Single-walled carbon nanotubes increase pandemic influenza A H1N1 virus infectivity of lung epithelial cells
- Pallab Sanpui†1,
- Xiao Zheng†1,
- Julia C Loeb1,
- Joseph H Bisesi Jr1,
- Iftheker A Khan2,
- A R M Nabiul Afrooz2,
- Keira Liu3,
- Appala Raju Badireddy3,
- Mark R Wiesner3,
- P Lee Ferguson3,
- Navid B Saleh2,
- John A Lednicky1 and
- Tara Sabo-Attwood1Email author
© Sanpui et al.; licensee BioMed Central. 2014
Received: 24 April 2014
Accepted: 13 October 2014
Published: 14 December 2014
Airborne exposure to nanomaterials from unintended occupational or environmental exposures or as a consequence of product use may lead to adverse health effects. Numerous studies have focused on single-walled carbon nanotubes (SWCNTs) and their ability to cause pulmonary injury related to fibrosis, and cancer; however few studies have addressed their impact on infectious agents, particularly viruses that are known for causing severe disease. Here we have demonstrated the ability of pristine SWCNTs of diverse electronic structure to increase the susceptibility of small airway epithelial cells (SAEC) to pandemic influenza A H1N1 infection and discerned potential mechanisms of action driving this response.
Small airway epithelial cells (SAEC) were exposed to three types of SWCNTs with varying electronic structure (SG65, SG76, CG200) followed by infection with A/Mexico/4108/2009 (pH1N1). Cells were then assayed for viral infectivity by immunofluorescence and viral titers. We quantified mRNA and protein levels of targets involved in inflammation and anti-viral activity (INFβ1, IL-8, RANTES/CCL5, IFIT2, IFIT3, ST3GAL4, ST6GAL1, IL-10), localized sialic acid receptors, and assessed mitochondrial function. Hyperspectral imaging analysis was performed to map the SWCNTs and virus particles in fixed SAEC preparations. We additionally performed characterization analysis to monitor SWCNT aggregate size and structure under biological conditions using dynamic light scattering (DLS), static light scattering (SLS).
Based on data from viral titer and immunofluorescence assays, we report that pre-treatment of SAEC with SWCNTs significantly enhances viral infectivity that is not dependent on SWCNT electronic structure and aggregate size within the range of 106 nm – 243 nm. We further provide evidence to support that this noted effect on infectivity is not likely due to direct interaction of the virus and nanoparticles, but rather a combination of suppression of pro-inflammatory (RANTES) and anti-viral (IFIT2, IFIT3) gene/protein expression, impaired mitochondrial function and modulation of viral receptors by SWCNTs.
Results of this work reveal the potential for SWCNTs to increase susceptibility to viral infections as a mechanism of adverse effect. These data highlight the importance of investigating the ability of carbon-nanomaterials to modulate the immune system, including impacts on anti-viral mechanisms in lung cells, thereby increasing susceptibility to infectious agents.
SWCNTs are emerging as one of the most commercially important and technologically-relevant nanomaterials in research and consumer industries. There are numerous types of SWCNTs , each with unique properties that allow for molecular level manipulation and thus have enabled them to be used in a variety of industrial and consumer products , and are being intensely investigated for their use in diverse biomedical applications -. While SWCNTs are well suited for many of these applications, there is emerging concern regarding the potential for adverse health effects associated with unintended occupational or environmental exposures or intended product use such as application of biomedical or personal care products. Data regarding the potential for exposure to SWCNTs and related health risks is sparse at best. Two recent reviews anticipate that the greatest risk of exposure to SWCNTs will likely occur from product release during manufacturing, subsequent processing and product wear and tear (tires, recycling, textiles) ,. With increasing manufacture and use of SWCNTs, it is imperative to thoroughly investigate nanomaterial biotoxicity in order to better evaluate associated health implications .
