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Black carbon particles and ozone-oxidized black carbon particles induced lung damage in mice through an interleukin-33 dependent pathway
Science of the Total Environment 644 (2018) 217–228
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Black carbon particles and ozone-oxidized black carbon particles induced lung damage in mice through an interleukin-33 dependent pathway Hongqian Chu a,b, Weidong Hao a,b, Zhiyuan Cheng a,b, Yao Huang a,b, Siqi Wang a,b, Jing Shang d, Xiaohong Hou a,b, Qinghe Meng a,b, Qi Zhang a,b, Lixia Jia a,b, Wenjuan Zhou a,b, Pengmin Wang a,b, Guang Jia c, Tong Zhu d, Xuetao Wei a,b,⁎ a
Department of Toxicology, School of Public Health, Peking University, Beijing 100191, PR China Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, PR China c Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, Beijing 100191, PR China d State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, PR China b
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• oBC has stronger potency in inducing lung inflammation in mice than BC. • IL-33 neutralizing antibody prevented BC and oBC induced lung damage. • IL-33 neutralizing antibody prevented BC and oBC induced lung damage through MAPK and PI3K/AKT pathways.
a r t i c l e
i n f o
Article history: Received 25 April 2018 Received in revised form 25 June 2018 Accepted 26 June 2018 Available online xxxx Editor: Jay Gan Keywords: BC oBC
a b s t r a c t Black carbon (BC) is a key component of atmospheric particles which has adverse effects on human health. Oxidation could lead to chemical property and toxicity potency changes of BC. The key cytokines participating in lung damage in mice induced by BC and ozone-oxidized BC (oBC) particles have been investigated in this study. It was concluded that oBC has stronger potency of inducing lung damage in mice comparing to BC. IL-6 and IL-33 were hypothesized to play important roles in this damage. Accordingly, IL-6 and IL-33 neutralizing antibodies were used to explore which cytokine might play a key role in lung inflammation induced by BC and oBC. As a result, IL-6 neutralizing antibody did not alleviate the lung damage induced by BC and oBC. However, IL-33 neutralizing antibody prevented BC and oBC induced lung damage. Furthermore, IL-33 neutralizing antibody treatment reduced IL-6 mRNA expression. It is hypothesized that MAPK and PI3K-AKT pathways might be
Abbreviations: α-IL-33, IL-33 neutralizing antibody; Akt, v-akt murine thymoma viral oncogene homolog; BALF, bronchoalveolar lavage fluid; BC, Black carbon; BET, BrunauerEmmett-Teller; CXCL, chemokine (C-X-C motif) ligand; ERK, Extracellular signal-regulated kinase; H&E, hematoxylin and eosin; IL-6, Interleukin 6; IL-33, Interleukin 33; JNK, c-Jun Nterminal kinase; MAPK, Mitogen-activated protein kinase; MWCNT, Multi-walled carbon nanotube; oBC, Ozone-oxidized black carbon; PM, Particulate matter; ROS, Reactive oxygen species; TEM, Transmission electron microscopy. ⁎ Corresponding author at: Department of Toxicology, School of Public Health, Beijing 100191, PR China. E-mail address: [email protected] (X. Wei).
https://doi.org/10.1016/j.scitotenv.2018.06.329 0048-9697/© 2018 Elsevier B.V. All rights reserved.
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involved in the oBC particles caused lung damage. It was concluded that IL-33 plays a key role in BC and oBC induced lung damage in mice. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Atmospheric particulate matter (PM) pollution is one of the most pressing issues in worldwide air quality regulation and represents one of the biggest sources of uncertainty in current climate simulations (Fuzzi et al., 2015). PM has drawn a wide attention in the past decades because of its adverse health effects. Black carbon (BC, also called soot) is an important constituent of PM, mainly produced by incomplete combustion, which is the second-largest contributor to global warming (Jacobson, 2001; Li et al., 2013a). The release of BC increased dramatically in recent years due to the increased biomass burning and fossil fuel consumption. BC is a traffic-related combustion product and is a new indicator for the adverse effects of traffic-related air pollution (Dons et al., 2012; Janssen et al., 2011; Wilker et al., 2013). Almost 75% of the PM products from diesel mobile is BC (Ramanathan, 2013). In addition to the effects to environment, BC or BC-related pollutants also reveal health detriments such as cardiovascular disease (Clougherty and Kubzansky, 2009; McCracken et al., 2010; Wittkopp et al., 2016; Nichols et al., 2013) and respiratory disease (McCracken et al., 2010; Geng et al., 2013; Smith et al., 2009). BC has large specific surface area that can react with other substances and it has been studied extensively (Xu et al., 2015; Han et al., 2013). BC undergoes an aging process when emitted into atmosphere which leads to the change of particle morphology, chemical features and redox activity (Li et al., 2009; Rattanavaraha et al., 2011; Verma et al., 2009). Ozone (O3) is a common urban air pollutant that is harmful to human health (EPA, 2006) and an important oxidant in atmospheric aging which can react with soot or BC in a mode of heterogeneous reaction (Han et al., 2012a; Han et al., 2012b). Even though there were lots of studies about health effects caused by O3 or BC, only few toxicological studies were about the health effects of ozone-particle (Jerrett et al., 2009). Interleukin (IL)-6 is a cytokine secreted by various types of cells such as macrophages and lymphocytes (Yudkin et al., 2000). IL-6 participates in a broad spectrum of biological events, such as immune responses, hemopoiesis and acute-phase reactions. Elevated IL-6 contributes to pathogenesis of various inflammatory diseases (Mihara et al., 2012). Exposure to diesel exhaust particles caused high level of IL-6 in mice and rats (Holland et al., 2015; Totlandsdal et al., 2015; Yanagisawa et al., 2014). Also, various of in vitro experiments revealed that diesel exhaust particles could induce the high expression of IL-6 in many different kinds of cells such as PBMC (peripheral blood mononuclear cell), human bronchial epithelial cells, peripheral blood mononuclear cells (Sarkar et al., 2014; Srivastava et al., 2014; Vaughan et al., 2014; Totlandsdal et al., 2010). Furthermore, in our previous research, we
found that C57BL/6 mice treated by BC and oBC particles for 4 weeks resulted in the increased expression of IL-6 mRNA in lung tissue and high level of IL-6 in BALF (Chu et al., 2016a; Jin et al., 2016). IL-33 is a newly discovered pro-inflammatory cytokine belonging to the IL-1 family. IL33 plays an important role in regulating both Th1 and Th2 cell responses (Liew, 2012) and was found involved in human inflammatory diseases (Beltran et al., 2010; Milovanovic et al., 2012; Chu et al., 2016b). Multi-wall carbon nanotube (MWCNTs) instillation impaired pulmonary function in C57BL/6 mice and leads to the increase of IL-33 amount in bronchoalveolar lavage fluid (BALF) (Wang et al., 2011). When treated ICR mice with MWCNTs for 34 weeks, the IL-33 mRNA expression in lung tissue was significantly increased (Yamaguchi et al., 2012). In our previous research, when mice were treated with BC and ozone-oxidized BC (oBC), the IL-33 mRNA expression in lung tissue and the content of IL-33 in BALF both clearly increased (Chu et al., 2016a; Jin et al., 2016). To date, the studies of IL-33 and its relation to particulate matter were mainly focused on allergy diseases such as asthma caused by PM exposure (Li et al., 2013b; Lee et al., 2014; Shadie et al., 2014; Leon Hsu et al., 2015; Li and Buglak, 2015). Whether chronic inflammation in lung is involved after PM exposure or not is still ambiguous. Herein, based on our previous research findings, it is important to explore which cytokine plays a key role in mice lung inflammation induced by BC and oBC, and the potential mechanism involved in those processes. 2. Materials and methods 2.1. Preparation and characterization of BC and oBC BC and oBC particles were provided by Professor Jing Shang (College of Environmental Sciences and Engineering, Peking University). Fig. 1 presents a schematic diagram of oBC. In brief, ozone was generated by the photolysis of oxygen (99.9% purity) with an ultraviolet lamp (ZW 18D 15Y-Z356, Cnlight, China) with a wavelength of 185 nm. A mixture of O2, O3 and N2 with a flow rate of 200 ml/min was made to flow through the tube in which BC powder was uniformly spread. O3 reacted with BC for 120 min at room temperature. N2 was purged through the tube before and after the reaction (Fig. 1). The features and characteristics of BC and oBC were also analyzed by Professor Jing Shang's group (Li et al., 2013a; Chu et al., 2016c; Gao et al., 2016). BC and oBC particles were stored at 4 °C in a black container. Both BC and oBC have the size of approximately 30 nm. The TEM images of original BC and O3-oxidized (100 ppm, 120 min) BC were shown in Fig. S1. Brunauer–Emmett–Teller (BET) specific surface areas of BC
Fig. 1. Schematic diagram of oBC production.
