Nanoparticle uptake by airway phagocytes after fungal spore challenge in murine allergic asthma and chronic bronchitis

Murine models were used to investigate particle uptake in conditions representing allergic asthma and COPD as well as in corresponding healthy controls (Figure 1). Recovery, treatment and analysis of BAL cells were identical in all animal groups.

Allergic airway inflammation was induced using an adjuvant-free experimental asthma protocol as previously described [27]. Briefly, animals (ten female BALB/c mice aged 6 - 8 weeks purchased from Harlan Winkelmann, Borchen, Germany) were sensitized by subcutaneous injection of 10 μg OVA (grade VI, Sigma, Steinheim, Germany, containing 1 ng of endotoxin per 10 μg OVA [dose used for sensitization]) in 200 μL phosphate buffered saline (PBS, Sigma, Steinheim, Germany) or sham injections of PBS on days 0, 7 and 14. Allergic airway inflammation was induced by 20 minutes of 1% OVA or sham PBS aerosol exposure on days 26, 27 and 28. BAL cell isolation was performed 24 h after the last allergen challenge.

The Scnn1b-Tg mouse is an established model of chronic inflammatory lung disease exhibiting key features of COPD and CF such as chronic airway inflammation, mucus obstruction and structural lung damage [28‐32]. Five Scnn1b-Tg mice (airway targeted overexpression of the epithelial Na⁺ channel β subunit Scnn1b; line 6608 generated and maintained on a mixed C3H/HeN:C57BL/6N genetic background as previously described [30] and five WT littermates aged 16 - 18 weeks, were bred and reared at the University of Heidelberg, Heidelberg, Germany. Mouse progeny were genotyped by tail biopsy polymerase chain reaction (PCR) as previously described [33]. Animals were housed under a 12 h light/dark cycle, controlled humidity of 55% and temperature of 22°C, with access to food and water ad libitum.

For cell retrieval, animals were deeply anesthetized by intraperitoneal injection of 300 mg/Kg ketamine hydrochloride plus 30 mg/Kg xylazine (Rompun, Bayer Health Care, Leverkusen, Germany) prior to exsanguination by cutting the abdominal aorta and lung lavage.

All experiments were performed under federal guidelines for the use and care of laboratory animals and were approved by the responsible local authorities (Regierungspräsidium Giessen, Germany; Regierungspräsidium Karlsruhe, Germany; and the Cantonal Veterinary Office, Bern, Switzerland) and included ethical evaluation.

Basidiospores of the Calvatia species were selected, since these spores have been well characterized in terms of their morphology and surface properties as well as their distribution, retention and clearance by macrophages in rodent lungs upon inhalation [26, 34]. In addition, all analyses were performed in comparison to other particle types of the same size [9, 34]. Fungal spores of Calvatia excipuliformis (3.5 μm in diameter), collected from dried mushrooms as previously described [26], were suspended at a density of 2 × 10⁷ particles/mL in fluorocarbon FC-72 (3 M, Neuss, Germany).

Similarly, valuable background information on AuNP for this in-vitro study was retrieved from very recent inhalation experiments with spark generated 20-nm AuNP using the same COPD animal model [18]. Colloidal gold particles (AuNP, 20 nm in diameter), suspended at 1 × 10¹¹ particles/mL in citrate buffer, were obtained from Plano (Wetzlar, Germany).

Lungs were lavaged with 5 × 1 mL sterile PBS as described previously [11]. Total number of BAL cells was determined using a Neubauer haemocytometer chamber. Differential cell counts (200 BAL cells per animal) were performed either on cytospin preparations (OVA-allergic and sham-sensitized mice) fixed and stained with Diff-Quick (Merz & Dade AG, Düdingen, Switzerland) or directly on filter-insert cell preparations (Scnn1b-Tg and WT mice) fixed with phosphate buffered 2.5% glutaraldehyde (EMS, Fort Washington, PA, USA). Macrophages, lymphocytes, eosinophils and neutrophils were differentiated by standard morphologic criteria.

