¤ Nanoparticles in polluted air, smoke and nanotechnology products have serious impact on health

Source : http://phys.org/news/2012-06-nanoparticles-polluted-air-nanotechnology-products.html

June 11, 2012

New groundbreaking research by scientists at Trinity College Dublin has found that exposure to nanoparticles can have a serious impact on health, linking it to rheumatoid arthritis and the development of other serious autoimmune diseases. The findings that have been recently published in the international journal Nanomedicine have health and safety implications for the manufacture, use and ultimate disposal of nanotechnology products and materials. They also identified new cellular targets for the development of potential drug therapies in combating the development of autoimmune diseases.

Environmental pollution including carbon particles emitted by car exhaust, smoking and long term inhalation of dust of various origins have been recognised as risk factors causing chronic inflammation of the lungs. The link between smoking and autoimmune diseases such as rheumatoid arthritis has also been established. This new research now raises serious concerns in relation to similar risks caused by nanotechnology products which if not handled appropriately may contribute to the generation of new types of airborne pollutants causing risks to global health.

In their research, the Nanomedicine and Molecular Imaging team at Trinity College Dublin’s School of Medicine led by Professor of Molecular Medicine, Yuri Volkov investigated whether there was a common underlying mechanism contributing to the development of autoimmune diseases in human cells following their exposure to a wide range of nanoparticles containing different physical and chemical properties.

The scientists applied a wide range of nanomaterials including ultrafine carbon black, carbon nanotubes and silicon dioxide particles of different sizes, ranging from 20 to 400 nanometres, to human cells derived from the lining of the airway passages, and to the cells of so-called phagocytic origin − those cells that are most frequently exposed to the inhaled foreign particles or are tasked with cleaning up our body from them. At the same time, collaborating researchers from the Health Effects Laboratory Division, National Institute for Occupational Safety & Health (Morgantown, WV, USA) have conducted the studies in mice exposed to chronic inhalation of air contaminated with single walled carbon nanotubes.

The result was clear and convincing: all types of nanoparticles in both the TCD and US study were causing an identical response in human cells and in the lungs of mice, manifesting in the specific transformation of the amino acid arginine into the molecule called citrulline which can lead to the development of autoimmune conditions such as rheumatoid arthritis.

In the transformation to citrulline, human proteins which incorporate this modified amino acid as building blocks, can no longer function properly and are subject to destruction and elimination by the bodily defence system. Once programmed to get rid of citrullinated proteins, the immune system can start attacking its own tissues and organs, thereby causing the autoimmune processes which may result in rheumatoid arthritis.

Commenting on the significance of the findings, TCD’s Professor Volkov says: « The research establishes a clear link between autoimmune diseases and nanoparticles. Preventing or interfering with the resulting citrullination process looks therefore as a promising target for the development of future preventative and therapeutic approaches in rheumatoid arthritis and possibly other autoimmune conditions. »

More information: The paper’s full title published in the ‘Nanomedicine‘ journal (Future Medicine journals group) is « Citrullination of proteins: a common post-translational modification pathway induced by different nanoparticles in vitro and in vivo » http://www.futurem … 7/nnm.11.177

Provided by Trinity College Dublin

FULL TEXT « NANOMEDICINE » ARTICLE :

Full Text Nanomedicine Posted online on May 25, 2012. (doi:10.2217/nnm.11.177)   Citrullination of proteins: a common post-translational modification pathway induced by different nanoparticles in vitro and in vivo Bashir M Mohamed‡, Navin K Verma‡, Anthony M Davies, Aoife McGowan, Kieran Crosbie Staunton, Adriele Prina-Mello, Dermot Kelleher,Catherine H Botting, Corey P Causey, Paul R Thompson, Ger JM Pruijn, Elena R Kisin, Alexey V Tkach, Anna A Shvedova* & Yuri Volkov* * Author for correspondence ‡Authors contibuted equally Sections: ChooseABSTRACTMaterials & methodsResultsDiscussionFuture perspectiveReferences   ABSTRACT ChooseTop of pageABSTRACT <<Materials & methodsResultsDiscussionFuture perspectiveReferences   Aim: Rapidly expanding manufacture and use of nanomaterials emphasize the requirements for thorough assessment of health outcomes associated with novel applications. Post-translational protein modifications catalyzed by Ca2+-dependent peptidylargininedeiminases have been shown to trigger immune responses including autoantibody generation, a hallmark of immune complexes deposition in rheumatoid arthritis. Therefore, the aim of the study was to assess if nanoparticles are able to promote protein citrullination. Materials & methods:Human A549 and THP-1 cells were exposed to silicon dioxide, carbon black or single-walled carbon nanotubes. C57BL/6 mice were exposed to respirable single-walled carbon nanotubes. Protein citrullination, peptidylargininedeiminases activity and target proteins were evaluated.Results: The studied nanoparticles induced protein citrullination both in cultured human cells and mouse lung tissues. Citrullination occurred via the peptidylargininedeiminase-dependent mechanism. Cytokeratines 7, 8, 18 and plectins were identified as intracellular citrullination targets. Conclusion: Nanoparticle exposure facilitated post-translational citrullination of proteins. Original submitted 18 March 2011; Revised submitted 10 Novemeber 2011   View larger version(122K) View larger version(91K)