There has been remarkable interest in inhalation as a route of exposure as several in vivo studies report that SWCNTs can induce adverse pulmonary effects - such as subchronic tissue damage, fibrogenesis, granulomatous changes, impaired clearance, robust inflammation, airway hyper-reactivity and airflow obstruction, and cardiopulmonary effects . The cellular and molecular mechanisms that contribute to these outcomes include oxidative stress, modulation of inflammatory mediators (cytokines, chemokines), genotoxicity, altered expression of stress genes, mitotic disruption, changes in biotransformation enzymes, phospholipid peroxidation, epithelial mesenchymal transition, and altered arterial baroreflex function -. The majority of these data originate from studies designed to assess the toxicity of carbon nanomaterial exposures in isolation of other imposed stressors.
It is well recognized that heightened and, in some cases, distinct biological responses can occur with co-exposure to multiple inhaled agents as is the case for synergistic free radical generation by diesel exhaust and bacterial lipopolysaccharide (LPS) . Although reports are controversial, certain viruses may also act as disease co-factors with toxicants - as is postulated for SV40 and asbestos in mesotheliomas ,. Only a few studies have investigated sequential exposure of nanoparticles and pathogens. These reports collectively show that co-exposure with bacteria leads to enhanced pulmonary inflammation and fibrosis and decreased pathogen clearance highlighting the potential impacts of combined exposures ,. More recently, carbon nanotubes have been shown to exacerbate ovalbumin- induced airway remodeling and allergic asthmatic responses in mice ,,-.
While there are intense ongoing research efforts focused on using nanoparticles for viral detection and vaccine development ,, we are unaware of studies performed to date that investigate the toxicological impact of pristine SWCNTs on viral infectivity. Historical evidence highlights the causal relationship between inhaled particulates and associated lung diseases including fibrosis, cancers and exacerbation of asthma and bronchitis, conditions that may also render individuals susceptible to the pathogenicity of infectious agents, chiefly bacteria and viruses . Conversely, these biologic agents may be capable of modulating the pulmonary response to inhaled particles at the nanometer scale. This can have immense consequences as infectious agents, such as influenza A, are notorious for causing global pandemics that carry heavy mortality burdens. As realistic exposure scenarios involve multiple agents, triggering of conserved mechanisms may lead to detrimental responses that contribute to more severe, and in some cases unexpected health outcomes. This underscores the critical need to understand how nanoparticles influence cell behavior, alone and in combination with familiar pathogens, acknowledging that many of these changes could have a significant impact on cell/organ function in vivo. Studies that define the immune defense response to nanoparticles and their modulation of anticipated pathogenic outcomes are critically lacking and have implications for long-term health effects.
Herein, we have focused our efforts on pristine non-surface functionalized SWCNTs, allotropes of carbon that are defined by the length and chiral angle of the tube roll-up vector . We will use the term ‘electronic structure’ to describe the combined properties of diameter and chiral wrapping angles. We investigated the impact of three distinct types of SWCNTs that differ in their electronic structure on the infectivity of lung epithelial cells to pandemic influenza A H1N1 virus (IAV). The hypothesis that SWCNT electronic structure could affect viral infectivity is based on the assumption that the diverse surface energies associated with conductive versus semi-conductive SWCNTs could influence binding of these nanoparticles to biomolecules such as proteins or viruses. In fact, our research team has previously shown that chirality does influence both fractal structure  and aggregation  of SWCNTs in biologically relevant conditions. It is also reported that peptide binding is highly dependent on the diameter variation of the SNCNTs, which is one of the two parameters that control their electronic structure.
Results of this work reveal that SWCNTs can enhance viral infectivity of lung cells independent of electronic structure. We also provide evidence that SWCNTs inhibit IAV-induced expression of anti-viral molecules IFIT2 and IFIT3 and the cytokine RANTES, increase viral attachment receptors, and impair mitochondrial function. Taken together, these studies highlight the potential for SWCNTs to influence pathogenic infections in the lung by altering the normal anti-viral immune response of epithelial cells.
