Induction of Inflammatory Responses in Human Bronchial Epithelial

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Ecotoxicology and Human Environmental Health

Induction of Inflammatory Responses in Human Bronchial Epithelial Cells by Pb2+-Containing Model PM2.5 Particles via Downregulation of a Novel Long Non-Coding RNA lnc-PCK1-2:1 Xiujiao Pan, Xiaoru Yuan, Xin Li, Sulian Gao, Hainan Sun, Hongyu Zhou, Lujian Hou, Xiaowu Peng, Yiguo Jiang, and Bing Yan Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 26, 2019

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Environmental Science & Technology

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Induction of Inflammatory Responses in Human Bronchial Epithelial Cells by

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Pb2+-Containing Model PM2.5 Particles via Downregulation of a Novel Long Non-

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Coding RNA lnc-PCK1-2:1

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Xiujiao Pan,1 Xiaoru Yuan,1, 2 Xin Li,3 Sulian Gao,4 Hainan Sun,1 Hongyu Zhou,2

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Lujian Hou,4 Xiaowu Peng,6 Yiguo Jiang,3, * Bing Yan,2, 5, *

6

1School

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China.

8

2Key

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of Education, Institute of Environmental Research at Greater Bay, Guangzhou

of Chemistry and Chemical Engineering, Shandong University, Jinan 250100,

Laboratory for Water Quality and Conservation of the Pearl River Delta, Ministry

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University, Guangzhou 510006, China.

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3State

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Guangzhou Medical University, Guangzhou 511436, China.

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4Environmental

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5School

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250100, China.

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6South

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Protection, Guangzhou 510655，China.

Key Laboratory of Respiratory Disease, Institute for Chemical Carcinogenesis,

Monitoring Center, Jinan 250102, China.

of Environmental Science and Engineering, Shandong University, Jinan

China Institute of Environmental Sciences, Ministry of Environmental

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*To whom correspondence should be addressed.

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Professor Bing Yan

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E-mail: [email protected]

22

Professor Yiguo Jiang 1

ACS Paragon Plus Environment


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E-mail: [email protected]

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Graphic TOC

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Abstract

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Air-borne particular matter (PM2.5) contain complex mixtures of pollutants and

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their compositions are also varying with time and location. Inhalation of PM2.5 may

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cause a number of diseases, such as bronchial and lung inflammation and lung cancer.

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So far, how different components of PM2.5 contribute to inflammation and toxicity is

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still not known. To identify key PM2.5 components that are responsible for inflammation,

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here we took a reductionism approach and synthesized a model PM2.5 library containing

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20 carbon nanoparticle-based members with loadings of As (III), Pb2+, Cr (VI) and BaP

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individually or in combination at environment relevant concentrations. We discovered 2


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that only carbon nanoparticle-Pb2+ adducts, not other pollutants or adducts, induced

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inflammation in human bronchial cells by suppressing the expression of a novel long

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non-coding RNA lnc-PCK1-2:1, while lnc-PCK1-2:1 routinely plays a regulatory role

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in inhibiting inflammation. This finding was further substantiated by varying Pb2+

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loadings on carbon nanoparticles and overexpressing lnc-PCK1-2:1. The success of this

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approach opens an avenue for further elucidation of molecular mechanisms of PM2.5-

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induced inflammation and toxicity.

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Introduction

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Fine particulate matter with aerodynamic diameters less than 2.5 μm (PM2.5) are

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small enough to penetrate human lungs and cross air-blood barriers. Long-term

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exposures to PM2.5 are linked to acute lower respiratory infections,1 chronic obstructive

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pulmonary disease,2 stroke,3, 4 ischemic heart attack5, 6 and lung cancer.7 In 2016, air

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pollution was responsible for 7.5% of global deaths, in which China and India together

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account for nearly half of it.8-10 Toxicological studies have shown that PM2.5 particles

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induce cytotoxicity,11, 12 immunotoxicity,11, 12 oxidative stress13 and DNA damages in

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human cells.13 Inflammation is a crucial trigger for most PM2.5-induced diseases.

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However, because of the complexity of PM2.5 composition and its region- and time-

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dependence, very little is known about how different components contribute to PM2.5-

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induced overall inflammatory responses.

