Animals and Experimental Setup
Male C57BL/6J mice were received (Charles River, 97633 Sulzfeld, Germany) at an age of four weeks. Upon entry into our animal facility, animals were kept in isolated ventilated cages (IVC-Racks; BioZone, Margate, UK) supplied with filtered air, in a 12-hr light/12-hr dark cycle (lights on from 06:00 - 18:00) and allowed to adapt to conditions for approximately three weeks (19-20 days). Food (standard chow) and water were available ad libitum. After acclimatisation period animals were divided into three weight-matched groups and fed different diets (SSniff Spezialdiäten GmbH, 59494 Soest, Germany) for a period of six weeks, respectively. One group remained on a standard low fat diet (Low Fat; LF, gross energy content 16.27 kJ/g dry mass; n = 25) isocaloric to the chow fed during acclimatisation period, the second group (n = 23) was fed a carbohydrate-rich Cafeteria-diet (Cafeteria; CA; sodium/rich biscuit/sugar; gross energy content 18.94 kJ/g dry mass). The three different cafeteria diets were mixed in equal proportions. The third group (n = 26) was fed a high-caloric fat-diet (High Fat; HF; gross energy content 23.54 kJ/g dry mass). All procedures for animal handling and experiments were performed in accordance with protocols approved by the Regierung von Oberbayern (District Government of Upper Bavaria). Experiments were performed at the German Mouse Clinic phenotyping platform (GMC) [36, 37].
Body Mass and Body Composition
Body mass of animals was monitored at the day of delivery (4 weeks old) and the day the diet challenge started (7 weeks old). Further body mass development was monitored in weekly intervals until the end of six weeks' feeding period. Body composition was determined at delivery; at the beginning of the diet challenge as well as every second week during the diet-feeding (after week 2, 4, 6, respectively) using a whole animal body composition analyzer (Minispec Bruker, Ettlingen Germany) based on Time Domain Nuclear Magnetic Resonance (TD-NMR) which provides a precise method for the measurement of lean tissue and body fat in live mice without anaesthesia. To conduct the measurement single mice were placed in a plastic restrainer that was introduced into the magnet of the scanner for up to 5 minutes to collect data. Data acquisition was based on a calibration using dissected lean muscle and fat tissue. For the statistical analysis, a linear regression model was applied including diet as main factor and body mass as covariate to adjust for body mass differences.
Particle Challenge Design and Group Setup
Particle challenge was done directly after six weeks' feeding period in the 13 weeks old animals. In a counterbalanced system, animals of all three groups (LF, CA, HF) were either instilled with an aqueous suspension of Printex 90 carbon-nanoparticles (CNP) as previously described  (zeta potential: 33 mV; agglomerate diameter in suspension: 0.17 μm), or pyrogene-free distilled water (SHAM exposed) respectively or were left undisturbed and served as controls (Home Cage Control; HCC). Printex 90 was chosen as frequently used commercially available pigment black (Degussa, Frankfurt, Germany) (diameter [nm]: 14; organic content [%]: 1; surface area [m2/g]: 272); as characterized earlier [30, 39]. For details on group setup and sample size, see Table 1.
Prior to instillation, mice were anesthetized by intraperitoneal injection of a mixture of Medetomidin (0.5 mg/kg body mass), Midazolam (5.0 mg/kg body mass) and Fentanyl (0.05 mg/kg body mass). The animals were then intubated by a nonsurgical technique (Brown et al. 1999). Using a cannula inserted 10 mm into the trachea, a suspension containing 20 μg CNPs particles, respectively, in 50 μL pyrogene-free distilled water was instilled, followed by 100 μL air; the suspension of poorly soluble CNPs was sonicated on ice for 1 min prior to instillation, using a SonoPlus HD70 (Bachofer, Berlin, Germany) at a moderate energy of 20 W resulting in a mean agglomerate size of 0.17 μm (Zetasizer Nano ZS, Malvern Instruments, Herrenberg, Germany). SHAM animals were instilled 50 μL pyrogene-free distilled water only . After instillation animals were antagonized by subcutaneous injection of a mixture of Atipamezol (2.5 mg/kg body mass), Flumazenil (0.5 mg/kg body mass) and Naloxon (1.2 mg/kg body mass) to guarantee their awakening and well-being. Animals were treated humanely and with regard for alleviation of suffering; experimental protocols were reviewed and approved by the Bavarian Animal Research Authority.
