Inhalation and Oropharyngeal Aspiration Exposure to Rod-Like

Inhalation and Oropharyngeal Aspiration Exposure to Rod-Like Carbon Nanotubes Induce Similar Airway Inflammation and Biological Responses in Mouse Lungs Pia Kinaret,†,‡ Marit Ilves,†,∇ Vittorio Fortino,‡,∇ Elina Rydman,§ Piia Karisola,† Anna Laḧ de,∥ Joonas Koivisto,⊥ Jorma Jokiniemi,∥ Henrik Wolff,§ Kai Savolainen,§ Dario Greco,‡ and Harri Alenius*,†,# †

Department of Bacteriology and Immunology and ‡Institute of Biotechnology, University of Helsinki, Helsinki 00100, Finland § Finnish Institute of Occupational Health, Helsinki 00251, Finland ∥ Fine Particle and Aerosol Technology Laboratory, Department of Environmental and Biological Sciences, University of Eastern Finland, Kuopio 80100, Finland ⊥ National Research Centre for the Working Environment, Copenhagen DK-2100, Denmark # Institute of Environmental Medicine (IMM), Karolinska Institutet, Stockholm 171 77, Sweden S Supporting Information *

ABSTRACT: Carbon nanotubes (CNTs) have the potential to impact technological and industrial progress, but their production and use may, in some cases, cause serious health problems. Certain rod-shaped multiwalled CNTs (rCNTs) can, in fact, induce severe asbestos-like pathogenicity in mice, including granuloma formation, fibrosis, and even cancer. Evaluating the comparability between alternative hazard assessment methods is needed to ensure fast and reliable evaluation of the potentially adverse effects of these materials. To compare two alternative airway exposure methods, C57BL/6 mice were exposed to rCNTs by a state-of-the-art but laborious and expensive inhalation method (6.2− 8.2 mg/m3, 4 h/day for 4 days) or by oropharyngeal aspiration (10 or 40 μg/day for 4 days), which is cheaper and easier to perform. In addition to histological and cytological studies, transcriptome analysis was also carried out on the lung tissue samples. Both inhalation and low-dose (10 μg/day) aspiration exposure to rCNTs promoted strong accumulation of eosinophils in the lungs and recruited also a few neutrophils and lymphocytes. In contrast, the aspiration of a high-dose (40 μg/day) rCNT caused only a mild pulmonary eosinophilia but enhanced accumulation of neutrophils in the airways. Inhalation and low-dose aspiration exposure promoted comparable giant cell formation, mucus production, and IL-13 expression in the lungs. Both exposure methods also exacerbated similar expression alterations with 154 (56.4%) differentially expressed, overlapping genes in microarray analyses. Of all differentially expressed genes, up to 80% of the activated biological functions were shared according to pathway enrichment analyses. Inhalation and low-dose aspiration elicited very similar pulmonary inflammation providing evidence that oropharyngeal aspiration is a valid approach and a convenient alternative to the inhalation exposure for the hazard assessment of nanomaterials. KEYWORDS: carbon nanotubes, inhalation, aspiration, immune system, transcriptomics, inflammation, allergic airway inflammation

T

CNTs, with length to diameter ratio being >3:1, in other words CNTs with high aspect ratio, has been compared to asbestos fibers, raising concern that their widespread use may lead to serious health consequences.2 Multiwalled CNTs (MWCNTs),

he distinct properties of carbon nanomaterials and their versatile applications have the potential for a remarkable technological and economic growth worldwide.1 They are currently used in electronics, optics, and material science, and innovations are quickly spreading in sensor technology and medicine. Carbon nanotubes (CNTs) constitute a class of carbon nanomaterials with properties that differ significantly from other forms of carbon such as graphite and diamond. However, the rod-shape of certain types of © 2017 American Chemical Society

Received: August 22, 2016 Accepted: January 3, 2017 Published: January 3, 2017 291

DOI: 10.1021/acsnano.6b05652 ACS Nano 2017, 11, 291−303

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Figure 1. (a, b) Inhalation and aspiration (10 μg) of rCNTs induce strong pulmonary eosinophilia in mice on day 5. BAL cell counts show the ability of rCNTs to cause a decrease in the number of alveolar macrophages and induce recruitment of inflammatory cells, especially eosinophils after inhalation, and aspiration of low amounts of rCNTs. (b) The aspiration of 40 μg of rCNTs induce enhanced recruitment of neutrophils and lymphocytes but decreased recruitment of eosinophils as compared to 10 μg of rCNT. (c, d) H&E-stained mouse lung sections show accumulation of eosinophils and neutrophils, a foreign-body giant cell formation, and macrophages undergoing “frustrated phagocytosis” after rCNT inhalation and (e, f) low dose (10 μg) of aspiration. Columns and error bars in (a) and (b) represent mean values ± standard error of mean (SEM; n = 8−11). Images (c) and (e) are shown by 500× magnification, of which the images (d) and (f) show more detailed pictures. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. C, vehicle-treated control group; HPF, high-power field; UT, untreated control group.

