2.1 Cell culture
Human iPS cells (DYR0100) were cultured under feeder-free culture conditions in chemically defined mTeSR™1 medium (Stem Cell Technologies, Inc., Vancouver, BC, Canada) on matrigel-coated (Corning, Inc., NY, USA) dishes; and maintained in our laboratory in the Department of Biochemistry and Molecular Biology, College of Life Science and Medicine, Zhejiang Sci-Tech University (Hangzhou, ZJ, China). The cells were cultured in 5% CO2 with 95% humidity at 37℃ in mTeSR™1 medium, supplemented with 10μM Rho-associated kinase (ROCK) inhibitor Y27632 (Stem Cell Technologies, Inc., Vancouver, BC, Canada). The hiPS were passaged when they reached 80‑90% con-fluency. For passage, the cells were incubated with Accutase™ (Stem Cell Technologies, Inc., Vancouver, BC, Canada) to a single-cell mass and replated on matrigel-coated dishes according to the appropriate split ratio. The medium was replaced daily. Human monocyte cell line THP-1 cells were provided by Dr. Caiyun Fu, Zhejiang Sci-Tech University and maintained in our laboratory in the Department of Biochemistry and Molecular Biology, College of Life Science and Medicine, Zhejiang Sci-Tech University (Hangzhou, ZJ, China). The THP-1 cells were cultured in 5% CO2 with 95% humidity at 37℃ in RPMI 1640 medium (Gibco, NY, USA), supplemented with 10% Fetal Bovine Serum (FBS, Gibco, NY, USA) and 1% Penicillin-Streptomycin (PSA, Gibco, NY, USA).
2.2 Macrophages differentiation from hiPS
hiPS-Mφ were generated by using a previously established protocol in our previous work50. The hiPS were gently digested with Accutase™ (StemCell Technologies, Inc., Vancouver, BC, Canada), and 2×106 cells were resuspended in Knockout-DMEM medium (KO-DMEM, Gibco, NY, USA) supplemented with 10% Knockout-serum replacement (KSR, Gibco, NY, USA), 1% Non-essential amino acids (NEAA, Gibco, NY, USA), 1 mM L-Glutamine (Sigma-Aldrich, St. Louis, MO, USA), 50 μM β-mercaptoethanol (β-ME, Solarbio, BJ, China) and 10 μM ROCK-inhibitor Y-27632 and then cultured on 6-well ultra-low attachment plates (Corning, SH, China) for 24 h. The medium was changed daily. After 8–11 days, EB were seeded onto gelatin-coated 24-well plates in DMEM medium (Gibco, NY, USA) supplemented with 10% FBS, 1 mM L-Glutamine, 50 μM β-ME, 50 ng/mL human macrophage colony-stimulating factor (M-CSF, PeproTech, Rocky Hill, NJ, USA), and 25ng/mL human IL-3 (Gibco, NY, USA) at a scale of 6–8 EB per well. The medium was changed every 3 days. Continuous monocyte production was cultured for 17–19 days. Non-adherent monocytes were collected every 4 days and other cells were continuously cultured, the adherent cells were further cultured for 2-3 weeks to collect suspended cells. Non-adherent monocytes were cultured in RPMI 1640 medium supplemented with 10% FBS, 100ng/mL M-CSF, 50 ng/mL interleukin (IL)-3, and 50 μM β-ME, and then used for identification experiments after 10 days.
2.3 Macrophages differentiation from THP-1
THP-1 cells were centrifuged at 1000 rpm for 5 min, the supernatant were discarded, and then THP-1 cells were resuspended in medium supplemented with 100 ng/mL PMA (Beyotime, SH, China) and incubated for 24 hours in 5% CO2 with 95% humidity at 37℃. After that, cells were cultured with the original medium and the medium was changed every two days.
2.4 Giemsa stain assay.
The slides were stained for 30 min with a working solution of Giemsa stain prepared from a commercially available stock solution (Beijing Solarbio Science & Technology Co., Ltd., BJ, China) according to recommendations of the manufacturer. The slides were then washed 2×1 min in ddH2O and air dried, then detected with microscope.
2.5 Phagocytosis assay. The medium of hiPS-Mφ and THP-1-Mφ was aspirated, and then washed cells once with PBS. The Indian ink was diluted to the medium at a ratio of 1:1000 and mixed, the reagent was added to the cell culture dish and incubated in 5% CO2 with 95% humidity at 37℃ for 1 h , and cells were observed under the microscope.
2.6 BCG infection
Fully dissolve BCG lyophilized powder with sterile physiological saline and the concentration of BCG strain after constant volume is 1×107 CFU/mL. hiPS-Mφ and THP-1-Mφ were infected with 10 MOI of BCG indicated condition priorto were harvested for analysis.