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Fig. 2. Total cell counts in BALF of mice. (A) Mice instilled with BC, oBC, BC/α-IL-6, oBC/α-IL-6. (B) Mice instilled with BC, oBC, BC/α-IL-33, oBC/α-IL-33. ##p b 0.01 versus the control group, **p b 0.01,*p b 0.05. n = 7.
and oBC were 96.39 ± 0.089 m2/g and 97.63 ± 0.064 m2/g respectively. BC showed a broad hydrodynamic size distribution (675.9 ± 17.6 nm), and the size distribution of oBC was relatively narrow (319.3 ± 7.9 nm) (ZetaSizer Nano ZS90, Malvern, UK). Also, Oxygen content was increased and more oxygen-containing functions were produced in oBC. Furthermore, oBC exhibited a strong EPR signal and improved generation of free radicals (Li et al., 2013a). Compared with BC, oBC tended
to be more dispersive in water. The particles were suspended in PBS, and then sonicated for 10 min before intratracheal instillation. 2.2. Animals C57BL/6 mice (female, 18–22 g) were purchased from Vital River Laboratory Technology Co. Ltd. (Beijing, China). All mice were kept in
Fig. 3. IL-6 and IL-33 mRNA expression in lung tissue of mice. (A) Mice instilled with BC, oBC, BC/α-IL-6, oBC/α-IL-6. (B) Mice instilled with BC, oBC, BC/α-IL-33, oBC/α-IL-33. ##p b 0.01 versus control group *p b 0.05, **p b 0.01. n = 7.
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specific pathogen-free conditions. Standard mice feed and tap water were available. Permission of the local ethics committee was obtained and all animal experiments were approved by the Animal Care Committee of Peking University according to Chinese law.
CD4-PECY5, CD8-FITC, FOXP3-FITC, and IL-17-PE, were purchased from eBioscience. Cells sub-type was analyzed by Beckman Coulter Flow Cytometer. The details of staining and blocking procedures were shown in supplementary.
2.3. Experimental design
2.9. Histological assessment of lung tissue
Animals were divided into 6 groups: negative control, neutralizing antibody control (α-IL-6: Biolegend, USA; α-IL-33: R&D systems, USA), particles treated groups (200 μg of 30 nm BC or oBC, n = 7) and particles together with neutralizing antibody groups.
Lung tissues were separated and fixed in 10% buffered formalin, and then embedded in paraffin, at last sectioned and stained the tissue with hematoxylin and eosin. The infiltration of lymphocytes and macrophages together with the swelling of alveolar walls were used to determine the severity of inflammation. The method of histology score of lung tissue were shown in supplementary.
2.4. Intratracheal instillation 5% chloral hydrate was intraperitoneal injected to each mouse (0.1 ml/10 g body weight) for anesthesia. BC and oBC suspensions were sonicated for 15 min prior to intratracheal instillation. The vehicle control mice were instilled with 0.06 ml of PBS and the antibody control mice were instilled with 20 μg α-IL-6 or α-IL-33 dissolved in 0.06 ml PBS. The mice were instilled twice a week for 4 weeks. The body weight of each mouse was measured before sacrificed by cervical dislocation after isoflurane anesthesia.
2.10. Western blot analysis
Mice were euthanized and lavaged with two separate 0.8 ml aliquots of PBS and collect at least 1.2 ml lavage. The lavages were centrifuged (300 ×g, 10 min) and supernatants were collected and then stored at −80 °C for cytokines testing. The cell pellets from lavages were resuspended in 1 ml of PBS containing 1% BSA. Cells were counted using Counter Star (Shang Hai, China). Cells were smeared to the slides and then stained with Diff Quik (American Scientific Inc., Sewickly, PA). About 300 cells were counted and two kind of cell subtypes (lymphocytes, neutrophils) were identified.
Lung tissue was weighted and homogenized in ice-cold lysis buffer (50 mmol/l Tris, 150 mmol/l NaCl, 1% NP-40, 50 mmol/l NaF, 1 mmol/l Na3VO4, 5 mmol/l EDTA, 50 mmol/l NaPPi, 1 mmol/l DTT, 1 mmol/l PMSF, 1% (v/v) protease inhibitors, 1% (v/v) phosphatase inhibitors) with 1% PMSF (Beyotime, China) and lysed for 30 min at 4 °C. Supernatants were collected after centrifugation (13,000 rpm for 5 min). Protein concentration was determined using Bradford Protein Quantitation Kit (Beyotime, China). Proteins were quantified and mixed with 2 × loading buffer 1:1. Equal amounts of protein were subjected to SDS/PAGE under reducing conditions, then transferred proteins to nitrocellulose filters. 5% milk (Sigma-Aldrich, USA; Dissolved in TBS with 0.1% Tween 20) was used to block the membrane for 2 h at room temperature. The blots were incubated with the primary antibody overnight at 4 °C and then incubated for 2 h with secondary antibody. Immunoreactive bands were visualized by chemiluminescence detection. β-actin was used as a loading control. Band densities were then quantified by densitometry (Tanon-4500). All experiments were performed independently at least three times.
2.6. ELISA analysis
2.11. Statistical analysis
The level of IL-6 and IL-33 in BALF was determined using ELISA kits. All of the kits were purchased from eBioscience Co. Ltd. (USA) and the procedures were performed according to the manufacturer's instructions.
Data are presented as mean ± S.D. One-way analysis of variance (ANOVA) followed by Dunnett t-test (SPSS 13.0 Peking University) were used to compare the difference more than two groups. A p value of b0.05 was considered significant.
2.7. RT-PCR analysis
3. Results
At the real-time PCR step, 2 μl cDNA was used for quantitative realtime PCR using SYBR Green kit (TaKaRa, DRR041A). The reaction was performed according to the standard protocol in the IQTM5 Multicolor Real-Time PCR Detection System (Bio-RAD). The endogenous control in the detection was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each sample was analyzed in three duplicates. The relative changes of gene expression were calculated using the following formula: fold change in gene expression, 2 − ΔΔCt = 2 − {ΔCt (vehicle or particles − treated samples) − ΔCt (untreated control)}, where ΔCt = Ct (detected genes) − Ct (GAPDH) and Ct represents the threshold cycle number. The details of PCR procedures were shown in supplementary. The primers used are described in TableS1.
3.1. Image of lung after instillation
2.5. Bronchoalveolar lavage and cell counts
2.8. Organ coefficient and flow cytometry analysis Mediastinal lymph nodes were ground into single cell suspensions after separated and weighted. Organ coefficient was the weight of the organ divided by the body weights. Cells were stained with a monoclonal antibody. Florescence-conjugated antibodies, including CD3-PE,
The change of lung after instillation of BC or oBC can be seen in Fig. S2. It was visible that BC and oBC dispersed in the lung after instillation. Also, BC and oBC could be found in mediastinal lymph nodes. 3.2. Total cell counts in BALF Total cell counts in BALF were calculated using Counter Star (Shang Hai, China). As shown in Fig. 2, mice treated with 200 μg BC or oBC presented an increase of total cell counts in BALF (p b 0.01). And the total cell counts in BALF of mice treated with oBC were significantly higher than that of BC-treated group (p b 0.05). Total cell counts in BALF of mice treated with BC/α-IL-6 or oBC/α-IL-6 were significantly lower than that of BC or oBC-treated group respectively but were still higher than that of PBS groups (p b 0.05) (Fig. 2A). However, mice treated with BC/α-IL-33 or oBC/α-IL-33 showed no increase of cell counts in BALF compared with control groups (Fig. 2B). Furthermore, the counts of lymphocytes and neutrophils in BALF of oBC group were higher
Fig. 4. CXCL1, CXCL2, CXCL5 and CXCL10 mRNA expression in lung tissue of mice. (A) Mice treated with BC, oBC, BC/anti-IL/6, oBC/α-IL-6. (B) Mice treated with BC, oBC, BC/anti-IL/33, oBC/ α-IL-33. Data are representative of three independent experiments. #p b 0.05 versus control group, ##p b 0.01 versus control group, *p b 0.05, **p b 0.01. n = 7.
