Experimental Gerontology 57 (2014) 29–40

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Age-related activation of MKK/p38/NF-κB signaling pathway in lung: From mouse to human Xiaoxia Ren a,b, Huadong Du c, Yan Li a,b, Xiujuan Yao a,b, Junmin Huang a,b, Zongli Li a,b, Wei Wang d, Junfa Li e, Song Han e, Chen Wang a,b, Kewu Huang a,b,⁎ a Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Department of Pulmonary and Critical Care Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, PR China b Beijing Institute of Respiratory Medicine, Beijing 100020, PR China c Department of Thoracic Surgery, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, PR China d Department of Immunology, Capital Medical University, Beijing 100069, PR China e Department of Neurobiology, Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069, PR China

a r t i c l e

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Article history: Received 22 December 2013 Received in revised form 27 April 2014 Accepted 29 April 2014 Available online 5 May 2014 Section Editor: B. Grubeck-Loebenstein Keywords: P38 Lung Inflammaging Lipopolysaccharide Cytokines

a b s t r a c t We and others previously reported that the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 significantly accumulate with age in mouse lung. This is accompanied by elevated phosphorylation of p38. Here, we further investigate whether aging affects activation of p38 signaling and the inflammatory reaction after exposure to lipopolysaccharide (LPS) in the lungs of mice in vivo and humans ex vivo. The data showed that activation of p38 peaked at 0.5 h and then rapidly declined in young (2-month-old) mouse lung, after intranasal inhalation challenge with LPS. In contract, activation of p38 peaked at 24 h and was sustained longer in aged (20-month-old) mice. As well as altered p38, activations of its upstream activator MKK and downstream substrate NF-κB were also changed in the lungs of aged mice, which corresponded with the absence in the early phase but delayed increases in concentrations of TNF-α, IL-1β and IL-6. Consistent with the above observations in mice, similar patterns of p38 signaling also occurred in human lungs. Compared with younger lungs from adult–middle aged subjects, the activation of p38, MKK and NF-κB, as well as the production of pro-inflammatory cytokines were significantly increased in the lungs of older subjects ex vivo. Exposure of human lung cells to LPS induced rapid activation of p38, MKK and NF-κB in these cells from adult–middle aged subjects, but not older subjects, with increases in the production of the pro-inflammatory cytokines. The LPS-induced rapid activation in the lung cells from adult–middle aged subjects occurred as early as 0.25 h after exposure, and then declined. Compared with adult–middle aged subjects, the LPS exposure did not induce marked changes in the early phase, either in the activation of p38, MKK and NF-κB, or in the production of TNF-α, IL-1β or IL-6 in the lung cells from older subjects. In contrast, these changes occurred relatively late, peaked at 16 h and were sustained longer in the lungs of older subjects. These data support the hypothesis that the sustained activation of the p38 signaling pathway at baseline and the absence in the early phase but delayed of p38 signaling pathway response to LPS in the elderly may play important roles in increased susceptibility of aged lungs to inflammatory injury. © 2014 Elsevier Inc. All rights reserved.

Abbreviations: ALI, acute lung injury; ANOVA, analysis of variance; BALF, bronchoalveolar lavage fluid; COPD, chronic obstructive pulmonary disease; DAB, 3, 3′-diaminobenzidine tetrahydrochloride; DAPI, 4′, 6-diamidino-2-phenylindole; DMSO, dimethyl sulphoxide; ERKs, extracellular signal-regulated kinases; FBS, fetal bovine serum; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; H&E, Hematoxylin and eosin; IL-1β, interleukin-1β; IL-6, interleukin-6; i.n, intranasal inhalation; INF-γ, interferon-γ; JNKs, c-Jun N-terminal kinases; LPS, lipopolysaccharide; MAPKs, mitogen activated protein kinases; MKK, mitogen activated protein kinase kinase; MKPs, MAPK phosphatases; NF-κB, nuclear factor-kappa B; OCT, optimum cutting temperature compound; PBS, phosphate-buffered saline; PLSD, protected least significant difference; P-MKK, phosphorylated-MKK; P-p38, phosphorylated-p38; PVDF, polyvinylidene floride; SA β-gal, senescence-associated β-galactosidase; SAPKs, stress-activated protein kinases; SEM, standard error of mean; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-α; W/D, wet/dry. ⁎ Corresponding author at: Department of Pulmonary and Critical Care Medicine, Beijing Chao-Yang Hospital, Capital Medical University, No. 8, Gongti South Road, Chaoyang District, Beijing 100020, PR China. Tel.: +86 10 85231167. E-mail address: [email protected] (K. Huang).

http://dx.doi.org/10.1016/j.exger.2014.04.017 0531-5565/© 2014 Elsevier Inc. All rights reserved.

