Helical Carbon Nanotubes Enhance the Early Immune Response and Inhibit Macrophage-Mediated Phagocytosis of Pseudomonas aeruginosa

Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.


HCNT characterization
Bright field transmission electron microscopy (TEM) images were acquired on a JEOL JEM2100 microscope operated at 200 kV with a spherical aberration coefficient of 2.0 mm (Fig. S1). The nanotubes were prepared by sonicating in ethyl alcohol for 5 minutes on ice, vortexed, and dispersed on copper grids coated with Formvar plastic. A Hitachi S-4800 field emission scanning electron microscope (SEM) was used to analyze the structural features of HCNTs. HCNTs were pressed onto double-sided carbon tape and imaged under a 5.0 kV accelerating voltage at varying magnifications ( Fig. S2-A iiv). HCNTs were also dispersed in 2% wt/vol sodium dodecyl sulfate (SDS) by centrifugation followed by vacuum filtration onto nitrocellulose membranes. The HCNTs were then transferred to a SiO 2 /Si supporting substrate for SEM analysis (Fig. S2-A v and vi). The diameter distribution was then obtained by importing the SEM files into Gwyddion (http://gwyddion.net/) [1]. Line profiles were taken perpendicular to the longitudinal axis of the HCNTs, ensuring that the profile ended at the HCNT edge. An example line profile is encircled in Fig. S2-A v. The HCNTs have a diameter distribution of 50-500 nm, normally distributed about ~200 nm (n = 80) ( Fig. S2-B).
Additionally, we determine the lengths of the helical HCNTs by edge extraction with NeuronJ [2]. We use SEM images gathered in the same way as Fig. S1A i-vi to extract the HCNT length. By analyzing the distance between maxima in the HCNTs' helices, we calculate the helical pitch to be b ≈ 12 nm. With the SEM images in Fig. S3A i-vi and NeuronJ, the apparent HCNT length is L app = 1.40 ± 0.53 µm (n = 50). To determine the actual HCNT length, that is, the length of the unraveled helix, we employ 3 the expression where a is the HCNT radius (~100 nm) and b is the helical pitch (~12 nm). Therefore, the helical length distribution is L HCNT = 1.9 ± 0.8 µm, as shown in Fig. S3-B. The difference between the helical length L HCNT and the apparent length L app is ΔL = L HCNT -L app . ΔL ranges from 0 to 1.46 µm, with a mean at 0.5 µm.
Using the diameter and length information, we can estimate the specific surface area (SSA) of the HCNTs. The interior core of HCNTs has previously been shown to be hollow [3]. There is a dense array of multi-walled CNTs (MWNTs) that surrounds this hollow core, and the hollow core is ~2 nm in dimension [3,4]. The thickness of a MWCNT is that of graphene (0.34 nm) and the separation between walls in a MWNT is shape for the helical HCNT. This is based upon fits of the C-1s of highly ordered pyrolytic graphite (HOPG) [6]. The presence of Cl and Al (Figs. S5-C, D) may be due remnant nanoparticles or residues from the synthesis process. 5 Raman spectroscopy was performed using a Renishaw micro-Raman spectrometer with a 632 nm excitation laser. Raman data were collected under a 50× long working distance objective at 1.3 mW incident power for HCNT powders on glass slides. The Raman spectra (Fig. S6) show two distinct peaks at ~1334 and ~1592 cm -1 , commonly referred to as the D and G peaks in carbon nanomaterials. The G peak is attributed to the in plane stretching of the sp 2 hybridized C-C sigma bond. The D peak is attributed to disorder in the crystal structure which breaks translational symmetry, allowing for breathing of the carbon hexagons in the lattice [7].
Size distribution and zeta potential of the HCNTs was performed using a Zetasizer Nano ZS(Red Badge) ZEN3600 (Malvern Instruments, Paris, France) with the nanotubes dispersed in the dispersal media. Particle size distribution of dispersed samples demonstrated a mean diameter of 532 nm (Fig. S7). The zeta potential of dispersed HCNTs in dispersal media was determined to be -3.04 mV. HCNT exposure and P. aeruginosa infection in mice 6 Six-week old wild-type CD-1 mice (Charles River Laboratories) were housed in positively ventilated microisolator cages with automatic recirculating water, located in a room with laminar, high efficiency particle accumulation-filtered air. The animals received autoclaved food, water, and bedding. Mice were handled in accordance with approved protocols through the Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign. HCNTs (50 µg in 50 µl of PBS/0.01% Tween-80) was intranasally inoculated into the lungs of isoflurane anaesthetized CD-1 mice either once or twice/week for 3 weeks. Control mice were exposed to 50 µl of PBS/0.01% Tween-80. At designated time points, mice were removed and the lungs were lavaged or collected for histopathology.

