SiO2, which has a diameter of approximately 2-5 μm, was purchased from Sigma (S5631). The silica was sterilized overnight (200 °C for 16 h)41 and then dissolved in sterile normal saline (NS) at a concentration of 5 mg/ml. The dose of SiO2 used in vivo and in vitro was based on previous studies.23 Antibodies against α-SMA (14395-1-AP, rabbit), CCR2 (16153-1-AP, rabbit), BECN (11306-1-AP, rabbit), ATG5 (60061-1-lg, mouse) and LC3 (14600-1-AP, rabbit) were obtained from ProteinTech, Inc. Antibodies against collagen I (BS1530, rabbit) and GAPDH (MB001, mouse) were obtained from BioWorld, Inc.
C57BL/6 mice (6-8 weeks old) were obtained from Dr. Tao Cheng at Nanjing Medical University Laboratories (Nanjing, China). All animals were male and housed (4 per cage) in a temperature-controlled room (25 °C, 50% relative humidity) with a 12-h light/dark cycle. All animal procedures were performed in strict accordance with ARRIVE guidelines, and animal protocols were approved by the Institutional Animal Care and Use Committee of Southeast University.
Single cell sequencing
1. Sample collection
For the model group, mice with significant lesions on CT were included. Lesions were removed from the lungs of mice representing the NS-7d, SiO2-7d, NS-56d, and SiO2-56d groups and were used for single-cell sequencing. Each lung was removed in 2 min and quickly washed in precooled PBS 3 times.
2. Single-Cell RNA Sequencing
2.1 Cell capture and cDNA synthesis
Using a single-cell ‘5’ Library and Gel Bead Kit (10x Genomics, 1000169) and Chromium Single-Cell G Chip Kit (10x Genomics, 1000120), the cell suspension (300-600 living cells per microliter determined by Countstar) was loaded onto a Chromium single-cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. In short, single cells were suspended in PBS containing 0.04% BSA. Approximately 20,000 cells were added to each channel, and the target cell recovered was estimated to be approximately 10,000 cells. Captured cells were lysed, and the released RNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53 °C for 45 min, followed by 85 °C for 5 min, and hold at 4 °C. cDNA was generated and then amplified, and quality was assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing).
2.2 Single cell RNA-Seq library preparation
According to the manufacturer’s instructions, single-cell RNA-seq libraries were constructed using the Single Cell 5’ Library and Gel Bead Kit, Single Cell V(D)J Enrichment Kit, Human T Cell (1000005) and Single Cell V(D)J Enrichment Kit. The libraries were finally sequenced using an Illumina NovaSeq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with a paired-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).
3. Data preprocessing
3.1 Cell Ranger pipeline
Cell Ranger software (v.4.0.0) was obtained from the 10x Genomics website https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest. Pipeline coupled with mouse reference version mm10. Alignment, filtering, barcode counting, and UMI counting were performed with the Cell Ranger count module to generate a feature-barcode matrix and determine clusters. Dimensionality reduction was performed using PCA, and the first ten principal components were used to generate clusters by the K-means algorithm and graph-based algorithm.
3.2 DEGs identification and Enrichment Analysis
Differentially expressed genes were analyzed using sc-Seq with negative binomial models to estimate the false discovery rate (FDR). For each cluster, genes with adjusted log2-fold change >3 and P < 0.001 were considered significantly upregulated. GO enrichment and KEGG enrichment of cluster markers were performed using the R package clusterProfiler, using the top significantly upregulated genes of each cluster. The results were visualized using R package.
3.3 Cell Type Annotation
Cell type was annotated by Cell Marker.
Isolation of mouse primary monocytes
Cells were collected from mouse bone marrow, treated with ACK lysis buffer, and routinely maintained in RPMI (10% FBS, 1% penicillin/streptomycin) at 37 °C with 5% CO2. The suspension cells were transferred into new medium for one day as the experiment required.
The THP-1 cell line was purchased from ATCC®, routinely maintained in RPMI (10% FBS, 1% penicillin/streptomycin) and incubated at 37 °C and 5% CO2. HPF-a cells were purchased from ATCC®, routinely maintained in DMEM (10% FBS, 1% penicillin/streptomycin) and incubated at 37 °C and 5% CO2.