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than that of BC group (p ≤ 0.05). α-IL-33 could inhibit BC and oBC induced increase of lymphocytes and neutrophils in BALF whereas α-IL6 not. There were no differences between neutralizing antibody group and negative control group (Fig. S3). 3.3. Cytokines mRNA expression in lung tissue As shown in Fig. 3, mice treated with BC or oBC both showed high expression of IL-6 and IL-33. Furthermore, IL-6 and IL-33 expression in oBC-treated group were significantly higher than BC-treated group (p b 0.05). However, IL-6 mRNA expression in BC/α-IL-6 or oBC/α-IL-6treated groups was significantly lower than that treated with BC or oBC respectively (p b 0.01). But IL-33 mRNA expression in mice treated with BC/α-IL-6 or oBC/α-IL-6 had no difference with BC or oBC-treated group, and much higher than control groups (p b 0.01 or p b 0.05) (Fig. 3A). However, IL-6 and IL-33 expression in mice treated with BC/ α-IL-33 or oBC/α-IL-33 were significantly lower than that treated with BC or oBC, and no differences could be found when compared with control groups (p b 0.01 or p b 0.05) (Fig. 3B). 3.4. Chemokines mRNA expression in lung tissue Chemokines mRNA expression in lung tissue was also detected. As shown in Fig. 4, mice treated with BC or oBC both showed high expression CXCL1, CXCL2, CXCL5 and CXCL10. Furthermore, chemokines mRNA expression in oBC-treated group were significantly higher than BC-treated group (p b 0.05). Surprisingly, chemokines mRNA expression in mice treated with BC/α-IL-6 or oBC/α-IL-6 had no difference compared with BC or oBC-treated group, and significantly higher than control groups (p b 0.01 or p b 0.05) (Fig. 4A). However, chemokines mRNA expression in BC/α-IL-33 or oBC/α-IL-33-treated mice was significantly lower than that of BC or oBC-treated group, and no differences could be found when compared with control groups (p b 0.01 or p b 0.05) (Fig. 4B). 3.5. Cytokines level in BALF and lung tissue homogenates Secretion of cytokines in BALF Cytokine levels in BALF and lung homogenates was detected using ELISA kits. As can be seen in Fig. 5, the level of IL-33 and IL-6 in BALF and lung tissue homogenates of BC group and oBC group were significantly higher than that of control groups (p b 0.01). The level of IL-33 and IL-6 in mice treated with the two particles together with α-IL-6 had no difference with BC or oBC group respectively and significantly higher than that of control groups (Fig. 5B). However, in BC/α-IL-33 or oBC/α-IL-33-treated group, the level of IL-33 and IL-6 in BALF and lung homogenates were significantly lower than that of BC group and oBC-treated group (p b 0.01) and had no difference compared with control groups (Fig. 5A). 3.6. Organ coefficient and T cell sub-types of mediastinal lymph nodes As shown in Fig. S4 the mediastinal lymph nodes coefficient of mice treated with BC and oBC was significantly higher than that of control groups (p b 0.01 or p b 0.05). The mediastinal lymph nodes coefficient of mice treated with BC/α-IL-6 or oBC/α-IL-6 was higher than that of control groups and had no difference compared with BC or oBCtreated group (Fig. S4A). However, the mediastinal lymph nodes coefficient of mice treated with BC/α-IL-33 or oBC/α-IL-33 had no difference compared with control groups (Fig. S4B). T cell sub-types of mediastinal lymph nodes were measured using Beckman Coulter Flow Cytometer. As shown in Fig. S5, the percentage of CD4+ T cells in mediastinal lymph nodes was increased after instilled with BC or oBC. But, the percentage of CD4+ T and Th17 cells in mediastinal lymph nodes of mice instilled with BC/α-IL-6 or oBC/α-IL-6 was
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significantly higher than that of control groups and had no difference compared with BC or oBC-treated group. The percentage of CD4+ T cells in mice instilled with BC/α-IL-33 or oBC/α-IL-33 was lower than that of BC or oBC-treated group. So was the result of Th17 cells. However, the percentage of CD8+ T cells and Treg cells had no difference of in mediastinal lymph nodes of each group (Fig. 6). 3.7. Histopathology analysis of lung tissue As shown in Fig. 7, inflammatory cells infiltration and alveolar swelling and thickening could be seen in the lung tissue of BC and oBCtreated mice and the dispersed particles in lung tissue could be clearly observed. The oBC-treated group showed much more severe lung inflammation when compared with BC groups. There were still lots of inflammatory cells infiltration and alveolar swelling and thickening in mice treated with BC/α-IL-6 or oBC/α-IL-6, which had no differences compared with BC or oBC-treated groups (Fig. 7A). However, even though the dispersed particles in lung tissues could be seen in mice treated with BC/α-IL-33 or oBC/α-IL-33, inflammatory cell infiltration and alveolar swelling and thickening were less observed when compared with BC or oBC-treated mice (Fig. 7B). The histopathology score results of the lung tissue were shown in Fig. S6. 3.8. Change of signaling pathway To confirm the hypothesis that MAPK is one of the important signal pathway involved in BC and oBC induced lung damage, the relative amount of signaling molecules were examined. As shown in Fig. S7A, the expression of ERK, JNK, P38 and their phosphorylated kinase were assessed. Phosphorylation of ERK, P38 and JNK increased after exposure to BC and oBC. Moreover, the phosphorylation of ERK, JNK and P38 in the oBC group occurred more than that in BC-treated group. Interestingly, phosphorylation of ERK, P38 and JNK in BC/α-IL-6 or oBC/α-IL6-treated group had no difference with that of BC or oBC group but still higher than control groups (Fig. S7A–C). However, phosphorylation of ERK, P38 and JNK were decreased after exposure to BC/α-IL-33 or oBC/α-IL-33 compared with BC or oBC group, and had no differences from control groups (Fig. 8A–C). Furthermore, phosphorylation of PI3K and AKT (Fig. S7D & E) increased after exposure to BC and oBC which was consistent with our pervious study. Similarly, the phosphorylation of PI3K and AKT in the oBC group was higher BC-treated group. Phosphorylation of PI3K and AKT in BC/α-IL-6 or oBC/α-IL-6 treatment group had no difference compared with BC or oBC group (Fig. S7D & E). On the contrary, Phosphorylation of PI3K and AKT decreased after exposure to BC/α-IL-33 or oBC/α-IL-33 compared with BC or oBC group (Fig. 8D & E). All these data indicated that the pro-inflammatory action of oBC is stronger than that of BC, and IL-33 neutralizing antibody inhibited BC and oBC induced lung inflammation in mice. IL-33 played a key role in BC and oBC induced lung damage in mice. 4. Discussion Ambient air pollution is a complex mixture of gaseous and particulate phase pollutants. The residence time of each pollutant in the atmosphere varies greatly. The major substances being monitored are O3 and PM (Madden et al., 2000). There are several reasons for investigating the health effects of BC and oBC. Black carbon is a key component of PM 2.5 and mainly exists in traffic-related particles. Many epidemiology studies have shown that atmospheric black carbon has negative relationship to human health (McCracken et al., 2010; Nichols et al., 2013; Geng et al., 2013; Smith et al., 2009). Ozone is a pollutant that coexists with PM, even though human mortality has been associated predominantly with
Fig. 5. Effects of α-IL-33 and α-IL-6 on BC and oBC induced secretion of cytokines in BALF and lung tissue homogenates. (A) Mice treated with BC, oBC, BC/anti-IL/6, oBC/α-IL-6. (B) Mice treated with BC, oBC, BC/anti-IL/33, oBC/α-IL-33. ##p b 0.01 versus control group, *p b 0.05, **p b 0.01. n = 7.
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Fig. 6. Effects of BC, oBC, BC/α-IL-33 and oBC/α-IL-33 on the percentage of CD4+ T cells and Th17 cells in mediastinal lymph nodes. ##p ≪ 0.01 versus control group, **p b 0.01. n = 7.
the concentration of PM, a relationship between ozone levels and cardiopulmonary deaths has also been observed (Kinney and Özkaynak, 1991). Furthermore, BC can react with ozone forming secondary pollution and yet the studies about health effects of ozone-oxidized BC are rare. Our previous studies have found/proved that IL-33 and IL-6 play important roles in BC and oBC induced lung inflammation (Chu et al., 2016a). To date, the underlying mechanism of BC or oBC induced lung damage remains unclear. In order to explore the potential effects of IL33 and IL-6 in the process of lung inflammation induced by the two particles, α-IL-33 and α-IL-6 were used to prevent the bioactivity of each cytokine so as to lift the veil enveloping the truth.
Previous studies have shown that IL-33 played a key role in the induction and perpetuation of the Th2-based immunological response such as allergic asthma (Liew et al., 2010; Cayrol and Girard, 2014). The IL-33 and IL-6 mRNA expression increased in lung tissue in mice models of allergic inflammation (Kurokawa et al., 2011; Bunting et al., 2013). Here we found that IL-33 and IL-6 mRNA expression in lung tissue and the secretion of IL-33 and IL-6 in BALF were significantly increased in mice instilled with BC and oBC. As our hypothesis presented, the mRNA expression and the level of two cytokines in BALF of mice treated with BC/α-IL-33 or oBC/α-IL-33 were lower than that of mice treated with BC or oBC and there were no difference
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Fig. 7. Histological assessment of lung damage (H&E). (A) Mice treated with BC, oBC, BC/anti-IL/6, oBC/α-IL-6. (B) Mice treated with BC, oBC, BC/anti-IL/33, oBC/α-IL-33. Arrows indicate the accumulation of inflammatory cells (Original magnification, 200×).