Blood, Serum, and Bronchoalveolar Lavage (BAL) sampling
Twenty-four hours after instillation, mice were anesthetized by intraperitoneal injection of a mixture of xylazine (4.1 mg/kg body weight) and ketamine (188.3 mg/kg body weight) and killed by exsanguination. Therefore, blood was drawn from the retroorbital plexus by a capillary and collected a.) in EDTA covered tubes (Sarstedt) for haematological analysis (ADVIA Hematology Systems (Bayer Diagnostics) and b.) non EDTA-covered tubes to gain blood serum. Subsequently BAL was performed by cannulating the trachea and infusing the lungs 10 times with 1.0 mL PBS without calcium and magnesium, as described previously . The BAL fluids from lavages 1 and 2 and from lavages 3-10 were pooled and centrifuged (425 g, 20 min at room temperature). The cell-free supernatant from lavages 1 and 2 were pooled and used for biochemical measurements such as lactate dehydrogenase (LDH), total protein, and cytokine concentration. The cell pellet was resuspended in 1 mL RPMI 1640 medium (BioChrome, Berlin, Germany) and supplemented with 10% foetal calf serum (Seromed, Berlin, Germany); the number of living cells was determined by the trypan blue exclusion method. We performed cell differentials on the cytocentrifuge preparations (May-Grünwald-Giemsa staining; 2 × 200 cells counted) and the number of polymorphonuclear leukocytes (PMNs) was used as a marker of inflammation.
BAL: Total Protein Content and Lactate Dehydrogenase (LDH) Assay
Total BAL protein content was determined spectrophotometrically with an ELISA reader (Labsystems iEMSReader MF, Helsinki, Finland) at 620 nm, applying the Bio-Rad Protein Assay Dye Reagent (no. 500-0006; BioRad, Munich, Germany), as a potential biological marker for pulmonary capillary leakage and lung injury . 5 μl BAL fluid/mouse was used for analysis.
For detection of the cytosolic enzyme lactate dehydrogenase (LDH) (U/ml), characteristic for membrane damaging effects, the Cytotoxicity Detection Kit (Roche Diagnostics, Germany) was used according to the manufacturer's instructions. LDH concentration in the BAL fluid (30 μl) was spectrophotometrically determined with an ELISA reader (Labsystems iEMS Reader MF, Helsinki, Finland) at a wavelength of 492 nm.
Cytokine and Adipokine Detection (Multiplexed immunoassays)
BAL fluid and blood serum adipokine concentrations were investigated using a Luminex xMAP system (Milliplex, mouse CVD Panel 2 and mouse adipocyte Panel, Millipore Corporation, 290 Concord Road, Billeria, MA 01821, USA) to simultaneously detect the concentration for the 4 following adipokine analytes: fibrinogen, leptin, adiponectin, PAI-1. Measurement was performed according to the manufacturer's instructions.
Accordingly to adipokine analysis, also cytokine/chemokine concentrations were investigated in BAL fluid and blood serum using Bio-Plex Pro Mouse 14Plex Cytokine Panel, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules CA 94547, USA).
Simultaneously the following 14 cytokines/chemokines were investigated in cell-free BAL fluid (50 μl) or blood serum (15 μl serum + 35 μl sample diluent). Analytes were as follows: IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12(p40), IL-12(p70), IFN-γ, TNF-α, CXCL2 (MIP2), G-CSF, CCL5 (RANTES), CXCL1 (KC).
For all measurements the mean fluorescence intensity (MFI) was detected by the Multiplex plate reader (Luminex System, Bio-Rad Laboratories, Germany). For each sample a minimum of 50 beads per region were analyzed. Multiplex plate reader software (Bioplex Manager, Version 4.1.1) was used to capture raw data (MFI). For data analysis, a four-parameter logistic curve fit was applied to each standard curve and sample.
We tested the effects of the two factors diet (3 levels: Low Fat diet (LF), Cafeteria diet (CA), High Fat diet (HF)) and treatment (3 levels: untreated home cage control (HCC), water-instilled SHAM group at 24 h (SHAM), and CNP instillation after 24 h (CNP)) on different response variables, as shown in Tables 2, 3, 4, 5 (Two-Way ANOVA). We included the interaction of the two factors (diet x treatment) in order to test whether the treatment showed differential effects within the different diet groups. If not statistically significant, the interaction term was reduced and the model was re-calculated. Response variables, which deviated from the normal distribution, were log-, or square-root-transformed. Normality of the model residuals was checked visually by normal probability plots and with the Shapiro-Wilk test, and we assured the homogeneity of variances and goodness of fit by plotting residuals versus fitted values and by the Levene test . Post-hoc comparisons were conducted with the Mann-Withney U test (MWU). In cases transformation of data failed to reach normal distribution MWU test was performed as statistical test. All statistical analyses were done using the software SPSS 11.0 (SPSS Inc., Chicago, IL).
Body mass, lean and fat mass data accordingly were tested by One-Way ANOVA for the factor diet only (Figure 1). Holm-Sidak method was used for post-hoc comparison between diet-fed groups.
All data are expressed as mean ± SEM. Within graphs significant P-values are shown by asterisks (*P < 0.050, **P < 0.010, ***P < 0.001) and trends are indicated by plus symbol (+
P < 0.10).