For each animal, 3 × 10⁵ BAL cells in 100 μL complete medium (Dulbecco’s Modified Eagle’s Medium, D-MEM, supplemented with 10% fetal calf serum, FCS) were seeded on a micro-porous filter insert (pore size: 0.4 μm, growth area: 0.3 cm², i.e. 1 × 10⁶ cells/cm²; Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) in 24-well plates (Falcon), with each well containing 800 μL complete D-MEM. Cells were then cultured for 2 h at 37°C, 5% CO₂ and 90% RH to select for adherent cells, i.e. macrophages and activated leukocytes (eosinophils, neutrophils, lymphocytes) that were freshly recruited to the lung surface. Non-adherent cells were washed off with serum-free D-MEM. Thereafter, sequential exposure of cells to the particles followed. Like this, concurrent exposure of micro- and nanoparticles was avoided to omit passive carriage of the smaller (nano)particles on the surface of the larger (micrometer sized) ones. To challenge phagocytosis, the remaining cells were first exposed to 1 × 10⁶ fungal spores (~ 3 spores/cell) in 100 μL serum-free D-MEM/insert for 2 h. Subsequently, non-adherent spores were washed off with 300 μL serum-free D-MEM/insert and BAL cells were then exposed to 1 × 10⁹ AuNP (~ 3 × 10³ NP/cell) in 100 μL serum-free D-MEM (zeta potential ~ 22 mV [35]) for 4 h. Non-adherent AuNP were rigorously washed off with 2 × 300 μL serum-free D-MEM/insert and 1 × 500 μL PBS. BAL cells were then successively fixed with 500 μL of 2.5% glutaraldehyde (EMS) in 0.03 M potassium-phosphate buffer, 1% osmium tetroxide in 0.1 M sodium-cacodylate buffer and 0.5% uranyl acetate in 0.05 M maleate buffer (Sigma-Aldrich, Buchs, Switzerland), dehydrated in a graded series of ethanol and embedded in epon resin (Sigma-Aldrich). Ultrathin sections of 50 nm nominal thickness were cut perpendicular to the filter-insert with an ultra-microtome (Ultratome, LKB, Stockholm, Sweden), mounted onto formvar-coated 200-mesh copper grids and post-stained with uranyl acetate and lead citrate (Ultrastain, Leica, Glattbrugg, Switzerland). For electron tomography, 150-nm sections were cut, mounted and stained as described above.

BAL cell morphology was first evaluated on toluidine blue stained sections of 1 - 2 μm nominal thickness using a Leica light microscope (Leica, Wetzlar, Germany). Ultrastructural analysis of cells and particles was performed on random ultrathin sections of the cells on filter inserts using a CM12 transmission electron microscope (TEM, Phillips, Eindhoven, The Netherlands) operated at 80 kV. For each animal model, approximately 150 cells were analyzed by TEM. Fungal spores and AuNP were unambiguously identified according to their size, shape and contrast. The diameters of macrophages, vesicles and particles are reported, since this is the common measure in cell biology. Equivalent diameters were calculated from section profile area measurements of the cell, vesicle or particle using the iTEM software (Olympus Soft Imaging Solutions, Münster, Germany).

For electron tomography, the 3D configuration of AuNP and surrounding structures was acquired by projection reconstruction of a multi-angular tilt series of thick sections (~ 150 nm) [36]. Electron tomography was performed on five vesicles containing AuNP using a Tecnai F20 field emission TEM (FEI Company, Eindhoven, Netherlands) operating at 200 kV. Typical tilt series were acquired at a magnification of 40,000× covering an angular range from -66° to 66° (with an increment of 2°) and a defocus of 0.53 μm. The pixel size in the tomogram was 0.51 nm. Tilt images were aligned semi-manually using the FEI software package (Inspect 3D, Amira, FEI Company, Eindhoven Netherlands). Three dimensional reconstructions of AuNP and segmentation of macrophage vesicles in the tomogram were performed manually with the segmentation editor implemented in Amira software.

BAL cells from all animal groups were treated the same way, i.e. first with spores and then with AuNP. Thus particle treatment was a within-subject (single measure) factor, while animal model (mouse strain) as well as allergy and COPD status was a between-animal factor. Due to the applied sampling design of analyzing all AuNP-containing vesicles in a macrophage, the number of data points obtained varied between macrophages and animals. Data of BAL cell number, macrophage size and distance of particles to vesicle membrane were analyzed by the non-parametric Mann-Whitney Rank Sum Test for two group comparisons and one-way analysis of variance (ANOVA) followed by Dunn or Holm-Sidak post hoc tests for pairwise multiple group comparisons. These analyses were performed using SigmaPlot 12 software (Systat Software Inc., San Jose, CA, USA). Data of vesicle to particle size ratios, R_V/P were analyzed for differences in the mechanism of AuNP uptake. A linear mixed effects model with a random intercept for each animal and fixed effects for health status, disease model/mouse strain and their interaction health status*disease model and vesicle class was used to model the logarithm of R_V/P (log[R_V/P]), where the log transformation was chosen for linearization and diagnostic reasons. These analyses were performed with R software version R-2.13.0. A P-value < 0.05 was considered to be statistically significant. Descriptive statistics such as number of observations, mean, standard error of the mean (SEM) and range (minimum, maximum) are used to summarize the continuous outcome variables.

As shown in Figure 2, fungal spores are sphere-shaped with spikes and a geometric diameter of 3.5 ± 0.2 μm (n = 120) [26]. Single AuNP are spheres with a geometric diameter of 20.8 ± 3.4 nm (n = 100). AuNP in stock solution were mostly single particles, whereas we found agglomerates of different sizes, when AuNP were suspended in serum-free cell culture medium.