Results and discussion
SWCNT characterization under biological conditions
We also performed trace metal analysis on cell culture media incubated with each type of SWCNT for 24 h as a simulated measure of leachability and thus, bioavailability. All three types of SWCNTs tested leached higher levels of Mo into the media compared to Co. Greater levels of Mo were observed in media incubated with SG76 (125.62 ppb) and CG200 (117.28 ppb) compared to SG65 (23.81 ppb) SWCNTs. For Co, the levels for SG65 SWCNTs were 5.430 ppb followed by CG200 (1.82 ppb) and SG76 (non-detectable). While the levels of Co and Mo are quite low and not likely to be a major driver of the cellular effects described below, it can not be entirely ruled out that these metals do not contribute to surface reactivity of SWCNTs as previously suggested .
Viral infectivity of SAEC
Number of virus particles after 24 h of infection in the presence or absence of SWCNT pre-treatment as determined by titer assay
Titer (24 h post-infection)
Fold increase in titer
SWNT (type, 50 ug/ml)
5.2 × 105TCID50/ml
1.8 × 106TCID50/ml
3.2 × 106TCID50/ml
1.8 × 107TCID50/ml
3.2 × 106TCID50/ml
1.6 × 107TCID50/ml
3.2 × 106TCID50/ml
1.6 × 107TCID50/ml
3.2 × 106TCID50/ml
3.2 × 106TCID50/ml
Morphological changes in cell architecture were also noted as SAEC exposed only to SWCNTs appear more rounded (Figure 3B) compared to classic cuboidal shaped control cells, where those infected with virus are more spindle shaped with granular cytoplasm and enlarged pronounced nuclei (Figure 3C). Cells with the combined exposures of SWCNTs and IAV present with a mixture of the aforementioned morphological traits (Figure 3D). At this point it is not clear what the consequence of this observed cellular ‘roundness’ is or related cellular changes at the molecular level. Based on the dye-based cell viability assay we did not observe severely compromised cell membranes although these results do not rule out more subtle changes in membrane function caused by exposure to the SWCNTs. To examine if there were differential impacts of other types of chirally-enriched SWCNTs compared to SG65 SWCNTs on IAV infectivity, additional experiments were performed where SAEC were pre-exposed to SG76 and CG200 SWCNTs (50 μg/mL) 24 h prior to IAV infection. Results from these studies show that both types of SWCNTs increased virus infectivity comparably to SG65 SWCNTs, resulting in a 5 fold increase in infectivity in the presence of SWCNTs compared to the virus alone (high dose, MOI 5.0). The presence of the control particle, carbon black, failed to increase virus infectivity in any of the exposure scenarios (Table 1).
The results of these experiments reveal two novel observations: (1) they are the first studies to support the hypothesis that pristine SWCNTs have the capability to enhance the infectivity of lung epithelial cells with a strain of influenza A H1N1 virus; (2) that the influence of SWCNTs on viral infectivity does not seem particularly dependent on electronic structure, aggregate size, or stability. These results have significant implications for pathogen susceptibility and they highlight a relatively understudied area of nanomaterial toxicity. Only a few reports to date have probed the ability of nanomaterials to influence pathogen behavior and biological effects. Two studies performed in rodent models showed that sequential exposure of carbon nanotubes and bacteria led to enhanced pulmonary inflammation and fibrosis and decreased pathogen clearance, highlighting the potential impacts of combined exposures to these agents ,. Another study recently reported that pre-exposure to SWCNTs through pharyngeal administration does not modulate the immune response to the parasite T. gondii suggesting that the influence of carbon nanotubes on infectious agents may be pathogen specific. Other types of nanomaterials have been shown to possess innate antiviral activity. For example, silver nanoparticles have the ability to inhibit infectivity of HIV-1 by interfering with viral fusion and entry into cells . Carbon nanotubes have also been studied in this capacity and appear to bind HIV-1 in modeled simulations . Greater attention has been given to research devoted to the utility of nanoparticles, including carbon-based materials, for viral detection, vaccine development and drug delivery. However, in most cases, the nanomaterials are specifically engineered for such applications.