compared with PBS and α-IL-33 treatment group. Furthermore, the result of histopathology showed that lung inflammation was alleviated significantly after α-IL-33 treatment. These results indicated that IL-33 is very likely to play an important role in lung damage caused by BC or oBC. IL-6 is a cytokine produced by inflammatory cells and lung epithelial cells in response to various stimuli such as allergens and respiratory viruses (Yokoyama et al., 1995; Stadnyk, 1994; Marini et al., 1992). A recent study showed that IL-6 mRNA expression was high in mouse primary lung epithelial but not in immune cells resident in lung (Neveu et al., 2011). Here in our study, IL-6 and IL-33 mRNA expression in lung tissue and IL-6 concentration in BALF significantly increased in BC and oBC-treated groups which is in line with our previous findings (Chu et al., 2016a; Jin et al., 2016). The astonishing phenomenon is that IL-6 mRNA expression was reduced significantly after α-IL-6 treatment when exposed to two particles but IL-33 had not been influenced by α-IL-6 treatment. From the histopathology results, instilled mice with BC or oBC together with α-IL-6 didn't decrease lung inflammation compared with mice only instilled BC or oBC. BC and oBC could increase the chemokines mRNA expression in lung tissue and α-IL-33 treatment could inhibit this increase whereas α-IL-6 treatment had no effects which further indicated that IL-33 may play an important role in BC or oBC induced lung inflammation. Th17 cells have been described as a kind of Th cells which are different from Th1 and Th2 cells (Park et al., 2005; Harrington et al., 2005). It has been showed that IL-17 and IL-17-producing (Th17) cells act a pivotal part in inflammatory responses. Mice treated with PM 2.5 exhibited an apparent increase of Th17 cells (Xie et al., 2013), and fine particulate matters (PM 2.5) could change Th17/Treg balance as well as related
cytokines (Yang et al., 2014). We found that the percentage of Th 17 cells in mediastinal lymph nodes was significantly increased in mice treated with BC and oBC, but not in BC/α-IL-33 and oBC/α-IL-33treated groups. Interestingly, percentage of Th 17 cells in mice instilled with BC/α-IL-6 or oBC/α-IL-6 was still as high as BC or oBC-treated group. This may be due to the potential of IL-6 inducing Th 17 cells and IL-33 can induced IL-6 synthesis (Miller et al., 2008). IL-33 cannot induced Th17 cells but it can induce IL-6 thus further influence Th17 cells (Miller et al., 2008; Xu et al., 2008; Oboki et al., 2010). It has been reported that urban particles (UP) could increase the phosphorylation of ERK, P38 in rat pulmonary artery (Li et al., 2005). Also, cigarette smoking particles can also activate MAPK in rat cerebral arteries (Sandhu et al., 2010). Extensive studies have declared that particles such as ultrafine carbon particles, diesel exhaust particles could activate MAPK in vivo and vitro (Nel et al., 1998; Kim et al., 2005; Baulig et al., 2003; Totlandsdal et al., 2010). We have already found that BC and oBC could activate the MAPK pathway (Chu et al., 2016a). Here we found that BC and oBC together with α-IL-33 can alleviate this activation but α-IL-6 treatment does not show similar efficiency. This indicated that IL-33 plays an important role in this MAPK activation. Additionally, we explored effects of BC and oBC together with αIL-33 on PI3K/AKT pathway. In our previous results, BC and oBC could increase the phosphorylation of PI3K and AKT (Chu et al., 2016a). Here we observed that the activation of BC and oBC on PI3K/AKT pathway could be abrogated by α-IL-33 treatment. Meanwhile, histopathology score has not been reduced by α-IL-6 treatment during two particles exposure. All information from α-IL-33 treatment suggested that IL-33 decrease could alleviate the lung inflammation induced by BC and oBC. In this process, IL-6 was regulated by IL-
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Fig. 8. Neutralizing IL-33 inhibited BC and oBC activated MAPK and PI3K/AKT signaling pathways. (A & C) MAPK signaling pathway. (B & D) PI3K/AKT signaling pathway. *p b 0.05 versus the control group, **p b 0.01 versus the control group. #p b 0.05 versus the corresponding BC or oBC group, ##p b 0.01 versus the corresponding BC or oBC group.
33. Combined with the results from IL-6 intervention, it has been demonstrated that IL-33 is a critical initiator for chronic inflammation in lung exposed to BC and oBC. Whether IL-6 involved in this kind of chronic inflammation remains to be extrapolated.
It could be drawn the conclusion that MAPK and PI3K/AKT signal pathways are the downstream of IL-33. Whether both of these two pathways are the upstream of other cytokines involved in the inflammation still need to be proved.
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5. Conclusions In summary, the data from this study demonstrated that oBC caused much more lung inflammation than BC in mice and IL-33 plays a key role in this process. In addition, these effects might involve in the activation of MAPK and PI3K/AKT pathway. The specific mechanism of IL-33 in lung damage induced by BC or oBC needs further investigation. Acknowledgements We would particularly like to thank Professor Jing Shang (College of Environmental Sciences and Engineering, Peking University) for her encouraging support and valuable facilities to carry out this work. The authors acknowledge support from the Major Program of National Natural Science Foundation of China (Grant No. 21190051). Disclosure statement The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of this article. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.06.329. References Baulig, A., Garlatti, M., Bonvallot, V., Marchand, A., Barouki, R., Marano, F., Baeza-Squiban, A., 2003. Involvement of reactive oxygen species in the metabolic pathways triggered by diesel exhaust particles in human airway epithelial cells. Am. J. Phys. Lung Cell. Mol. Phys. 285, L671–L679. Beltran, C.J., Núñez, L.E., Díaz Jiménez, D., Farfan, N., Candia, E., Heine, C., Lopez, F., González, M.J., Quera, R., Hermoso, M.A., 2010. Characterization of the novel ST2/IL33 system in patients with inflammatory bowel disease. Inflamm. Bowel Dis. 16, 1097–1107. Bunting, M.M., Shadie, A.M., Flesher, R.P., Nikiforova, V., Garthwaite, L., Tedla, N., Herbert, C., Kumar, R.K., 2013. Interleukin-33 drives activation of alveolar macrophages and airway inflammation in a mouse model of acute exacerbation of chronic asthma. Biomed. Res. Int. 2013. Cayrol, C., Girard, J., 2014. IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy. Curr. Opin. Immunol. 31, 31–37. Chu, H., Shang, J., Jin, M., Li, Q., Chen, Y., Huang, H., Li, Y., Pan, Y., Tao, X., Cheng, Z., 2016a. Comparison of lung damage in mice exposed to black carbon particles and ozoneoxidized black carbon particles. Sci. Total Environ. 573, 303–312. Chu, H., Li, J., Huang, H., Hao, W., Duan, L., Wei, X., 2016b. Protective effects of tranilast on oxazolone-induced rat colitis through a mast cell-dependent pathway. Dig. Liver Dis. 48, 162–171. Chu, H., Shang, J., Jin, M., Li, Q., Chen, Y., Huang, H., Li, Y., Pan, Y., Tao, X., Cheng, Z., Meng, Q., Jia, G., Zhu, T., Wei, X., Hao, W., 2016c. Comparison of lung damage in mice exposed to black carbon particles and ozone-oxidized black carbon particles. Sci. Total Environ. 573, 303–312. Clougherty, J.E., Kubzansky, L.D., 2009. A framework for examining social stress and susceptibility to air pollution in respiratory health. Environ. Health Perspect. 117, 1351–1358. Dons, E., Panis, L.I., Van Poppel, M., Theunis, J., Wets, G., 2012. Personal exposure to black carbon in transport microenvironments. Atmos. Environ. 55, 392–398. EPA, 2006. Air Quality Criteria for Ozone and Related Photochemical Oxidants., EPA/600/R -05/004aF-cF 2006. Fuzzi, S., Baltensperger, U., Carslaw, K., Decesari, S., Denier Van Der Gon, H., Facchini, M.C., Fowler, D., Koren, I., Langford, B., Lohmann, U., Nemitz, E., Pandis, S., Riipinen, I., Rudich, Y., Schaap, M., Slowik, J.G., Spracklen, D.V., Vignati, E., Wild, M., Williams, M., Gilardoni, S., 2015. Particulate matter, air quality and climate: lessons learned and future needs. Atmos. Chem. Phys. 15, 8217–8299. Gao, X., Xu, H., Shang, J., Yuan, L., Zhang, Y., Wang, L., Zhang, W., Luan, X., Hu, G., Chu, H., 2016. Ozonized carbon black induces mitochondrial dysfunction and DNA damage. Environ. Toxicol. 32, 944–955. Geng, F., Hua, J., Mu, Z., Peng, L., Xu, X., Chen, R., Kan, H., 2013. Differentiating the associations of black carbon and fine particle with daily mortality in a Chinese city. Environ. Res. 120, 27–32. Han, C., Liu, Y., Ma, J., He, H., 2012a. Effect of soot microstructure on its ozonization reactivity. J. Chem. Phys. 137, 84507. Han, S., Jing, S., Tong, Z., Li, Z., Jun-Hui, Y., 2012b. Heterogeneous oxidation of SO2 by ozone on the surface of black carbon particles. Chem. J. Chin. Univ. 33, 2295–2302. Han, C., Liu, Y., He, H., 2013. Role of organic carbon in heterogeneous reaction of NO2 with soot. Environ. Sci. Technol. 47, 3174–3181.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Black carbon particles and ozone-oxidized black carbon particles induced lung damage in mice through an interleukin-33 dependent pathway Hongqian Chu a,b, Weidong Hao a,b, Zhiyuan Cheng a,b, Yao Huang a,b, Siqi Wang a,b, Jing Shang d, Xiaohong Hou a,b, Qinghe Meng a,b, Qi Zhang a,b, Lixia Jia a,b, Wenjuan Zhou a,b, Pengmin Wang a,b, Guang Jia c, Tong Zhu d, Xuetao Wei a,b,⁎ a
Department of Toxicology, School of Public Health, Peking University, Beijing 100191, PR China Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Beijing 100191, PR China c Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, Beijing 100191, PR China d State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, PR China b
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• oBC has stronger potency in inducing lung inflammation in mice than BC. • IL-33 neutralizing antibody prevented BC and oBC induced lung damage. • IL-33 neutralizing antibody prevented BC and oBC induced lung damage through MAPK and PI3K/AKT pathways.
