The DEP powder was purchased from the National Institute of Standards and Technology (SRM 2975). To obtain the water–soluble (WS–DEP) and –insoluble (WIS–DEP) fractions at a concentration equivalent to that of DEP, the DEP powder was suspended in sterile phosphate–buffered saline (PBS, Fisher scientific) at a concentration of 2 mg/ml, vortexed for 30 min, and then centrifuged for 30 min at 12,000 g. The supernatant was transferred to a new vial and adjusted to the same volume as the initial suspension using sterile PBS. The pellet was also suspended in the same volume as the initial suspension using sterile PBS.
Male C57Bl/6J mice (3‐week‐old) were purchased from the Animal Center of Shanghai Medical School, Fudan University. After 1 week of acclimation, 10/group mice were randomized into four groups and intratracheally instilled with PBS, DEP, WS–DEP, or WIS–DEP, 3 times per week (Monday, Wednesday, and Friday), and for 15 weeks in total. To minimize the aggregation of particles, suspensions were sonicated (Clifton Ultrasonic Bath) for 20 min and vortexed for 30 s before each instillation. Instillation was performed as described previously.(12) In brief, mice were anesthetized with 3% isoflurane, and placed supine with extended neck on an angled board. An 18-gauge cannula (Becton Dickinson) was inserted into the trachea. Suspensions (50 μl PBS, WS–DEP, WIS–DEP, or DEP) were then intratracheally administrated via a sterile syringe, followed by an air bolus of 150 μl. The incubation catheter was removed, and the mouse was transferred to a vertical hanging position with the head up for 5 min, to make sure that the delivered materials were maintained in the lung. During the whole experimental period, all mice were housed in the animal facilities of Fudan University, which maintained a 12 h light/12 h dark cycle, a room temperature of 20–25 °C, and a relative humidity of 40–70%. All animal–related procedures were approved by the institutional animal care and use committees of Fudan University, and all the animals were treated humanely and with regard for alleviation of suffering.
Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT)
IPGTT was performed after 12 weeks of instillation. Briefly, after a 16–hour fasting, the basal blood glucose levels of mice were determined using an automatic glucometer (Glucotrend 2, Roche Diagnostics). The mice were then intraperitoneally injected with glucose (2 g/kg of body weight) and the blood glucose levels at 15, 30, 60, and 120 min after injection of glucose were measured as described above. ITT was performed after 13 weeks of instillation. Briefly, after a 4–hour fasting, the basal blood glucose levels of mice were determined using an automatic glucometer (Glucotrend 2, Roche Diagnostics). The mice were then intraperitoneally injected with insulin (0.5 U/kg of body weight), and the blood glucose levels at 15, 30, 60, and 120 min after injection of insulin were measured as described above.
Fecal sample collection, DNA extraction and sequencing
After 14 weeks of instillation, mice were transferred to clean cage without any bedding (individually housed) and allowed to defecate normally. 24–h fecal pellets of each mouse were transferred to sterile Eppendorf tubes and stored at –80 °C until ready to extract DNA. The total genomic DNA of fecal sample was extracted using a MoBio PowerFecal DNA extraction kit (Qiagen) per the manufacturer’s instructions. In brief, samples were firstly homogenized in a tube with garnet beads. The lysis of microbial cells and host cells was facilitated by both chemical disruption of cell membranes and mechanical collisions between beads to ensure efficient extraction. The extracted genomic DNA was captured in a silica spin column and eluted out with elution buffer (50 μl). To generate the sequencing libraries, the concentration and quality of extracted DNA was assessed using a NanoDrop 1000 spectrophotometer (Thermo Scientific) and agarose gel electrophoresis (1% wt/vol agarose in tris–acetate–EDTA buffer) respectively. The bacterial 16s rRNA V4 region was amplified by PCR using the barcoded primers: 515F (5’–GTG CCA GCM GCC GCG G–3’) and 907R (5’–CCG TCA ATT CMT TTR AGT TT–3’). Thermal cycling was performed using a 9700 PCR system (ABI, GeneAmp 9700), and all the PCR products were subjected to agarose gel electrophoresis (2%) followed by purification using the AXYGEN gel extraction kit (Axygen). The purified amplicons were quantified using the Quant–iT PicoGreen dsDNA Assay kit (Thermo Fisher) and QuantiFluorTM–ST Blue–florescence quantitative system (Promega), and then sequenced using the Illumina MiSeq system (Illumina) per the manufacturer’s instructions.