Figure 1. Cellular uptake of nanomaterials and induction of protein citrullination (continued overleaf).(A) Transmission electron microscopy image of A549 cells exposed to 40 nm SiO2 NPs for 3 h. The arrow indicates internalized SiO2 NPs. (B) Fluorescent image of A549 cells exposed to tetramethyl rhodamine iso-thiocynate-labeled SiO2 NPs (30 nm) for 3 h was acquired by an IN Cell Analyzer 1000 automated microscope using 20× objective. Arrows indicate internalized SiO2 NPs. (C & D) A549 and THP-1 cells were exposed to indicated concentrations of SiO2NP (20, 30, 40, 80 or 400 nm), ufCB or SWCNT for 1–24 h. Cells were immunostained with anticitrulline antibody (cat#ab6464), imaged using automated microscope, and protein citrullination was quantified as presented in Heatmaps. Heatmaps were generated for the above indicated protein citrullination and their colorimetric gradient table spans from: dark green: lower than 15% of maximum value measured; bright green: 30%; yellow: 50%; bright orange: 60%; dark orange: 75%; red: higher than 75% of the maximum value. (E) A549 cells unexposed (N/T), exposed to 80 nm SiO2 NPs or PAD (positive control) for 24 h and fixed in 3% paraformaldehyde. Cells were immunostained with anticitrulline antibody (cat#ab6464) (green) and nuclei were stained with Hoechst (blue). Protein citrullination was visualized by IN Cell Analyzer 1000 using a 20× objective lens. (F) A549 cells (N/T) were exposed to 500 µg/ml SiO2 NPs (20, 30, 40, 80 or 400 nm), 40 µg/ml ufCB or 40 µg/ml SWCNTs for 24 h and were then lysed. Cell lysates (50 µg each) were resolved by SDS-PAGE and after western blotting were probed for citrullinated proteins (Millipore, cat# 07-377) or tubulin. Results shown are representative of three independent experiments.

NP: Nanoparticle; N/T: No treatment; PAD: Peptidylargininedeiminase; SiO₂: Silicon dioxide; SWCNT: Single-walled carbon nanotube; ufCB: Ultra-fine carbon black.

 

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Figure 2. Immunohistochemical analysis of citrullinated proteins.Mice were exposed to SWCNT by pharyngeal aspiration and sacrificed on days 7, 28, 2 months or 6 months. Lung tissue sections (5 µm thick) were prepared and stained immunohistochemically with anticitrulline antibody (cat#ab6464). (A) Lung tissue section from control mice. (B) Lung tissue section from SWCNT treated mice. Arrows indicate the anticitrulline antibody positive cells. (C) The induction of protein citrullination observed in the lung of mice following SWCNT exposure. The stained slides were digitally scanned and quantified as described in the ‘Materials & methods’ section. Statistical analysis was carried out by two-way analysis of variance with Bonferroni post-test analysis.

 

*Statistically significant data p < 0.001.

N/T: No treatment; SWCNT: Single-walled carbon nanotube.

 

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Figure 3. Peptidylargininedeiminase activity assays.(A & B) A549 and (C & D) THP-1 cells were exposed to 500 µg/ml 80 nm SiO₂ NPs or 40 µg/ml SWCNTs for 24 h and were then lysed. (A & C) The PAD enzyme activity in cell lysates (5 µg each) was determined and presented (mean OD_{450 nm} ± standard error of mean). (B & D) Cells were lysed, the lysates (50 µg each) were resolved by SDS-PAGE and after western blotting were probed for PAD2, PAD4 or tubulin. Results shown are representative of three independent experiments.

 

*Statistically significant data is p < 0.05.

NP: Nanoparticle; N/T: No treatment; OD: Optical density; PAD: Peptidylargininedeiminase; SiO₂: Silicon dioxide; SWCNT: Single-walled carbon nanotube.

 

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Figure 4. Effect of peptidylargininedeiminase inhibition on nanomaterial-induced protein citrullination.(A) A549 and (B) THP-1 cells were pretreated with 50, 100 or 200 µM Cl-amidine for 2 h and then exposed to SiO₂ NPs or SWCNTs for 24 h. The peptidylargininedeiminase enzyme activity in cell lysates (5 µg each) was determined by the ELISA method using ModiQuest antibody-based assay. Absorbance (OD_{450 nm}) was measured and presented. Untreated or Cl-amidine pretreated (C) A549 or (D) THP-1 cells were exposed to various sizes of SiO₂ NPs or ultra-fine carbon black. Cells were immunostained with anticitrulline antibody (cat#ab6464) and imaged using automated microscope IN Cell Analyzer 1000 and quantified. Data are mean ± standard error of mean of three independent experiments performed in triplicates.

 

NP: Nanoparticle; OD: Optical density; PAD: Peptidylargininedeiminase; RFU: Relative fluorescence units; SiO₂: Silicon dioxide; SWCNT: Single-walled carbon nanotube; ufCB: Ultra-fine carbon black.

 

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Figure 5. Intracellular Ca²⁺ levels in cells exposed to nanomaterials and the effect of blocking Ca²⁺ channels on protein citrullination.(A & B) A549 and (C &D) THP-1 cells were exposed to various sized SiO₂ NPs in the presence or absence of a Ca²⁺ CCB verapamil (10 µM). (A & B) Intracellular levels of free Ca²⁺ were measured and plotted as RFU. Verapamil treated or untreated cells were exposed to SiO₂ NP (30, 40, 80 or 400 nm) for 24 h, protein citrullination levels were analyzed and plotted as relative fluorescence units (RFU; C & D). Data are mean ± standard error of mean of three independent experiments performed in triplicates.