a r t i c l e
i n f o
Article history: Received 25 April 2018 Received in revised form 25 June 2018 Accepted 26 June 2018 Available online xxxx Editor: Jay Gan Keywords: BC oBC
a b s t r a c t Black carbon (BC) is a key component of atmospheric particles which has adverse effects on human health. Oxidation could lead to chemical property and toxicity potency changes of BC. The key cytokines participating in lung damage in mice induced by BC and ozone-oxidized BC (oBC) particles have been investigated in this study. It was concluded that oBC has stronger potency of inducing lung damage in mice comparing to BC. IL-6 and IL-33 were hypothesized to play important roles in this damage. Accordingly, IL-6 and IL-33 neutralizing antibodies were used to explore which cytokine might play a key role in lung inflammation induced by BC and oBC. As a result, IL-6 neutralizing antibody did not alleviate the lung damage induced by BC and oBC. However, IL-33 neutralizing antibody prevented BC and oBC induced lung damage. Furthermore, IL-33 neutralizing antibody treatment reduced IL-6 mRNA expression. It is hypothesized that MAPK and PI3K-AKT pathways might be
Abbreviations: α-IL-33, IL-33 neutralizing antibody; Akt, v-akt murine thymoma viral oncogene homolog; BALF, bronchoalveolar lavage fluid; BC, Black carbon; BET, BrunauerEmmett-Teller; CXCL, chemokine (C-X-C motif) ligand; ERK, Extracellular signal-regulated kinase; H&E, hematoxylin and eosin; IL-6, Interleukin 6; IL-33, Interleukin 33; JNK, c-Jun Nterminal kinase; MAPK, Mitogen-activated protein kinase; MWCNT, Multi-walled carbon nanotube; oBC, Ozone-oxidized black carbon; PM, Particulate matter; ROS, Reactive oxygen species; TEM, Transmission electron microscopy. ⁎ Corresponding author at: Department of Toxicology, School of Public Health, Beijing 100191, PR China. E-mail address: [email protected] (X. Wei).
https://doi.org/10.1016/j.scitotenv.2018.06.329 0048-9697/© 2018 Elsevier B.V. All rights reserved.
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involved in the oBC particles caused lung damage. It was concluded that IL-33 plays a key role in BC and oBC induced lung damage in mice. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Atmospheric particulate matter (PM) pollution is one of the most pressing issues in worldwide air quality regulation and represents one of the biggest sources of uncertainty in current climate simulations (Fuzzi et al., 2015). PM has drawn a wide attention in the past decades because of its adverse health effects. Black carbon (BC, also called soot) is an important constituent of PM, mainly produced by incomplete combustion, which is the second-largest contributor to global warming (Jacobson, 2001; Li et al., 2013a). The release of BC increased dramatically in recent years due to the increased biomass burning and fossil fuel consumption. BC is a traffic-related combustion product and is a new indicator for the adverse effects of traffic-related air pollution (Dons et al., 2012; Janssen et al., 2011; Wilker et al., 2013). Almost 75% of the PM products from diesel mobile is BC (Ramanathan, 2013). In addition to the effects to environment, BC or BC-related pollutants also reveal health detriments such as cardiovascular disease (Clougherty and Kubzansky, 2009; McCracken et al., 2010; Wittkopp et al., 2016; Nichols et al., 2013) and respiratory disease (McCracken et al., 2010; Geng et al., 2013; Smith et al., 2009). BC has large specific surface area that can react with other substances and it has been studied extensively (Xu et al., 2015; Han et al., 2013). BC undergoes an aging process when emitted into atmosphere which leads to the change of particle morphology, chemical features and redox activity (Li et al., 2009; Rattanavaraha et al., 2011; Verma et al., 2009). Ozone (O3) is a common urban air pollutant that is harmful to human health (EPA, 2006) and an important oxidant in atmospheric aging which can react with soot or BC in a mode of heterogeneous reaction (Han et al., 2012a; Han et al., 2012b). Even though there were lots of studies about health effects caused by O3 or BC, only few toxicological studies were about the health effects of ozone-particle (Jerrett et al., 2009). Interleukin (IL)-6 is a cytokine secreted by various types of cells such as macrophages and lymphocytes (Yudkin et al., 2000). IL-6 participates in a broad spectrum of biological events, such as immune responses, hemopoiesis and acute-phase reactions. Elevated IL-6 contributes to pathogenesis of various inflammatory diseases (Mihara et al., 2012). Exposure to diesel exhaust particles caused high level of IL-6 in mice and rats (Holland et al., 2015; Totlandsdal et al., 2015; Yanagisawa et al., 2014). Also, various of in vitro experiments revealed that diesel exhaust particles could induce the high expression of IL-6 in many different kinds of cells such as PBMC (peripheral blood mononuclear cell), human bronchial epithelial cells, peripheral blood mononuclear cells (Sarkar et al., 2014; Srivastava et al., 2014; Vaughan et al., 2014; Totlandsdal et al., 2010). Furthermore, in our previous research, we
found that C57BL/6 mice treated by BC and oBC particles for 4 weeks resulted in the increased expression of IL-6 mRNA in lung tissue and high level of IL-6 in BALF (Chu et al., 2016a; Jin et al., 2016). IL-33 is a newly discovered pro-inflammatory cytokine belonging to the IL-1 family. IL33 plays an important role in regulating both Th1 and Th2 cell responses (Liew, 2012) and was found involved in human inflammatory diseases (Beltran et al., 2010; Milovanovic et al., 2012; Chu et al., 2016b). Multi-wall carbon nanotube (MWCNTs) instillation impaired pulmonary function in C57BL/6 mice and leads to the increase of IL-33 amount in bronchoalveolar lavage fluid (BALF) (Wang et al., 2011). When treated ICR mice with MWCNTs for 34 weeks, the IL-33 mRNA expression in lung tissue was significantly increased (Yamaguchi et al., 2012). In our previous research, when mice were treated with BC and ozone-oxidized BC (oBC), the IL-33 mRNA expression in lung tissue and the content of IL-33 in BALF both clearly increased (Chu et al., 2016a; Jin et al., 2016). To date, the studies of IL-33 and its relation to particulate matter were mainly focused on allergy diseases such as asthma caused by PM exposure (Li et al., 2013b; Lee et al., 2014; Shadie et al., 2014; Leon Hsu et al., 2015; Li and Buglak, 2015). Whether chronic inflammation in lung is involved after PM exposure or not is still ambiguous. Herein, based on our previous research findings, it is important to explore which cytokine plays a key role in mice lung inflammation induced by BC and oBC, and the potential mechanism involved in those processes. 2. Materials and methods 2.1. Preparation and characterization of BC and oBC BC and oBC particles were provided by Professor Jing Shang (College of Environmental Sciences and Engineering, Peking University). Fig. 1 presents a schematic diagram of oBC. In brief, ozone was generated by the photolysis of oxygen (99.9% purity) with an ultraviolet lamp (ZW 18D 15Y-Z356, Cnlight, China) with a wavelength of 185 nm. A mixture of O2, O3 and N2 with a flow rate of 200 ml/min was made to flow through the tube in which BC powder was uniformly spread. O3 reacted with BC for 120 min at room temperature. N2 was purged through the tube before and after the reaction (Fig. 1). The features and characteristics of BC and oBC were also analyzed by Professor Jing Shang's group (Li et al., 2013a; Chu et al., 2016c; Gao et al., 2016). BC and oBC particles were stored at 4 °C in a black container. Both BC and oBC have the size of approximately 30 nm. The TEM images of original BC and O3-oxidized (100 ppm, 120 min) BC were shown in Fig. S1. Brunauer–Emmett–Teller (BET) specific surface areas of BC
Fig. 1. Schematic diagram of oBC production.
