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      Multi-cellular human bronchial models exposed to diesel exhaust particles: assessment of inflammation, oxidative stress and macrophage polarization

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          Abstract

          Background

          Diesel exhaust particles (DEP) are a major component of outdoor air pollution. DEP mediated pulmonary effects are plausibly linked to inflammatory and oxidative stress response in which macrophages (MQ), epithelial cells and their cell-cell interaction plays a crucial role. Therefore, in this study we aimed at studying the cellular crosstalk between airway epithelial cells with MQ and MQ polarization following exposure to aerosolized DEP by assessing inflammation, oxidative stress, and MQ polarization response markers.

          Method

          Lung mucosa models including primary bronchial epithelial cells (PBEC) cultured at air-liquid interface (ALI) were co-cultured without (PBEC-ALI) and with MQ (PBEC-ALI/MQ). Cells were exposed to 12.7 μg/cm 2 aerosolized DEP using Xpose ALI ®. Control (sham) models were exposed to clean air. Cell viability was assessed. CXCL8 and IL-6 were measured in the basal medium by ELISA. The mRNA expression of inflammatory markers ( CXCL8, IL6, TNFα), oxidative stress ( NFKB, HMOX1, GPx) and MQ polarization markers ( IL10, IL4, IL13, MRC1, MRC2 RETNLA, IL12 and IL23) were measured by qRT-PCR. The surface/mRNA expression of TLR2/TLR4 was detected by FACS and qRT-PCR.

          Results

          In PBEC-ALI exposure to DEP significantly increased the secretion of CXCL8, mRNA expression of inflammatory markers ( CXCL8, TNFα) and oxidative stress markers ( NFKB, HMOX1, GPx). However, mRNA expressions of these markers ( CXCL8, IL6, NFKB, and HMOX1) were reduced in PBEC-ALI/MQ models after DEP exposure. TLR2 and TLR4 mRNA expression increased after DEP exposure in PBEC-ALI. The surface expression of TLR2 and TLR4 on PBEC was significantly reduced in sham-exposed PBEC-ALI/MQ compared to PBEC-ALI. After DEP exposure surface expression of TLR2 was increased on PBEC of PBEC-ALI/MQ, while TLR4 was decreased in both models. DEP exposure resulted in similar expression pattern of TLR2/TLR4 on MQ as in PBEC. In PBEC-ALI/MQ, DEP exposure increased the mRNA expression of anti-inflammatory M2 macrophage markers ( IL10, IL4, IL13, MRC1, MRC2).

          Conclusion

          The cellular interaction of PBEC with MQ in response to DEP plays a pivotal role for MQ phenotypic alteration towards M2-subtypes, thereby promoting an efficient resolution of the inflammation. Furthermore, this study highlighted the fact that cell–cell interaction using multicellular ALI-models combined with an in vivo-like inhalation exposure system is critical in better mimicking the airway physiology compared with traditional cell culture systems.

          Electronic supplementary material

          The online version of this article (10.1186/s12989-018-0256-2) contains supplementary material, which is available to authorized users.

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          Most cited references53

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          Optimized THP-1 differentiation is required for the detection of responses to weak stimuli.

          The differentiation of THP-1 monocytes into macrophages is mainly conducted at a phorbol 12-myristate 13-acetate (PMA) concentration of 10-400 ng/ml. However, this concentration might be high enough to upregulate the expressions of some genes in differentiated macrophages, which could overwhelm gene expression increases induced by other stimuli. The present study was performed to optimize the PMA concentration required to differentiate monocytes whilst minimizing gene upregulation. THP-1 cells were treated with 2.5-100 ng/ml PMA and analyzed for the extent of cell adherence, the surface marker of macrophages, and stable differentiation without undesirable gene upregulation. The stably differentiated THP-1 cells at the minimum PMA concentration were treated with 10 ng/ml LPS or 125 nM amyloid beta (Abeta(1-42)). The treatment of THP-1 with 5 ng/ml PMA was found to be sufficient to induce stable differentiation without undesirable gene upregulation. These macrophages differentiated at 5 ng/ml responded well to secondary weak stimuli like 10 ng/ml LPS or 125 nM of amyloid beta (Abeta(1-42)). This finding suggests that THP-1 cells are well differentiated by 5 ng/ml PMA, and that the resulting differentiated macrophages respond well to secondary weak stimuli without being overwhelmed by undesirable gene upregulation induced by PMA.
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            Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers.