 

CCB: Ca²⁺ channel blocker; NP: Nanoparticle; RFU: Relative fluorescence units; SiO₂: Silicon dioxide.

 

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Figure 6. Proteomic analysis of citrullinated proteins.A549 cells (N/T) were exposed to 500 µg/ml 80 nm SiO₂ NPs or 40 µg/ml ufCB for 24 h and were then lysed. (A)Cell lysates (1 mg each) were immunoprecipitated using anticitrulline antibody (Millipore, cat#07-377), resolved by SDS-PAGE (10% gel) and visualized by colloidal coomassie blue staining. Bands of interest, as indicated, were excised and processed for subsequent mass spectrometric analysis. (B & C) Cell lysates (500 µg each) were immunoprecipitated with anticitrulline antibody (immunoprecipitated:anticitrulline) or IgG (isotype control; immunoprecipitated:IgG). Immunoprecipitates and WCLs (used as a control 20 µg each) were WB and probed with (B) anticytokeratin 7 or (C) anticytokeratin 8/18. Relative densitometric analysis of the individual band was performed and presented. Data are mean ± standard error of mean of three independent experiments.

 

*Statistically significant data is p < 0.05.

NP: Nanoparticle; N/T: No treatment; SiO₂: Silicon dioxide; ufCB: Ultra-fine carbon black; WB: Western blotted; WCL: Whole-cell lysate.

 

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Figure 7. Mechanism(s) of nanomaterials-induced protein citrullination.Exposure to nanomaterials increases intracellular Ca²⁺. Elevation of cytosolic Ca²⁺ results in subsequent activation of PADs, which in turn cause citrullination of target proteins (cytokeratins and plectins). Blocking of Ca²⁺ channels or inhibition of PAD activity downregulates protein citrullination.

 

PAD: Peptidylargininedeiminase.

Recent progress in nanotechnology has enabled the manufacturing of nanosized materials for various applications ranging from information technologies to advanced composite materials, consumer products, healthcare and life sciences. Because of their fascinating physicochemical properties, nanomaterials exhibit unique bioactivity. However, there remains considerable uncertainty regarding the potential risk to human health related to the widespread production and use of nanomaterials. Numerous epidemiological studies have associated exposure to ambient ultra-fine carbonaceous particles in air pollution to various diseases, including chronic obstructive pulmonary disease, pneumonia, heart attacks, autoimmune disorders and all-cause mortality increased with longer time scales [1–9]. It has been reported earlier that silica exposure is associated with increased risk of developing rheumatoid arthritis (RA) [10]. Moreover, smoking, which has long since been considered a nonspecific risk factor causing chronic inflammation, is now known to be associated with autoimmune diseases such as RA [9,11,12].

Chronic inflammatory diseases affect millions of people across the globe leading to untold suffering, economic burden and premature death. Recent studies have identified inflammation and the recruitment of immune cells to the site injury with a unique role for IL-1β activating platforms, known as inflammasomes, in the regulation/induction and pathogenesis of multiple autoimmune and inflammatory disorders [13]. Inflammation caused by airborne particles is associated with respiratory ailments including chronic obstructive pulmonary disease and autoimmune diseases, for example, RA [1,8,9,14]. It has been well documented that nanoparticles (NPs) are efficiently internalized by epithelial cells and professional phagocytes. Size- and dose-dependent cellular uptake, toxicity, stimulation/release of proinflammatory mediators and formation of nucleoplastic protein aggregates were reported in BEAS2, THP-1, and A549 cells treated with nanosized amorphous silica [15–18]. Single-walled carbon nanotubes (SWCNTs) functionalized by phosphatidylserine, were efficiently internalized by different phagocytic cells, such as murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells and rat brain microglia, for example [19]. It was also documented that SWCNTs localized in lysosomal compartments of alveolar macrophages after pulmonary exposure [19]. Oxidative stress induced by micro-/nano-sized particles has been reported to cause protein modifications leading to compromised protein recognition, perhaps contributing to autoimmunity [20].

Antibodies to citrullinated proteins have a high diagnostic value in RA and are linked to the pathogenesis of several autoimmune diseases[12,21–25]. Citrullinated proteins are generated by a post-translational deimination of polypeptide-bound arginine by a family of Ca²⁺-dependent enzyme peptidylarginine deiminase (PAD) [22,26,27]. Several isotypes of PAD exist, each with different tissue distribution [26–28]. PAD2 and PAD4 are most important as they are widely expressed in a variety of tissues, including hematopoietic cells [26–28]. Citrullination results in a loss of net positive charge of molecules and causes significant biochemical changes and/or protein conformational changes [22]. Citrullinated proteins/peptides are recognized as nonself proteins, and subsequently induce an autoimmune response [21,22,24,25,29]. Citrullination is essential for the formation of neutrophil extracellular traps [30]. Site-specific citrullination was reported to alter chemokine function [31–33]. We hypothesize that exposure of amorphous silicon dioxide (SiO₂), ultra-fine carbon black (ufCB) and SWCNTs to A549 epithelial and professional phagocyte THP-1 cells cause enhanced PAD activity via the increase of extracellular Ca²⁺, thus facilitating protein citrullination. Here, we demonstrate that nanomaterials of distinct origin, morphology and physicochemical properties are able to induce protein citrullination via increased Ca²⁺-mediated PAD activity in human cells and animals.