Molecular, cytotoxic, and immunological assays
For infection models, HCNTs or PBS/0.01% Tween-80 exposed mice were given a single intranasal dose of PAO1 (10 7 cfu in 50 µl) 72 hours following the last HCNT administration [8]. After 24 hours of PAO1 infection, mouse lungs were harvested for bacterial enumeration, histopathology, or lavaged for cell enumeration and for ELISA.
For in vivo phagocytosis enumeration, mice were exposed to HCNTs or dispersal media for 3 weeks as described above. Mice were given a single intranasal dose of PAO1-GFP (1 x 10 7 cfu in 50 µl) 72 hours after the last HCNT treatment. After 24 hours, leukocytes from the lavage were collected. Leukocytes were concentrated onto glass slides using cytospin and cells were fixed with 1% paraformaldehyde overnight at 4ºC.
Macrophages were examined with confocal microscopy as described above. The % of cytoplasm occupied by HCNTs was measured using Adobe Photoshop (San Jose, CA).
For neutrophil depletion, mice were exposed to HCNTs or dispersal media for 3 weeks as described above. 24 hours before infection, mice were given 0.2 mg of the 7 anti-Ly6g antibody (BioXCell, West Lebanon, NH) intraperitoneally. Mice were given a single intranasal dose of PAO1 (10 4 cfu in 50 µl). After 24 hours, mouse lungs were harvested for bacterial enumeration, histopathology, or lavaged for cell enumeration.
For macrophage depletion, mice were exposed to HCNTs or dispersal media for

Bronchoalveolar lavage (BAL)
BAL of mouse lungs was performed as described [9,10]. The trachea was exposed and intubated with an 18 gauge needle. Four 1 ml aliquots of cold PBS were instilled. The first aliquot was centrifuged at 500 g for 5 minutes at 4ºC and the supernatant was removed and stored separately at -80ºC for ELISA. Cells from the first aliquot were then pooled with the remaining aliquots. Cell numbers were counted by hemocytometer and cell differential was determined microscopically following cytospin 8 preparation of cells stained with Diff-Quik stain. At least 200 cells per slide were counted.

Histopathology evaluation of mouse lung tissues
Mouse lungs were collected for histopathological analyses as described [11].
Mouse lungs were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned and stained with hematoxylin and eosin (H&E) or with Alcian blue. Tissue sections were examined by a pathologist at the Department of Pathobiology, University of Illinois at Urbana-Champaign.

Statistical analysis
Normality of the data was evaluated using the Anderson-Darling normality test with rejection of normality when p-value < 0.05. Data were then analyzed for statistical significance by Student's t-tests, with differences between means considered significant when p-value < 0.05. For comparing the means of groups of three or more, data were analyzed for statistical significance by ANOVA followed by Tukey's tests for comparison between the means.

Results
Cytologic evaluation of leukocyte cytospin preps from BAL of mice collected 24 hours after exposure to HCNTs shows 71.5% of macrophages have phagocytized HCNTs after one exposure and 88% of macrophages have phagocytized HCNTs after 3 weeks of exposure ( Figure S8).