            Several epidemiologic studies have demonstrated a consistent association between levels of particulate matter (PM) in the ambient air with increases in cardiovascular and respiratory mortality and morbidity. Diesel exhaust (DE), in addition to generating other pollutants, is a major contributor to PM pollution in most places in the world. Although the epidemiologic evidence is strong, there are as yet no established biological mechanisms to explain the toxicity of PM in humans. To determine the impact of DE on human airways, we exposed 15 healthy human volunteers to air and diluted DE under controlled conditions for 1 h with intermittent exercise. Lung functions were measured before and after each exposure. Blood sampling and bronchoscopy were performed 6 h after each exposure to obtain airway lavages and endobronchial biopsies. While standard lung function measures did not change following DE exposure, there was a significant increase in neutrophils and B lymphocytes in airway lavage, along with increases in histamine and fibronectin. The bronchial biopsies obtained 6 h after DE exposure showed a significant increase in neutrophils, mast cells, CD4+ and CD8+ T lymphocytes along with upregulation of the endothelial adhesion molecules ICAM-1 and VCAM-1, with increases in the numbers of LFA-1+ cells in the bronchial tissue. Significant increases in neutrophils and platelets were observed in peripheral blood following DE exposure. This study demonstrates that at high ambient concentrations, acute short-term DE exposure produces a well-defined and marked systemic and pulmonary inflammatory response in healthy human volunteers, which is underestimated by standard lung function measurements.
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              The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte-macrophage differentiation.

              J Auwerx (1991)
              THP-1 is a human monocytic leukemia cell line. After treatment with phorbol esters, THP-1 cells differentiate into macrophage-like cells which mimic native monocyte-derived macrophages in several respects. Compared to other human myeloid cell lines, such as HL-60, U937, KG-1, or HEL cell lines, differentiated THP-1 cells behave more like native monocyte-derived macrophages. Because of these characteristics, the THP-1 cell line provides a valuable model for studying the mechanisms involved in macrophage differentiation, and for exploring the regulation of macrophage-specific genes as they relate to physiological functions displayed by these cells.
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                Author and article information

                Contributors
                +46 72 558 7110 , jie.ji@ki.se
                +46 72 558 7110 , swapna.upadhyay@ki.se
                eileenmiao2000@hotmail.com
                maria.malmlof@inhalation.se
                thomas.sandstrom@umu.se
                per.gerde@inhalation.se
                lena.palmberg@ki.se
                Journal
                Part Fibre Toxicol
                Part Fibre Toxicol
                Particle and Fibre Toxicology
                BioMed Central (London )
                1743-8977
                2 May 2018
                2 May 2018
                2018
                : 15
                : 19
                Affiliations
                [1 ]ISNI 0000 0004 1937 0626, GRID grid.4714.6, Institute of Environmental Medicine, , Karolinska Institute, ; Box 210, SE-171 77 Stockholm, Sweden
                [2 ]Inhalation Sciences Sweden AB, Stockholm, Sweden
                [3 ]ISNI 0000 0004 0623 991X, GRID grid.412215.1, Department of Public Health and Clinical Medicine, , University Hospital, ; Umeå, Sweden
                Article
                256
                10.1186/s12989-018-0256-2
                5930819
                29716632
                95b34d6b-ebae-47f0-b234-3431e00b8911
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 7 December 2017
                : 20 April 2018
                Funding
                Funded by: Swedish Fund for Research without Animal Experiments
                Award ID: 22/10, 40/11, F35/12, F25/13, F34-14 and F36/15
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004359, Vetenskapsrådet;
                Award ID: (VR 521 2010-2801 and 2014-02767)
                Award Recipient :
                Funded by: Swedish Heart-lung foundation
                Award ID: (20100180, 20120376, 20120818, 20150328, 20150329 and 20150330
                Award Recipient :
                Funded by: European Respiratory Society
                Award ID: (ERS: ERS LTRF 2014 - 3567
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                Toxicology
                diesel exhaust particles,air-liquid interface multicellular model,inflammation,macrophage polarization,oxidative stress

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