Preparation of Nanomaterial Suspensions
MWCNTs (MWCNT-7) were obtained from Mitsui Chemicals Co., Ltd. Tokyo, Japan; TiO2 (rutile type, with a mean primary size of 20 nm) was provided by Japan Cosmetic Association, Tokyo, Japan; SiO2 (with a primary size of 10–20 nm) was purchased from Sigma-Aldrich, USA; and Fullerene C60 (with a mean primary size of 1 nm) was provided by Frontier Carbon Corporation, Japan. 10 mg of these 4 types of ENMs was suspended in saline containing 0.5% (w/v) Pluronic® F-68 (a non-ionic detergent from Sigma-Aldrich, USA) to a final concentration of 500 µg/ml. The MWCNT-7 suspension was homogenized four times, with 1 minute each time, using the Polytron PT1600E bench-top homogenizer (Kinematica, Switzerland) at a speed of 3000 rpm. The prepared four nanomaterial suspensions were sonicated at 600W for 30 minutes using the JY92-2 sonicator (Scientz Co., Ltd, Ningbo, China), 5 minutes for 6 times with 2 minute-interval rests. To ensure the dispersion and suspension of the nanomaterials, the suspensions were further sonicated for 5 minutes for 4 times just before use. Characterization of the suspended 4 nanomaterials, including shape, size distribution and element analysis, was shown in the Figure S1, S2 and S3.
Eight weeks-old female wild-type Sprague-Dawley (SD) rats were obtained from and housed in the Animal Center of Anhui Medical University, and received Oriental MF basal diet and water ad libitum. The animal experiment protocols were approved by the Institutional Animal Care and Use Committee.
Intratracheal spraying of nanomaterial suspensions
Twenty-five female SD rats were divided in to 5 groups and 0.5 ml of the suspensions were intratracheally sprayed with 0.5 ml of the vehicle, 500 µg/ml MWCNT-7, TiO2, SiO2, or C60 suspensions, using an intratracheal aerosolizer (series IA-1B, Penn-century, Philadelphia, USA), as previously described , 2 times per week for 2 weeks. The total amount of the administered nanomaterials was 1 mg per rat. Three days after the last spraying, the animals were sacrificed under isoflurane anesthesia, and the lung was excised, and then fixed in 4% paraformaldehyde solution in phosphate-buffered saline (PBS) adjusted to pH 7.3 and processed for light microscopic examination and transmission electron microscopy (TEM) or scanning electron microscopy.
Light microscopy and electron microscopy
Hematoxylin-eosin (HE) stained sections of the lung tissues treated with the 4 ENM suspensions were used to observe lung inflammation and localization of the nanomaterials. For transmission electron microscopic (TEM) observation of TiO2, SiO2 and C60, paraffin blocks were deparaffinized and small pieces of the lung tissues were embedded in epon resin and processed for nanomaterial observation using the JEM-2100 transmission electron microscope (JEOL Co. Ltd, Tokyo, Japan). Since MWCNT-7 are hard to be cut by the electronic microtome, scanning electron microscopy (SEM) was used to observe the MWCNT-7-treated lung tissues. Briefly, the HE-stained slides of the lung tissues were immersed in xylene for 3 days to remove the cover glass, immersed in 100% ethanol for 10 min to remove the xylene, and then air-dried for 2 hours at room temperature. The slides were then coated with platinum for observation using Model S-4700 Field Emission SEM (Hitachi High Technologies Corporation, Tokyo, Japan) at 5–10 kV.
Preparation of BALF and isolation of rat Alveolar Macrophages
Eight weeks-old female wild-type SD rats were sacrificed under anesthesia with intraperitoneally injected sodium pentobarbital, the lung was excised under aseptic conditions and injected with 5 ml of saline through the trachea. After gently shaking the lung, the fluid in the lung was taken out. Repeat the washing steps for another 2 times. The collected fluid was centrifuged at 1800g for 5min at 4℃, and the supernatant was BALF, concentrated with a concentrator tube (Millipore), and then stored at -80℃ for later use.