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X. Ren et al. / Experimental Gerontology 57 (2014) 29–40

1. Introduction Inflammaging is an emerging concept based on a progressive increase in pro-inflammatory status during the aging process (Franceschi et al., 2000). Some studies have shown that pulmonary inflammation progressively increases with age (Rahman and Adcock, 2006) and that there is an age-dependent increase of interleukin-6 (IL-6), interferon-γ (INF-γ), and tumor necrosis factor-α (TNF-α) in the airways of humans and rodents (Aoshiba and Nagai, 2007; Sharma et al., 2009). We previously also reported that the concentrations of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 were elevated in lung tissues and in bronchoalveolar lavage fluid (BALF) of mice with age (Li et al., 2011). These mediators might contribute to the increased neutrophils observed in the lower respiratory tract of healthy elderly individuals (Nikolaidis et al., 2011; Patel et al., 2012; Pignatti et al., 2011), suggesting that “inflammaging” naturally occurs in the lung with age. Recent studies indicated that aging can impair lung ability to repair the damaged lung tissues underlying inflammaging (Rahman et al., 2012), leading to aggressive lung destruction when exposed to extrinsic environmental factors. The elderly individuals have increased vulnerability to inflammatory injury, including acute lung injury (ALI) (Meyer, 2010; Rubenfeld et al., 2005), which is characterized by acute inflammation of alveoli resulting in diffuse alveolar injury, recruitment of inflammatory cells including neutrophils to lung parenchyma, release of cytokines, and the disruption of the alveolar-epithelial barrier (Fanelli et al., 2013). Similar observations have also been reported in mice. Exposure to cigarette smoke or bacterial products (such as LPS), or extra stimulations (such as cecal ligation and puncture) potentially promotes a relatively exacerbated pulmonary inflammatory response in aged mice compared with the young controls, and may contribute to pulmonary dysfunction (Gould et al., 2010; Ito et al., 2007; Saito et al., 2003). These studies confirmed that aging may potentially worsen acute lung injury induced by extrinsic environmental factors, although the underlying mechanisms are poorly understood. P38 signaling pathway plays a key role in stress (Gong et al., 2012). Activated p38 enhances pro-inflammatory responses through modulation of transcription factors, such as NF-κB (Wong et al., 2012), or by altering the stability and translation of the relevant signaling factors at the mRNA level (Buxade et al., 2008; Ronkina et al., 2008). A major role of p38 is to promote the production of some pro-inflammatory cytokines, particularly TNF-α, IL-1β, IL-6 and IL-8 (Chung et al., 2009). Thus, p38 activation in stress response to environmental stimuli, like LPS, may be a key factor in triggering inflammation in the lung. It has been reported that aging affects the activation of p38 signaling in neutrophils when replying to stress, contributing to a pro-inflammatory state in elderly individuals (Chaves et al., 2009; Larbi et al., 2005). Data also showed that there is an alteration with aging in the p38 activation in T cell from the elderly subjects following stimulation (Di et al., 2011; Douziech et al., 2002). Furthermore, we and others previously found that activated p38, but not total p38, was apparently increased in the lungs from aged mice (Li et al., 2011) and elderly individuals (Gaffey et al., 2013). However, it is still unclear whether aging affects the activation of p38 as part of a stress response to an inflammatory stimulation in the lung. In this study, we attempted to determine whether aging influences the p38 signaling pathway during stress response to LPS in the mouse lung and human lung. Our data demonstrate that aging impairs the p38 signaling pathway in stress response to environment stimuli in the lung, suggesting a novel mechanism by which the lung from the elderly is susceptible to inflammation. 2. Materials and methods 2.1. Animal and subject characteristics Young (2 months old) and aged (20 months old) male C57BL/6J mice were fed ad libitum with a pelleted stock diet and housed under

pathogen-free conditions at Capital Medical University, PR China. The protocol was approved by the Animal Care and Use Committee of Capital Medical University and followed the “principles of laboratory animal care” (NIH Publication No. 86-23, revised 1985). In addition, a cohort of 11 adult–middle aged subjects (age range, 29–47 years) and 14 older subjects (age range, 62–74 years) with normal lung function undergoing surgical resection for suspected or confirmed lung cancers were recruited from Beijing Chao-Yang Hospital (Table 1). Subjects who had hormone replacement and preexistent conditions such as inflammatory disorders, including connective tissue, tuberculosis and diabetes were excluded. “Normal lung tissue” was collected from an area of the lung as far distal to the tumor as possible, and processed as described previously (Plumb et al., 2009). The study was approved by the Ethics Committee of Beijing Chao-Yang Hospital.

2.2. Administration of LPS and SB203580 in mice LPS (Escherichia coli, serotype O55:B5, Sigma, St. Louis, MO, USA) was dissolved in sterile physiological saline (0.9% NaCl) at a concentration of 200 μg/50 μl. Animals exposed to LPS received a single intranasal (i.n.) inhalation at a dosage of 200 μg. Control animals received only administration of the vehicle control (normal saline). For the time-course of the study, mice received a single i.n. inhalation of 200 μg LPS and were assessed at 0.5, 4, 24 or 72 h later, which were based on the other previous studies (Ito et al., 2005; Maijó et al., 2012; Qiu et al., 2011) and our own preliminary experiments. To test whether targeting p38 inhibits activation of its downstream, NF-κB in aged mice after LPS challenge at 72 h, SB203580 (Biosource International, Camarillo, CA, USA), a specific inhibitor of p38 was given to aged mice (200 μg/50 μl, i.n.) at various time points including 0.5 h prior and 4, 16 and 36 h post LPS inhalation. All the animals were sacrificed and examined at 72 h after LPS inhalation.