Bioinformatics analysis and bacterial diversity and richness analysis
The primary sequencing data were saved in Fastq format at SRA (Sequence Archive, http://www.ncbi.nlm.nih.gov/Traces/sra). All pyrosequencing reads were pre-processed based on the barcode and primer–end readers using the Usearch software (http://drive5.com/uparse/). All recruited reads had the barcode, a minimal average quality score of 20, and a maximum of 2 mismatches within the primers. All the overlapped reads with a minimal overlap of 10 bps with a mismatched rate ≤ 0.2 were merged. The repetitive sequences were extracted and discarded (http://drive5.com/usearch/manual/singletons.html) to avoid unnecessary computations. The optimized sequences were clustered into operational taxonomic unit (OTU) with UCLUST followed by de novo OTU picking. The chimeras were removed using RDP gold database of Usearch software (http://drive5.com/uparse/). The bacterial taxonomy was assigned via the Naïve Bayesian classifier in QIIME platform using the SILVA database (Release 119, http://www.arbsilva.de).
The α-diversity estimators including ACE, CHAO-1, Shannon, and Simpson were obtained using matrices of Mothur (www.mothur.org/wiki/ Schloss_SOP#Alpha_diversity). ACE and Chao–1 are quantitative indicators of the richness of a sample, and Shannon and Simpson are quantitative indicators of biodiversity (a smaller Shannon estimator or a larger Simpson estimator indicates a lower community diversity). The β-diversity of bacterial community was assessed by UniFrac distance analysis, principal component analysis (PCA), and principal co–ordinates analysis (PCOA) using the relative abundance of OTU. The linear discriminant effect size (LEfSe) analysis was performed to find OTUs differentially represented in-between the experimental groups: a nonparametric factorial Kruskal–Wallis sum–rank test was used to detect significantly (p< 0.05) differential taxa, and the identified taxa were subjected to a linear discriminant analysis (LDA) to evaluate the effect size of each single differential taxon.
Mouse euthanasia and tissue harvesting
Mice were fasted overnight and assessed for body weight and head-to-tail length on the experiment day. In 20 min after i.p. injection of insulin (10 U/kg), the mice were euthanized, and the blood was harvested from the orbital venous plexus. The mouse trachea was then cannulated, and the right primary bronchus was closed off with a ligation. 0.5 mL of sterile PBS with 0.1 mM EDTA was instilled through the tracheal cannula and withdrawn to recover the bronchoalveolar lavage fluid (BALF). This process was repeated for three times. The cell number in BALF was determined using a hemocytometer and the protein in BALF was quantified by the bicinchoninic acid assay (BCA, Biyotime) per the manufacturer’s instruction. The ligation of right primary bronchus was removed after harvesting BALF and the right lung was ligated in the middle. The lower half was fixed using 4% paraformaldehyde through the trachea and the upper half was snap–frozen in liquid nitrogen and stored at –80 °C. Other tissues including brain, heart, liver, kidney, pancreas, spleen, testis, epididymis, skeletal muscle, perirenal adipose tissue, epididymal adipose tissue, subcutaneous adipose tissue, and brown adipose tissue were harvested, weighted, and fixed in 4% paraformaldehyde for morphological analysis and/or snap–frozen in liquid nitrogen and then stored at –80 °C for further processing.