The cell pellet was resuspended in RPMI 1640 medium containing 10% fetal bovine serum (Gibco, USA), seeded in a six-well plate and cultured at 37℃ for 90 minutes. The cells were washed with PBS three times to remove red blood cells, other cells and cell debris, and the remaining adherent cells were stained with immunofluorescence for CD68, a macrophage marker, to confirm their identity. Briefly, the adherent cells were fixed in 4% paraformaldehyde and treated with 0.2% Triton X-100 containing 10% fetal bovine serum (Gibco)/1% bovine serum albumin in PBS at room temperature for 15 minutes, and then incubated with rabbit anti-CD68 (1:50 dilution, Bioss, Beijing, China) overnight at 4°C and added with Cy3 labelled anti-rabbit IgG (1:100 dilution, Proteintech, Wuhan, China). After washing, the cells were counter-stanning with DAPI (Sigma-Aldrich). Images were captured with a florescence microscope (ZEISS LSM880 + Airyscan, Germany). As shown in Figure S4, more than 95% of the adherent cells were positive for CD68. About 5×105-106 alveolar macrophages per rat were isolated.
Binding of nanomaterials to BALF proteins, SDS-PAGE and LC-MS
1.6 ml of the four ENM suspensions (equal to 800 µg of each ENM) were incubated with 1ml of the 20-fold concentrated rat BALF at 37℃ in a shaker at 200 rpm for 4–6 hours, and then centrifuged at 20000×g for 30 minutes to separate the nanomaterials from the supernatants. The precipitates were re-suspended in PBS, and the suspensions were centrifuged 20000×g for 10 minutes and the supernatants were discarded. The washing steps were repeated for another 2 times. The final ENM precipitates with their bound proteins were resuspended in 50 µl of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer (10% glycerol, 62.5 mM Tris-HCl [pH 6.8], 2% 2-mercaptoethanol, 2% SDS), heated for 5 minutes at 95℃, and then centrifuged at 20000×g for 30 minutes to dissociate the bound proteins from the nanomaterial precipitates.
After protein quantification with BCA (BestBio, Shanghai, China), 10 µl of the final suspensions was subjected for SDS-PAGE and the gels was silver-stained with a silver staining kit (BestBio). 20 µl of the remaining SDS-PAGE buffer-dissociated protein solution was added DTT to a final concentration of 100 mM and boiled 5 minutes. The samples were subjected for Liquid Chromatography Method with Tandem Mass Spectrometry (LC-MS) analysis (Shanghai Applied Protein Technology Company, Ltd, Shanghai, China).
Exposure of ENMs to primary alveolar macrophages in vitro
1×106 rat primary alveolar macrophages (PAMs) were cultured at 37℃ overnight in a 6-well plate in RPMI 1640 culture medium containing 10% fetal bovine serum (Gibco), washed with PBS for 3 times, further cultured in the X-VIVO™ serum-free medium (Lonza, Belgium) containing 1 µg/ml of recombinant human SP-A or SP-D, or 10% concentrated BALF for 1 hours, and the cells were treated with 1 µg/ml MWCNT-7, TiO2, SiO2, or C60 and cultured for 12 hours. The cells were harvested for RNA isolation and quantified polymerase chain reaction (qPCR) analysis of cytokine expression, and the culture supernatants were collected for ELISA.
qPCR and ELISA analyses of IL-1β, IL-6 and TNF-α production
Total RNA in rat PAMs was extracted with Trizol (Magen, Guangzhou, China) according to the manufacturer’s instruction. 500 ng of the RNA samples was reverse transcribed using the HiScriptⅡRT SuperMix kit (Vazyme Biotech, Nanjing, China), and qPCR analyses of IL-1β, IL-6 and TNF-α were then performed using the AceQqPCR SYBR Green Master Mix (Vazyme). GAPDH was used as an internal reference, and the relative expression of each gene was analyzed by 2 −△△CT method. The detailed sequences of the primer pairs (forward/backward) are described as follows: CAGCAGCATCTCGACAAGAG/CATCATCCCACGAGTCACAG for IL-1β; AGTT- GCCTTCTTGGGACTGA/TCCAAGATCTCCCTGAGAACA for IL-6; ACTCCCAGA- AAAGCAAGCAA/CGAGCAGGAATGAGAAGAGG for TNF-α; and GACATGCCG- CCTGGAGAAAC/AGCCCAGGATGCCCTTTAGT for GAPDH. ELISA detection of IL-1β, IL-6, and TNF-α in the supernatants of rat primary alveolar macrophage culture was performed with the rat ELISA kits (MLBio, Shanghai, China), as described by the manufacturer.