Materials & methods	ChooseTop of pageABSTRACTMaterials & methods <<ResultsDiscussionFuture perspectiveReferences

▪ NanomaterialsThe SiO₂ NPs; 30, 80 and 400 nm (Glantero Ltd, Cork, Ireland), positively-charged alumina-coated chloride ion-stabilized SiO₂ NPs (40 nm; LUDOX CL 420891, Sigma-Aldrich, MO, USA), sodium counterion stabilized SiO₂ NPs (20 nm; LUDOX CL 420883, Sigma-Aldrich, ufCB [Printex 90, Degussa, Germany]), and SWCNTs (Carbon Nanotechnologies Inc., TX, USA) were used. The physicochemical properties of SiO₂ NPs have previously been described [34]. The SWCNTs were manufactured using the high-pressure carbon monoxide disproportionation process. Purity assessment of high-pressure carbon monoxide disproportionation process SWCNTs was carried out by using several standard analytical techniques, including thermogravimetric analysis with differential scanning colorimetry, thermoprograming oxidation, and Raman and near infrared spectroscopy [35]. All the nanomaterials were dispersed in culture media. SWCNTs were sonicated (Branson Sonifier 450, CT, USA) before treatments.

▪ Cell cultureHuman lung epithelial cell line A549 and a phagocytic cell line THP-1 (ATCC, VA, USA) were cultured in DMEM and RPMI 1640 medium, respectively. Both the media were supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) L-glutamine/penicillin/streptomycin. Cells were grown in a humidified incubator at 37^oC in 5% CO₂. THP-1 cells were stimulated with phorbol 12-myristate 13-acetate (25 ng/ml) before experimentation.

▪ AnimalsAnimal studies were carried out under the experimental protocol (#07-AS-M-010) approved by the Institutional Animal Care and Use Committee at the National Institute for Occupational Safety and Health (WV, USA). The National Institute for Occupational Safety and Health facilities are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care–International Committee. Animals were supplied, housed and exposed as described previously [35]. Briefly, 20.0 ± 1.9 g C57BL/6 pathogen-free adult female mice (Jackson Laboratory, ME, USA) were housed in solitary cages supplied with high efficiency particulate-filtered air. Mice were given water and certified chow 7913 (Teklad, IN, USA) ad libitum in compliance with the policies of the Institute of Laboratory Animal Resources (National Research Council). Experimental and control groups were given equal volumes of SWCNTs (40 µg/mouse) or sterile magnesium- and calcium-free phosphate-buffered saline (PBS) solution by pharyngeal aspiration. Experimental and control groups were euthanized at 7 days, 28 days, 2 months and 6 months following exposures.

▪ Antibodies & reagentsRabbit polyclonal anticitrulline (cat#ab6464), rabbit monoclonal anticytokeratin 7, rabbit polyclonal anticytokeratin 8/18 and fluorescein isothiocyanate (FITC)-linked goat antirabbit antibodies were from Abcam (Abcam plc., Cambridge, UK). Immunoaffinity purified rabbit polyclonal anticitrulline antibody used for western blot analysis was from Millipore (cat#07-377, MA, USA). The specificity data of anticitrulline antibodies provided by the manufacturer showed high reactivity with citrulline, compared with very low cross-reactivity with homocitrulline (10³-times higher for citrulline; Abcam plc., Cambridge, UK). Furthermore, in-house validation was assessed against the manufacturer’s data(Supplementary Figure S1). Rabbit antihuman PAD2 and antihuman PAD4 antisera were produced by GJM Pruijn and WJ van Venrooij, Nijmegen, The Netherlands. Horseradish peroxidase-conjugated anti-rabbit IgG and antimouse IgG antibodies were from Dako A/S (Glostrup, Denmark). Cl-amidine was synthesized as described previously [36]. Unless attributed specifically, all the reagents were from Sigma-Aldrich, and plastic wares were from Nunc (Thermo Fisher Scientific Inc., MA, USA).

▪ Analysis of protein citrullinationCells were seeded in 96-well plates (1 × 10⁴ cells/well), exposed to various concentrations of nanomaterials for multiple time points and fixed using 3% paraformaldehyde as described [37]. After gentle washing with PBS, cells were incubated with anticitrulline antibody (cat#ab6464, 1:200 dilution) for 1 h at room temperature. Cells were washed three times with PBS and then incubated with FITC-linked goat anti-rabbit antibody and Hoechst 33342 for 1 h. Plates were scanned using IN Cell Analyzer 1000 automated microscope (GE Healthcare, Buckinghamshire, UK). Images were acquired in a stereology configuration of five randomly selected fields per well at 20× magnification using two detection channels. These included a 4´,6-diamidino-2-phenylindole filter (channel 1: λ = 461 nm), which detected blue fluorescence indicating nuclear staining and FITC filter (channel 2: λ = 509 nm), which detected green fluorescence indicating citrullinated proteins. Protein citrullination was quantified using the dual area object analysis module of the IN Cell Investigator software (GE Healthcare, Buckinghamshire, UK). The module allows for simultaneous quantification of subcellular inclusions that are marked by different fluorescent labels and measures fluorescence intensity associated with predefined nuclear and cytoplasmic compartments.

▪ Immunohistochemical staining & analysisThe lung tissue preparation including preservation and fixation was performed as previously described [35,38]. Immunohistochemical investigation for the presence of citrullinated proteins was conducted on 5-µm thick lung sections using the avidin–biotin–peroxidase complex detection procedure [39]. Stained tissue sections were digitally scanned using Aperio ImageScope™ and the images were analyzed automatically by ImageScope software (Aperio Technologies Inc., CA, USA), as described [40].