As shown in Table 1, the mean number of total BAL cells obtained from OVA-allergic mice (n = 5) was significantly higher (P = 0.001) compared to healthy controls (n = 5). BAL cells from allergic mice consisted of 56.6 ± 3.8% eosinophils, 25.9 ± 2.4% macrophages, 10.4 ± 0.5% neutrophils and 9.1 ± 1.6% lymphocytes. In sham controls, 99.6 ± 0.4% of BAL cells were macrophages. The mean number of total BAL cells from Scnn1b-Tg animals (n = 5) was not significantly different from WT mice (n = 5). BAL cells from Scnn1b-Tg mice consisted of 97.8 ± 0.3% macrophages and 2.2 ± 0.3% lymphocytes (Table 1). In WT animals, 99.8 ± 0.2% of the cells were macrophages.

As shown in Figure 3, BAL macrophages from OVA-allergic and sham-sensitized mice appeared structurally normal, although, those of OVA-allergic animals were significantly larger (27.0 ± 1.3 μm in diameter, range 7.1 - 46.3 μm) compared to sham controls (15.8 ± 0.5 μm, range 8.2 - 25.2 μm) and any other animal group analyzed (P < 0.001). The macrophage population of Scnn1b-Tg mice appeared morphologically heterogeneous; some cells were abnormally large and many had a “foamy” cytoplasm as previously demonstrated [30, 32]. WT macrophages showed normal morphology. As Figure 3 also demonstrates, Scnn1b-Tg macrophages were significantly larger (17.7 ± 0.5 μm diameter, range 5.3 - 36.2 μm) than those of WT mice (15.9 ± 0.6 μm, range 7.5 - 26.7 μm) (P = 0.014).

Electron microscopic and morphometric analysis of AuNP uptake by BAL cells revealed that a similar percentage of macrophages from OVA-allergic animals and sham controls contained AuNP (61.0 ± 21.4% vs. 62.1 ± 5.7% respectively). The percentage of BAL macrophages from Scnn1b-Tg mice containing AuNP tended to be lower (74.9 ± 7.2%) than in WT animals (82.0 ± 3.3%), though not statistically significant with the number of animals included in this study. Comparison of AuNP uptake by macrophages obtained from healthy control groups revealed significantly higher percentages of macrophages containing AuNP in naïve WT, i.e. C3H/HeN:C57BL/6N mice compared to sham-sensitized BALB/c mice (P = 0.016).In all animal groups, AuNP were found either as single particles in vesicles largely exceeding their size and containing other electron dense material of mostly unknown origin (Figure 4A), as agglomerates in vesicles of similar size (Figure 4B) or co-localized with spores in phagosomes (Figure 4C).

As shown in Table 2 and Figure 5, the sizes of AuNP clusters in macrophage vesicles increased according to the number of agglomerated particles, while the sizes of the vesicles enclosing the AuNP remained constant, irrespective of AuNP cluster size in all experimental groups (Figure 5A). Thus, the ratio of vesicle to particle diameter R_V/P (Figure 5B) was large, i.e. ~ 20 for single AuNP and ~ 10 for small clusters of 2 - 5 AuNP; it then decreased to ~ 5 when clusters contained 6 - 10 AuNP and approached R_V/P ~ 1, i.e. the value representing phagocytic particle uptake, when clusters consisted of more than 10 AuNP. The supplementary statistical analysis of R_V/P data (Table 3) revealed no significant difference in AuNP uptake between macrophages from (i) the healthy and diseased animals, (ii) the two mouse strains or (iii) the diseased animals of the two groups. However, a significant vesicle class effect is found. The three coefficients given in Table 3 correspond to the effect of not being in vesicle class A (single AuNP) but in the respective category B (2 - 5 AuNP), C (6 -10 AuNP) or D (> 10 AuNP). All these three differences to vesicle class A are significant and the predicted log(R_V/P), thus also the R_V/P itself, decreases with increasing number of nanoparticles.

In all groups studied, fungal spores (2.9 μm ± 0.2 μm in diameter, measured on ultrathin sections) were found internalized as single particles. They were localized in macrophage phagosomes of 3.4 μm ± 0.1 μm diameter (Figure 5A), with the vesicle membrane closely surrounding the spores (Figure 4C), resulting in a R_V/P = 1.4 ± 0.1. AuNP were mostly found adjacent to the vesicle membrane. Thereby, the distance of AuNP to the membrane tended to be larger in diseased animals of both murine models compared to the healthy mice, though not statistically significant (Figure 6A). The mean distance to the vesicle membrane was 38.5 ± 4.1 nm in BAL cells of OVA-allergic mice vs. 36.9 ± 3.5 nm in sham-sensitized controls and 40.3 ± 2.7 nm in Scnn1b-Tg mice vs. 31.4 ± 2.5 nm in WT littermates. Tomographic reconstructions (n = 3) of single AuNP and small agglomerates, as shown in Figure 6B, confirmed the proximity of AuNP to the vesicle membrane. Rarely, we found AuNP that were not enclosed by a membrane, i.e. localized in the macrophage cytoplasm.In BAL cells from OVA-allergic mice, AuNP were also found in 13.9 ± 8.3% of all observed eosinophils (n = 36), localized in vesicles exceeding NP size and together with other electron dense material of unknown origin (Figure 4D). The few lymphocytes (n = 6) and neutrophils (n = 1) observed in OVA-allergic mice, did not contain any AuNP.