Quartz disrupts iron homeostasis in alveolar ... - ACS Publications

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Quartz disrupts iron homeostasis in alveolar macrophages to impact a pro-inflammatory effect Andrew J. Ghio, Joleen M. Soukup, Jacqueline Stonehuerner, Haiyan Tong, Judy Richards, M. Ian Gilmour, Michael C. Madden, Zhiwei Shen, and Stephen P. Kantrow Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00301 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019

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Chemical Research in Toxicology

Quartz disrupts iron homeostasis in alveolar macrophages to impact a pro-inflammatory effect

Andrew J. Ghio,†* Joleen M. Soukup,† Jacqueline Stonehuerner,† Haiyan Tong,† Judy Richards,† M. Ian Gilmour,† Michael C. Madden,† Zhiwei Shen, ‡ and Stephen P. Kantrow‡

From the †National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Chapel Hill NC 27514 and the ‡Section of Pulmonary and Critical Care Medicine, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA

*Correspondence

should be addressed to:

Andrew Ghio, Human Studies Facility, 104 Mason Farm Road, Chapel Hill NC 27599-7315 Telephone #: (919)-966-0670; FAX #: (919)-966-6271; Email: [email protected]

1 ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract The biological response of bronchial epithelial cells to particles is associated with a sequestration of cell metal by the particle surface and a subsequent disruption in host iron homeostasis. The macrophage is that cell type resident in the respiratory tract which inhaled particles are most likely to initially contact. We tested the postulates that 1) silica, a prototypical particle, disrupts iron homeostasis in alveolar macrophages (AMs) and 2) the altered iron homeostasis results in both an oxidative stress and pro-inflammatory effects. Human AMs (1.0x106/mL) demonstrated an increased import of iron following particle exposure with nonheme iron concentrations of 0.57±0.03, 1.72±0.09, 0.88±0.09, and 3.21±0.11 ppm in cells exposed for 4 hr to media, 500 µM ferric ammonium citrate (FAC), 100 µg/mL silica, and both silica and FAC respectively. Intracellular ferritin concentrations and iron release were similarly increased after AM exposure to FAC and silica. Silica increased oxidant generation by AMs measured using both dichlorofluorescein diacetate fluorescence and reduction of nitroblue tetrazolium salt. Concentrations of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α in macrophage supernatant increased following 100 µg/mL silica exposure for 24 hr. Treatment of AMs with 500 µM FAC decreased both oxidant generation and cytokine release associated with silica exposure supporting a dependence of these effects on sequestration of cell metal by the particle surface. We conclude that 1) silica exposure disrupts iron homeostasis resulting in increased import, accumulation, and release of the metal and 2) the altered iron homeostasis following silica exposure impacts oxidant generation and pro-inflammatory effects.

Keywords: Silicon dioxide; ferritin; inflammation; oxidative stress; interleukins Introduction



Inhalation of particulate matter (PM) presents a challenge to human health. Several of the most prominent causes of global deaths currently reported by the World Health Organization are associated with PM exposure including respiratory infections, chronic obstructive pulmonary disease, respiratory cancers, coronary heart disease, and stroke 1. The biological effects of particle exposure in respiratory epithelial cells can result from an alteration in host iron homeostasis 2. Oxygen-containing functional groups at the particle surface provide a capacity to bind cations. Among the cellular cations available for complexation by the particle surface, iron is abundant and kinetically preferred as a result of its electropositivity and high affinity for oxygen-containing functional groups 3, 4. Surface complexation of iron from sources in respiratory epithelial cells by a particle is associated with cell and mitochondrial superoxide production, activation of mitogen-activated protein (MAP) kinases and transcription factors, and release of inflammatory mediators culminating in an inflammatory injury 2. Macrophages function in lung defense against inhaled microorganisms and environmental pollutants including particles. Inhaled materials are phagocytosed by macrophages residing on the epithelial surfaces of the airways and the alveoli. Of the many types of macrophages found in the body, those in the respiratory tract have the most frequent contact with external stimuli. Subsequently, these cells have a pivotal role in the pathogenesis of numerous pulmonary diseases including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma, and pulmonary sarcoidosis 5, 6. The macrophage is adept at iron uptake 7. Comparable to respiratory epithelial cells, iron import by the macrophage is predicted to impact efficient function after particle exposure. Accordingly, for the cell to maintain maximal utility in the lower respiratory tract, metal export must follow its import. Despite pathways for its release, macrophages can accumulate


