Alveolar Epithelial Inter-alpha-trypsin Inhibitor Heavy Chain 4 Deciency Associated With Senescence-regulated Apoptosis by Particulate Air Pollution

Background: Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is considered a type II acute-phase protein; however, the role of ITIH4 in the lungs after exposure to ne particulate matter (PM 2.5 ) remains unclear. The objective of this study was to investigate the role of ITIH4 in the lungs in response to PM 2.5 exposure. Results: ITIH4 expression in bronchoalveolar lavage uid (BAL) of 47 healthy subjects and of SD rats exposed to PM 2.5 was determined, and the underlying anti-apoptotic and matrix-stabilizing pathways in A549 cells by diesel exhaust particles (DEPs) were also investigated. First, we observed that an interquartile range (IQR) increase in PM 2.5 accounted for a decrease of 2.673 ng/mL in ITIH4, an increase of 1.104 pg/mL in 8-isoprostane, and an increase of 6.918 pg/mL in interleukin (IL)-6 in human BAL. Increases in 8-isoprostane and IL-6 in the lungs and decreases in ITIH4 in the BAL, lungs, and serum were observed after PM 2.5 exposure. ITIH4 was correlated with lung lysates and BAL samples (r=0.377, p<0.01), whereas ITIH4 was correlated with IL-6 in BAL (r=-0.420, p<0.01). ITIH4 expression was signicantly reduced in alveolar epithelial cells by PM 2.5 . ITIH4 expression decreased after DEP exposure in a dose-dependent manner. A decrease in sirtuin 1 (Sirt1) and increases in phosphorylated extracellular signal-regulated kinase (p-ERK) and caspase-3 were observed after DEP exposure. Conclusions: In conclusion, PM 2.5 decreased ITIH4 in the lungs, which was associated with alveolar epithelial cell senescence and apoptosis. ITIH4 could be a vital protein in regulating alveolar destruction, and its deciency occurs due to PM 2.5 . ×g 4


Background
The acute phase of a systemic response in reaction to injury and in ammation is characterized by alterations in white blood cells, secretion of glucocorticoids, and activation of complements, proteins and cytokines [1]. Generally, acute-phase proteins are categorized into types I and II. Type I acute-phase proteins include serum amyloid A, C-reactive protein (CRP), α1-acid glycoprotein, and complement C3, which are induced by pro-in ammatory cytokines. Type II acute-phase proteins include brinogen, haptoglobin, α2-macroglobulin, α1-antichymotrypsin, and α1-antitrypsin, which are induced by interleukin (IL)-6-like cytokines [2]. In addition, acute-phase proteins are divided into positive and negative acutephase proteins. Positive acute-phase proteins are those which increase with the in ammatory response, whereas negative acute-phase proteins are those which decrease the in ammatory response [3,4].
Numerous acute-phase proteins have been linked to exposure to air pollution. For example, the positive acute-phase proteins, IL-6, CRP, and brinogen, were increased by air pollution in myocardial infarction survivors [5]. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), a negative acute-phase protein, decreased in serum after exposure to particulate matter (PM) of less than 10 μm in aerodynamic diameter (PM 10 ) in subjects with chronic obstructive pulmonary disease (COPD) [6]. ITIH4 is an anti-in ammatory protein that is encoded by a gene that is dominantly expressed by the liver [7]. ITIH4 is secreted into the blood [8], where it is cleaved by plasma kallikrein into two smaller forms. It was reported that ITIH4 expression is induced by different pathological conditions [6,9], and it functions as an anti-apoptotic and matrixstabilizing molecule.
We found an association between PM 10 and circulating ITIH4 in COPD subjects. However, ITIH4 expression in the lung environment due to ne PM (<2.5 μm in aerodynamic diameter; PM 2.5 ) exposure and the underlying pathways remain unclear. The objective of this study was to investigate the role of ITIH4 in the lungs in response to PM 2.5 exposure. ITIH4 expression in the lungs of humans and rodents and the underlying anti-apoptotic and matrix-stabilizing pathways were examined in the present study.