▪ Cell lyses, immunoprecipitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis & western immunoblottingThe cell lysis, immunoprecipitation and western immunoblotting were performed as described previously [41,42].

▪ Nano-liquid chromatography-electrospray ionization mass spectrometry/mass spectrometryBands of interest on colloidal coomassie blue-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis gel were excised and the proteins were identified by Nano-liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry as described [43]. Ten or more matching peptides and a significant probability score (p < 0.05 = MOWSE score >150) were required for a secure identity assignment.

▪ PAD enzyme activity measurementsCells were exposed to the above indicated nanomaterials in triplicates for indicated time points and were lysed. PAD enzyme activity was determined by the ELISA method using ModiQuest Antibody Based Assay for PAD Activity (ABAP) kit according to the manufacturer’s instructions (ModiQuest Research, Nijmegen, The Netherlands).

▪ Measurements of intracellular Ca²⁺Cells were seeded in 96-well plate and allowed to adhere overnight. Following exposure to nanomaterials, cells were gently washed with prewarmed fresh medium. They were then incubated with 1 µM Hoechst 33342 and 1 µM Fluo-4 (fluo-4 acetoxymethyl ester) for 1 h. Plates were scanned using IN Cell Analyzer 1000 automated microscope (GE Healthcare). Images were acquired in a stereology configuration of five randomly selected fields per well at 10× magnification using two detection channels. Hoechst 33342 was visualized in the blue channel (4´,6-diamidino-2-phenylindole filter) while Fluo-4 was visualized in the green channel (FITC filter). Intracellular free Ca²⁺ concentration was measured by the fluorescence intensity of Fluo-4 in a circular region (cellular compartment) centered at the nucleus and quantified using the dual area object analysis module of the IN Cell Investigator software (GE Healthcare).

▪ Statistical analysisResponse of each cell type to various NPs was analyzed by two-way analysis of variance with Bonferroni post-test analysis. A p-value of <0.05 was considered to be statistically significant (GraphPad Prism 4, GraphPad Software, CA, USA). Konstanz Information Miner [101] data exploration platform and the screening module HiTS [102] were used to visualize the data [44–46]. The protein citrullination level induced by the nanomaterials was normalized using the percent of the positive controls. The Z score was used for scoring the normalized values. These scores were summarized using the mean function as follows: Z score = (x-mean)/standard deviation, as from previous work [47]. As previously described, Heatmap-type graphical illustration in a colorimetric gradient table format was adopted as the most suitable schematic representation to report on any statistical significance and variation from normalized controls based on their Z score values [46].

Results	ChooseTop of pageABSTRACTMaterials & methodsResults <<DiscussionFuture perspectiveReferences

▪ Nanomaterials induced protein citrullination in human cellsHuman lung epithelial A549 cells and phagocytic THP-1 cells were employed in this study. Transmission electron microscopy images showed internalization of SiO₂ NPs (40 nm) by A549 cells after exposure (3 h) (Figure 1A). Similar results were observed when A549 cells were exposed to fluorescently labeled SiO₂NP (30 nm) and examined by fluorescent microscopy (Figure 1B). To investigate the potential effect of nanomaterials on protein citrullination, A549 and THP-1 cells were exposed to various concentrations (10, 50, 100 or 500 µg/ml) of SiO₂ NPs (20, 30, 40, 80 or 400 nm), ufCB (1, 4, 10 or 40 µg/ml) or SWCNTs (1, 4, 10 or 40 µg/ml) for multiple time-points. High content analysis for the induction of protein citrullination was performed and heatmaps were generated (Figures 1C & 1D). The data indicated a significant increase in protein citrullination in both cell lines found in a time- and dose-dependent manner with maximum effects seen at 24 h (Figures 1C & 1D andSupplementary Figure S2–S4). Immunofluorescent images of cells exposed to silica or carbon nanomaterials showed citrullinated proteins in the cytoplasm and around the nucleus (Figure 1E & Supplementary Figure S5).To further validate if citrullinated proteins were expressed after 24 h in the cells treated with nanosized SiO₂ NPs, ufCB or SWCNTs, the cells were lysed and cell lysates were electrophoretically separated and probed with anticitrulline antibody (Millipore, cat#07-377). Four distinct citrullinated protein bands were detected by this method (Figure 1F and Supplementary Figure S6), while a smear-like pattern appeared when the cell lysate was preincubated with PAD (Supplementary Figure S7). Densitometric quantitation of citrullinated protein bands were normalized by tubulin content indicating a significant fourfold increase in protein citrullination after NP exposures compared with those of respective unexposed controls (Figure 1F).

▪ SWCNT exposure induced protein citrullination in mouse lung tissuesTo investigate if exposure to respirable SWCNTs facilitated protein citrullination in vivo, C57BL/6 mice were given SWCNTs by pharyngeal aspiration. Lungs were harvested from mice during recovery time up to 6 months postexposure and the lung tissue sections were used for immunohistochemical staining against citrullinated proteins. Images were analyzed using Aperio ImageScope. Citrullinated proteins were not present in the lung tissue sections of control mice (Figure 2A), while images of lung tissues from SWCNT-exposed mice revealed immunoreactivity of citrullinated proteins (Figure 2B). Quantification of the immunostained tissues using ImageScope software indicated a significant increase in the number of cells bearing citrullinated proteins. The induction of protein citrullination was observed on day 7 following SWCNT exposure and the highest number of cells with citrullinated proteins was observed at day 28 (Figure 2C).