2.7 Preparation and analysis of transcriptome sequencing samples
After BCG-infected cells were cultured for 24 h, the cells were digested by 0.25% trypsin (Gibco, NY, USA) and harvested. The total RNA of the cells was extracted according to the Trizol product specification, and the purity and concentration of the RNA samples were determined by an Agilent 2100 Analyzer (USA). After the total RNA of the sample had passed the test, the sequencing library was established according to the standard procedure and was sequenced using Illumina Hiseq4000 with a sequencing read length of 150 bp (PE150). According to the standard transcriptome sequencing analysis process, sequencing data output statistics, gene expression level analysis, differential gene expression analysis, differential gene KEGG enrichment analysis and differential gene GO enrichment analysis were performed.
2.8 Scanning electron microscope
The sample was immersed in Gluta fixative at 4℃ overnight and then post-fixed for1-2 h in1% citric acid solution, dehydrated in ethanol. And the sample was dried using a Hitachi HCP-2 critical point dryer to reach the dry critical point. The above-prepared sample was observed by a scanning electron microscope (SU-8010 type, Hitachi), and photographed.
2.9 Transmission electron microscope
The sample was immersed in Gluta fixative at 4℃ overnight, post-fixed for 1-2 h in1% citric acid solution, dehydrated in ethanol and embedded with a mixture of Spurr embedding agent and acetone (V/V = 1/1).The sample was sliced using a LEICA EM UC7 ultrathin slicer with a thickness of 70-90 nm; the obtained sections were stained with lead citrate solution, uranyl acetate and 50% ethanol saturated solution for 5-10 min. Finally, the sections were observed and photographed in a transmission electron microscope (H-7650,Hitachi).
2.10 Real-time Quantitative PCR Detecting System (qPCR)
RNA was extracted by using TRIzolTM Reagent, and cDNA was obtained by reverse transcription using the PrimeScript RT Reagent Kit according to the instructions. The qPCR analysis was set up in duplicate with SYBR Premix Ex Taq (Takara Bio, Inc., BJ, China) and performed using the 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cDNA was used as a template for qPCR, using β-actin as the internal reference gene, the relative expression of mRNA was calculated by 2-ΔΔCT method, and the results were statistically plotted. The mRNA fluorescent quantitative PCR primer information is shown in Table3. All primers were synthesized by Bio-Bioengineering Co., Ltd (Shanghai, China) .
2.11 Laser confocal detection of autophagy protein expression
The Ad-mCherry-GFP-LC3B was purchased from Beyotime (Shanghai, China) .The cells were seeded at 1 × 105 cells/well into 24-well plates. The Ad-mCherry-GFP-LC3Bwas added at an MOI of 20, and we observed the expression of fluorescent proteins after 24 h and 48 h of infection by laser scanning confocal microscope. Red puncta represented autolysosome, and yellow puncta overlaid by green and red appearing in the images indicated autophagosomes formation. they were counted to determine the autophagy level.
2.12 Western blot analysis
The cells were homogenized in lysis buffer (IP Buffer, 10 μg/μL Aprotinin,10 μg/μL Leupeptin, 100 mM PMSF,200 mM Vanadate (pH 10.0)overnight at 4℃, the lysates were centrifuged at 13,000 × g for 30 min at 4℃, and the supernatants were collected.The soluble protein concentration was determined via the Quick Start Bradford Protein Assay kit. And then the cell extracts (20 μg) were separated by 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and transferred toa nitrocellulose membrane (EMD Millipore). The membrane was blocked by 5% fat-free dry milk in PBS with 0.1% Tween-20 and probed by antibodies against LC3B (Abcam),GABARAPL2 (Abcam), Cathepsin D (Abcam), RAB7B (Abcam), DDIT4 (Abcam), EXOC8 (Novus), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech), GAPDH(Proteintech), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech), mTOR (Cell signaling), p-mTOR (Cell signaling), Akt (Cell signaling), p-Akt (Cell signaling), Beclin1 (MBL BEIJING BIOTECH CO.,LTD) at a dilution of 1:1,000,followed by horseradish peroxidase (HRP)‑conjugated secondary anti-rabbit IgG-HRP antibodiesor anti-mouse IgG-HRP antibodies (1:1,000, Beyotime, Shanghai, China).The blots were then developed using enhanced chemiluminescence (ECL) (Amersham; GE Healthcare Life Sciences, Piscataway, NJ, USA)or the ECL Tanon 5500 system (Tanon Science and Technology Co., Ltd., Shanghai, China).
2.13 Statistical analysis
Statistical analysis was conducted with GraphPad Prism 7 software. One-way ANOVA with uncorrected Fisher’s least significant differences test was applied for the analysis of two independent variables. Comparison between two samples was done with the unpaired t test. More details are described in the figure legends. Mean values of three independent experiments are shown. Error bars are shown as mean ± SD. ns, non-significant,*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.