Results
Associations between PM 2.5 and BAL biomarkers in healthy subjects Baseline characteristics of the 47 healthy subjects are presented in Table 1. Their FEV 1 /FVC ratio was 85.8% ± 5.8%. BAL ITIH4, 8-isoprostane, and IL-6 concentrations were 2.3 ± 3.1 ng/mL, 4.3 ± 1.3 pg/mL, and 15.9 ± 8.2 pg/mL, respectively. Exposure levels of air pollutants and associations between these pollutants and BAL biomarkers are shown in Table 2. A decrease in ITIH4 and increases in 8-isoprostane and IL-6 were signi cantly associated with PM 2.5 exposure in the BAL of healthy subjects. An IQR increase in PM 2.5 (4.6 μg/m 3 ) accounted for a 2.673 ng/mL decrease in ITIH4, a 1.104 pg/mL increase in 8-isoprostane, and a 6.918 pg/mL increase in IL-6. However, there were no statistically signi cant associations observed for NO 2 or O 3 with these BAL biomarkers.
Decrease of ITIH4 in alveolar epithelium after chronic exposure to PM 2.5 in vivo Figure 1 shows alterations in 8-isoprostane, IL-6, and ITIH4 in rats in the control, HEPA, and PM 2.5 groups after 3 and 6 months of exposure. First, we observed that 8-isoprostane was signi cantly increased by 3and 6-months exposure in the HEPA and PM 2.5 groups (p<0.05) (Figure 1a). We next determined levels of ITIH4 in BAL, lung lysates, and serum between the groups. ITIH4 in BAL and lung lysates had signi cantly decreased after 6 months of PM 2.5 exposure compared to the control group (p<0.05). Levels of ITIH4 in serum had decreased with both 3 and 6 months of PM 2.5 exposure compared to the control group (p<0.05). Together, ITIH4 expression in lung lysates was correlated with levels in BAL and serum ( Figure   1b). We found that there was a statistically signi cant correlation of ITIH4 levels between lung lysates and BAL samples (r=0.377, p<0.01). But the correlation of ITIH4 was not identi ed between lung lysates and serum samples (r=0.184, p=0.21). We then examined correlations of ITIH4 with 8-isoprostane and IL-6 in BAL samples (Figure 1c). There was a negative correlation between ITIH4 and IL-6 in BAL (r=-0.420, p<0.01), but no correlation was observed between ITIH4 and 8-isoprostane (r=-0.128, p=0.386). For the ITIH4 intensity in lung sections, we observed that ITIH4 expression had decreased in the lungs after 6 months of PM 2.5 exposure (Figure 1d). Notably, ITIH4 was highly expressed by alveolar epithelial cells in control rats, which had decreased with 6 months of PM 2.5 exposure.
Decrease in ITIH4 associated with activation of alveolar epithelial senescence-induced apoptosis by DEPs in vitro To determine the underlying pathways of ITIH4 as affected by PM 2.5 , human alveolar epithelial A549 cells were exposed to tra c-related DEPs. Expression of ITIH4 decreased with DEP exposure in a dosedependent manner (p<0.05) ( Figure 2a). Next, we found that Sirt1 had signi cantly decreased with 50 μg/mL DEPs (p<0.05; Figure 2b), while p-ERK and caspase-3 had increased with 50 and/or 100 μg/mL DEPs (p<0.05) (Figure 2c). There were no signi cant alterations in MMP9 or MMP12 after DEP exposure.