The timing of SWCNT exposure is important in driving increased viral infectivity
Number of virus particles after 24 h of infection with various treatment scenarios with SWCNTs as determined by titer assay
Titer (24 h post-infection)
Fold increase in titer
8.8 × 106TCID50/ml
SG65 24 h then IAV
3.8 × 107TCID50/ml
SG65 2 h then IAV
8.8 × 106TCID50/ml
IAV 2 h then SG65
8.8 × 106TCID50/ml
Mix SG65 + IAV then add to cells
8.8 × 106TCID50/ml
Changes in gene and protein expression of immune and viral genes by SWCNTs and pandemic influenza H1N1
SWCNTs alter mitochondrial function in SAEC
It has been well documented that the response of the lung to SWCNTs administered to the respiratory tract can result in robust inflammation, fibrosis, granulomas and pre-cancerous lesions -. These events are hallmarks of injury induced by other causative particles that lead to fibrosis and cancer, both in humans and animal models. While these studies highlight the potential health impacts of exposure to SWCNTs in isolation, few studies have addressed toxicological impacts associated with combined exposures of nanomaterial and pathogenic agents, with a particular focus on viruses. Results presented here show for the first time that SWCNTs can increase susceptibility of SAEC to IAV infection, possibly by modulating key genes important for viral replication and inflammatory responses, cell receptors that bind IAV and mitochondrial function. Certainly these results warrant translation of these effects to model systems in vivo, an emerging focus of our ongoing work. In addition, these studies lay the groundwork for investigating other pathogens and nanomaterials and further elucidating molecular mechanisms of action.
Materials and methods
SWCNT and viral preparations
Three types of pristine SWCNTs (SG65, SG76 and CG200) were provided by SouthWest Nanotechnologies Inc. (SWeNT). All SWCNT suspensions were prepared in 1% pluronic F68 (v/v in deionized water, Sigma) using protocols similar to methods previously described . To make suspensions, dry tubes were weighed and added to an appropriate volume of 1% pluronic, sonicated at 30–50 watt power for 25 minutes (Sonifier™ S-450 Digital Ultrasonic Cell Disruptor/Homogenizer, Branson Ultrasonics, Danbury, CT) in an ice bath. SWCNTs were serially diluted in 1% pluronic, their absorbance read at 775 nm (Synergy H1, Biotek). The rest of the stock was centrifuged at 17860 × g for 20 min and the resultant supernatant was sonicated at 30 watt power, diluted, and the absorbance was measured as mentioned above. The concentration of SWCNTs in the supernatant was calculated as follows: [supernatant] = [stock] × [Abssupernatant/Absstock]. All working stocks were re-sonicated at 30 watt power for 1 min immediately before using in the experiments. Carbon black particles (Sigma Aldrich) <50 nm in size was employed as a particle control in select studies.
Influenza virus H1N1 strain A/Mexico/4108/2009 was kindly provided by Drs. Gary Heil and Gregory Gray at the University of Florida and was propagated in MDCK cells in serum-free medium with 1 μg/ml L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin.
Dynamic light scattering, static light scattering and metal analysis of SWCNT
Dynamic light scattering (DLS) analysis using a goniometer system (ALV-CGS/3, ALV-GmbH, Langen, Germany) was employed to evaluate stability of SWCNTs suspended in pluronic F68 solution over a 24 h period. A detailed measurement procedure has been described elsewhere ,. In brief, a SWCNT sample was introduced to the sample chamber in a cleaned borosilicate vial (Fisher Scientific, Pittsburg, PA). A 632.8 nm laser was shined through the sample and scattered light was collected at an angle of 90° in 15 s intervals for 1 h. Hydrodynamic radii (HR) of the nanoparticles were calculated by the software interface using the collected time-dependent scattering data. HR of the particles were then plotted against measurement time to see the stability in this time period. This procedure was repeated in every 10 s for 24 h to monitor aggregate size change.