Airways are the most relevant route for exposure of engineered nanomaterials (ENMs) especially in occupational settings. Experimental inhalation closely mimics the real-life exposure and is therefore the preferred method to investigate ENM-induced pulmonary inflammation. However, only a few studies are available due to the large amounts of ENM needed for aerosolization as well as the demanding requirements for methodological and technological implementation. In contrast, oropharyngeal aspiration is a much simpler airway exposure

in fact, are able to induce asbestos-like pathologies and biopersistence in mice when introduced into intraperitoneal cavity, acting as a surrogate tissue for mesothelial lining of the lung.2 Moreover, exposure to a MWCNT aggravates allergeninduced airway inflammation, and inhalation of high-dose of the CNT induces subpleural fibrosis in ovalbumin-sensitized mice.3 These observations merit immediate attention as they have a very remarkable impact on the risk assessment of the nanomaterials. 292


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Figure 2. (a, b) The inhalation and aspiration of 10 μg of rCNTs cause a significant increase in the number of PAS-positive cells in the BAL cells, indicating an enhanced production of mucus. (c, d) The expression of Th2-type cytokine, IL-13, is significantly increased after rCNT inhalation and oropharyngeal aspiration (10 μg). (e) Representative images of PAS-stained lung tissue show activation of mucin-producing cells in lungs after inhalation treatment and (f) after pharyngeal aspiration of 10 μg of the rCNT. (g) A foreign-body giant cell formation is seen after inhalation and (h) after aspiration of 10 μg of rCNTs. Figures (e) and (f) are taken at 500× magnification, and (g) and (h) at 1000× magnification. Columns and error bars in (a−d) represent mean values ± standard error of mean (SEM; n = 8−11). NS > 0.05,*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. C, vehicle-treated control group; RU, relative unit; UT, untreated control group.

procedure, as it does not require expensive and special laboratory equipment and necessitates only a small amount of nanomaterial, as compared to inhalation. Nevertheless, the efficacy of this methods has been questioned, since the ENM is introduced in the airways as liquid dispersions, requiring

additional proteins to facilitate a more homogeneous and stable distribution of nanoparticles in the liquid phase, especially in the case of CNT. This results in drastic differences in the biocorona formation compared to inhalation exposure, where CNTs are aerosolized in powder form or generated in situ in an 293


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distinct peaks at Raman shift of G band at 1566 and 1583 cm−1 with 200 μg/mL and 800 μg/mL, respectively, and the 2D band at 2594 and 2687 cm−1, respectively, indicating the typical G and D2 bands of MWCNT and graphite spectras.12 The band at 1102 cm−1 can be assigned to υ(CN) of albumin (Figure S4).13 The lower aspiration exposure dosage of 10 μg, totaling in 40 μg after 4 days, is relatively high, but still adjustable for human occupational settings, and also causing similar lung burden as inhalation exposures as reported by Porter et al. by exposing the same rCNT for 4 days by both inhalation (5 h/day) and aspiration (10 μg in 50 μL/day).14,15 According to their calculations, 10 μg per day for 4 days is equivalent to 800 μg rCNT/m2 alveolar epithelium (mouse alveolar epithelium surface area 0.05 m2). This dosage would respond to human deposition for a person exposed to rCNT for more than 3 months in an environment with a MWCNT aerosol of 400 μg/ m3. Inhalation for 4 days, 5 h per day would result in approximately 33 μg MWCNT lung burden (620 μg/m2 alveolar epithelium) as calculated by Porter et al.15 Assuming that the aspirated rCNT (totaling to 40 μg) is largely directed to lung, with a minute fraction perchance staying in the larger airways, the lung burden between both methods would be in a range varying from 600 to 800 μg/m2 alveolar epithelium. Cross sections from the lungs stained with picrosirius red reveal the material distribution throughout the lower airways and movement across the lungs also toward the pleura (Figure S2d−g). Both exposure methods caused histological changes compatible with an asthmatic phenotype, with an increase in mucin production and the presence of eosinophils. In addition to this, the aspiration of the rCNT induced a pronounced macrophage activation. After inhalation exposure, small aggregates of rCNT fibers are located in alveolar macrophages at the luminal site of the alveoli, whereas larger aggregates of rCNT fibers are seen in contact with alveolar macrophages after aspiration (Figures 1d,f). Mercer et al. have also shown that the effects on bronchoalveolar lavage fluid (BAL) cell number and fibrotic responses are very similar after aspiration or inhalation of MWCNTs, with the exception that particle clearance is slower after inhalation than after aspiration.16,17 Excessive mucus secretion in the airways is another important feature of allergic asthma.18 Four days of inhalation exposure to rCNT elicited an abundant mucus production in mice (Figure 2a,e). Likewise, aspiration to a low-dose (10 μg/ mice) of rCNTs elicited substantial goblet cell hyperplasia in the bronchial epithelia, whereas only a very minor mucus production could be observed after aspiration exposure to a high-dose (40 μg/mice) of rCNTs (Figure 2b,f). The T helper 2 (Th2) cytokine interleukin (IL)-13 is characteristically found in the asthmatic airways.11 Inhalation of rCNTs triggered a strong expression of IL-13 mRNA in the lung tissue (Figure 2c). Aspiration exposure to low-dose rCNTs also induced robust induction of IL-13 expression, while a high-dose elicited significantly lower expression of IL-13 mRNA in the lungs (Figure 2d). Furthermore, CNT exposure is reported to cause a formation of foreign body giant cells in vivo.2 The inhalation exposure to rCNT as well as a low-dose of rCNT by oropharyngeal aspiration exposure elicited comparable giantcell formation of macrophages in BAL (Figures 2g,h). Taken together, these findings suggest that repeated oropharyngeal aspiration exposure to low-dose (10 μg/mice), but not to highdose (40 μg/mice), rCNTs induces an allergic-type pulmonary inflammation with high eosinophilia, mucus secretion, and