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Inflammation induction is orchestrated by sophisticated cell signaling networks

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involving various biomolecules such as proteins, DNA and RNA molecules.14-17 3



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However, the involvement of non-coding RNAs in PM2.5-induced inflammation is not

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well known. Noncoding RNAs are non-protein-coding transcripts. About 75-90%

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mammalian genomes are transcribed as noncoding RNAs.18 There is growing evidence

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that long noncoding RNAs (lncRNAs), which have more than 200 nucleotides in length,

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contribute to the regulation of gene expression in immune cells (such as macrophages,19,

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20

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proteins to alter the expression of NF-κB related genes.19, 22 A lncRNA THRIL interacts

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with hnRNPL (heterogenous nuclear ribonucleoprotein L) and regulates the expression

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of tumor necrosis factor alpha (TNF-α) following the activation of toll-like receptor 2

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in human monocytes.20 Our previous work has found that PM2.5 upregulated lncRNA

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LINC00341 in 16HBE cells and induced cell cycle arrest.23 However, PM2.5-induced

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epigenetic changes that regulate inflammation are not understood.

DCs,21 and epithelial cells22). It is reported that lncRNA-Cox2 interacts with other

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Human bronchial epithelial (16HBE) cells participate in the initial interactions

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with inhaled PM2.5 particles and, therefore, induce early inflammatory responses.24 In

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this investigation, we use 16HBE as a cell model. By taking a reductionism approach

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we synthesized a carbon nanoparticle-based model PM2.5 library of 20 members with

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controlled loadings of pollutants either individually or in combination at environment

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relevant concentrations. We discovered that only Pb2+-loaded model PM2.5 particles,

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not with other tested pollutants, induced inflammation by suppressing the expression

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of a novel long non-coding RNA lnc-PCK1-2:1.

81 82

Materials and methods 4


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PM2.5 collection and analysis

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The sampling method was reported in our previous work. Briefly, PM2.5 particles

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were collected from city of Jinan, China, using a high volume ambient particulate

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sampler (Tianhong Instruments, TH-150CⅢ, Wuhan, China), at an air flow rate of 100

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L/min. After sampling, the filter membranes were cut into small pieces and soaked in

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150 mL ultrapure H2O. The mixture was sonicated for an hour on an ice bath, then the

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membranes were soaked in 100 mL of dimethyl sulfoxide and sonicated for another

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hour on an ice bath. Finally, all the extract and blank particles were suspension and

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dried in a vacuum freeze dryer. The resulting dried, gray, flocculent, particles were

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weighed and resuspended in H2O, then placed in a refrigerator at 4°C for future use.

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An array of techniques was used to analyzed components in PM2.5-JN. Ionic

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chromatography (Dionex ICS-3000, MA, USA) was used to analyze water-soluble

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components, inductively coupled plasma-mass spectrometry was used to analyze

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inorganic metal elements, Thermal/Optical Carbon Analyzer (Desert Research Institute，

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DRI Model 2001A, Reno, USA) was used to analyzed organic carbon/elemental carbon

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(OC/EC), and gas chromatography-mass spectrometry (Agilent, GC-MS, 7890 A/5975

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C, Santa Clara, USA) was used to analyzed polycyclic aromatic hydrocarbons (PAHs).

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Synthesis of the model PM2.5 library

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Detailed procedure for synthesizing the model PM2.5 library showed in SI. In short,

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carbon nanoparticles carrying pollutants represent model insoluble part of PM2.5

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particles. We synthesized a model PM2.5 library containing 20 different model PM2.5

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particles (Table 1, 2) through different combinations of 4 pollutants. The mass fraction 5



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of each pollutant loaded on model PM2.5 particles were consistent with PM2.5-JN (Cr

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(VI): 0.14%, Pb2+: 0.60%, As (III): 0.08%, BaP: 0.10%). The concentrations of loaded

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pollutants were 0.30 μg/mL (K2Cr2O7), 0.82 μg/mL (Pb(CH3COO)2·3H2O), 0.080

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μg/mL (As2O3) and 0.075 μg/mL (BaP) in model PM2.5 solutions.