2.3. Preparations of BALF and lung tissue homogenate BALF was performed as previously described (Li et al., 2011). Briefly, lungs dissected from anesthetized animals were lavaged via the trachea with initial 0.8 ml ice-cold isotonic phosphate-buffered saline (PBS, pH 7.2) followed by a consecutive aliquot of PBS (total of 1.5 ml). The two rinses were pooled and centrifuged at 1500 rpm for 10 min at 4 °C, and then processed for cell counts and differentials. The cell-free supernatant was collected and stored at − 20 °C for analysis of cytokines. Lung tissues were harvested after vascular perfusion. To determine the protein levels of MKK, p38 and NF-κB p65, the right cranial lobe of lungs was homogenized at 4 °C in lysis buffer A, B or C. Cytosolic proteins was extracted by Buffer A (50 mM Tris–HCl, pH 7.5, 1 mM dithiothreitol, 2 mM ethylene diamine tetraacetic acid, 2 mM ethylene glycol tetraacetate, 5 mg/ml each of leupeptin, aprotinin, pepstatin A, and chymostatin, 50 mM potassium fluoride, 50 nM okadaic acid, 5 mM sodium pyrophosphate, and 100 μM sodium vanadate). The nuclear pellets were resuspended in buffer B containing all reagents in buffer A and 0.5% NP-40. Buffer C (buffer A and 2% SDS) was used for total cell protein extraction. The protein concentration was determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). 2.4. Lung wet/dry weight ratio assessment in mice The water content of lung tissue was determined by calculating the tissue wet/dry weight ratio. The right middle lobe of lungs was excised, rinsed briefly in PBS, blotted dry and weighed to obtain the “wet” weight. The lung was then immediately dried at 80 °C for 72 h in an oven to obtain the “dry” weight. The ratio of wet lung to dry lung was calculated to assess lung tissue edema (Patel et al., 2012).

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Table 1 Subject demography. Subjects

Male/female

Age (years)

Smoking history pack years

Current/ex-smokers

FEV1 %pred

FEV1/FVC %

Adult–middle aged (n = 11) Older (n = 14)

9/2 14/0

35 ± 5 66 ± 4

12 ± 2 36 ± 4

7/4 3/11

87 ± 6 83 ± 2

80 ± 2 76 ± 5

Data are expressed as mean ± SEM. FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity.

2.5. Histopathological staining of lung tissue The left lobe of the lungs were fixed in 10% neutral formalin for 24 h, then placed in 30% sucrose for 48 h, before they were finally embedded in optimum cutting temperature compound (OCT) and sliced. A commercial kit was employed to determine activity of senescenceassociated β-galatosidase (SA β-gal) according to the manufacturer's protocol (Cell Signal Technology, Danvers, MA, USA). Using this kit, SA β-gal activity, detectable at PH 6.0, permits the identification of senescent cells in culture and mammalian tissues (Debacq-Chainiaux et al., 2009). Hematoxylin and eosin (H&E) staining was also included for testing pathological changes of lung tissues under a light microscope. 2.6. Ex vivo stimulation of human lung cells Explanted human lung tissues were collected aseptically and disrupted to yield a cell suspension in ice-cold PBS containing 0.5% fetal bovine serum (FBS) (Robbiano et al., 2006). The cell numbers were counted using a cytometer and the cell viability was measured by Trypan blue exclusion assay. The percentage of viable cells was above 85%. Cells were cultured with RPMI-1640 (Sigma Aldrich, Poole, Dorset, UK) for 24 h. After washing with RPMI-1640 medium, cells (macrophages: 80–85%, epithelial cells: 15–20%, according to the morphology of the cells and the phenotypic markers in the double staining) were stimulated with the medium containing LPS (100 ng/ml) for further 0.25, 0.5, 1, 4, 16 and 24 h. Cells exposed to medium alone were used as controls. After culturing, cell debris was collected for the signaling analysis, while supernatants were harvested and stored at − 20 °C for the measurement of cytokines. Some cells of lung tissues were incubated in chamber slides for identifying phenotypes of p-p38+ cells. 2.7. Immunohistochemistry and immunofluorescence Immunohistochemistry was performed to determine phosphorylated-p38 (p-p38) in murine and human lung (Li et al., 2011). Briefly, slides were incubated with a rabbit monoclonal primary antibody against p-p38 (dilution 1:100; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, followed by horseradish peroxidase-conjugated second antibody. The positive staining was detected by 3, 3′-diaminobenzidine tetrahydrochloride (DAB). The number of p-p38 positive cells within the lung was computed using a light microscope (Olympus BX51, Olympus Optical, Tokyo, Japan), connected to a video recorder linked to a computerized image system (Image-Pro Plus. Media Cybernetics, Bethesda, MD, USA). A total of 10 fields, distributed randomly across the slide, were evaluated for each subject or mouse and the results were expressed as the number of positive cells/mm2. To determine the cellular distribution of p-p38 in the lung, a dual label immunofluorescence technique was performed to co-localize phenotypes of cells expressing p-p38. Briefly, slides were incubated with a rabbit antip-p38 antibody (dilution 1:50; Cell Signaling Technology) and a mouse monoclonal primary antibody against E-cadherin (5 μg/ml) for identifying epithelial cells (1:50; Abcam, Cambridge, London, UK), or a rat antimouse F4/80 antibody for identifying macrophages (1:50; Abcam, Cambridge, London, UK), or a goat polyclonal antibody against mouse