Discussion
ITIH4 is a type II acute-phase protein that is dominantly expressed in the liver [7]. We demonstrated that serum ITIH4 is associated with exposure to PM 10 in COPD patients; however, the underlying mechanisms of ITH4 in response to PM 2.5 remain unclear. The present study showed that PM 2.5 was associated with an ITIH4 de ciency and increases in 8-isoprostane and IL-6 in BAL of healthy subjects. Next, we demonstrated increases in 8-isoprostane and IL-6 in the lungs and reductions in ITIH4 in BAL, the lungs, and serum of rats after PM 2.5 exposure. Signi cant downregulation of ITIH4 occurred by PM 2.5 in alveolar epithelial cells of rats. Therefore, we next examined ITIH4 levels in alveolar epithelial A549 cells. p-ERK and caspase-3 were activated along with downregulation of ITIH4 and Sirt1 in A549 cells by DEPs. Our results suggest that activation of apoptosis may be through ITIH4-associated senescence of alveolar epithelial cells produced by particulate air pollution.
The BAL analysis represents direct alterations of the lung microenvironment, and it is commonly used in air pollution studies [12,13]. Our results demonstrated that exposure to PM 2.5 was associated with increases in oxidative stress and in ammatory responses in BAL samples of healthy subjects, which is consistent with a previous report [12]. Our previous study indicated that exposure to PM 10 was associated with a decrease in serum ITIH4 levels in COPD patients [6]. The reduction was correlated with in ammatory CRP levels of COPD. Notably, in the present study, we found that the decrease in ITIH4 in BAL was associated with PM 2.5 in healthy subjects. The results suggest that ITIH4 was produced by lung cells after PM 2.5 exposure and was captured by BAL, even if ITIH4 is mainly present in the liver and released into the circulation. This type II acute-phase protein (ITIH4) in BAL could be an indicator of PM 2.5 exposure. However, ITIH4 expression in lung tissues and the underlying mechanisms are still unclear.
Next, we conducted a controlled in vivo experiment to determine the role of ITIH4 in the lung environment after long-term exposure to air pollution. We found that oxidative stress and in ammatory markers in BAL of rats had increased by PM 2.5 exposure, which is in line with the BAL results of healthy subjects. In addition, BAL, lung, and serum levels of ITIH4 decreased after 3 and/or 6 months of exposure to PM 2.5 .
These results con rmed that ITIH4 is a type II negative acute-phase protein. To understand the alterations in ITIH4 in BAL secreted from the lungs and serum in circulation, we examined correlations of ITIH4 levels between the lungs and BAL and between the lungs and serum. We found a positive correlation of ITIH4 in the lungs with BAL levels, but no correlation was observed between lung and serum ITIH4 levels. Thus, we suspected that BAL ITIH4 may be a lung-speci c protein in response to PM 2.5 exposure. Circulating ITIH4 may be related to the systemic response after PM 2.5 exposure. Our previous report indicated that serum ITIH4 had a good correlation with serum CRP levels in COPD patients after PM 10 exposure. Circulating ITIH4 may be associated with liver regulation into the systemic circulation as reported previously [14,15], and it was also correlated with circulating CRP levels. If lung-speci c ITIH4 has the same function as the acute-phase response, it may be associated with the in ammatory response in BAL or lung samples. Therefore, we examined the correlation of ITIH4 with oxidative stress and in ammation in BAL of rats. A good negative correlation between ITIH4 and IL-6 in BAL of rats was identi ed after PM 2.5 exposure. This result suggests that an ITIH4 de ciency may be involved in regulation of in ammatory responses in the lung environment after PM 2.5 exposure.
Our in vivo IHC results showed that pulmonary ITIH4 was mainly expressed in alveolar epithelial cells of rats after 6 months of PM 2.5 exposure. Thus, alveolar epithelial A549 cells were then used to investigate the underlying mechanisms in reaction to DEP exposure in the present study. Consistent with the in vivo results, ITIH4 expression decreased with DEP exposure in a dose-dependent manner. Previous evidence indicated that ITIH4 is involved in anti-apoptosis and matrix-stabilizing molecules [14,16]. Therefore, alterations in senescence, apoptosis, and matrix-stabilizing proteins were determined by tra c-related DEP exposure in A549 cells. Our results showed that DEPs not only reduced ITIH4, but also suppressed Sitr1 expression. In addition, downregulation of Sirt1 may have activated senescence-apoptotic p-ERK and caspase-3. Activation of p-ERK can downregulate Srti1 expression leading to upregulation of caspase-3 [17]. However, regulation of MMP9 and MMP12 was not observed in A549 cells by DEP exposure. Taking the in vivo and in vitro results together, PM exposure suppressed alveolar epithelial ITIH4 expression and activated cellular senescence and apoptosis pathways.
There are some limitations of this study that should be noted. We investigated BAL levels of ITIH4 in healthy subjects; however, ITIH4 expression in the lungs, especially alveolar epithelial cells, remains unclear. Further investigation into lung sections should be conducted. We observed that gaseous pollution reduced ITIH4 levels in the lungs and serum of rats after exposure. Although the associations between ITIH4 and gaseous pollutants were not identi ed in healthy human subjects, the gaseous effects on ITIH4 alterations should be examined in future work. Chemical effects of PM 2.5 on ITIH4 expression in the lungs were not examined in this study, and should be determined in the future.

Conclusion
In summary, our ndings provide evidence that PM 2.5 decreased ITIH4 in the lungs, which was associated with alveolar epithelial cell senescence and apoptosis. We further observed that there were different correlation results of ITIH4 between BAL and serum and with ITIH4 in the lungs. This implies that serum and BAL ITIH4 could originate from different organs. An ITIH4 de ciency could be a vital protein in regulating alveolar destruction due to PM 2.5 .

Subjects and bronchoalveolar lavage (BAL) collection
The study was approved by the Taipei Medical University-Joint Institutional Review Board (TMU-JIRB no. 201310027), and informed consent was obtained from all human subjects. In total, 47 healthy subjects (female: male of 24:23) with an average age of 53 years were recruited for the study. All subjects underwent a physical examination, including a pulmonary function test and blood collection. None of the subjects had a history of allergies or had suffered an airway infection within 3 months before recruitment into the study. The pulmonary function test was conducted immediately before bronchoscopy and included parameters of the forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV 1 ), taken with a spirometer. Bronchoscopy was performed with a exible beroptic bronchoscope under local anesthesia. A bronchial wash was performed by instilling 100 ml of normal saline at 37 °C into the proper lung segment, and then the uid was gently aspirated. Bronchoalveolar lavage uid (BAL) was thus collected. The BAL was ltered through a single layer of loose sterile surgical gauze and collected followed by centrifugation at 400 ×g for 10 min at 4 °C, and the supernatant was collected for biochemical analysis.
Air pollution exposure Exposure to PM 2.5 , NO 2 , and O 3 was estimated using a hybrid kriging-land-use regression (LUR) approach as described in previous reports [18][19][20]. Brie y, in situ observations of PM 2.5 , NO 2 , and O 3 were collected from Taiwan EPA air quality monitoring stations (https://airtw.epa.gov.tw/ENG/default.aspx). Predictor variables with an absolute value of a Spearman correlation coe cient of >0.4 with a prior direction of effect on the air pollution concentration were maintained and entered into stepwise linear regression procedures. Next, a set of interpolated pollutant levels were generated through a leave-one-out ordinary kriging function and added into the LUR model as a variable to improve the model performance. We obtained adjusted model R 2 values of 0.89 for PM 2.5 , 0.87 for NO 2 , and 0.74 for O 3 using the resultant hybrid kriging-LUR models. The models provided reliable air pollution exposure assessments in all subjects.