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Characterization of model PM2.5 and PM2.5-JN particles

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The morphology of model PM2.5 and PM2.5-JN particles were analyzed by

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transmission electron microscope (TEM) (JEM-1011, JEOL, Tokyo, Japan) after a 30

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min sonication. Hydrodynamic diameters and zeta potentials of model PM2.5 and PM2.5-

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JN solutions (75 μg/mL) in ultrapure water (18.2 MΩ) or cell culture medium

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supplemented with 10% fetal bovine serum were measured by a laser particle size

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analyzer (Malvern Nano ZS, Malvern, UK).

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LncRNA microarray

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RNA was obtained from 16HBE cells after incubating with C, C-BaP-Cr-Pb-As,

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or PM2.5-JN at 75 μg/mL for 48 h. Trizol reagent (Invitrogen, Carlsbad, CA, USA) was

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used to extract total RNA according to the manufacturer’s instructions. The quality and

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concentrations of total RNA were measured by a spectrophotometer (NanoDrop ND-

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2000, Thermo Scientific, MA, USA).

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Agilent Human lncRNA Microarray V6 (Agilent Technologies, Santa Clara, USA)

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was used for lncRNA microarrays detection, and lncRNA microarray experiments were

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completed by OE Biotechnology Company (Shanghai, China). This protocol involves

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the transcription of total RNA into double-stranded cDNA, labeling cRNA with

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Cyanine-3-CTP, followed by hybridizing the cRNA to the microarray. Agilent Scanner 6


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G2505C (Agilent Technologies, Santa Clara, USA) was used to scan the array. An

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analysis of the raw data was carried out by Genespring (Agilent Technologies, Santa

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Clara, USA). Differentially expressed lncRNAs or genes were set as fold change ≥ 2.0

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and p ≤ 0.05. Finally, hierarchical cluster analyses were performed to classify the

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distinguishable expression patterns of lncRNAs and genes in different samples.

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Quantitative real-time polymerase chain reaction (qRT-PCR)

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GoscriptTM Reverse Transcription System (Promega, Madison, WI, USA) was

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used to reverse transcribe RNAs to cDNAs. Then, qRT-PCR was carried out by

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QuantStudioTM 5 System (Thermo Fisher Scientific, MA, USA) using Go Taq® qPCR

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Master Mix (Promega, Fitchburg, USA). β-actin was used as an internal control to

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normalize the expression levels. The relative expression of lncRNAs and genes were

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quantified by the 2−ΔΔCT method.25 ΔΔCT = (CTTarget

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(CTTarget gene - CTβ-actin)Control group. All primers were synthesized by Invitrogen (Waltham,

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MA, USA) and sequences were listed in Table S2.

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RNA overexpression

gene-CTβ-actin)Experimental group

-

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Overexpression vectors of lnc-PCK1-2:1 and control empty vectors were

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constructed by BersinBio (Guangzhou, China). BamHI and XhoI were jointly

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connected to express vector pcDNA 3.1 through double enzyme connection.

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Overexpression sequence of lnc-PCK1-2:1 was shown in Table S3. Cells were

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transfected as previously reported.26 In brief, after a 15-25 min equilibration, the mixed

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Endofectin™-Max reagent (Genecopoeia), plasmids and Opti-MEM® I Reduced

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Serum Medium (Gibco) were added to the wells. After incubation at 37℃ with 5% CO2 7



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for 6 h, the serum-free medium was replaced with fresh medium containing model

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PM2.5 or PM2.5-JN (75 μg/mL).

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Cell viability measurements

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16HBE cells were cultured in minimum essential medium (MEM) (Genom,

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Hangzhou, China) supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou,

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China), 1% penicillin-streptomycin antibiotics (Sigma-Aldrich, St. Louis, Mo, USA) at

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37 °C with 5% CO2. The CCK-8 kit (Dojindo, Japan) was used to access relative cell

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viability. 16HBE cells were harvested in logarithmic phase, seeded in a 96-well plate

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with the density of 9000 cells per well. After incubation at 37℃ with 5% CO2 for 24 h,

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medium in each well were replaced with fresh culture medium containing same or

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different amounts of model PM2.5 or PM2.5-JN. Cells were incubated with particles for

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48 h. The absorbance was read by a microplate reader (BioTek, Winooski, VT) at 450

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nm after adding 10 μL per well CCK-8 solution for 1-2 h.