neutrophil elastase (1:50; Santa Cruz Biotechnology, CA, USA) overnight at 4 °C, followed by Alexa Fluor 488-conjugated anti-rabbit IgG at 1:800 dilution (green color) (Cell Signaling Technology) and rhodamineconjugated anti-mouse or rat or goat IgG at 1:100 dilution (red color) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The nuclei were counterstained using 4′, 6-diamidino-2-phenylindole (DAPI; blue color) (Vector Laboratories, Burlingame, CA, USA). Images were captured by using a microscope (Olympus BX51. Olympus Optical, Tokyo, Japan). 2.8. Western blotting The expression of MKK, phosphorylated (p)-MKK, p38, p-p38 and NF-κB p65 in the lungs was analyzed by Western blot analysis as reported previously (Long et al., 2006). Each sample was boiled in SDS-sample buffer for 10 min followed by brief centrifugation. Samples were loaded on to 10% SDS polyacrylamide gels, separated, and then transferred onto the polyvinylidene floride membranes (PVDF membrane) (Bio-Rad Laboratories, Hercules, CA, USA) at 4 °C, 400 mA for 3 h using standard methods. Western blot analysis was performed using commercially available antibodies for MKK, p-MKK, p38, p-p38 and NF-κB p65 (Cell Signal Technology). For the secondary antibodies, detection was performed using peroxidase conjugated goat anti-rabbit antibody (Sigma-Aldrich, St. Louis, MO, USA). Membranes were developed using ECL-plus Kit (Chemicon International, Temecula, CA, USA), and band density was quantified using NIH ImageJ software. β-Actin and nuclear matrix protein (ab487) were determined using antibodies against β-actin (Sigma-Aldrich, St. Louis, MO, USA) and ab487 (Abcam, Cambridge, London, UK) for loading controls. 2.9. Cytokine measurements The right caudal lobe of mouse lung was homogenized in phosphatebuffered saline containing protease inhibitors before analysis of cytokine content. The concentrations of TNF-α, IL-1β and IL-6 in BALF, lung homogenates and supernatants of cultured cells were measured using ELISA kit (R&D Systems, Minneapolis, MN, USA) and Cytometric Bead Array (BD Biosciences, Rockville, MD, USA) according to the manufacturers' instructions. 2.10. Statistical analysis Data were analyzed using a statistical package (GraphPad Software, La Jolla, CA, USA). Two-way analysis of variance (ANOVA) was performed to compare 2-month with 20-month old mice or compare adult–middle aged subjects and older subjects followed by Fisher's protected least significant difference (PLSD) to compare all groups at various time points. Statistical significance was considered to be p b 0.05. 3. Results 3.1. Increased activity of SA β-gal, lung wet/dry weight ratio and inflammation in aged mice SA β-gal activity was significantly elevated in the lungs of 20-month mice (Fig. 1A). The wet/dry weight lung ratios were increased by 24 and

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Fig. 1. Effect of LPS challenge in young and aged mice. (A) Increased SA β-gal (blue, original magnification ×200) in lung sections from 20-month old mice compared with the controls. (B) LPS challenge increased the lung wet/dry (W/D) weight ratio in aged mice compared with the controls. (C) Typical histological changes in lungs from young and aged mice at various time points after LPS challenge (H&E staining, ×200). (D) Total number of cells and cellular differential counts in the BALF from 2- and 20-month-old mice at various time points after LPS challenge (n = 4–6 for each group). Saline (0 h) was used as baseline control. Data were expressed as the mean ± SEM. *p b 0.05, **p b 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

72 h after LPS challenge in both 2- and 20-month-old mice. Lung wet/dry weight ratio of 20-month-old mice was ~ 1.3-fold and ~ 1.5fold higher than that of 2-month-old mice after LPS inhalation at 24 and 72 h, respectively (Fig. 1B). H&E staining showed that there was significantly greater infiltration of inflammatory cells in lung tissues from aged mice compared with young mice, particularly at 24 and 72 h after LPS challenge (Fig. 1C). However, there were no significant changes between two groups at the earlier time points (such as 0.5 and 4 h) after LPS challenge. Cell counts of BALF showed that the total number of BALF cells significantly increased in aged mice compared with the control mice at the baseline (Fig. 1D). LPS stimulation induced significant infiltration of inflammatory cells into the airway lumen in both groups of mice and this increase remained up to 72 h (Fig. 1D). At the later time points (24 and 72 h), the inflammatory cell infiltration was significantly higher in the aged mice compared with the young mice. The majority of increased inflammatory cells were neutrophils and macrophages, but not lymphocytes or eosinophils (Fig. 1D). In addition, LPS stimulation also induced