ENM reactor.4,5 As a consequence, corona formation takes place in the airways naturally when aerosolized CNTs come in contact with proteins and biomolecules of the bronchial epithelium.5 Moreover, CNT aggregation and agglomeration stages differ in aspiration and inhalation methods which may result in dissimilar and uneven deposition of CNTs in different airway regions. In the present study, we compared the pulmonary effects of mice to rod-shaped MWCNTs (rCNTs) after inhalation and oropharyngeal aspiration exposures, respectively. In addition to state-of-the-art immunological and histological examination, we also performed global messenger ribonucleic acid (mRNA) expression analysis of the lung, which has not been used previously to compare the effects of aspiration and inhalation at the gene and pathway level. Our results reveal that, when the dose is adequately adjusted, inhalation and aspiration exposures elicit very similar pulmonary inflammation, which is also evidenced by activation of essentially similar biological pathways. Our results have a potentially important impact on the assessment of the hazard potential of ENM, which is crucial for their risk assessment.

RESULTS AND DISCUSSION We have previously examined pulmonary effects induced by repeated exposure to an aerosol of rCNT (aerosol mass concentrations of 6.2−8.2 mg/m3) for 4 h, for a total of 4 days, thus mimicking one-week occupational exposure.6 In this study, we expanded the setup and compared the differences of inhalation and oropharyngeal aspiration exposures with the same rCNT causing inflammatory and immunological responses (Figure S1).3,7−9 Inhalation exposure induced a significant decrease of macrophages (Figures 1a and S2a) and a minor increase of neutrophils and lymphocytes, but elicited a drastic infiltration of eosinophils into the lungs, which is a hallmark of allergic asthma (Figures 1a,c,d).10,11 In order to investigate pulmonary inflammation to the same rCNT after oropharyngeal aspiration, mice were exposed to 10 μg (lowdose) or 40 μg (high-dose) of dispersed rCNTs (in 50 μL/ mice) for 4 consecutive days, and samples were collected 24 h after the last administration (Figure S1). Aspiration exposure to higher doses (40 μg) of rCNT resulted in decrease of macrophages and a minor increase of lymphocytes, but a moderate increase of neutrophils and eosinophils with comparable cell counts (Figures 1b and S2b,c). In contrast, aspiration to a low-dose (10 μg) of rCNT caused allergic pulmonary inflammation comparable to that observed in the inhalation exposure set up with strong eosinophilia and a minor increase in neutrophils and lymphocytes (Figure 1b,e,f). The rCNT (Table S1) and produced dispersions were extensively characterized by transmission (TEM) and scanning (SEM) microscopying, electrophoretic mobility analysis (DLS), and by Raman spectroscopy. The morphology of the suspended rCNTs in phosphate buffered saline/bovine serum albumin (PBS/BSA) suspensions and aerosolized rCNTs were highly similar, indicating rigid, tangled bundles of fibers/tubes (SEM, Figure S3a−c) and multiple layers of graphene sheets (TEM, Figure S3d−f). The measured zeta potentials of 200 μg/mL and 800 μg/mL dilutions resulted in −18.6 mV and −16.8 mV, respectively, indicating that the materials are well dispersed but will not stay stable over time. The sedimentation and agglomeration were reduced by preparing fresh solutions every day prior to treatment and vortexing the mixture always before adminstrations. Raman spectroscopy resulted in two 294


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Figure 3. Differential gene expression analysis and hierarchical clustering heat map of lung tissue reveal rapid activation of an innate immune system in response to rCNTs. (a) Heat map of differentially expressed genes (linear fold change (FC) > |1.5|, post hoc adjusted P value = 0.05) in lungs of mice which are exposed by inhalation or by oropharyngeal aspiration (10 μg/mouse) to rCNTs. Genes are arranged by hierarchical clustering analysis, and each of the test groups forms separate clusters. The rCNT-inhaled and aspirated groups as well as control clusters share similar expression patterns, respectively. The red color indicates a higher expression while the blue denotes a lower expression. (b) Venn diagram demonstrates differentially up- and down-regulated genes (post hoc adj. P < 0.05, linear FC > |1.5|) in lung tissue after 4 days exposure to rCNTs by inhalation or aspiration. Both exposure routes to rCNT share remarkable expression of the same DEGs after 4 days of exposure (154 genes), of which most are up-regulated (144) and only a few are down-regulated (10 genes). The number of genes that are specific to aspiration (95 genes) and inhalation (24 genes) are notably lower than the common ones. DEGs are listed in Table S2; C, vehicletreated control group; UT, untreated control group.

expression of Th2 cytokine IL-13 mRNA. The differences between higher (40 μg) and lower dose (10 μg) in eosinophilia, mucus production, and in IL-13 expression might be due to the fact that the higher dose of 40 μg might yield more aggregates and cause more acute, initial lung injury responses, as suggested by Oberdörster et al., whereas a lower dose of 10 μg causes weaker/suboptimal activation of immune system, which is more optimal for promoting the development of allergic inflammation.19 This is noted also in the lung cross cut sections of 10 μg per day compared to 40 μg per day, with larger aggregates with the higher dose (as seen in hematoxylin and eosin (H&E) stainings in Figures 1 and S2c). This low-dose rCNT aspiration-induced pulmonary inflammation highly resembles the pulmonary inflammation induced by 4 days of inhalation with the same rCNT. In contrast to the

present study, Park and collaborators reported that neutrophilic inflammation was induced 1 day after intratracheal administration of MWCNTs.20 Similarly, Morimoto et al. reported MWCNT-induced neutrophilia peaking at day 3 after intratracheal instillation.21 Oropharyngeal aspiration studies have shown that MWCNTs induce an influx of both neutrophils and eosinophils, especially at low doses.22,23 Moreover, we recently reported that a single aspiration of rCNTs induced strong pulmonary neutrophilia accompanied by the proinflammatory cytokines and chemokines already 16 h after the exposure in mice.24 However, 7 days after the exposure, neutrophilia had essentially disappeared, but remarkable pulmonary eosinophilia peaked in rCNT exposed animals.24 Based on these findings, it can be concluded that exposure dose and time critically control 295