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Fig. 2. Total cell counts in BALF of mice. (A) Mice instilled with BC, oBC, BC/α-IL-6, oBC/α-IL-6. (B) Mice instilled with BC, oBC, BC/α-IL-33, oBC/α-IL-33. ##p b 0.01 versus the control group, **p b 0.01,*p b 0.05. n = 7.
and oBC were 96.39 ± 0.089 m2/g and 97.63 ± 0.064 m2/g respectively. BC showed a broad hydrodynamic size distribution (675.9 ± 17.6 nm), and the size distribution of oBC was relatively narrow (319.3 ± 7.9 nm) (ZetaSizer Nano ZS90, Malvern, UK). Also, Oxygen content was increased and more oxygen-containing functions were produced in oBC. Furthermore, oBC exhibited a strong EPR signal and improved generation of free radicals (Li et al., 2013a). Compared with BC, oBC tended
to be more dispersive in water. The particles were suspended in PBS, and then sonicated for 10 min before intratracheal instillation. 2.2. Animals C57BL/6 mice (female, 18–22 g) were purchased from Vital River Laboratory Technology Co. Ltd. (Beijing, China). All mice were kept in
Fig. 3. IL-6 and IL-33 mRNA expression in lung tissue of mice. (A) Mice instilled with BC, oBC, BC/α-IL-6, oBC/α-IL-6. (B) Mice instilled with BC, oBC, BC/α-IL-33, oBC/α-IL-33. ##p b 0.01 versus control group *p b 0.05, **p b 0.01. n = 7.
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specific pathogen-free conditions. Standard mice feed and tap water were available. Permission of the local ethics committee was obtained and all animal experiments were approved by the Animal Care Committee of Peking University according to Chinese law.
CD4-PECY5, CD8-FITC, FOXP3-FITC, and IL-17-PE, were purchased from eBioscience. Cells sub-type was analyzed by Beckman Coulter Flow Cytometer. The details of staining and blocking procedures were shown in supplementary.
2.3. Experimental design
2.9. Histological assessment of lung tissue
Animals were divided into 6 groups: negative control, neutralizing antibody control (α-IL-6: Biolegend, USA; α-IL-33: R&D systems, USA), particles treated groups (200 μg of 30 nm BC or oBC, n = 7) and particles together with neutralizing antibody groups.
Lung tissues were separated and fixed in 10% buffered formalin, and then embedded in paraffin, at last sectioned and stained the tissue with hematoxylin and eosin. The infiltration of lymphocytes and macrophages together with the swelling of alveolar walls were used to determine the severity of inflammation. The method of histology score of lung tissue were shown in supplementary.
2.4. Intratracheal instillation 5% chloral hydrate was intraperitoneal injected to each mouse (0.1 ml/10 g body weight) for anesthesia. BC and oBC suspensions were sonicated for 15 min prior to intratracheal instillation. The vehicle control mice were instilled with 0.06 ml of PBS and the antibody control mice were instilled with 20 μg α-IL-6 or α-IL-33 dissolved in 0.06 ml PBS. The mice were instilled twice a week for 4 weeks. The body weight of each mouse was measured before sacrificed by cervical dislocation after isoflurane anesthesia.
2.10. Western blot analysis
Mice were euthanized and lavaged with two separate 0.8 ml aliquots of PBS and collect at least 1.2 ml lavage. The lavages were centrifuged (300 ×g, 10 min) and supernatants were collected and then stored at −80 °C for cytokines testing. The cell pellets from lavages were resuspended in 1 ml of PBS containing 1% BSA. Cells were counted using Counter Star (Shang Hai, China). Cells were smeared to the slides and then stained with Diff Quik (American Scientific Inc., Sewickly, PA). About 300 cells were counted and two kind of cell subtypes (lymphocytes, neutrophils) were identified.
Lung tissue was weighted and homogenized in ice-cold lysis buffer (50 mmol/l Tris, 150 mmol/l NaCl, 1% NP-40, 50 mmol/l NaF, 1 mmol/l Na3VO4, 5 mmol/l EDTA, 50 mmol/l NaPPi, 1 mmol/l DTT, 1 mmol/l PMSF, 1% (v/v) protease inhibitors, 1% (v/v) phosphatase inhibitors) with 1% PMSF (Beyotime, China) and lysed for 30 min at 4 °C. Supernatants were collected after centrifugation (13,000 rpm for 5 min). Protein concentration was determined using Bradford Protein Quantitation Kit (Beyotime, China). Proteins were quantified and mixed with 2 × loading buffer 1:1. Equal amounts of protein were subjected to SDS/PAGE under reducing conditions, then transferred proteins to nitrocellulose filters. 5% milk (Sigma-Aldrich, USA; Dissolved in TBS with 0.1% Tween 20) was used to block the membrane for 2 h at room temperature. The blots were incubated with the primary antibody overnight at 4 °C and then incubated for 2 h with secondary antibody. Immunoreactive bands were visualized by chemiluminescence detection. β-actin was used as a loading control. Band densities were then quantified by densitometry (Tanon-4500). All experiments were performed independently at least three times.
2.6. ELISA analysis
2.11. Statistical analysis
The level of IL-6 and IL-33 in BALF was determined using ELISA kits. All of the kits were purchased from eBioscience Co. Ltd. (USA) and the procedures were performed according to the manufacturer's instructions.
Data are presented as mean ± S.D. One-way analysis of variance (ANOVA) followed by Dunnett t-test (SPSS 13.0 Peking University) were used to compare the difference more than two groups. A p value of b0.05 was considered significant.
2.7. RT-PCR analysis
3. Results
At the real-time PCR step, 2 μl cDNA was used for quantitative realtime PCR using SYBR Green kit (TaKaRa, DRR041A). The reaction was performed according to the standard protocol in the IQTM5 Multicolor Real-Time PCR Detection System (Bio-RAD). The endogenous control in the detection was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each sample was analyzed in three duplicates. The relative changes of gene expression were calculated using the following formula: fold change in gene expression, 2 − ΔΔCt = 2 − {ΔCt (vehicle or particles − treated samples) − ΔCt (untreated control)}, where ΔCt = Ct (detected genes) − Ct (GAPDH) and Ct represents the threshold cycle number. The details of PCR procedures were shown in supplementary. The primers used are described in TableS1.
3.1. Image of lung after instillation
2.5. Bronchoalveolar lavage and cell counts
2.8. Organ coefficient and flow cytometry analysis Mediastinal lymph nodes were ground into single cell suspensions after separated and weighted. Organ coefficient was the weight of the organ divided by the body weights. Cells were stained with a monoclonal antibody. Florescence-conjugated antibodies, including CD3-PE,
The change of lung after instillation of BC or oBC can be seen in Fig. S2. It was visible that BC and oBC dispersed in the lung after instillation. Also, BC and oBC could be found in mediastinal lymph nodes. 3.2. Total cell counts in BALF Total cell counts in BALF were calculated using Counter Star (Shang Hai, China). As shown in Fig. 2, mice treated with 200 μg BC or oBC presented an increase of total cell counts in BALF (p b 0.01). And the total cell counts in BALF of mice treated with oBC were significantly higher than that of BC-treated group (p b 0.05). Total cell counts in BALF of mice treated with BC/α-IL-6 or oBC/α-IL-6 were significantly lower than that of BC or oBC-treated group respectively but were still higher than that of PBS groups (p b 0.05) (Fig. 2A). However, mice treated with BC/α-IL-33 or oBC/α-IL-33 showed no increase of cell counts in BALF compared with control groups (Fig. 2B). Furthermore, the counts of lymphocytes and neutrophils in BALF of oBC group were higher
Fig. 4. CXCL1, CXCL2, CXCL5 and CXCL10 mRNA expression in lung tissue of mice. (A) Mice treated with BC, oBC, BC/anti-IL/6, oBC/α-IL-6. (B) Mice treated with BC, oBC, BC/anti-IL/33, oBC/ α-IL-33. Data are representative of three independent experiments. #p b 0.05 versus control group, ##p b 0.01 versus control group, *p b 0.05, **p b 0.01. n = 7.