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Quantification of inflammatory cytokines

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The interleukin-6 (IL-6) and interleukin-8 (IL-8) levels in cell culture supernatants

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were quantified by commercially available human uncoated ELISA kits (ThermoFisher

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scientific,

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Lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, USA) at 400 ng/mL was used as

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a positive control.

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Cellular uptake

MA,

USA)

according

to

the

manufacturer’s

recommendations.

169

To study cellular uptake of particles, 16HBE cells were cultured in 12-well plates

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at a density of 120000 cells per well for 24 h, and the solutions of particles in cell culture 8


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medium (75 μg/mL) were added. After incubation for 48h at 37℃ with 5% CO2, the

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cells were washed 3 times with phosphate buffer saline (PBS) to remove free particles.

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The cells were then fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer

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(pH 7.4) for one hour at room temperature. After rinsing, the cells were fixed in 2%

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osmium tetroxide containing 3% potassium ferrocyanide for one hour and rinsed.

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Finally, the thin (70 nm) sections were cut on a Leica UC6 ultramicrotome and images

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were taken on TEM using an AMT 2k digital camera.

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Statistical Analysis

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Data are presented as mean values ± s.d. of at least 3 independent determinations.

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One-way ANOVAs followed by Tukey’s tests were used to analyze differences

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between control and experimental groups. A P-value of < 0.05 was considered

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significant. All statistical calculations were carried out using Sigma Plot 12.0.

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Results and discussion

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Water-insoluble PM2.5 particles are main contributors for induction of

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cytotoxicity and inflammation

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Components of PM2.5 include carbon, silica, inorganic and organic compounds,

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and even biological pollutants. Some components of PM2.5, like ammonium salts,

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sulfates, and liquid pollutants are water soluble, while others, such as carbon and silica

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particles with adsorbed pollutants are insoluble in aqueous solution. Because PM2.5

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particles are quickly suspended in lung fluid after inhalation, we need to know cellular

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effects of the dissolved and insoluble parts of PM2.5 particles. The complete PM2.5 9



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particles were weighed and then fully suspended in water for 24 hours. After careful

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removal of the supernatant, the lower suspension was lyophilized and this insoluble

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portion was weighed. The water-soluble portion was obtained by subtracting the

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insoluble part. Our results showed that the mass ratio of insoluble and soluble

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components of PM2.5 particles from Jinan city (PM2.5-JN) was 0.46:0.54 or roughly 1:1

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(Figure 1A).

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When PM2.5 particles are inhaled into respiratory systems, it is the bronchial

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epithelial cells, rather than alveolar macrophages, that are involved in the initial

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interactions with foreign particles and induce early inflammatory responses.24

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Therefore, normal human bronchial epithelial (16HBE) cells were used as a cell model

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for investigating early lung inflammation. In 16HBE cells, soluble components of

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PM2.5-JN exhibited very low cytotoxicity, while the whole PM2.5-JN suspension

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induced more severe cytotoxicity at both 75 and 150 μg/mL (Figure 1B), suggesting

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that the insoluble components were the main cause of cytotoxicity. Similarly, insoluble

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components contributed to the majority of inflammatory responses induced by PM2.5-

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JN as evaluated by the release of inflammatory factors IL-6 and IL-8 proteins (Figure

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1C, D). Therefore, our data demonstrated that insoluble components with the adsorbed

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toxic pollutants are the major contributors to both cytotoxicity and inflammation

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induced by PM2.5-JN.

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Figure 1. Cytotoxicity and inflammatory responses of water-insoluble PM2.5-JN in

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16HBE cells. (A) Weight percentage of water-soluble and water-insoluble components

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of PM2.5-JN collected in December, 2017. (B) Water-soluble components of PM2.5-JN

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particles are less toxic compared to the whole PM2.5-JN particles. (C, D) Water-soluble

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parts of PM2.5-JN do not induce inflammation, unlike the whole particles. 16HBE cells

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were incubated with solution or particle suspension at various concentrations for 48h.

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The dotted lines represent cellular responses to the addition of cell culture medium.

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(n=3, mean ± SD, **p

Induction of Inflammatory Responses in Human Bronchial Epithelial

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