increased numbers of sloughed epithelial cells, particularly in aged mice at the later time points (Fig. 1D). 3.2. The effects of aging on activation of p38 in murine lung induced by LPS The results of p-p38 expression in murine lung are shown in Fig. 2. Representative immunohistochemistry of p-p38 are shown in Fig. 2A, and the quantitative analysis is depicted in Fig. 2B. At baseline, the number of p-p38 immunoreactive cells was ~ 3.6-fold higher in aged mice relative to the young mice (Fig. 2B). Intriguingly, LPS challenge rapidly increased the activation of p38 in the lungs of young mice, with a peak at 0.5 h. This LPS-induced increase in activation of p38 then sharply decreased by 4 h and progressively dropped to baseline by 72 h (Fig. 2B). In contrast, the phosphorylation of p38 in aged mice was delayed relative to that seen in young mice, with a peak at 24 h after LPS exposure and persisting for up to 72 h (Fig. 2B). The p-p38 immunoreactive cells were mainly located in alveolar walls in both young and aged mice.

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Fig. 2. Effect of aging on the activation of the p38 in the lungs of 2- and 20-month old mice before and after LPS challenge. (A) Immunoreactive cells of p-p38 (brown) in the lungs of young and aged mice at 0.5 h and 24 h after LPS challenge (original magnification ×400). (B) Quantitative analysis of the total number of p-p38 immunoreactive cells in the lung sections from 2and 20-month-old mice (n = 4–6 for each group). Data are expressed as the mean ± SEM of number of p-p38+ cells per unit of whole sections. **p b 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.3. Phenotypes of p-p38 positive cells Double immunofluorescent staining showed that LPS-induced phosphorylation of p38 in mouse lung was mainly located in E-cadherin+ alveolar epithelial cells (Fig. 3A) or F4/80+ macrophages (Fig. 3B), but not neutrophils (data not show). Similar patterns of p-p38 distribution were also observed in isolated cells of human lungs (data not shown).

3.4. Effects of aging on p38 signaling pathways and pro-inflammatory cytokine production in mouse lung in response to LPS At baseline, increased activation of p38 itself, the upstream (MKK, the activator of p38) and the downstream (NF-κB, the p38 substrate) was observed in the lung tissues of aged mice compared to the young mice (Fig. 4A). LPS exposure induced rapid phosphorylation of p38, MKK and translocation of NF-κB p65 in the young mice, which occurred as early as 0.5 h after challenge, then declined and came back to baseline by 24 or 72 h after exposure (Fig. 4A). In contrast to the young control

mice, LPS-induced activation of p38 signaling pathways occurred relatively late, with a peak at 24 h and remaining elevated levels by 72 h after challenge in the lungs from aged mice (Fig. 4A). Furthermore, enhanced p-p38 and p-MKK was accompanied by increased nuclear expression with decreased cytoplasmic NF-κB p65 in aged mice compared to the young mice after LPS inhalation (Fig. 4A). Taking the changes at various time points together, Fig. 4B shows that the p-MKK, p-p38 and nuclear NF-κB p65 significantly increased in aged murine lung compared with the controls, which was accompanied by decreased cytoplasmic NF-κB p65 (Fig. 4B). In agreement with the elevated activities of p38 signaling pathways, the concentrations of TNF-α, IL-1β and IL-6 were higher in lung homogenates and BALF from aged mice compared with that of young controls (Fig. 4C). The young mice had a robust production of TNF-α, IL-1β and IL-6 by 0.5 and 4 h after LPS inhalation, which then rapidly declined, although they were slightly higher than the baseline at later time points; whereas the aged mice showed a late phase increased expression of these pro-inflammatory cytokines, with peaks at 24 and 72 h (Fig. 4C).

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Fig. 4. Effects of aging on p38 signaling and pro-inflammatory cytokine production in response to LPS in mouse lungs. (A) Western blot analysis of p-p38, p-MKK and nuclear translocation of NF-κB p65 in lungs of young and aged mice at various time points after LPS challenge. β-Actin and nuclear marker (ab487) were used as loading controls. Time point 0 h was used as the baseline control. (B) Taking the changes at various time points together, the total level of p38 signaling was significantly higher in the lung from 20-month old mice compared with those of 2-month old controls. (C) The concentrations of TNF-α, IL-1β and IL-6 in BALF and lung tissues of young and aged mice. Data are expressed as the mean ± SEM (n = 4–6 mice for each group). *p b 0.05, **p b 0.01.

3.5. Inhibition of p38 suppresses NF-κB activation and pro-inflammatory cytokine production in lung of aged mice after LPS challenge

3.6. Aging increases the activity of SA β-gal and p38 signaling pathways and production of pro-inflammatory cytokines in human lung

Pre-treatment with SB203580 0.5 h before LPS inhalation significantly suppressed LPS-induced activation of p38 and translocation of NF-κB p65 (Fig. 5A). Treatment with SB203580 after LPS inhalation at 4 or 16 h also abolished the p38 activation and NF-κB p65 translocation by LPS. However, SB203580 administration after LPS challenge at 36 h had no effect on p38 activity and NF-κB p65 translocation induced by LPS (Fig. 5A). Correspondingly, administration with SB203580 0.5 h before LPS administration or 4 and 16 h after LPS treatment also almost completely abolished LPS-induced production of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 both in BALF (Fig. 5B, top panel) and lung homogenates (Fig. 5B, bottom panel). However, SB203580 inhalation 36 h after LPS administration was unable to inhibit pro-inflammatory cytokine production induced by LPS challenge (Fig. 5B).