Chronic exposure to air pollution in vivo
The animal study was approved by the Animal and Ethics Review Committee of the Laboratory Animal Center at Taipei Medical University (no. LAC-2015-0290). Six-week-old male Sprague-Dawley (SD) rats obtained from the National Laboratory Animal Center (Taipei, Taiwan) were exposed to tra c-dominated urban air pollution. Rats were whole-body exposed to (1) clean air in the Laboratory Animal Center of Taipei Medical University (as the control group), (2) high-e ciency particulate air (HEPA)-ltered tra crelated urban air (as the HEPA group), and (3) tra c-dominated urban PM 2.5 (as the PM 2.5 group) for 3 and 6 months. The whole-body exposure system to unconcentrated particulate pollution with a 2.5-μm particle size classi er for rodents was previously described [21,22]. Gaseous pollutants used for rat exposure were referenced from the nearest Yonghe EPA air quality monitoring station. After 3 and 6 months of exposure, rats were euthanized followed by collection of BAL, lung tissues, and serum (n=8 in each group).

In vitro experiment
Human lung alveolar epithelial A549 cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in RPMI medium containing 10% fetal bovine serum, penicillin, and streptomycin. Cells were incubated at 37 C with 95% humidity and 5% CO 2 . Standard Reference Material (SRM) diesel exhaust particles (DEPs) obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) were used to expose cells at 0 (control), 50, and 100 μg/mL at 37 C for 24 h in a humidi ed atmosphere with 5% CO 2 (n=6 per group). Supernatants and cell lysates were collected for biochemical analyses.

Enzyme-linked immunosorbent assay (ELISA)
An ELISA was used to determine levels of 8-isoprostane (Cayman, Ann Arbor, MI, USA) and IL-6 (R&D Systems, Minneapolis, MN, USA) in BAL of humans and rats and in cell supernatants. ITIH4 in human BAL, rat BAL, lung lysates, and serum was examined using an ELISA (MyBioSource, San Diego, CA, USA). Details of the analytical procedures followed the manufacturer's instructions. Results obtained from lung lysates are presented after adjusting for the total protein.

Western blotting
Expressions of ITIH4, sirtuin 1 (Sirt1), phosphorylated extracellular signal-regulated kinase (p-ERK), caspase-3, matrix metallopeptidase 9 (MMP9), and MMP12 were examined using Western blotting as described in our previous report [23]. ). An HRP-labeled secondary antibody was incubated and washed with TBST after blocking. Enhanced chemiluminescence Western blotting reagents were used. Images were taken using the ChemiDoc MP imager (Bio-Rad, Hercules, CA, USA). Quantitative data were obtained using Image-Pro vers. 4 (Media Cybernetics, MD, USA) for Windows. All data were adjusted to the control (multiples of change of the control) as previously reported [24].

Statistical analysis
We applied a linear regression model to examine associations between air pollutants and BAL biomarkers in healthy subjects. Exposure variables were 1-year average PM 2.5 , NO 2 , and O 3 , and dependent variables were ITIH4, 8-isoprostane, and IL-6. Each regression model included age, sex, and current smoker (yes or no). The effects of air pollutants on BAL biomarkers are expressed as unit changes (β) multiplied by the interquartile range (IQR). A nonparametric Kruskal-Wallis test with Dunn's post-hoc test was used to compare multiple variables. Pearson's correlation was used to determine correlations of lung ITIH4 with BAL ITIH4 and serum ITIH4. All of the statistical analyses in this study were conducted with SPSS 15.0 software (SPSS, New York, NY, USA). The level of signi cance for all of the statistical analyses was set to p<0.05.

Declarations
Ethical Approval and Consent to participate Human study was approved by the Taipei Medical University-Joint Institutional Review Board (TMU-JIRB no. 201310027), and informed consent was obtained from all human subjects.
All the animal protocols were prepared in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Center at Taipei Medical University (approval no. 20130531; Taipei, Taiwan).

Consent for publication
Not applicable.

Availability of supporting data
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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