Western blot analysis
Cells were collected in polyethylene tubes and briefly washed with ice-cold phosphate-buffered saline (PBS) twice before being lysed. The protein concentrations of the lysates were measured with a bicinchoninic acid (BCA) kit (Beyotime, China), and 30 μg of total protein was resolved via SDS-PAGE. Then, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h and then incubated overnight (16 h) with primary antibodies against Col1A1, α-SMA, CHOP, BIP, BECN, ATG5 and LC3B (1:1000). After being washed with TBST, the membrane was incubated for 1 h with secondary antibodies conjugated to horseradish peroxidase. Protein bands were visualized using a chemiluminescence detection system. All Western blots are representative of three or more independent experiments. The protein bands were quantified using ImageJ 1.52v software.
Cell viability assay
Cell viability was measured using CCK-8 assays. Briefly, the cells were seeded in 96-well plates at a density of 1 × 105 cells/well (for THP-1 cells) or 5 × 104 cells/well (for HPF-a cells). The cells were treated with conditioned media for 24 h (for HPF-a cells) or with 3-MA or rapamycin for 24 h (for THP-1 cells). The cells were then exposed to CCK-8 solution (10 μL), and the plates were incubated for an additional 30 min to 4 h. The absorption values were measured at 450 nm.
Cells were fixed with 4% paraformaldehyde in PBS on ice for 2 h. The fixed samples were permeabilized for 30 min at room temperature (RT) in PBS containing 0.3% Triton X-100 (PBST) and then blocked with 10% normal goat serum (NGS; Life Technologies) in PBST at RT for 2 h. The blocked samples were incubated for 4 h on ice with primary antibodies diluted in PBST plus 10% NGS. The samples were then washed three times with PBS and incubated with donkey anti-rabbit (conjugated to Alexa Fluor® 488) and donkey anti-mouse (conjugated to Alexa Fluor® 576) secondary antibodies for 2 h at RT. After being washed three times in PBS, the samples were mounted with Prolong® Gold antifade reagent containing DAPI, and the slides were examined using a fluorescence microscope.
Detection of autophagic flux
THP-1 cells were seeded in 6-well plates and transfected with mRFP-green fluorescent protein (GFP)-LC3 adenoviral vectors according to the manufacturer’s instructions (HanbioInc, Shanghai, CN, USA). Successfully transfected cells expressed LC3 protein tagged with RFP and GFP. GFP is acid-sensitive, and the green fluorescence is quenched in the acidic environment of a lysosome. However, in contrast, RFP is relatively stable within lysosomes. Therefore, the numbers of GFP and RFP puncta were examined and quantified by confocal microscopy. The red and yellow (i.e., a combination of red and green) spots indicate autophagosomes and autolysosomes, respectively.42
HPF-a cells were treated with conditioned media, IL-8 or IL-10 for 48 h in 24-well plates. To assess fibroblast motility, a scratch assay was performed as previously described.43
Total RNA was isolated from cells and subjected to reverse transcription using a Prime Script RT master mix kit (TaKaRa, RR036). Real-time PCR was performed by a StepOneTM Real-Time PCR System (Life Technologies, 4376357, Singapore) using primers for human IL-10 (forward primer: 5’-GTGATGCCCCAAGCTGAGA-3’; reverse primer: 5’-CACGGCCTTGCTCTTGTTT -3’) and human IL-8 (forward primer: 5’-CTGATTTCTGCAGCTCTGTG-3’; reverse primer: 5’-GGGTGGAAAGGTTTGGAGTATG-3’).
Twelve inflammatory cytokines were analyzed by Human Inflammatory Cytokines Multi-Analyte ELISArray™ Kits (QIAGEN, MEH-004A). Human IL-10 and human IL-8 ELISA kits were purchased from JinYiBai® (Nanjing, China). All cytokines were measured according to the manufacturer’s protocol.
The data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc.). Unpaired numerical data were analyzed by unpaired Student’s t-tests (2 groups) or ANOVA (2 groups). The level of significance was set at 0.05; values of P<0.05 indicated statistical significance.