The number of SA β-gal positive cells was significantly higher in the lungs from older subjects compared with the adult–middle aged subjects (Fig. 6A) (p b 0.01). Immunohistochemistry showed that the number of p-p38 immunoreactive cells was significantly elevated in the lungs of older subjects compared with the adult–middle aged subjects (Fig. 6B) (p b 0.05). These immunoreactive cells were mainly located in alveolar walls and spaces in the lungs of both groups (Fig. 6B). We next determined the p-p38, p-MKK and nuclear translocation of NF-κB p65 in the lung tissues of older subjects compared with those in adult–middle aged subjects. Western blotting showed that there were significant increases in the p-p38, p-MKK and nuclear translocation of NF-κB p65 in the lung homogenates from the older subjects in comparison to the adult–middle aged subjects (Fig. 6C). As was observed in

Fig. 3. Phenotypes of p-p38 immunoreactive cells. Double-labeled immunofluorescent staining of p-p38 positive signals (green) co-located with the lung epithelial cells (E-cadherin positive, red) (Fig. 3A) and BALF macrophages (F4/80 positive, red) (Fig. 3B) from 2- and 20-month-old mice at 0.5, 24 h after LPS challenge, respectively. DAPI was used for nuclear staining. Original magnification ×200.

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Fig. 5. Effects of SB203580 on p38 signaling and cytokine production. SB203580, but not DMSO (vehicle control) pretreatment suppressed the activation of p38 and translocation of NF-κB p65 from cytoplasm into nuclei in lung homogenate (A) and production of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in BALF (B, top panel) and lung homogenate (B, bottom panel) of aged mice after LPS challenge at various time points. Data represented as the mean ± SEM of 4–6 mice for each group. *p b 0.05; **p b 0.01 vs controls; #p b 0.05, ## p b 0.01 vs LPS treated mice.

mice, the expression of TNF-α, IL-1β and IL-6 in lung tissue was elevated significantly in older subjects compared with controls (Fig. 6D) (TNF-α: p b 0.05, IL-1β: p b 0.05, IL-6: p b 0.01).

3.7. Aging impairs the activation of p38 signaling pathways and production of pro-inflammatory cytokines after LPS exposure to human lung cells in vitro Western blotting showed that rapid activation of p38 signaling was observed in the cells of adult–middle aged subjects' lungs at 0.25 h after exposure. Compared with the cells from adult–middle aged subjects, the LPS exposure did not induce marked changes in the activation of p38 signaling in those lung cells from older subjects in the early phase. In contrast, such activation of p38 occurred relatively late and peaked at 16 h (Fig. 7A). Taking the changes at various time points together, Fig. 7B shows that the p-MKK, p-p38 and nuclear NF-κB p65 significantly increased in the lung cells from older subjects compared with the adult–middle aged subjects, which was accompanied by decreased cytoplasmic NF-κB p65 (Fig. 7B). Correspondingly, the concentrations of TNF-α, IL-1β and IL-6 in the supernatants were associated with the dynamic activation of p38 signaling of lung cells from adultmiddle aged subjects or older subjects after LPS exposure (Fig. 7C).

4. Discussion It is believed that p38 plays a prominent role in the chronic stress response and tissue aging, possibly through increasing basal levels of phosphorylated-p38 (Hsieh et al., 2003, 2010; Wu et al., 2009). In the present study, we tested whether aging influences the lung's ability in the activation of p38 signaling pathway in stress response to extrinsic stimulation, such as LPS, and whether this contributes to an exacerbated pulmonary inflammatory injury. Our data showed that after LPS challenge the number of pulmonary neutrophils, macrophages and epithelial cells, concentrations of inflammatory cytokines and lung wet/dry weight ratio were significantly elevated in aged mice compared with young mice. These observations further support the notion that aging exacerbates pulmonary inflammatory injury after stimulus (Boyd et al., 2012; Geller and Zenick, 2005; Gomez et al., 2007; Ito et al., 2007). Although LPS challenge is not an optimal model of trauma and sepsis for failing to fully reproduce the condition (Nomellini et al., 2009), it is still widely used to mimic the changes of acute lung injury or airway inflammation by intranasal administration (Hiroshima et al., 2014; Mercer et al., 2014; Tang et al., 2014). Increased activity of p38 has been observed in aged lung (Gaffey et al., 2013; Li et al., 2011). However, it is still little known whether aging impairs the response of p38 signaling to environmental stress.