The ALV-CGS/3 goniometer system (ALV-GmbH, Langen, Germany) was also utilized to perform angle-dependent static light scattering (SLS) of pluronic suspended SWCNTs. For these measurements SWCNTs were added to a borosilicate glass vial and angle-dependent scattering was performed for an angular range of 12.5° to 100° with 0.5° increment. Fractal dimension was computed from log-log profiles of scattering intensity and wave vector. Slope of the fractal regime profile was then computed using linear fit which represented the fractal dimension . Data was collected initially and after 2, 6, 12 and 24 h upon exposure of the SWCNTs to the media. Analysis of trace metal composition within SWCNTs and in cell culture media exposed to SWCNT leachate was performed by inductively coupled plasma-mass spectrometry (ICP-MS) using methods previously described . Analysis was performed on triplicate SWCNT samples (20 mg dry weight) and in cell media after incubation with SWCNTs for 48 h. See supplemental data section for complete results of analysis (Additional file 1: Table S1).
Cell viability assay
Human small airway epithelial cells (SAEC) were obtained from Dr. Brooke Mossman and previously characterized by Hei and colleagues . Cells were seeded in 24-well plates in RPMI 1640 advanced medium supplemented with 10% FBS. Stock solutions of SWCNTs were added at final concentrations of 12.5 μg/ml, 25 μg/ml, 50 μg/ml and 100 μg/ml. After 24 h, cell viability was assessed using a standard trypan blue dye exclusion technique where the number of live and dead (blue) cells were counted under a light microscope with a hemocytometer. A total of 200 cells were counted per treatment (n = 3) and the entire experiment was repeated 3 times. Data are graphed as the mean and standard error of all experiments combined.
SWCNT and virus co-exposures to lung cells
SAEC were seeded onto cover-slips in 6-well plates in complete RPMI media and grown overnight. When the cells reached ~90% confluency, they were washed and 2 ml of serum free media were added. Appropriate volume of SG65 SWCNTs was added for a final concentration of 50 μg/mL. In control wells, equal volume of pluronic F68 was added. After 24 h, two different doses of virus (at MOI of 0.1 and 0.5) were added in designated wells. After 24 h of exposure the media was collected and stored for viral titer assays. The cells were fixed with cold 80% acetone and 20% methanol for immunofluorescence staining (see below). Similar exposures were performed where cells were pretreated with 50 μg/mL SG76 and CG200 SWCNTs followed by viral infections (high dose only).
Three additional treatment regimens were performed with SG65 SWCNTs; cells were pretreated with the SWCNTs for 2 h followed by infection with the high dose of virus; cells were first infected with virus for 2 h followed by treatment with 50 μg/mL SG65 SWCNTs; SG65 SWCNTs and virus were mixed together and incubated at 33°C for 3 h on a rotating platform and then added to the cells. Viral titers were performed after 24 h. Duplicates were assayed for each treatment. The entire experiment was repeated 3 times and data presented as the mean titer value for the experiments combined.
Immunofluorescence staining of cells for virus
For detection of IAV, the fixed cells were first stained for nucleoprotein A (Anti-Influenza A nucleoprotein, Clone A1, A3 Blend, Millipore Cat# MAB8251) by incubating the cells with antibody (1:100 diluted in PBS) for 1 h at 37°C. Next, the cells were washed three times with PBS and then incubated with the fluorescent labeled secondary antibody (Anti-Mouse IgG, FITC conjugated, Millipore Cat # AQ303F, 1:100 diluted in PBS) for 1 h at 37°C. Finally, the cells were washed three times in PBS and the cover slips were mounted onto microscope slides for observation under a fluorescence microscope. Triplicates were assayed for each treatment where 200 cells were counted per slide (n = 2) and the experiment was repeated 3 times. Data are presented as the mean and standard error for each treatment for the 3 experiments combined.
Viral titers (endpoint dilution assay)
To quantify the number of virus particles we calculated TCID50 values which is the dilution of virus required to infect 50% of inoculated cells. To perform these assays, MDCK cells were seeded in 96 well plates. Serial dilutions (10−1, 10−2, 10−3, 10−4, 10−5, 10−6) of virus stock and supernatant from exposed SAEC as described above were added to MDCK monolayers. Cells were incubated at 33°C and monitored for cytopathic effects by light microscopy (CPE) at 5 days post infection. Each well that displayed CPE were scored as positive. The TCID50 is calculated to be the dilution of virus at which 50% of the cell cultures are infected. Duplicates were assayed for each treatment. The entire experiment was repeated 5 times (Table 2) or 3 times (Table 1) and data presented as the mean titer value for the experiments combined.