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Figure 4. Pulmonary eosinophilia is regulated by chemokine ligands CCL11 (eotaxin-1) and CCL24 (eotaxin-2), whereas recruitment of Th2 cells, induction of airway hyperreactivity, and mucus secretion are mainly driven by Th2-type cytokines IL-13 and IL-4. Comparison of mRNA expression levels of cytokines/chemokines in lung tissue shows that the expression of IL-13 and IL-4 cytokines is highly enhanced after rCNT exposure, and this induction is very similar after inhalation and low doses of aspiration (10 μg/mouse). Exposure to rCNTs enhances also eosinophil chemoattractants CCL11 and CCL24 in a very similar manner both after inhalation and aspiration. The expression levels of RNA in the inhalation groups are presented as fold changes relative to the untreated (UT) group (n = 6−8). In aspiration groups, the values indicate fold changes compared to the vehicle-treated control group (C) (n = 8). **P ≤ 0.01, ***P ≤ 0.001. Two mouse samples are pooled for the microarray analysis.

the type and the magnitude of the rCNT-induced pulmonary inflammation. Transcriptomics is an effective mechanism-based method to characterize the exposure to nanomaterials.25,26 We hence performed genome-wide expression analysis by DNA microarrays to compare inhalation (aerosolized rCNT) and aspiration exposed (dispersed low-dose rCNT) lungs from control and rCNT exposed mice (NCBI GEO accession number GSE85711). The rCNT caused consistent and robust changes in the gene expression patterns of the exposed lungs as compared to the nontreated samples (Figure 3a). A total of 178 and 249 genes were significantly differentially expressed (≥1.5fold change, adjusted P value ≤0.01) in inhalation and aspiration exposed mice, as compared to controls, respectively (Table S2). 154 (56.4%) differentially expressed genes (DEGs) were common for inhalation and aspiration exposed groups (Figure 3b), and 144 DEG were up-regulated and 10 downregulated (Figure 3b). 20 (8.1%) of the up-regulated DEGs were specific for the inhalation group and 82 (33.3%) DEGs were specific for the aspiration groups (Figure 3b). To focus on allergy and asthma related genes, we compared the expression levels of Th2-type cytokines IL-13 and IL-4 mRNA and Th2 chemokines CCL11 (eotaxin-1) and CCL24 (eotaxin-2) in our study groups. The expression patterns of these Th2 cytokines and chemokines were essentially identical between the inhalation and aspiration exposed groups (Figure 4), which shows that these responses to rCNTs are developing in similar ways. We further performed pathway enrichment analysis of the DEGs, confirming a high degree of consistency

between the transcriptional landscapes of the lungs after the different exposure methods (Figure 5). Innate immunity pathways, e.g., NOD-like and Toll-like receptor signaling pathways, chemokine signaling pathways, and cytokinecytokine receptor interaction pathways, were significantly and comparably induced in both groups (Figure 5), while commonly down-regulated pathways included drug, retinol, and cytochrome P450 metabolism pathways (Figure 5). A few pathways were specifically enriched only in one exposure group. Pathways associated with viral myocarditis or cardiomyopathy were down-regulated in the inhalation exposure group, and pathways with prion diseases, immunoglobulin A (IgA) network, and hematopoietic cell lineage were up-regulated only in the aspiration exposure group. However, the number of DEGs in these pathways was very small. Taken together, these results strongly support our histological and cytological findings, suggesting that 4 days of inhalation and 4 days of aspiration exposure to rCNTs induce essentially similar alterations. We further focused on the 154 DEGs overlapping between the exposures, by performing gene ontology (GO) enrichment analysis (Figure 6a). Overrepresentation of GO biological processes revealed several common GO terms including inflammatory response, mitotic cell cycle, chemotaxis, cell chemotaxis, and taxis (Figure 6b). Moreover, the trend of gene expression alteration of the 144 commonly upregulated genes was also consistent across the exposure methods (Figure S5). Clustergram of the top 40 input genes within the top 5 GO terms revealed a cluster of 15−18 chemokines, chemokine 296


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Figure 5. Functional annotation plot (BACA) compares the most significant Kyoto Encyclopdia of Genes and Genomes pathways (post hoc adj. P < 0.05, with a minimum number of 5 genes per pathway) associated with the significant up- and down-regulated genes found in inhalation and oropharyngeal aspiration (10 μg) of rCNTs. This pathway bubble-plot shows that both exposure routes upregulate common pathway clusters including innate immunity-associated pathways and cytokine/chemokine pathways and that they downregulate cellular metabolic pathways. The diameter of each circle represents the number of genes annotated in each pathway (legend “Counts”). The red color indicates up-regulated pathways, and the green color indicates down-regulated pathways compared to untreated controls.