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significant concentrations of iron. Consequently, an exposure of the macrophage which overwhelms the capacity to export the metal is associated with production of hemosiderin (or a comparable product), which stains in the Perl’s Prussian blue reaction (i.e., a sideromacrophage or siderophage). We tested the postulate that 1) silica, a prototypical particle, disrupts iron homeostasis in alveolar macrophages (AMs) with increased import and export and 2) the altered iron homeostasis results in both an oxidative stress and pro-inflammatory effects.

Experimental procedures Materials. Reagents were from Sigma Co. (St. Louis, MO) unless specified otherwise. Silica (SiO2) was chosen as a prototypical particle for study because of its historical and public health significance and the ease with which its surface metal concentration can be manipulated. The silica used was Min-U-Sil-5 (U.S. Silica, Berkeley Springs, WV) which is a quartz particle with a 5µ diameter and subsequently considered both respirable and relevant to human health. For selected endpoints, the effect of silica was compared to silica with surface-complexed iron. Silica demonstrated a capacity to complex iron from solutions of ferric chloride, ferric sulfate, and ferric ammonium sulfate but not from solutions of either ferric citrate or ferric ammonium citrate. Subsequently, acid-washed silica (Silica-H) and iron-loaded silica (Silica-Fe) were prepared by washing Min-U-Sil in 1 N HCl for 1 hr and agitating the particle in either H2O or 1000 µM ferric ammonium sulfate in H2O for 1 hr (x4). The particle was centrifuged (1000 g x 10 min), washed in H2O, and dispersed into PBS at 20 mg/mL. The ionizable iron concentration of the particles was quantified as that displaced into 1.0 N HCl after 1 hr agitation. Iron concentrations were measured using inductively coupled plasma optical emission



spectroscopy (ICPOES; Model Optima 4300D, Perkin Elmer, Norwalk, CT) operated at a wavelength of 238.204 nm. The iron concentration associated with Silica-H particle was below detectable limits while the Silica-Fe particle contained 97.2 ± 2.6 µg/g. The diameters of Silica-H and Silica-Fe particles were measured using dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). The particle was suspended in distilled water at 300 µg/mL and the optical density at 633 nm was determined. The refractive index of silica was taken as 1.547. The sizing protocol recorded 5 independent measurements of three Silica-H and three Silica-Fe particles. Mean diameters (± standard deviations) for Silica-H and Silica-Fe were not significantly different at 1.40 ± 0.42 and 1.99 ± 0.53 µm respectively. Cell Culture. The protocol and consent form for the acquisition of AMs through fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) were approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects 8. AM incubations and exposures were cell suspensions in RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 2% fetal calf serum (FCS) (Invitrogen) and gentamicin solution (20 µg/ml) (Sigma, St. Louis, MO). Cytotoxicity of AM was determined using trypan blue exclusion. This dye exclusion test is used to determine the number of viable cells present in a cell suspension with live cells possessing intact cell membranes that exclude the dye. THP1 cells are a monocyte-like cell line employed in investigations requiring exposures with duration greater than 24 hr. THP1 cell incubations and exposures were cell suspensions in RPMI-1640 supplemented with 10% FCS and gentamicin solution (20 µg/ml). Cell iron import after exposures to iron and silica. AMs (1.0 x 106 cells/mL) were exposed to 0, 250, and 500 µM ferric ammonium citrate (FAC) both with and without 100 µg/mL silica. After 4 hr incubation, the cells were centrifuged (300g x 10 min), washed with PBS, and placed