▪ Exposure to nanomaterials activated PAD in human cellsSignificantly higher PAD activity was observed in A549 and THP-1 cells exposed to SiO₂ NPs or ufCB (Figure 3). In particular, in A549 cells exposed to SWCNTs or SiO₂ NPs, activity of PAD was detectable after 3 h and peaked at 24 h (Figure 3A). In THP-1 cells exposed to SWCNTs or SiO₂ NPs significantly higher PAD activity was detected only at 24 h (Figure 3B). No changes in the expression levels of PAD isoforms, PAD2 and PAD4, after SiO₂ NP or SWCNT treatments were observed in A549 cells assessed by western blotting (Figure 3C). In THP-1 cells, PAD2 expression was unchanged (Figure 3D).

▪ Inhibition of PAD reduced protein citrullination in the cells treated with nanosized particlesTo further validate the role of PAD activity in nanomaterial-induced protein citrullination in human cells, we used a synthetic inhibitor of PAD Cl-amidine. Cl-amidine is a well-characterized irreversible inhibitor of all PAD isozymes including PAD4 [48,49]. Cl-amidine was also shown to suppress PAD activity in vivo [30,50,51]. Pretreatment of A549 and THP-1 cells with various concentrations of Cl-amidine (50, 100 or 200 µM) resulted in a dose-dependent inhibition of SiO₂ NP- or SWCNT-induced PAD activity (Figures 4A & 4C). Since 100-µM concentration of Cl-amidine showed significant reduction in PAD activity with minimal effect on cell viability (Supplementary Figure S8), we selected this concentration for the following experimentation. Preincubation of A549 cells with C1-amidine resulted in significant reduction in SiO₂ NP- or SWCNT-induced protein citrullination (Figure 4B). However, preincubation of THP-1 cells with C1-amidine failed to abrogate NP-induced protein citrullination (Figure 4D).

▪ Ca²⁺ channel blocker inhibited nanomaterial-induced protein citrullinationExpression and the activation of PAD is required for protein citrullination. This activation requires local concentration of Ca²⁺ that are much higher than those in normal cytosolic conditions [52]. When THP-1 and A549 cells were exposed to SiO₂ NPs for 24 h, we observed an increased intracellular Ca²⁺ concentration compared with control cells (Figure 5). Employment of Ca²⁺ channel blocker (10 µM) and verapamil(Figure 5A) resulted in the inhibition of nanomaterial-induced protein citrullination observed in A549 cells (Figure 5C). In THP-1 cells inhibition of Ca²⁺ influx by verapamil (Figure 5B) did not fully block protein citrullination (Figure 5D).

▪ Identification of proteins undergoing citrullination by proteomicsA549 cells exposed to SiO₂ NPs or ufCB for 24 h were lysed; cell lysates were immunoprecipitated by anticitrulline antibody (Millipore, cat#07-377) and resolved by electrophoresis. Differentially citrullinated protein bands that were clearly present in SiO₂ NP- or ufCB-exposed cell lysates – four bands exhibiting strong intensity were excised from the gel (Figure 6A). These protein bands were subjected to mass spectrometry analysis as described under ‘Materials & methods’ section. Evaluation of the mass spectrometry/mass spectrometry data resulted in the identification of cytoskeletal proteins cytokeratin 7, 8, 18 and plectins (Figure 6A), and their citrullination sites (Supplementary Table 1). The presence of citrullination on cytokeratins was further validated biochemically. For this purpose, cell lysates from A549 control cells or those exposed to SiO₂NP (24 h) were separately immunoprecipitated with anticitrulline or anti-IgG (isotype control) antibodies and thereafter were assessed by western blotting using the specific antibodies against cytokeratin 7 (Figure 6B) or cytokeratin 8/18 (Figure 6C). As seen inFigure 6, the western blot analysis was consistent with the results obtained by mass spectrometry.

Discussion	ChooseTop of pageABSTRACTMaterials & methodsResultsDiscussion <<Future perspectiveReferences

Post-translational citrulination of proteins has been shown to alter their structure, antigenicity and functions. In RA, antibodies to cyclic citrullinated peptides are now well established for clinical diagnostics. The most commonly accepted molecular mechanism for autoimmunity associated with citrullinated peptides/proteins is that the self-proteins modified by virtue of cell damage and/or uncontrolled apoptosis turn out to be capable of priming autoimmune responses. Our study is the first report demonstrating that NPs of different origin were capable of promoting citrullination of proteins. We have shown that exposure to SiO₂ NPs, ufCB and SWCNTs accelerated accumulation of citrullinated proteins found both in cultured human cells and in the lungs of mice exposed to respirable SWCNTs. Furthermore, we demonstrated that the protein citrullination occurred due to activation of PAD. Several proteins, for example, cytokeratines 7, 8 and 18, and plectins were identified as targets for citrullination, potentially acquiring antigenic properties. These findings provide evidence that NPs could be quite immune-reactive and possibly able to facilitate autoimmune responses. Therefore, keeping in mind the broad application of nanomaterials for drug delivery, implants and medical devices, assessments of their ability to provoke post-translational protein citrullination are warranted. Thus, further investigations are necessary to fully explore the mechanisms of immune outcomes elicited by nanomaterials.