significant enrichment mainly in pathways such as DNA strand elongation, DNA replication initiation, and mitotic recombination. The aspiration-specific 95 DEGs revealed a significant enrichment of several GO terms that were involved in cell activation, cell migration, and cytokine-mediated signaling pathways (Figure S7). We further investigated the functions and the interactions of the 154 genes commonly regulated in inhalation and aspiration groups, by GeneMANIA (http:// www.genemania.org).29 We could extrapolate a coordinated regulation of genes belonging to several relevant functions such as nuclear division, cell chemotaxis, and regulation of cytokine production (Figure 7). Genes associated with cell chemotaxis and regulation of cytokine production were substantially overlapping, whereas genes associated with nuclear division belonged to a separate cluster in the inferred gene network (Figure 7). In order to study the similarities of the pathways, we utilized different enrichment tools (based on overrepresentation analysis on CPDB and functional annotation on DAVID). Interestingly, additional functional annotation analyses revealed that the inhalation-induced and aspiration-induced pathways included 80% the same biological processes (Figure S8a), and depending on the enrichment tool, only 0−20% of the pathways ended up being specific for the inhalation or aspiration alone (Figure S8b,c and Table S3).

receptors, and cytokines, and another separate cluster of 15 genes included in mitotic cell cycle GO term (Figure 6c). These results correlate well with our histological and cytological results, as both inhalation and aspiration of rCNTs attracted a large number of eosinophils as well as neutrophils and lymphocytes to the lungs, which are programmed to divide and defend the surrounding environment. In the first cluster, serum amyloid A1 (SAA1) is a major acute-phase protein, and IL-1β is the major proinflammatory cytokine in the body, whereas CCR5 and CCR2 are chemokine receptors on the surface of white blood cells that bind to cytokines CCL3-CCL5 to recruit leukocytes to the site of inflammation. Additionally, chemokines CXCL3 macrophage inflammatory protein ((MIP)-2β) and CXCL10 (IP-10) attract monocytes, and CXCL5 attracts mainly neutrophils.27 The second mitotic cell cycle cluster includes genes playing a role in transcription (TOP2A), cell division (KNTC1, CENPP/K, CDC6/A8, NCAPD2, NSL1, SPC24), and replication (MCM2, RFC4). Although the rCNTs used in this study are known to be toxic and cause cell death and mutations, the number of dying cells might be so small when compared to the invading army of leukocytes that the genes associated with cell division machinery are therefore highly present in the DEG list.14,24,28 The 24 DEGs whose expression was specifically altered by inhalation exposure only (Figure S6) showed statistically 297


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Figure 6. Inhalation and aspiration of rCNTs share 154 common DEGs, which are mainly involved in inflammatory responses, chemotaxis, and cell division. (a) Venn diagram of the microarray results summarizes the numbers of DEG (linear FC > |1.5|, post hoc adjusted P value = 0.01) in lung tissue after 4 days of inhalation and oropharyngeal aspiration (10 μg) exposure to rCNTs. (b) The enrichment analysis using Enrichr revealed several significantly enriched pathways from the 154 overlapping DEGs including inflammatory response, mitotic cell cycle, chemotaxis, cell chemotaxis, and taxis. (c) Clustergram of the top 40 input genes within the top five GO pathways revealed a cluster of 15−18 chemokines, chemokine receptors, and cytokines and another separate cluster of 15 genes included in mitotic cell cycle pathways.

A cluster of chemokines, chemokine receptors, and cytokines found both in inhalation and aspiration exposed mice is in line with highly similar allergic pulmonary inflammation. The CCL11 (eotaxin-1) and CCL24 (eotaxin-2) are important chemokines regulating pulmonary eosinophilia, while IL-4, IL13, and CCL17 (TARC) are major Th2-type cytokines and chemokines involved in recruitment of Th2 lymphocytes, airway hyperreactivity, and mucus secretion from the bronchial epithelium.30−34 Additionally, the same rCNT is shown to induce IL-13 and IL-4 and also their downstream signaling molecules signal transducer and activator of transcription

(STAT6) and GATA-3 after oropharyngeal aspiration in a genome-wide microarray analysis.35 Although eosinophils are the major force in the allergic inflammation and are able to cause direct damage to supporting cells and tissue fibrosis at the site of inflammation, recent studies suggest that eosinophils might have homeostasis-maintaining roles, which may play a role after CNT exposure.36 In addition, a separate cluster of genes involved in events of mitotic cell cycle and nuclear division was induced after inhalation and aspiration exposure to rCNT. It is possible that these genes are observed as up-regulated consequently to the 298


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Figure 7. Gene interactions of the 154 common DEGs and their functions were predicted by GeneMANIA program. Three significant functional gene groups emerged including nuclear division (striped yellow nodes), cell chemotaxis (striped red nodes), and regulation of cytokine production (striped blue nodes). The nonstriped nodes represent functionally similar genes but are not found within the 154 DEGs in the intersection of inhalation and aspiration exposures. The purple edges correspond to coexpression between the genes, and the light beige lines correspond to genes with shared protein domains. The small gray nodes are complex components having high weight coexpression or are involved in the same processes with the queried DEGs.

these two exposure approaches, possibly impacting the nanobio interactions.4 Indeed, we also appreciated larger aggregates of ENM in the interstitial space after aspiration, whereas after inhalation, the particles formed smaller aggregates (Figures 1 and S2). In this study, we have been able to compare the cellular and transcriptomic responses and their interrelated, functional pathways between the two methods. Together our analyses emphasize that inspite of the different exposure methods, which activate slightly different gene expressions, they finally end up activating the same biological pathways. The multiwalled carbon nanotube (rCNT) was chosen as a model particle for being well characterized and known to cause major inflammatory effects. Nevertheless, it should be noted that the current study focuses on only one type of a carbon nanomaterial, whereas materials with not so clear inflammatory responses might not provide clear responses at the cellular level, but should be further studied at the transcriptomic level. Remarkably, despite this, both techniques exacerbated very similar histological, immunological, and molecular responses.