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into 1.0 mL 3 N HCl/10% trichloroacetic acid (TCA). After hydrolysis at 70o C for 24 hr with precipitation of heme in TCA, non-heme iron concentrations were determined using ICPOES. AMs (1.0 x 106 cells/mL) were exposed to media, 100 µg/mL silica, 500 µM ferric ammonium citrate (FAC), and both FAC and silica. After 1, 4, and 24 hr incubation, cell nonheme iron was determined after hydrolysis using ICPOES. A small aliquot of macrophages (50,000 cells/200 µL) at the 24 hr time point were cytocentrifuged and stained for iron (Perls’ Prussian blue stain). THP1 cells (1.0 x 106 cells/mL) were exposed to either media or 100 µg/mL silica. Twenty-four hours later, cells were centrifuged, resuspended in fresh media, and either media or FAC added to the incubation. After 4 hr, cell non-heme iron was determined after hydrolysis. AMs (1.0 x 106 cells/mL) were exposed to media, 100 µg/mL silica, 500 µM FAC, and both FAC and silica. Incubations included either media or 100 µM apocynin, an inhibitor of NADPH oxidase activity. After 4 hr incubation, cell non-heme iron was determined after hydrolysis using ICPOES. Cell ferritin concentrations. AM (1.0 x 106 cells/mL) exposures to media, 500 µM FAC, 100 µg/mL silica, and both FAC and silica were repeated for 24 hr. After centrifugation (300g x 10 min) and removal of the media, cells were washed with PBS, placed in 1.0 mL PBS, and disrupted using five passes through a small gauge needle. The ferritin concentrations in the lysates were quantified using an immunoturbidimetric assay (Kamiya Biomedical Company, Seattle, WA). Cellular oxidant generation. Oxidant generation by AMs was determined using 2,7dichlorofluorescein diacetate (DCF) fluorescence. AMs were treated with and without 500 µM FAC for 4 hr. Cells were loaded with the dye at 20 µM for 20 min prior to exposure. The cells



were washed twice with 1 mL/tube PBS. Following the second wash, 50,000-100,000 macrophages were placed in each of 96 wells in a white walled plate (Corning Cat. No. 07-200568 Corning, NY). Silica was added to the appropriate wells in 100 µL PBS and the plate was read on a Perkin Elmer HTS7000 Bioassay Plate Reader using an excitation filter of 485nm and emission filter at 535nm at various time points. The reported value is fold change over control cells which have been pre-loaded with dye and exposed to PBS only (time= 0 min). The nitro blue tetrazolium (NBT) reduction reaction was employed for colorimetric assay of oxidoreductase activity. AMs (200,000/0.2 mL RPMI 1640 with 2% serum) were treated with and without 500 µM FAC for 4 hr at 37o C in polypropylene tubes and centrifuged. Fresh media was provided and cells were incubated with and without 100 µg silica/mL in 0.2 mg NBT/0.2mL for 30 minutes at 37o C. Cells (approximately 50,000) were cytocentrifuged onto slides, counterstained with Nuclear Fast Red, dried, and micrographs obtained. Release of inflammatory mediators. Following AM (1.0 x 106 cells/mL) exposures to media, 500 µM FAC, 100 µg/mL silica, and both FAC and silica for 24 hr, interleukin (IL)-1β, IL-6, IL8, and tumor necrosis factor (TNF)-α concentrations in cell supernatant were measured using the MSD Human Pro-Inflammatory 4-Plex II kits (catalog number K15025B-2 MesoScale Discovery Rockville, MD). Kits were run according to package directions with no dilution of the samples. AM iron export and ferritin release after exposures to iron and silica. AMs (1.0 x 106 cells/mL) were exposed to media, 500 µM FAC, 100 µg/mL silica, and both FAC and silica for 4 hr, centrifuged (300g x 10 min), washed with PBS, and placed into 1.0 mL RPMI 1640 with 2% FCS. After 4, 8, and 12 hr, the cells were centrifuged (300g x 10 min) and both the supernatant and the cells were obtained. Iron concentrations in the cell supernatants were determined using