Enhanced darkfield hyperspectral imaging microscopy
An optical microscope (Olympus BX41) equipped with a CytoViva optical condenser and a hyperspectral imaging (HSI) spectrometer (CytoViva Hyperspectral Imaging System, Auburn, AL) was used to collect darkfield and hyperspectral images of SAEC treated with viruses and/or SWCNTs. HSI analysis was performed using a previously described procedure ,. Using a 100× objective the samples were scanned (512 lines with a step size of 10 nm) in the visible and near-infrared wavelengths (VNIR: 400–1000 nm) with a bandwidth of 1.5 nm. The spatial and spectral information was derived from each image using the Environment for Visualization (ENVI) v4.8 software. For each sample, three to four images were collected at a constant high gain with 2 s exposure time and background subtracted using 10 dark-current images obtained prior to the sample scan. Using ENVI Spectral Hourglass Wizard the endmembers (spectrally dominant pixels) of IAV and SWCNTs were derived from hyperspectral images of SAEC co-exposed with viruses or SWNTs (controls). These endmembers were used for mapping. Spectral analysis of endmembers from controls (IAV or SWNTs) and the experiment (IAV + SWCNTs) confirmed the presence of spectral fingerprints of viruses and SWCNTs in the experiment. The hyperspectral classification images were generated using spectral angle mapper method with threshold set at 0.25. Analysis was done on one experiment with triplicate coverslips per treatment. A total of 10 images per slide was analyzed.
Expression levels of immune and viral-related genes in lung cells
Changes in mRNA expression in SWCNT and virus exposed lung cells as described above was performed by qRT-PCR using methods previously described . Briefly, RNA was isolated from cells using the RNeasy Mini Kit (Qiagen), quantified and reverse transcribed (Promega). The resultant cDNA was amplified using validated primers and probes specific to each gene target; RANTES, IL-8, IFIT2. IFIT3, RANTES, INFβ1, ST6GAL1, ST3GAL4, IL-10 (Applied Biosystems). Expression of GAPDH was employed for a standard housekeeping gene and all data is presented as normalized fold change in expression compared to controls using the delta delta Ct method. Since no IL-10 was detected in SAEC, to validate the IL-10 primers RNA from Namalwa cells was tested as a positive control. Namalwa cells are B lymphocytes that are immortalized by Epstein-Barr virus (EBV) and express IL-10. For this experiment the cells were cultured in advanced RPMI 1640 medium with 2 g/L D-Glucose, 1.0 mM Sodium Pyruvate, 2 g/L Sodium Bicarbonate, 1% GlutaMax (Invitrogen), 1% PSN (Invitrogen), and 7.5% fetal bovine serum (Thermo Fisher Scientific Inc.). Cells were collected and their RNA was extracted for qRT-PCR as described above. For all genes, triplicate samples were assayed for each treatment. The entire experiment was repeated 3 times and data presented as the mean and standard error for the combined experiments.
Secretion of IL-8 and RANTES in SAEC media
Levels of IL-8 and RANTES in cell culture supernatants were measured using a human CXCL8/IL-8 Quantikine ELISA kit (R&D Systems, MN) and human CCL5/RANTES Quantikine ELISA Kit (R&D Systems), respectively, following the manufacturer’s instructions. Absorbance was measured using a Synergy H1 hybrid multi-mode microplate reader with Gen 5 software (BioTek, VT). Triplicates were assayed for each treatment. The entire experiment was repeated 3 times and data presented as the mean and standard error for the experiments combined.