infiltration of vigorously proliferating inflammatory cells to the inflammation site. Alternatively, these DEGs may also be related to rCNT-induced genototoxicity or cell damage. In line with this, Catalan et al. recently reported a dose-dependent increase in DNA strand breaks in the lung cells of C57Bl/6 mice 24 h after a single oropharyngeal aspiration of rCNTs.37 Additionally, DNA strand breaks and micronuclei were also triggered in lung and BAL cells exposed to aerosolized rCNTs.37 Thus, inhalation and aspiration exposure to rCNTs induces not only regulating pulmonary inflammation but also genes involved in cell division and possible interaction of ENM with DNA. Although clearly valuable, the inhalation exposure method is very laborious, technologically demanding and expensive, and requires large amounts of ENM for aerosolization. Therefore, hazard assessment of all the ENM constantly designed and produced is not feasible by using the inhalation exposure approach. Oropharyngeal aspiration is cheaper and a technologically simpler alternative to the inhalation method, but its validity has been questioned due to clear differences in the form that ENM assumes when introduced in the airways.5,38 One of the arguments in this sense highlights the difference in biomolecule corona around the nanoparticles in

CONCLUSION In conclusion, here we report that short-term inhalation exposure to rCNTs and repeated oropharyngeal aspiration to 299


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with 0.6 mg/mL of BSA. The stock dispersions were vortexed for 1 min and sonicated for 20 min at 30 °C in bath sonicator (Elmasonic, USA). Further dilutions (200 and 800 μg/mL) were prepared in PBS and sonicated for additional 20 min before the exposures. Mice were exposed to 10 μg/mouse or 40 μg/mouse of rCNT in 50 μL of vehicle by oropharyngeal aspiration on four consecutive days under isoflurane anesthesia. All mice were sacrificed by isoflurane overdose 24 h after the exposure(s). Sample Collection. After sacrificing, the lungs were lavaged with 800 μL of PBS for 15 s by cannulating the trachea with a blunt syringe. The left pulmonary lobe was removed and divided into subsamples that were kept in RNAlater solution (Ambion, Life Technologies, CA, USA) and stored at −70 °C for real-time quantitative polymerase chain reaction (PCR) and microarray analysis. The right lung was formalin-fixed, embedded in paraffin, cut, affixed on slides, and stained with H&E and periodic acid-Schiff (PAS) staining solutions. BAL Cell Counts. BAL samples were cytocentrifuged onto slides and stained with May Grünwald-Giemsa (MGG) stain. Macrophages, neutrophils, eosinophils, and lymphocytes were counted from three high-power fields (HPF) under the light microscope (Leica DM 4000B, Leica, Wetzlar, Germany). Cytokine and Chemokine Expression. Total RNA was isolated from the lung samples by a phenol/chloroform isolation method. Tissue samples in RNAlater were thawed and moved to the lysing matrix D tubes (MP Biomedicals, Illkirch, France), containing 1 mL of TRIsure reagent (Bioline reagents, Ltd., London, UK). Samples were homogenized in a FastPrep FP120 homogenizer (BIO 101, Thermo savant, Waltham, MA, USA), and mRNA was extracted and purified according to the instructions by Bioline Reagents. Quantity and purity were measured with NanoDrop spectrophotometer (ND-1000, Thermo Fisher Scientific Inc., Wilmington, NC, USA). Complementary DNA (cDNA) was synthesized from 500 ng of total RNA in a 25 μL reaction, utilizing MultiScribe Reverse Transcriptase and random primers (The High Capacity cDNA Archive Kit, Applied Biosystems) according to the manufacturer’s protocol. The synthesis was performed in a 2720 Thermal Cycler (Applied Biosystems, Carlsbad, CA, USA) with thermal cycles of 25 °C for 10 min and 37 °C for 120 min (Thermal Cycler, Applied Biosystems). Primers and probes (18S rRNA, IL-13) for PCR analysis were purchased as predeveloped assay reagents from Applied Biosystems. The PCR assays were performed with a Relative Quantification 7500 Fast System (7500 Fast Real-Time PCR system, Applied Biosystems, Foster City, CA, USA). Amplifications were done in 11 μL reaction volume containing TaqMan universal PCR master mix and primers by Applied Biosystems and 1 μL of cDNA sample. Ribosomal 18S was utilized as a housekeeping gene. Statistical Analysis of Cell Counts and mRNA Levels. Graphs were built, and data were analyzed using GraphPad Prism 7 Software (GraphPad Software Inc., San Diego, CA, USA). The Nonparametric Mann−Whitney U tests were used for BAL cell counts and cytokine and chemokine mRNA levels between two-group analyses. Results were conducted by mean with standard error of the mean. P value 8 were used for microarray hybridization. Two-Color Microarray-Based Gene Expression Analysis protocol (version 6.5) was used for all the samples. 70 ng of pooled total RNA was primed using oligo-dT (T7) promoter primers and converted to cDNA with AffinityScript RNase Block (Quick Amp Labeling Kit, Two-color, Agilent). Primed cDNA was amplified and labeled using fluorescently labeled deoxynucleotide triphosphate (dNTP)-nucleotides (Cy3 and Cy5) and T7 RNA Polymerase. 300 ng of labeled sample (Cy5) and 300 ng of labeled reference sample (Cy3) were combined (total 600 ng). Clean-up of labeled and amplified samples was performed by using Qiagen’s