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ICPOES. Cells were washed with PBS and placed into 1.0 mL 3 N HCl/10% TCA. After hydrolysis, non-heme iron concentrations were determined using ICPOES. AMs (1.0 x 106 cells/mL) were exposed to media, 500 µM FAC, 100 µg/mL silica, and both FAC and silica for 24 hr, centrifuged (600g x 10 min), washed with PBS, and placed into 1.0 mL RPMI 1640 with 2% FCS. After 4, 8, and 12 hr, both the supernatant and the cells were collected. Ferritin concentration in the cell supernatants was quantified. Cells were placed into 1.0 mL PBS, and lysed using five passes through a 25-gauge needle. The ferritin concentrations in the lysates were quantified. THP1 cells were exposed to media, 500 µM FAC, 100 µg/mL silica, and both FAC and silica for 1 and 2 weeks (media with or without FAC was replaced weekly). The cells were collected, placed in tubes (1.0 x 106 cells/mL in replicates of 4), washed with PBS, and hydrolyzed using 1.0 mL 3 N HCl/10% TCA. After hydrolysis, non-heme iron concentrations were determined using ICPOES. THP1 cells were exposed to media, 500 µM FAC, 100 µg/mL silica, and both FAC and silica for 1 week. The cells were collected and placed into fresh media (without FAC) for 1 week. The cells were centrifuged (300g x 10 min), placed in tubes (1.0 x 106 cells/mL in replicates of 4), washed with PBS, and hydrolyzed using 1.0 mL 3 N HCl/10% TCA. AM iron export and ferritin and IL-8 release after exposures to Silica-H and Silica-Fe. AMs (1.0 x 106 cells/mL) were exposed to 100 µg/mL Silica-H and Silica-Fe for 4 hr. Both the supernatants and the cells were collected. After hydrolysis, non-heme iron concentrations in the cell lysates were determined using ICPOES. Iron concentrations in the cell supernatants were determined using ICPOES. AMs (1.0 x 106 cells/mL) were again exposed to 100 µg/mL Silica-H and Silica-Fe for 24



hr. Both the cells and the supernatants were collected. Ferritin concentrations in the cell lysates and the supernatants were quantified. IL-8 was measured in the supernatant following AM exposures to 100 µg/mL Silica-H and Silica-Fe for 24 hr. AM iron export and ferritin and IL-8 release after treatment with apocynin and exposure to Silica-Fe. AMs (1.0 x 106 cells/mL) were exposed to either media or 100 µg/mL Silica-Fe with and without 100 µM apocynin. Supernatants were obtained at 4 and 24 hr. Iron and ferritin concentrations were determined using ICPOES and immunoturbidimetric assay respectively. IL8 levels were also measured in the supernatant. AMs from wild-type and gp91phox deficient mice, iron export, and ferritin and cytokine release after exposure to Silica-Fe. Wild-type male C57BL/6 and gp91phox-deficient mice were purchased from Jackson Laboratories (Bar Harbor, ME), maintained on a standard laboratory diet, and housed in a controlled environment with a 12-hr light/12-hr dark cycle. Experiments were performed when mice were 8–12 weeks old. Approval for these experiments was obtained from the Louisiana State University Institutional Animal Care and Use Committee (New Orleans, LA). Mice were anesthetized by injection of pentobarbital sodium (80 mg/kg intraperitoneally), the trachea was exposed and cannulated, and the animals were euthanized by exsanguination through the abdominal aorta. BAL was performed with 0.5 ml volume of PBS for a total of 3 mL. The collected fluid was centrifuged and AMs isolated. AMs (0.5 x 106 cells/mL) from wild-type and gp91phox-deficient mice were exposed to 100 µg/mL Silica-Fe for 4 and 24 hr. Non-heme iron and ferritin concentrations and macrophage inflammatory protein (MIP)-1 release were measured in the supernatants. Statistics. Data are expressed as mean values ± standard deviation (SD) unless specified


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otherwise. AM from a minimum of three volunteers were used for all endpoints. Measurements using AMs were determined in replicates of three unless specified otherwise; the minimal n is therefore 9. Experiments using THP1 cells were done in replicates of eight. Differences between two groups were compared using T-tests of independent means and those between more than two groups were compared using analysis of variance (ANOVA; one-and two-way); the post-hoc test employed was the Tukey test. Two-tailed tests of significance were employed. Significance was assumed at p

Quartz disrupts iron homeostasis in alveolar ... - ACS Publications

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