Protein citrullination is one of the post-translational modifications where peptidylarginine residues of the target protein(s) are converted to peptidylcitrulline by PAD enzymes [26–28,53]. Post-translational modifications of proteins influence their structure and biological functions[22]. In fact, some of the post-translationally modified proteins may generate neoepitopes responsible for the pathogenesis of autoimmune diseases including RA. The conversion of arginine to citrulline has been shown to increase peptide–MHC affinity and activate T cells in transgenic mice [54], ultimately inducing immune responses. In our study, cytokeratins, the largest intermediate filament protein subgroup[55,56] were shown to undergo citrullination after treatment with nanomaterials. Another target of citrullination identified in this study was plectin, which is an important component of the cytoskeleton [57]. Post-translational citrullination of these proteins in response to nanomaterials has not previously been reported. Citrullination of vimentin, fliggrin and histone proteins has been reported in various autoimmune diseases [30,50,53,58].

In recent studies, post-translational protein/amino acid modifications, for example, carbamylation (CM) and/or homocitrullination were linked to inflammation, uremia, atherogenesis and autoimmune diseases including RA [59,60]. CM is the nonenzymatic irreversible reaction of cyanate with amino, hydroxy or thiol groups. In vivo, amino group modification resulting in altered function of proteins/amino acids has been observed in patients suffering from uremia due to urea-derived cyanate. These data indicated that CM could impair the free radical and hypochlorous acid scavenging of thiol-amino acids, reducing their protective property against low-density lipoprotein atherogenic modification by oxygen species [61]. Given that exposure to particles trigger an inflammatory response, including markedly increased levels of myeloperoxidase-rich neutrophils, one can assume that CM of protein lysines (with the formation of homocitrulline) can occur in nanosized particle exposed animals. However, in our experiments, neutrophil response found in BAL of mice exposed to respirable SWCNTs occurred on day 7 postexposure (63 × 10³ cells/BAL). On day 28 postexposure the amounts of neutrophils declined (17.3 × 10³ cells/BAL) [62]. Therefore, the detected marked (2.8-fold) increase of protein citrullination at this late time-point after the exposure was not likely to be dependent on neutrophils accumulation and/or myeloperoxidase activity. We have previously seen that exposure to respirable SWCNTs elicited inflammation, pulmonary damage, modified cytokine pattern in the lung and suppressed systemicimmunity. The mechanism(s) of altered systemic immunity was, to some extent, due to direct effects of SWCNTs on pulmonary dendritic cells [63].

Previously, in epidemiological studies, it was shown that exposure to nanosized SiO₂ NP, droplets of mineral-oil and ufCB increased the risk of RA [3,4,10,11,12]. It has been reported that exposure of mice to diesel exhaust particles (DEPs) augmented both the incidence and the severity of collagen-induced arthritis. DEPs increased production of anti-CII IgG, IgG_2a, and IgG₁ antibodies, as well as secretion of IFN-γ. These results suggested that Th1 but not Th2 response was triggered by DEPs in collagen-induced arthritis [64,65]. Combustion-derived ufCB, a major air pollutant in urban areas, has been linked to increased incidence of respiratory, cardio-vascular diseases and RA [1,2,6]. Notably, smokers with chronic obstructive pulmonary disease have a relatively high amount of citrullinated proteins [12,66]. Exposure to cigarette smoke was associated with higher expression of the PAD2 enzymes in the lungs [67]. Adjuvant properties of DEPs, ufCB and SWCNTs have previously been reported [64,65,67]. The presence of autoantibodies against citrulline-containing proteins is well documented in RA patients and serves as one of the accepted diagnostic tests [12,13,21–25,53]. Augmented levels of citrullinated proteins may certainly contribute to adjuvant properties of carbonaceous nanomaterials.

It is well-established that intracellular Ca²⁺ plays a vital role in PAD activation [26,51,57]. We observed increased intracellular Ca²⁺ in the cells exposed to SiO₂ NPs, SWCNTs and ufCB. Nanomaterials have been shown to accelerate extracellular Ca²⁺ influx via compromised cell membrane integrity [68–70].Nanosized titanium dioxide has been shown to increase levels of cytosolic Ca²⁺ in human bronchial ChaGo-K1 epithelial cells [68]. To explore if nanomaterials mediated Ca²⁺ influx led to PAD-dependent protein citrullination, human epithelial A549 and phagocytic THP-1 cells were exposed to SWCNTs, ufCB and SiO₂ NPs in the presence of a Ca²⁺channel blocker. Blocking of the Ca²⁺ channel with verapamil resulted in alleviated protein citrullination. Direct inhibition of PAD by Cl-amidine reduced protein citrullination. However, Cl-amidine and/or verapamil failed to fully inhibit protein citrullination in human THP-1 cells perhaps due to constitutively low PAD4 inherently found in these cells. Proposed mechanism(s) of nanomaterial-induced protein citrullination is outlined in Figure 7. Tissue-specific expression of various isoforms of PAD has been reported [26–28]. In humans, PAD2 is expressed in skeletal muscle, the uterus, brain, salivary glands and pancreas. PAD4 is primarily expressed in macrophages, neutrophils and eosinophils [26–28,52]. PADs are mainly localized in the cytosol of mammalian cells. Citrullination is essential for the formation of neutrophil extracellular traps [30] and site-specific citrullination was reported to alter chemokine function [31–33]. When professional phagocytes were incubated with unmodified hen egg lysozyme, citrullinated peptides were expressed on dendritic cells and peritoneal macrophages facilitating the stimulation of citrulline-specific T-cell responses [71]. Noteworthy, subcellular localization of PADs was found at the sites of inflammation, where citrullinated proteins were elevated [29]. Importantly, both transcriptional and translational regulation govern PAD2 and PAD4 expression in monocytes and macrophages, possibly depending on various stages of their development and cytokine milieu [26,27]. Further studies are required to fully explore intimate mechanism(s) involved in nanomaterial-mediated PAD activation.

