMP-12 and TCA-3 was observed in both MKK6 c.a.-TG mice and WT littermates. Most of them were
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Figure 4. Enhancement of emphysematous changes induced by CSS + LPS in MKK6 c.a.-TG mice. (A) Histopathological findings in the lung in response to CSS + LPS in MKK6 c.a.-TG mice and WT littermates. (B) MLI, an index of enlargement of alveolar air spaces. Data represent mean ± SE (n ¼ 7). The difference between the two groups was statistically significant (*p50.05) by Student’s t-test for unpaired values. (C) DI, a measurement of lung parenchymal destruction. Data represent mean ± SE (n ¼ 7). The difference between the two groups was statistically significant (*p50.05) by Student’s t-test for unpaired values. Table 1. Increase of COPD-related molecules in BALF from two genotypes of mouse. Induction index (Arbitrary unit)
Category
Protein name
WT
MKK6 c.a.
Function
IL-16 IL-27 KC Leptin MMP-12 TCA-3 Adiponectin CRP I-TAC Prolactin uPAR
6 9 6 4 4 7 () () () () ()
9.5 9 9 6 19 10 4 3 8 6 3
Cytokine Cytokine Chemokine Adipokine Protease Chemokine Adipokine Acute phase protein Chemokine Hormone Receptor
A
B *
* * * * * * * * * *
Category A: participation in COPD has been proved experimentally. Category B: biomarker for COPD (recognized in animal models and patients). *, means that each protein satisfies alternative category.
further upregulated in MKK6 c.a.-TG mice compared with WT littermates. In addition, an increase in expression of adiponectin, CRP (C-reactive protein), I-TAC (interferoninducible T cell a chemoattractant), prolactin and uPAR (urokinase-type plasminogen activator receptor) was observed specifically in BALF from MKK6 c.a.-TG mice. Adiponectin has been reported to be involved in the pathogenesis of COPD as follows: adiponectin stimulates airway epithelial cells in an autocrine/paracrine manner to produce IL-8 in COPD patients, and plasma adiponectin level is increased in COPD patients (27,28). Although the correlation between p38 activity and expression of adiponectin has never been
shown, our present data suggest that p38 mediates an increase in adiponectin expression. The p38/adiponectin/IL-8 axis is of interest in the pathogenesis of COPD. Previous reports have demonstrated that both human and mouse adiponectin promoter regions involve the cAMP-responsive element (CRE) and that the CRE functions as a positive regulator of the mouse adiponectin gene transcription (32,33). Moreover, we have demonstrated that p38 could activate the CRE in the proximal promoter region of IL17 gene through phosphorylation of CREB and ATF2 (9). These facts tempt us to consider that the CRE may play a crucial role in the p38-induced adiponectin expression. In addition to adiponectin, CRP, I-TAC, prolactin and uPAR are recognized as biomarkers of COPD (29–31). Taken together, these findings indicate that alveolar epithelial cells with high intrinsic p38 activity may be committed to efficiently produce pathophysiological substances leading to emphysema in response to CSS + LPS. Notably, the expression level of MMP-12 in BALF from MKK6 c.a.-TG mice was nearly five times higher than that in WT littermates. In chondrocytes, a possible relationship between p38 signaling and MMP-12 expression is suggested (34). Although a precise mechanism for p38-regulated MMP12 expression in the lung is still unknown, p38 can contribute to the development of emphysema at least through MMP-12 induction. Likewise, expression of TIMPs, antiproteolytic molecules, was not detected in BALF from either MKK6 c.a.TG mice or WT littermates. Thus, protease-antiprotease imbalance may be more enhanced in the bronchoalveolar space of MKK6 c.a.-TG mice with CSS + LPS instillation compared with WT littermates. The p38 signaling pathway is activated by a variety of extracellular stimuli including osmotic shock,
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inflammatory cytokines, and toll like receptor (TLR) agonist. Once activated, p38 phosphorylates a variety of substrates such as other downstream kinases and transcription factors, and plays a central role in inflammatory processes. p38 inhibitors efficiently reduce cytokine production from various types of cells including alveolar macrophages (8,35). By virtue of the anti-inflammatory potency of p38 inhibitors, several p38 inhibitors are in clinical trials for the treatment of COPD (36,37). However, little is known about the precise mechanism by which p38 mediates the pathogenesis of COPD. In this study, we clearly demonstrated that p38 in type II alveolar epithelial cells positively contributes to the pathogenesis of emphysema.
Conclusions We have established a mouse model of COPD that can be produced in a relatively short period of time. Furthermore, p38 in alveolar epithelial cells can play a crucial role as a driving force for emphysematous changes in our mouse COPD model. This mouse model is expected to be a useful tool for elucidating the pathogenesis of COPD and drug development.
Acknowledgements The human SPC/SV40 vector was kindly provided by Dr. Jeffrey A. Whitsett (Children’s Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio). We thank Dr. Wendy Gray for editing our manuscript.
Declaration of interest The authors declare no conflict of interest. This work was supported in part by Grants-in-Aid for Scientific Research [(B), 24390137 to Y.K.] and for Challenging Exploratory Research (25670256 to Y.K.), by the Hamaguchi Foundation for the Advancement of Biochemistry (to Y.K.), and by the Takeda Science Foundation for Visionary Research (to Y.K.).
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