Detection of IFIT proteins by western blot analysis
Cells were exposed to SWCNTs and IAV as describe above, the media removed and cells washed in PBS. The cells were then harvested in Pierce RIPA buffer (Thermo Fisher Scientific Inc., MA). The total protein concentration was quantified and equal amounts of protein for each sample (80 μg) was separated on a 10% polyacrylamide gel and transferred to nitrocellulose membrane. The blots were incubated with IFIT2 (F-12, 1:200 dilution, Santa Cruz Biotechnology, Inc., TX), IFIT3 (B-7, 1:200 dilution, Santa Cruz Biotechnology, Inc) or β-actin (AC-15, 1:5000 dilution, Sigma-Aldrich, MO) overnight at 4°C. After three washes with TBST, the blots were incubated with rabbit anti-Mouse IgG (H + L) HRP-conjugated secondary antibody (1:2500 dilution, Thermo Fisher Scientific Inc., MA) for 2 h at room temperature. Following three washes with TBST, standard detection was carried out using Novex® ECL chemiluminescent substrate reagent kit (Invitrogen, NY) according to manufacturer’s instruction. Images were scanned using a ChemiDoc™ MP Imaging System with Precision Melt Analysis™ software 1.2 (Bio-Rad, CA).
SAEC plated on coverslips were fixed in acetone, washed with PBS, and endogenous peroxidase was quenched by incubation BLOXALL™ Blocking Solution (Vector Laboratories, CA) for 10 min at room temperature. Cells were then washed three times with PBS and blocked in 3% (w/v) bovine serum albumin (BSA) for 1 h at 4°C prior to adding 20 μg/ml biotinylated Maackia amurensis lectin II (MAA, Vector Laboratories) or 10 μg/ml biotinylated Sambucus nigra (elderberry) bark lectin (SNA, Vector Laboratories) overnight at 4°C. After three washes with PBS, the slides were then incubated with 5 μg/ml horseradish peroxidase streptavidin (HRP, Vector Laboratories) for 1 h at room temperature. After another three washes with PBS, Vectastain® ABC kit (Cat # PK-4000, Vector Laboratories) was used to detect the biotinylated lectins following the manufacturer’s instructions. The cells were counterstained with Hematoxylin QS (Vector Laboratories) for 10 s, washed, and mounted with VectaMount® permanent mounting solution (Vector Laboratories). Images were taken using a Nikon ECLIPSE Ti-U microscope (Nikon Instruments Inc., NY) with QImaging camera (Q-Capture Pro 7.0.5 software installed, BC, Canada). Analysis was done on two experiments with duplicate coverslips per treatment. A total of 10 images per slide was captured and analyzed.
Assessment of mitochondrial function
Measurements of basal respiration rate and mitochondrial stress were performed on a seahorse XF24. For these experiments, SAEC were plated at 20,000 cells/well in XF 24 microplates, allowed to adhere overnight, and exposed to SG65 SWCNTs or 1% pluronic (control). After 24 h following exposure, media was removed, rinsed twice, and replaced with XF mito stress test media (DMEM, 2 mM L-glutamine, 2 mM sodium pyruvate, 10 mM glucose). Basal respiration rates were measured three times, followed by injection of oligomycin (1 μM final concentration). Oligomycin inhibits ATP synthase allowing for measurement of mitochondrial respiration associated with ATP production. Three respiration rates were again measured followed by injection of Carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP, 0.5 μM final concentration). FCCP collapses the mitochondrial proton gradient causing uninhibited electron flow, resulting in maximal oxygen consumption. Maximal respiration rates were measured three times followed by an injection of rotenone (ROT, 1 μM final concentration) and antimycin A (AA, 1 μM final concentration). These two inhibitors completely shut down mitochondrial respiration, allowing for measurement of non-mitochondrial respiration in the cell. The spare respiratory capacity was calculated by subtraction of basal respiratory rate from maximal respiratory rate
SigmaPlot version 12.0 (Systat Software Inc., San Jose, CA) software for Windows was used for all statistical analysis. ANOVA with Tukeys was used to analyze differences between treatments. Null hypothesis was rejected at a p-value < 0.05.
The authors would like to thanks Drs. Gregory Gray and Gary Heil for the influenza virus stocks. This work was supported by the National Institute of Health (R01HL114907 to TSA) and the University of Florida Research Foundation (to TSA) for funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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