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Fig. 6. The activity of SA β-gal, p38 signaling and the expression of pro-inflammatory cytokines in the human lungs. (A) SA β-gal (blue) increased in lung sections of older subjects compared with adult–middle aged subjects (original magnification ×200). (B) P-p38 immunoreactive cells (brown) increased in older subjects lungs compared with controls (original magnification ×400). (C) Western blot analysis of p-p38, p-MKK, nuclear translocation of NF-κB p65 in the lungs of older subjects (n = 3) compared with adult–middle aged subjects (n = 3). β-Actin and nuclear marker (ab487) were used as loading controls. (D) Concentrations of TNF-α, IL-1β and IL-6 in lung tissues obtained from adult–middle aged subjects and older subjects. Data are expressed as the mean ± SEM (n = 6–10 for each group). *p b 0.05, **p b 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Our data showed that the rapid activation and sharp inactivation of p38 to LPS exposure were only observed in the lungs of the 2-month, but not in the 20-month mice. Compared with youth, an absence in the early phase but delayed activation of the p38 signaling pathway was observed in the lungs of aged mice, which started at 24 h and was sustained for 72 h after LPS challenge. These findings suggest that aging might affect the activation pattern of p38 signaling pathway in response to environment stress, such as LPS stimulation. To the best of our knowledge this is the first report showing that aging impairs LPS-induced p38 signaling responses in the lung. Moreover, the lung epithelial cells and macrophages might be the major cellular sources in this process. Although neutrophils were the predominant cells after the LPS challenge, we didn't find co-localization of phospho-p38 with neutrophil-specific marker (elastase), suggesting that these cells might be less important in the changes of p38 signaling pathways in the lung induced by LPS, at least under our experimental conditions. MKK, a kinase enzyme, can phosphorylate MAPKs including p38, extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) (Chang and Karin, 2001; Hsieh and Papaconstantinou, 2002). Among MKK, MKK3 and MKK6 are particularly involved in the activation of p38 (Han et al., 1994; Lee et al., 1994; Rouse et al., 1994). In

addition, NF-κB, as a well established downstream of p38, plays a critical role in regulating pro-inflammatory cytokine expression. Our data showed that both LPS-induced activation of MKK and translocation of NF-κB p65 were dynamically associated with phosphorylation of p38 in young and aged murine lungs. This may signify that these docking processes requiring protein–protein interactions among MKK, p38 and NF-κB have not been altered in the aged lung tissues. These data raise an important question of why aging lung has an absent early phase but delayed p38 signaling response to LPS. Several speculative reasons might explain the observed phenomenon. First, LPS-binding protein is the principal plasma protein responsible for transporting endotoxin to cells. Gomez and colleagues had observed a marginal tendency toward lower levels of LPS-binding protein in the LPS-treated aged mice (Gomez et al., 2006). Second, it is well known that many extracellular stimuli such as reactive oxygen species, cytokines or Toll-like receptor (TLR) stimulation can trigger phosphorylation of p38 (Cuadrado and Nebreda, 2010), and then affects a variety of intracellular responses. Several studies have shown that aging negatively skews TLR-mediated pro-inflammatory responses in macrophages, possibly due to the decreased phosphorylation of p38 (Boehmer et al., 2004; Boehmer et al., 2005; Boyd et al., 2012).

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Fig. 7. Effects of aging on p38 signaling and pro-inflammatory cytokine production in response to LPS in human lungs in vitro. (A) Western blot analysis of p-p38, p-MKK and nuclear translocation of NF-κB p65 in lung cells from adult–middle aged subjects and older subjects at various time points after LPS challenge. β-Actin and nuclear marker (ab487) were used as loading controls. Time point 0 h was used as the baseline control. (B) Taking the changes at various time points together, the total level of p38 signaling was significantly higher in the lung cells from older subjects compared with those of adult–middle aged subjects. (C) The concentrations of TNF-α, IL-1β and IL-6 in the supernatants of lung cells of adult–middle aged subjects and older subjects after LPS stimulation. Data are expressed as the mean ± SEM (n = 4 for each group). *p b 0.05, **p b 0.01.

Thus, it is reasonable to speculate that impaired p38 signaling response to LPS in the present study might be partly because of the deficiencies in TLR in the aging mouse lung. Third, regulation of p38 signaling plays an important role to limit the action time and intensity of p38 activation, through dephosphorylation and inactivation by MAPK phosphatases (MKPs) (Cadalbert et al., 2010; Coulthard et al., 2009; Cuadrado and Nebreda, 2010). Dasgupta et al. showed that there was a decrease in MKP-1 in the old lung fibroblasts under 21% O2 exposure, which resulted in enhancing overall kinase signaling (Dasgupta et al., 2010). Therefore, in the aged lung sustained and increased p38 signaling response to LPS is, at least partly because of the decreased MKP activity, or of the inability of the MKPs to dock with the activated p38 complex, or both. Although the mechanisms of delayed activation of p38 with aging are still in mystery, some studies showed that the basal level of p-p38 was elevated in peripheral blood neutrophils and T cells of aged subjects (Di et al., 2011; Larbi et al., 2005), which is consistent with what we observed in the aged lungs. On the other hand, an inability of p-p38 has been observed up to 18 h after stimulation in aging cells and tissues (Hsieh and Papaconstantinou, 2002; Larbi et al., 2005). One possible explanation is that the p38 activated complex is stabilized to prevent new complex formation (Hsieh and Papaconstantinou, 2002), which might