Analysis of ENM phagocytosis by polarizing microscopy
1×106 rat PAMs were seeded in a 6-cm culture dish pre-placed a circle microscope cover glass (NEST, China) and cultured in the X-VIVO™ serum-free medium (Lonza) containing 1 µg/ml of recombinant human SP-A or SP-D, or 10% concentrated BALF for 1 hours. The cells were treated with 1 µg/ml MWCNT-7, TiO2, SiO2, or C60 and continuously cultured for 12 hours. The cells in the cover glass were fixed in 4% paraformaldehyde and stained with HE. The stained cover glass was observed under the ECLIPSE polarizing microscope (LV100NPOL, Nikon, Japan). Total number of cells and number of the cells with brightening phagocytosed ENMs under dark background in each 20× sight field was counted, and 5 sight fields of each cover glass were randomly selected. Finally, phagocytosis rates were calculated by number of the cells with brightening phagocytosed ENMs/total number of cells.
Knockdown of LRP1, CD14 and SIRPα with siRNAs and its influence on cytokine production and phagocytosis
Small interfering RNAs (siRNA) were used to knock-down the expression of potential receptors of SP-A/D, including LDL receptor related protein 1 (LRP1), CD14 and signal regulatory protein alpha (SIRPα). 3 pairs of siRNAs for each of the genes were provided by GenePharma Co. Ltd., Shanghai, China, and 1 pair of siRNAs with the best silencing efficacy determined by preliminary experiments were chosen for further use. The siRNA sequences are as follow: GCUAAACUCGCUCAAUCUATT/UAGAUUGAGCGAGUU- UAGCTT for CD14; CCAUCAAACGGGCAUUCAUTT/AUGAAUGCCCGUUUGAU- GGTT for LRP1; and GCUCUAUGUACUCGCCAAATT/UUUGGCGAGUACAUAG- AGCTT for SIRPα.
Briefly, 1×106 rat PAMs were seeded in each well of a 6-well plate and cultured at 37℃ overnight. Negative control RNA or siRNAs for LRP1, CD14 and SIRPα were transfected into the cells using Lipofectamine 2000 (Thermo Fisher, USA). 6 hours later, the culture media were changed with the X-VIVO™ serum-free medium (Lonza) containing 1 µg/ml of recombinant human SP-A or SP-D, or 10% concentrated BALF, and the cells were treated with 1 µg/ml MWCNT-7, TiO2, SiO2, or C60 and continuously cultured for 12 hours. The cells were harvested for RNA isolation, qPCR analysis of silencing efficacy and cytokine expression, and western blotting; the culture supernatants were collected for ELISA.
The silencing efficacy for LRP1, CD14 and SIRPα was analyzed by qPCR, as described above, and confirmed by western blotting. The specific primers are CCAGGA- ACTTTGGCTTTGCTC/ACCGATGGACAACTTTCAGG for CD14; CCAATTGTGC- ATTTTTGCAG/GAATCAGGGGCATAGGTGAA for LRP1; and GTGTCTGTTGCT- GCTGGAGA/GCATCTTCTGGGGTGACATT for SIRPα. The expression of LRP1, CD14 and SIRPα at protein level was detected by western blotting. The cells were lysed in RIPA buffer (150mM NaCl, 50mM Tris pH 7.4, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and 1mM PMSF) for 5 minutes and centrifuged at 4℃, 12000 rpm for 20min. After protein quantification with BCA kit (Bestbio), aliquots of the supernatants (20 µg protein) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane (Millipore, Boston, USA). After blocking in 5% nonfat milk, the PVDF membranes were incubated with primary antibodies (anti-GAPDH from Peprotech, USA, 1:500 dilution, as an internal control; anti-CD14 from Bioss, Beijing, China, 1:500 dilution; anti-SIRPα from Cell Signaling Technology, Danvers, USA, 1:1000 dilution; anti-LRP1 from Abcam, USA, 1:10000 dilution) at 4℃ overnight. After three washings, the PVDF membrane was incubated with peroxidase conjugated anti-mouse or anti-rabbit secondary antibodies (1:10,000) for 60 minutes. The protein was visualized with ECL (Thermo Fisher) detection solution in the GEL Imaging System (Tanon, Shanghai, China).
Influence of knockdown of LRP1, CD14 or SIRPα on IL-1β, IL-6 and TNF-α production was analyzed by qPCR and ELISA, and its effect on ENMs phagocytosis was assessed by the polarized microscopy, as described above.
Statistical analysis was performed using SSPS17 software. The statistical significance was analyzed using two tailed Student’s t test. A p value of < 0.05 was considered to be significant.