Overall, our study demonstrated that nanomaterials of distinct origin, morphology and physicochemical properties were able to induce protein citrullination via increased Ca²⁺-mediated PAD activity in human cells and animals.

Future perspective	ChooseTop of pageABSTRACTMaterials & methodsResultsDiscussionFuture perspective <<References

In the years to come, one can expect the increasing variety of nanomaterials to be introduced in everyday life and biomedical applications. Altered immune response developing as a result of human exposure to NPs via the citrullination-dependent mechanism could contribute to the pathogenesis of autoimmune diseases such as RA. Thus, further investigations exploring the nature and mechanisms of immune outcomes elicited by nanomaterials are warranted.

Executive summary

Background

▪ Exposure to silica nanoparticles has been reported to be associated with an increased risk of developing rheumatoid arthritis. Smoking, which has long since been considered a nonspecific risk factor causing chronic inflammation, is now also known to be associated with autoimmune diseases, including rheumatoid arthritis.

Materials & methods

▪ Several types of nanoparticles of different origin, including silicon dioxide, ultrafine carbon black and single-walled carbon nanotubes were applied to human cells in vitro and also investigated in the in vivo model of mice exposed to respirable single-walled carbon nanotubes.

Results

	▪ This is the first report demonstrating the induction of protein citrullination in human cells and in mouse lung tissues following exposure to nanosized silica or carbon-derived nanomaterials.
	▪ We have identified and validated the presence of citrulline residues in the cytoskeletal proteins: cytokeratins and plectins.
	▪ Nanomaterial-induced citrullination of proteins was consistent with Ca2+-mediated activation of PAD activity.

Conclusion

▪ It is proposed that nanomaterials facilitate post-translational citrullination of proteins, which can contribute to the development of autoimmune diseases, including rheumatoid arthritis.

DisclaimerThe findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.

AcknowledegmentsBM Mohamed and NK Verma both contributed equally. AA Shvedova and Y Volkov are both senior writers who contributed equally.Silica nanoparticles were kindly provided by K Dawson, Centre for BioNano Interactions, University College Dublin, Ireland. We are grateful to VE Kagan, L Tormey, Y Williams and J Conroy for their helpful discussions.

Financial & competing interests disclosureThis work was supported, in part, by the EU FP6 project NanoInteract (NMP4-CT-2006-0333231), SFI SRC Bionanointeract, HEA PRTLI cycles III–IV, NIH grant GM079357, NIOSH NORA/NTRC grant 1927ZHF, EU FP7 project NAMDIATREAM (NMP-2009-LARGE-3–246479) and EU FP7 project NANOMMUNE (EC-FP-7-NANOMMUNE-214281). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.

Ethical conduct of researchThe authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

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▪ Websites

101 .	Konstanz Information Miner. www.knime.org. (Acccessed 12 June 2010)
102 .	HiTS. http://code.google.com/p/hits,0.3.0 (Acccessed 12 June 2010)

Affiliations

Bashir M Mohamed

Department of Clinical Medicine, Trinity College Dublin, Ireland

Navin K Verma

Department of Clinical Medicine, Trinity College Dublin, Ireland and Centre for Research on Adaptive Nanostructures & Nanodevices, Trinity College Dublin, Ireland

Anthony M Davies

Department of Clinical Medicine, Trinity College Dublin, Ireland

Aoife McGowan

Department of Clinical Medicine, Trinity College Dublin, Ireland

Kieran Crosbie Staunton

Department of Clinical Medicine, Trinity College Dublin, Ireland and Centre for Research on Adaptive Nanostructures & Nanodevices, Trinity College Dublin, Ireland

Adriele Prina-Mello

Department of Clinical Medicine, Trinity College Dublin, Ireland and Centre for Research on Adaptive Nanostructures & Nanodevices, Trinity College Dublin, Ireland

Dermot Kelleher

Department of Clinical Medicine, Trinity College Dublin, Ireland

Catherine H Botting

BMS Mass Spectrometry & Proteomics Facility, University of St Andrews, Scotland, UK

Corey P Causey

Department of Chemistry & Biochemistry, University of South Carolina, SC, USA

Paul R Thompson

Department of Chemistry & Biochemistry, University of South Carolina, SC, USA and Department of Chemistry, The Scripps Research Institute, FL, USA

Ger JM Pruijn

Department of Biomolecular Chemistry, Radboud University Nijmegen, Nijmegen-Midden, The Netherlands

Elena R Kisin

National Institute for Occupational Safety & Health (NIOSH), WV, USA

Alexey V Tkach

National Institute for Occupational Safety & Health (NIOSH), WV, USA

Anna A Shvedova

National Institute for Occupational Safety & Health (NIOSH), WV, USA and Department of Pharmacology & Physiology, West Virginia University, WV, USA; Health Effects Laboratory Division, NIOSH, Morgantown, WV, USA. [e-mail]

Yuri Volkov

Department of Clinical Medicine, Trinity College Dublin, Ireland and Centre for Research on Adaptive Nanostructures & Nanodevices, Trinity College Dublin, Ireland. [e-mail]

Supplementary Material :

Supplementary information (.docx)