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than that of BC group (p ≤ 0.05). α-IL-33 could inhibit BC and oBC induced increase of lymphocytes and neutrophils in BALF whereas α-IL6 not. There were no differences between neutralizing antibody group and negative control group (Fig. S3). 3.3. Cytokines mRNA expression in lung tissue As shown in Fig. 3, mice treated with BC or oBC both showed high expression of IL-6 and IL-33. Furthermore, IL-6 and IL-33 expression in oBC-treated group were significantly higher than BC-treated group (p b 0.05). However, IL-6 mRNA expression in BC/α-IL-6 or oBC/α-IL-6treated groups was significantly lower than that treated with BC or oBC respectively (p b 0.01). But IL-33 mRNA expression in mice treated with BC/α-IL-6 or oBC/α-IL-6 had no difference with BC or oBC-treated group, and much higher than control groups (p b 0.01 or p b 0.05) (Fig. 3A). However, IL-6 and IL-33 expression in mice treated with BC/ α-IL-33 or oBC/α-IL-33 were significantly lower than that treated with BC or oBC, and no differences could be found when compared with control groups (p b 0.01 or p b 0.05) (Fig. 3B). 3.4. Chemokines mRNA expression in lung tissue Chemokines mRNA expression in lung tissue was also detected. As shown in Fig. 4, mice treated with BC or oBC both showed high expression CXCL1, CXCL2, CXCL5 and CXCL10. Furthermore, chemokines mRNA expression in oBC-treated group were significantly higher than BC-treated group (p b 0.05). Surprisingly, chemokines mRNA expression in mice treated with BC/α-IL-6 or oBC/α-IL-6 had no difference compared with BC or oBC-treated group, and significantly higher than control groups (p b 0.01 or p b 0.05) (Fig. 4A). However, chemokines mRNA expression in BC/α-IL-33 or oBC/α-IL-33-treated mice was significantly lower than that of BC or oBC-treated group, and no differences could be found when compared with control groups (p b 0.01 or p b 0.05) (Fig. 4B). 3.5. Cytokines level in BALF and lung tissue homogenates Secretion of cytokines in BALF Cytokine levels in BALF and lung homogenates was detected using ELISA kits. As can be seen in Fig. 5, the level of IL-33 and IL-6 in BALF and lung tissue homogenates of BC group and oBC group were significantly higher than that of control groups (p b 0.01). The level of IL-33 and IL-6 in mice treated with the two particles together with α-IL-6 had no difference with BC or oBC group respectively and significantly higher than that of control groups (Fig. 5B). However, in BC/α-IL-33 or oBC/α-IL-33-treated group, the level of IL-33 and IL-6 in BALF and lung homogenates were significantly lower than that of BC group and oBC-treated group (p b 0.01) and had no difference compared with control groups (Fig. 5A). 3.6. Organ coefficient and T cell sub-types of mediastinal lymph nodes As shown in Fig. S4 the mediastinal lymph nodes coefficient of mice treated with BC and oBC was significantly higher than that of control groups (p b 0.01 or p b 0.05). The mediastinal lymph nodes coefficient of mice treated with BC/α-IL-6 or oBC/α-IL-6 was higher than that of control groups and had no difference compared with BC or oBCtreated group (Fig. S4A). However, the mediastinal lymph nodes coefficient of mice treated with BC/α-IL-33 or oBC/α-IL-33 had no difference compared with control groups (Fig. S4B). T cell sub-types of mediastinal lymph nodes were measured using Beckman Coulter Flow Cytometer. As shown in Fig. S5, the percentage of CD4+ T cells in mediastinal lymph nodes was increased after instilled with BC or oBC. But, the percentage of CD4+ T and Th17 cells in mediastinal lymph nodes of mice instilled with BC/α-IL-6 or oBC/α-IL-6 was
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significantly higher than that of control groups and had no difference compared with BC or oBC-treated group. The percentage of CD4+ T cells in mice instilled with BC/α-IL-33 or oBC/α-IL-33 was lower than that of BC or oBC-treated group. So was the result of Th17 cells. However, the percentage of CD8+ T cells and Treg cells had no difference of in mediastinal lymph nodes of each group (Fig. 6). 3.7. Histopathology analysis of lung tissue As shown in Fig. 7, inflammatory cells infiltration and alveolar swelling and thickening could be seen in the lung tissue of BC and oBCtreated mice and the dispersed particles in lung tissue could be clearly observed. The oBC-treated group showed much more severe lung inflammation when compared with BC groups. There were still lots of inflammatory cells infiltration and alveolar swelling and thickening in mice treated with BC/α-IL-6 or oBC/α-IL-6, which had no differences compared with BC or oBC-treated groups (Fig. 7A). However, even though the dispersed particles in lung tissues could be seen in mice treated with BC/α-IL-33 or oBC/α-IL-33, inflammatory cell infiltration and alveolar swelling and thickening were less observed when compared with BC or oBC-treated mice (Fig. 7B). The histopathology score results of the lung tissue were shown in Fig. S6. 3.8. Change of signaling pathway To confirm the hypothesis that MAPK is one of the important signal pathway involved in BC and oBC induced lung damage, the relative amount of signaling molecules were examined. As shown in Fig. S7A, the expression of ERK, JNK, P38 and their phosphorylated kinase were assessed. Phosphorylation of ERK, P38 and JNK increased after exposure to BC and oBC. Moreover, the phosphorylation of ERK, JNK and P38 in the oBC group occurred more than that in BC-treated group. Interestingly, phosphorylation of ERK, P38 and JNK in BC/α-IL-6 or oBC/α-IL6-treated group had no difference with that of BC or oBC group but still higher than control groups (Fig. S7A–C). However, phosphorylation of ERK, P38 and JNK were decreased after exposure to BC/α-IL-33 or oBC/α-IL-33 compared with BC or oBC group, and had no differences from control groups (Fig. 8A–C). Furthermore, phosphorylation of PI3K and AKT (Fig. S7D & E) increased after exposure to BC and oBC which was consistent with our pervious study. Similarly, the phosphorylation of PI3K and AKT in the oBC group was higher BC-treated group. Phosphorylation of PI3K and AKT in BC/α-IL-6 or oBC/α-IL-6 treatment group had no difference compared with BC or oBC group (Fig. S7D & E). On the contrary, Phosphorylation of PI3K and AKT decreased after exposure to BC/α-IL-33 or oBC/α-IL-33 compared with BC or oBC group (Fig. 8D & E). All these data indicated that the pro-inflammatory action of oBC is stronger than that of BC, and IL-33 neutralizing antibody inhibited BC and oBC induced lung inflammation in mice. IL-33 played a key role in BC and oBC induced lung damage in mice. 4. Discussion Ambient air pollution is a complex mixture of gaseous and particulate phase pollutants. The residence time of each pollutant in the atmosphere varies greatly. The major substances being monitored are O3 and PM (Madden et al., 2000). There are several reasons for investigating the health effects of BC and oBC. Black carbon is a key component of PM 2.5 and mainly exists in traffic-related particles. Many epidemiology studies have shown that atmospheric black carbon has negative relationship to human health (McCracken et al., 2010; Nichols et al., 2013; Geng et al., 2013; Smith et al., 2009). Ozone is a pollutant that coexists with PM, even though human mortality has been associated predominantly with
Fig. 5. Effects of α-IL-33 and α-IL-6 on BC and oBC induced secretion of cytokines in BALF and lung tissue homogenates. (A) Mice treated with BC, oBC, BC/anti-IL/6, oBC/α-IL-6. (B) Mice treated with BC, oBC, BC/anti-IL/33, oBC/α-IL-33. ##p b 0.01 versus control group, *p b 0.05, **p b 0.01. n = 7.
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Fig. 6. Effects of BC, oBC, BC/α-IL-33 and oBC/α-IL-33 on the percentage of CD4+ T cells and Th17 cells in mediastinal lymph nodes. ##p ≪ 0.01 versus control group, **p b 0.01. n = 7.
the concentration of PM, a relationship between ozone levels and cardiopulmonary deaths has also been observed (Kinney and Özkaynak, 1991). Furthermore, BC can react with ozone forming secondary pollution and yet the studies about health effects of ozone-oxidized BC are rare. Our previous studies have found/proved that IL-33 and IL-6 play important roles in BC and oBC induced lung inflammation (Chu et al., 2016a). To date, the underlying mechanism of BC or oBC induced lung damage remains unclear. In order to explore the potential effects of IL33 and IL-6 in the process of lung inflammation induced by the two particles, α-IL-33 and α-IL-6 were used to prevent the bioactivity of each cytokine so as to lift the veil enveloping the truth.
Previous studies have shown that IL-33 played a key role in the induction and perpetuation of the Th2-based immunological response such as allergic asthma (Liew et al., 2010; Cayrol and Girard, 2014). The IL-33 and IL-6 mRNA expression increased in lung tissue in mice models of allergic inflammation (Kurokawa et al., 2011; Bunting et al., 2013). Here we found that IL-33 and IL-6 mRNA expression in lung tissue and the secretion of IL-33 and IL-6 in BALF were significantly increased in mice instilled with BC and oBC. As our hypothesis presented, the mRNA expression and the level of two cytokines in BALF of mice treated with BC/α-IL-33 or oBC/α-IL-33 were lower than that of mice treated with BC or oBC and there were no difference
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Fig. 7. Histological assessment of lung damage (H&E). (A) Mice treated with BC, oBC, BC/anti-IL/6, oBC/α-IL-6. (B) Mice treated with BC, oBC, BC/anti-IL/33, oBC/α-IL-33. Arrows indicate the accumulation of inflammatory cells (Original magnification, 200×).