low-dose rCNTs induce essentially similar pulmonary inflammation evidenced by pulmonary eosinophilia, goblet cell hyperplasia, and expression of Th2 cytokines in mice. Global transcriptomic analysis of the lung tissue confirms similarities in gene and pathway activation in response to rCNTs by both methods, including genes involved in inflammatory response, mitotic cell cycle, and cell chemotaxis. Altogether, our results suggest that when the exposure dose is adequately adjusted, oropharyngeal aspiration could be a valid alternative to the expensive and laborious inhalation for the hazard assessment of the pulmonary effects of nanomaterials.

METHODS MWCNT. Rod-like MWCNT (rCNT) were received from Mitsui & Co., Ltd. (Tokyo, Japan). Physico-chemical properties, morphology of the rCNTs, and their elemental composition have been characterized and described in a study by Rydman et al. and also in Table S1.6 Concentrations and size distributions of CNT aerosols as well as the scheme of the inhalation system (Figure S9) with essential parameters and typical flow values are described by Rydman et al. and in Figure S10.6,39 MWCNT Characterization. The samples of the freshly made MWCNTs suspensions were taken on a holey carbon copper grid (SPI Supplies, Inc.) The shape of the MWCNTs was studied with a fieldemission electron microscopy (SEM, Zeiss Sigma HD-VP) operated at 2 kV acceleration voltage. The morphology of the particles was determined with the transmission electron microscopy (TEM, Jeol JEM-2100F) operated at 200 kV acceleration voltage. Raman spectroscopy (Bruker Senterra 200 LX) was used to analyze the composition of the MWCNTs. The samples were prepared by pipetting a small drop of the freshly made MWCNT suspensions on the microscope glass slides. The samples were let to dry before the analysis. The the zeta potential of the freshly made suspensions was measured using an electrophoretic mobility analysis (ZetaSizer ZS DLS, Malvern Instruments). The measurements were carried out in triplicates, and the average value was calculated based on the measurements. Animals. Female C57BL/6, (7−8 weeks old) purchased from Scanbur A/S (Karslunde, Denmark) were quarantined for 1 week and housed in stainless steel cages in groups of four. Temperature was kept between 20 and 21 °C and humidity within a range of 40−45%, with 12 h dark/light cycle. Mice were bedded with aspen chip and received standard mouse chow diet (Altromin no. 1314 FORTI, Altromin Spezialfutter GmbH & Co., Germany) and water ad libitum. The experiments were performed in agreement with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg March 18, 1986, adopted in Finland May 31, 1990). The study was approved by the Animal Experiment Board and the State Provincial Office of Southern Finland (ESAVI-3241−04.10.07−2013). Exposure Methods. To understand whether different administration techniques lead to the development of similar inflammatory reactions in the lungs, a 5 day experimental model was adapted for two exposure techniques: inhalation and oropharyngeal aspiration (Figure S1). Inhalation exposures were performed in an earlier published study by Rydman et al. 2014.6 Briefly, rCNTs were aerosolized with a fluidized bed aerosol generator (FBAG; TSI Model 3400A) for which the material was used without any pretreatment.40 Mice were exposed to aerosolized rCNT for 4 h/day on 4 consecutive days in a wholebody inhalation chamber. Aerosol mass concentrations for individual experiments ranged from 6.2 to 8.2 mg/m3. During the experiments, untreated control mice were housed in the same room with CNTexposed animals. For oropharyngeal aspiration exposures, rCNT stock suspensions were made in glass tubes, by weighing 1 mg of ENM to 1 mL PBS (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented 300


Article

ACS Nano RNeasy mini spin columns (Qiagen, GmbH, Hilden, Germany). RNA Spike-In Kit was used to monitor the success of the labeling. Fragmented cRNA was hybridized to microarray slides (Agilent technologies, Sure Print G3Mouse GE 8 × 60K Kit, USA) according to the instructions of the manufacturer (Agilent Technologies, TwoColor Microarray-Based Gene Expression Analysis, Low Input Quick Amp Labeling). Slides were scanned with Agilent DNA microarray Scanner (Agilent Technologies, DNA Microarray Scanner with Surescan High-Resolution Technology, model G2505C, USA), and the data were extracted using Agilent Feature Extraction Software (V11.0.1.1). Microarray Data Analysis. Statistical Analysis. The median foreground intensities were imported to R software, log2 transformed, and quantile normalized with Bioconductor package limma.42 Batch effect removal with Combat was computed to remove known batches, such as dye, slide area, and array.43 Then, SVA was applied to find other hidden variables that were subsequently removed with Combat.44 The linear model was fitted to the data, and empirical Bayes was used for comparing the groups of samples. Genes were defined as being differentially expressed after satisfying a minimum fold change of ±1.5 and a maximum, Benjamini−Hochberg adjusted, P value of 0.01. Heat Maps and Venn Diagrams. Heat maps and Venn diagrams were created using the R packages made4 and VennDiagram.45,46 Heat maps were produced by color-coding genewise standardized log gene expression levels (mean zero standard deviation one). Genes were shown hierarchically clustered by similarity based on Pearson correlation and group average aggregation method. Venn diagrams showing the total number of genes up and down regulated in each experimental condition. Annotation Analysis. After extraction of the DEG, the R package BACA was used to find and compare biological pathways of up- and down-regulated genes associated with the inhalation and aspiration exposure to rod-like carbon nanotubes.47,48 First, BACA was used to query the DAVID knowledgebase for each treatment, retrieving separate functional annotation charts for up- and down-regulated genes.48 Then, BACA was exploited to generate a bubble plot summarizing the shared and different functional annotations found by enrichment. Each annotation in the chart is represented as a circle (or bubble) that has a size, indicating how many genes in a list of DEGs are associated with it (circle size), and a color indicating whether the genes are down- (default color is green) or up- (default color is red) regulated. Web-Based Tools. The network analyses were done by GeneMANIA program, and the pathway enrichment analyses were performed using Enrichr tool, ConsensusPathDB, CPDB, and The Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8.29,49−52