be also responsible for the delayed activation of p38 observed in the present study. As a stress-activated kinase, p38 signaling is critical in normal immune and inflammatory responses and regulates expression of proinflammatory cytokine by promoting transcription factors including NF-κB (Qian et al., 2012; Yeung et al., 2012). Thus, early activation of p38 signaling may help the host to effectively eliminate potential pathogenic invasion through producing pro-inflammatory cytokines. However, long term excessive local inflammation may also lead to lung injury, even at the baseline (Anzai, 2013). Our data showed that delayed production of pro-inflammatory cytokines was associated with the dynamic activation of the p38 signaling pathway in aged lung after LPS challenge. Thus, it is possible that such an impaired p38 signaling response to LPS might cause a deficient host defense in the early phase and an excessive production of pro-inflammatory cytokines in the later phase, contributing to an exacerbated inflammatory injury in aged mouse lungs. Previous studies have shown that targeting p38 with specific inhibitors or genetic deletion reduces LPS-induced release of proinflammatory cytokines in vivo and in vitro (Liu et al., 2008; Ronkina et al., 2010). To further test whether this occurs in aged lung, a specific p38 inhibitor SB203580 was used in the present study. We found that

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administration of the compound in aged mice significantly reduced the LPS-induced p38 activation at 0.5 h prior and 4 h, 16 h but not 36 h post LPS inhalation, which consequently resulted in the changes of the downstream of p38 signaling, including the decreased translocation of NF-κB p65 and expression of pro-inflammatory cytokines. We also found that SB203580 treatment in a certain period significantly inhibited the LPS-induced translocation of NF-κB p65, confirming the involvement of p38 in NF-κB activation during LPS-induced lung inflammation. These data suggest that administration of inhibitor of p38 can only exert its therapeutic effect before the activation of p38 pathway. This phenomenon possibly explains, at least partly, why some clinical trials showed that p38 inhibitors are not always effective for age-related lung diseases, such as COPD (Chung, 2011). Increased basal activity of p38 has been noted in the lungs of patients with COPD (Renda et al., 2008), although it is less known whether the increased p38 activity is a natural event occurring in aged human lungs. We observed that the basal activity of MKK and p38 increased in human lungs with aging, which might result in the over translocation of its downstream substrate NF-κB p65, leading to increased expression of pro-inflammatory cytokines in the older subjects compared to that of adult–middle aged subjects. Although LPS inhalation challenge in human is an ideal model for studying signaling, we had to use human lung cells as target cells for the LPS stimulation in vitro because of ethics limitations. The activation of p38 signaling was not observed in the lung cells of older subjects in the early phase but peaked at 16 h after exposure. These data confirm that an impaired p38 signaling response to exogenous stimuli exists in aged human lungs. Interestingly, the production of proinflammatory cytokines was also associated with the dynamic activation of p38 signaling in lung cells from adult–middle aged subjects or older subjects after LPS exposure. These suggest that this “relative delayed” activation of p38 signaling might contribute to some phenomena seen in clinic, at least partly. For instance, older patients normally have delayed and more severe symptoms in infectious respiratory diseases than those of younger subjects, possibly because the lack of effective response in the early phase of disease in aging lungs causes failure or ineffective immediate immune responses. This might result in further amplification of pathogenic microorganisms or severe damage in a clinical setting. One possible concern is that the 2 month old mouse is much younger than a 29–45 year old adult. It is generally believed that the mouse reaches sexual maturity at 2 months old (Vandenbergh, 1967), which might be equivalent to 14 years old in that of human (Luciano et al., 2013). Although adolescents may differ in psychological status compared to adult individuals (Kim and Yi, 2013), therapeutic regimens, at least in most conditions or diseases in clinic are much similar to that of the adult (Chervinsky et al., 1999; Deniz and Gupta, 2005; Freund et al., 1999; Straumann et al., 2010). In addition, it is quite difficult in the clinic to obtain explanted lung tissue from patients under 20 years old of age. Although it is not possible to draw direct causal links from the present study, our data do support the hypothesis that aging impairs the p38 signaling pathway during stress response to LPS in the lungs. Moreover, the results illustrate the potential underlying relationship between the p38 signaling response and lung inflammatory injury in aging lung after the LPS challenge. In addition, although the p38 signaling pathway appears to play a key role in inflammation, LPS can trigger the inflammatory response through other pathways, including the ERK signaling pathway. Whether there is a relationship between these signaling pathways during inflammatory injury in aged lungs needs further study. Additionally, although smoking can induce inflammatory responses in the lung, combining the data together, similarity of alterations in the aging lungs between mouse and human in the responses to LPS exposure may imply the major role of age in this study. In summary, aging affects the p38 signaling pathway and the production of pro-inflammatory cytokines in the lungs in vivo in mice as well as in humans ex vivo after the LPS challenge. This suggests that the elderly are susceptible to respiratory diseases through altered

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