compared with PBS and α-IL-33 treatment group. Furthermore, the result of histopathology showed that lung inflammation was alleviated significantly after α-IL-33 treatment. These results indicated that IL-33 is very likely to play an important role in lung damage caused by BC or oBC. IL-6 is a cytokine produced by inflammatory cells and lung epithelial cells in response to various stimuli such as allergens and respiratory viruses (Yokoyama et al., 1995; Stadnyk, 1994; Marini et al., 1992). A recent study showed that IL-6 mRNA expression was high in mouse primary lung epithelial but not in immune cells resident in lung (Neveu et al., 2011). Here in our study, IL-6 and IL-33 mRNA expression in lung tissue and IL-6 concentration in BALF significantly increased in BC and oBC-treated groups which is in line with our previous findings (Chu et al., 2016a; Jin et al., 2016). The astonishing phenomenon is that IL-6 mRNA expression was reduced significantly after α-IL-6 treatment when exposed to two particles but IL-33 had not been influenced by α-IL-6 treatment. From the histopathology results, instilled mice with BC or oBC together with α-IL-6 didn't decrease lung inflammation compared with mice only instilled BC or oBC. BC and oBC could increase the chemokines mRNA expression in lung tissue and α-IL-33 treatment could inhibit this increase whereas α-IL-6 treatment had no effects which further indicated that IL-33 may play an important role in BC or oBC induced lung inflammation. Th17 cells have been described as a kind of Th cells which are different from Th1 and Th2 cells (Park et al., 2005; Harrington et al., 2005). It has been showed that IL-17 and IL-17-producing (Th17) cells act a pivotal part in inflammatory responses. Mice treated with PM 2.5 exhibited an apparent increase of Th17 cells (Xie et al., 2013), and fine particulate matters (PM 2.5) could change Th17/Treg balance as well as related
cytokines (Yang et al., 2014). We found that the percentage of Th 17 cells in mediastinal lymph nodes was significantly increased in mice treated with BC and oBC, but not in BC/α-IL-33 and oBC/α-IL-33treated groups. Interestingly, percentage of Th 17 cells in mice instilled with BC/α-IL-6 or oBC/α-IL-6 was still as high as BC or oBC-treated group. This may be due to the potential of IL-6 inducing Th 17 cells and IL-33 can induced IL-6 synthesis (Miller et al., 2008). IL-33 cannot induced Th17 cells but it can induce IL-6 thus further influence Th17 cells (Miller et al., 2008; Xu et al., 2008; Oboki et al., 2010). It has been reported that urban particles (UP) could increase the phosphorylation of ERK, P38 in rat pulmonary artery (Li et al., 2005). Also, cigarette smoking particles can also activate MAPK in rat cerebral arteries (Sandhu et al., 2010). Extensive studies have declared that particles such as ultrafine carbon particles, diesel exhaust particles could activate MAPK in vivo and vitro (Nel et al., 1998; Kim et al., 2005; Baulig et al., 2003; Totlandsdal et al., 2010). We have already found that BC and oBC could activate the MAPK pathway (Chu et al., 2016a). Here we found that BC and oBC together with α-IL-33 can alleviate this activation but α-IL-6 treatment does not show similar efficiency. This indicated that IL-33 plays an important role in this MAPK activation. Additionally, we explored effects of BC and oBC together with αIL-33 on PI3K/AKT pathway. In our previous results, BC and oBC could increase the phosphorylation of PI3K and AKT (Chu et al., 2016a). Here we observed that the activation of BC and oBC on PI3K/AKT pathway could be abrogated by α-IL-33 treatment. Meanwhile, histopathology score has not been reduced by α-IL-6 treatment during two particles exposure. All information from α-IL-33 treatment suggested that IL-33 decrease could alleviate the lung inflammation induced by BC and oBC. In this process, IL-6 was regulated by IL-
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Fig. 8. Neutralizing IL-33 inhibited BC and oBC activated MAPK and PI3K/AKT signaling pathways. (A & C) MAPK signaling pathway. (B & D) PI3K/AKT signaling pathway. *p b 0.05 versus the control group, **p b 0.01 versus the control group. #p b 0.05 versus the corresponding BC or oBC group, ##p b 0.01 versus the corresponding BC or oBC group.
33. Combined with the results from IL-6 intervention, it has been demonstrated that IL-33 is a critical initiator for chronic inflammation in lung exposed to BC and oBC. Whether IL-6 involved in this kind of chronic inflammation remains to be extrapolated.
It could be drawn the conclusion that MAPK and PI3K/AKT signal pathways are the downstream of IL-33. Whether both of these two pathways are the upstream of other cytokines involved in the inflammation still need to be proved.
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5. Conclusions In summary, the data from this study demonstrated that oBC caused much more lung inflammation than BC in mice and IL-33 plays a key role in this process. In addition, these effects might involve in the activation of MAPK and PI3K/AKT pathway. The specific mechanism of IL-33 in lung damage induced by BC or oBC needs further investigation. Acknowledgements We would particularly like to thank Professor Jing Shang (College of Environmental Sciences and Engineering, Peking University) for her encouraging support and valuable facilities to carry out this work. The authors acknowledge support from the Major Program of National Natural Science Foundation of China (Grant No. 21190051). Disclosure statement The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of this article. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.06.329. References Baulig, A., Garlatti, M., Bonvallot, V., Marchand, A., Barouki, R., Marano, F., Baeza-Squiban, A., 2003. Involvement of reactive oxygen species in the metabolic pathways triggered by diesel exhaust particles in human airway epithelial cells. Am. J. Phys. Lung Cell. Mol. Phys. 285, L671–L679. Beltran, C.J., Núñez, L.E., Díaz Jiménez, D., Farfan, N., Candia, E., Heine, C., Lopez, F., González, M.J., Quera, R., Hermoso, M.A., 2010. Characterization of the novel ST2/IL33 system in patients with inflammatory bowel disease. Inflamm. Bowel Dis. 16, 1097–1107. Bunting, M.M., Shadie, A.M., Flesher, R.P., Nikiforova, V., Garthwaite, L., Tedla, N., Herbert, C., Kumar, R.K., 2013. Interleukin-33 drives activation of alveolar macrophages and airway inflammation in a mouse model of acute exacerbation of chronic asthma. Biomed. Res. Int. 2013. Cayrol, C., Girard, J., 2014. IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy. Curr. Opin. Immunol. 31, 31–37. Chu, H., Shang, J., Jin, M., Li, Q., Chen, Y., Huang, H., Li, Y., Pan, Y., Tao, X., Cheng, Z., 2016a. Comparison of lung damage in mice exposed to black carbon particles and ozoneoxidized black carbon particles. Sci. Total Environ. 573, 303–312. Chu, H., Li, J., Huang, H., Hao, W., Duan, L., Wei, X., 2016b. Protective effects of tranilast on oxazolone-induced rat colitis through a mast cell-dependent pathway. Dig. Liver Dis. 48, 162–171. Chu, H., Shang, J., Jin, M., Li, Q., Chen, Y., Huang, H., Li, Y., Pan, Y., Tao, X., Cheng, Z., Meng, Q., Jia, G., Zhu, T., Wei, X., Hao, W., 2016c. Comparison of lung damage in mice exposed to black carbon particles and ozone-oxidized black carbon particles. Sci. Total Environ. 573, 303–312. Clougherty, J.E., Kubzansky, L.D., 2009. A framework for examining social stress and susceptibility to air pollution in respiratory health. Environ. Health Perspect. 117, 1351–1358. Dons, E., Panis, L.I., Van Poppel, M., Theunis, J., Wets, G., 2012. Personal exposure to black carbon in transport microenvironments. Atmos. Environ. 55, 392–398. EPA, 2006. Air Quality Criteria for Ozone and Related Photochemical Oxidants., EPA/600/R -05/004aF-cF 2006. Fuzzi, S., Baltensperger, U., Carslaw, K., Decesari, S., Denier Van Der Gon, H., Facchini, M.C., Fowler, D., Koren, I., Langford, B., Lohmann, U., Nemitz, E., Pandis, S., Riipinen, I., Rudich, Y., Schaap, M., Slowik, J.G., Spracklen, D.V., Vignati, E., Wild, M., Williams, M., Gilardoni, S., 2015. Particulate matter, air quality and climate: lessons learned and future needs. Atmos. Chem. Phys. 15, 8217–8299. Gao, X., Xu, H., Shang, J., Yuan, L., Zhang, Y., Wang, L., Zhang, W., Luan, X., Hu, G., Chu, H., 2016. Ozonized carbon black induces mitochondrial dysfunction and DNA damage. Environ. Toxicol. 32, 944–955. Geng, F., Hua, J., Mu, Z., Peng, L., Xu, X., Chen, R., Kan, H., 2013. Differentiating the associations of black carbon and fine particle with daily mortality in a Chinese city. Environ. Res. 120, 27–32. Han, C., Liu, Y., Ma, J., He, H., 2012a. Effect of soot microstructure on its ozonization reactivity. J. Chem. Phys. 137, 84507. Han, S., Jing, S., Tong, Z., Li, Z., Jun-Hui, Y., 2012b. Heterogeneous oxidation of SO2 by ozone on the surface of black carbon particles. Chem. J. Chin. Univ. 33, 2295–2302. Han, C., Liu, Y., He, H., 2013. Role of organic carbon in heterogeneous reaction of NO2 with soot. Environ. Sci. Technol. 47, 3174–3181.
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