Table S2: Lists of the differentially expressed genes from the microarray experiment (XLSX) Table S3: CPDP overrespresentation enrichment analysis; GO biological process (XLSX)

AUTHOR INFORMATION Corresponding Author

*E-mail: Harri.Alenius@helsinki.fi. ORCID

Dario Greco: 0000-0001-9195-9003 Harri Alenius: 0000-0003-0106-8923 Author Contributions ∇

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The work was supported by grants from the Academy of Finland (139115, 275151 and 292307), from the European Community’s Seventh Framework Programme (FP7) under grant agreement no 309329 (NANOSOLUTIONS), the Finnish Work Environment Fund (109137), and the Research Fundation of the University of Helsinki. This study was also supported by the basic funding of the University of Eastern Finland and by Danish Centre for Nanosafety 2. The authors also wish to thank P. Alander, S. Hirvikorpi, S. Savukoski, and S. Tillander for their excellent technical assistance and C. Chen for the valuable and inspiring discussions. REFERENCES (1) De Volder, M. F.; Tawfick, S. H.; Baughman, R. H.; Hart, A. J. Carbon Nanotubes Present and Future Commercial Applications. Science 2013, 339, 535−539. (2) Poland, C. A.; Duffin, R.; Kinloch, I.; Maynard, A.; Wallace, W. A.; Seaton, A.; Stone, V.; Brown, S.; Macnee, W.; Donaldson, K. Carbon Nanotubes Introduced into the Abdominal Cavity of Mice Show Asbestos-Like Pathogenicity in a Pilot Study. Nat. Nanotechnol. 2008, 3, 423−428. (3) Ryman-Rasmussen, J. P.; Tewksbury, E. W.; Moss, O. R.; Cesta, M. F.; Wong, B. A.; Bonner, J. C. Inhaled Multiwalled Carbon Nanotubes Potentiate Airway Fibrosis in Murine Allergic Asthma. Am. J. Respir. Cell Mol. Biol. 2009, 40, 349−358. (4) Bhattacharya, K.; Mukherjee, S. P.; Gallud, A.; Burkert, S. C.; Bistarelli, S.; Bellucci, S.; Bottini, M.; Star, A.; Fadeel, B. Biological Interactions of Carbon-Based Nanomaterials: From Coronation to Degradation. Nanomedicine 2016, 12, 333−351. (5) Morimoto, Y.; Horie, M.; Kobayashi, N.; Shinohara, N.; Shimada, M. Inhalation Toxicity Assessment of Carbon-Based Nanoparticles. Acc. Chem. Res. 2013, 46, 770−781. (6) Rydman, E. M.; Ilves, M.; Koivisto, A. J.; Kinaret, P. A.; Fortino, V.; Savinko, T. S.; Lehto, M. T.; Pulkkinen, V.; Vippola, M.; Hameri, K. J.; Matikainen, S.; Wolff, H.; Savolainen, K. M.; Greco, D.; Alenius, H. Inhalation of Rod-Like Carbon Nanotubes Causes Unconventional Allergic Airway Inflammation. Part. Fibre Toxicol. 2014, 11, 48. (7) Nygaard, U. C.; Hansen, J. S.; Samuelsen, M.; Alberg, T.; Marioara, C. D.; Lovik, M. Single-Walled and Multi-Walled Carbon Nanotubes Promote Allergic Immune Responses in Mice. Toxicol. Sci. 2009, 109, 113−123. (8) Ronzani, C.; Casset, A.; Pons, F. Exposure to Multi-Walled Carbon Nanotubes Results in Aggravation of Airway Inflammation and Remodeling and in Increased Production of Epithelium-Derived Innate Cytokines in a Mouse Model of Asthma. Arch. Toxicol. 2014, 88, 489−499. (9) Sos Poulsen, S.; Jacobsen, N. R.; Labib, S.; Wu, D.; Husain, M.; Williams, A.; Bogelund, J. P.; Andersen, O.; Kobler, C.; Molhave, K.;

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b05652. Figures: The study setup; H&E and picrosirius red stained lung sections; SEM and TEM images from the rCNT suspensions; Raman peaks of the carbon-based materials; correlation of the gene expressions of the 144 up-regulated DEGs between inhalation and aspiration (10 μg/mouse) of rCNTs is statistically very significant; the 24 inhalation-specific genes to rCNTs involved in DNA recombination; replication and DNA repair pathways; the 95 aspiration-specific genes to rCNTs (10 μg) involved in cytokine responses and lymphocyte activation and migration; pathway analyses of the inhalation and aspiration DEGs; exposure setup for rCNT inhalation studies; particle size distribution and concentrations in inhalation aerosol (PDF) 301


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