Levels of aspartate transaminase (AST, Mouse AST Elisa Kit, JianCheng Bioengineering Institute, Nanjing, China), alanine transaminase (ALT, Mouse ALT Elisa Kit, JianCheng Bioengineering Institute, Nanjing, China), total triglycerides (TG, Mouse TG Elisa Kit, JianCheng Bioengineering Institute, Nanjing, China), total cholesterol (Mouse TC Elisa Kit, JianCheng Bioengineering Institute, Nanjing, China), low density lipoprotein (LDL) cholesterol (Mouse LDL–C Elisa Kit, JianCheng Bioengineering Institute, Nanjing, China), and high density lipoprotein (HDL) cholesterol (Mouse HDL–C Elisa Kit, JianCheng Bioengineering Institute, Nanjing, China) in serum or homogenized liver tissue were determined per manufacturer’s instructions.
Quantitative real–time RT–PCR (qPCR)
Total RNA was isolated from frozen lung tissues with Invitrogen™ TRIzol™ reagent (Invitrogen) per the manufacturer’s instruction. The quality and concentration of RNA was determined using a nanodrop spectrophotometer (ThermoFisher Scientific). 2 μg of DNase–treated RNA was then reverse–transcribed to cDNA using a High–Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Real–time RT–PCR was performed using SYBR qPCR Master Mix (Vazyme) with a 7500 Real–time PCR system (Applied Biosystems). The sequences of PCR primers were as follows: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5′–TGA ACG GGA AGC TCA CTG G–3′ and 5’–TCC ACC ACC CTG TTG CTG TA–3’; Interleukin (IL)–1β: 5’–ACG GAC CCC AAA AGA TGA AG–3’ and 5’–TTC TCC ACA GCC ACA ATG AG–3’; IL–6: 5’–ATC CAG TTG CCT TCT TGG GAC TGA–3’ and 5’–TAA GCC TCC GAC TTG TGA AGT GGT–3’; Tumor necrosis factor alpha (TNFα) 5’–TTC CGA ATT CAC TGG AGC CTC GAA–3’ and 5’–TGC ACC TCA GGG AAG AAT CTG GAA–3’. 2ΔCt was calculated as the relative expression level of detected genes as previously described.(13)
The fixed lung tissue was embedded in paraffin, cut into 5-μm sections. 6 consecutive lung sections/mouse were subjected to H & E staining and images covering all lung tissues were taken and analyzed by a pathologist who was blind to the grouping. The pulmonary inflammation level was quantitated per the scoring system in Table 3. An aliquot of liver was fixed at 4 °C in 4% paraformaldehyde for 24 hours. These liver tissues were embedded in paraffine and cut into 5-μm sections. 6 consecutive sections/mouse were subjected to H & E staining or Masson’s trichrome staining and images covering all liver tissues were taken and analyzed by a pathologist who was blind to the grouping. Hepatic steatosis, hepatocyte ballooning, lobular and portal inflammation, Mallory bodies, and fibrosis were examined and scored according to the modified Brunt scoring system for non-alcoholic fatty liver disease. Another aliquot of liver tissue was frozen in optimal cutting temperature compound, cut into 4-μm sections, and subjected to Oil O red staining. Images covering the entire liver tissue area were taken and analyzed by a laboratory technician who was blind to the grouping. The collagen areas on Masson’s trichrome staining images and the lipid areas on Oil O red staining images were assessed using ImageJ.
Western blotting analysis
Liver tissue lysates were prepared using radioimmunoprecipitation assay buffer (Sigma) supplemented with phosphatase and protease inhibitors (Sigma) per the manufacturer’s instruction, subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and electroblotted onto polyvinylidene fluroride membranes. The Akt phosphorylation was detected using rabbit anti–phospho–Akt (Thr308) (Cell Signaling) and horseradish peroxidase-conjugated secondary antibodies (Amersham). After stripping using striping buffer (Thermo Scientific), total Akt levels were detected using rabbit anti–AKT (Santa Cruz). The protein bands were visualized with chemiluminescence reagent (Amersham) and quantitated as previously described.(14)
All data were expressed as mean ± SEM unless specified otherwise. Statistical tests were performed using one–way analysis of variance (ANOVA) with Bonferroni correction or unpaired t–test using GraphPad Prism (version 8.0.2; GraphPad Software). The significance level was set at *p<0.05.