Antibodies and reagents
Double-antibody (Procell Life Science & Technology, Wuhan, China; PB180120); anti-CTSS (Abcam, Cambridge, UK; ab134157); anti-IRFs (Abcam, ab181553); anti-GSDMD (Abcam, ab219800); anti-trypsinogen (Abcam, ab166898); anti-CD11c (BD, Franklin Lakes, NJ, USA; 562454); anti-CD206 (Invitrogen, Carlsbad, CA, USA;17-2069-41); anti-CD63 (Invitrogen, 12-0639-41); anti-TSG101 (Invitrogen, MA1-23296); anti-ALIX (Abcam, ab225555); anti-caspase1 (Invitrogen, PA5-17570); anti-caspase-8 (Abcam, ab25901); anti-IL-1β (Abcam, ab2105); anti-ASC (Abcam, ab155970); anti-Actin (Abcam, ab8226).
Trypsase (Procell, PB180225); Ham’s F-12K medium (Procell, PM150910); high-sugar Dulbecco’s modified Eagle’s medium (DMEM; Procell, PM150210); Fetal bovine serum (FBS, Procell, 164210-500); polylysine (Procell, PB180523); phosphate-buffered saline (PBS, Procell, PB180327); DMSO (Sigma-Aldrich, St. Louis, MO, USA); CTSS ELISA kit (Abcam, ab155427); IL-1β, TNF-α，IL-10 ELISA kit (Sangon Biotech, Shanghai, China; D711047, D711114, D711045); Amylase, lipase ELISA kit (Sangon Biotech, D799323, D799801); Hematoxylin and eosin (HE) stain kit (Solarbio, Beijing, China; G1120-10); Trizol (Invitrogen); Tween-20 (Sinopharm, Beijing, China); Formaldehyde (Sinopharm); Lipofectamine™ RNAiMAX Transfection Reagent (13778075, Invitrogen); RT-RNA PCR Kit (Takara, Kyoto, Japan); Taqman Universal Master Mix II (Cat. No: 4440040, Thermo Fisher, Waltham, MA, USA); ExoQuick-TC (SBI, Palo Alto, CA, USA); T4 DNA ligase (Takara); Lipofectamine 3000 (Invitrogen).
Pancreatic acinar cells, AR42J, were purchased from the American type culture collection (ATCC, Rockville, MD, USA; ATCC CRL1492). Cells were maintained in Ham’s F-12K complete growth medium with 20% FBS, at 37°C with 5% CO2. The culture medium was renewed every 2–3 days. Murine macrophage cells were isolated from mice peritoneum. The mice (6–8 weeks old) were purchase from Shanghai Lab Animal Research Center (Shanghai, China). The cell isolation and culture steps were as follows. The mice were killed by cervical dislocation; 5–8 mL of high-sugar DMEM was aspirated with a sterile syringe, injected into the abdominal cavity, and gently massaged for 10 min. The abdominal cavity was opened; pale yellow abdominal fluid was aspirated with a syringe then placed in a centrifuge tube, and centrifuged at 400 × g for 10 min. The supernatant was discarded and the cellular precipitate was retained. Cells were re-suspended in complete culture medium with mouse abdominal macrophage and inoculated in a culture dish at 37°C, 5% CO2 at room temperature. The solution was changed after 24 h.
Peripheral blood was collected from 64 patients with HP at the Tenth People’s Hospital of Shanghai, China. Blood samples from AP patients (57) and normal (7). The blood was obtained without any pretreatment by using a vacuum blood tube. Then stored overnight at 2–4°C and centrifuged at 3,600 × g for 15 min, the supernatant was collected and placed in liquid nitrogen and the precipitation was discarded. All patients signed informed consent and the study was approved by the Institute Research Ethics Committee of the Tenth People’s Hospital of Shanghai. The study methodologies conformed to the standards set by the Declaration of Helsinki.
Animal model of hyperlipidemia pancreatitis
Twenty-four (6–8 weeks, 32±2 g) C57BL/6J mice were purchase from the Shanghai Lab Animal Research Center (Shanghai, China). The mice were randomly housed in steel cages in a pathogen-free room with a 12 h light/12 h dark cycle and fed with a standard diet. All animal studies followed the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care Committee of the Centers for Disease Control and Prevention (Shanghai, China). The study methodologies conformed to the standards set by the Declaration of Helsinki.
After 1–2 weeks of acclimation, the mice were randomly divided into 4 groups with six individuals per group. One group was chosen as a blank control (Normal) and the mice were fed a standard chow diet throughout the whole experiment. The other 3 groups were induced as hyperlipidemia rats by Poloxamer 407 (P-407, Pluronic® F-127, P2443, Sigma-Aldrich, USA,) administration. P-407 was dissolved in PBS (PH=7.4) and stored in refrigerator with 4℃ overnight. For long-term hyperlipidemia induction, 4 weeks consecutive dosing (300mg/kg, intraperitoneal injection) administration needed. Acute pancreatitis was induced after hyperlipidemia induction with injection of 5% taurocholic acid sodium (145-42-6, Solarbio, USA). Briefly, mice were fast 12h (free water) before anesthesia with 3% phenobarbital. Next, the abdomen was opened and taurocholic acid sodium was injected via pancreatic duct carefully; then closed abdomen and resuscitation with normal saline (40mL/kg) subcutaneous injection. After 48 hours, hyperlipidemia pancreatitis model was validated by amylase and triglyceride level in the serum. Afterwards, CTSS inhibitor, Z-FL-CHCHO (5 mg/kg; intraperitoneal injection; three times per day for one weeks), was used in one of these three hyperlipidemia pancreatitis groups; the other two groups were administrated with PBS or none. Finally, all the mice were sacrificed and the blood and pancreatic tissues were collected for RT-PCR, western blot, and pathologic analysis.
Construction of gene inhibition and overexpression vectors
For knockdown specific gene expression, shRNA sequences of target genes and the negative control shRNAs (scr) without sequence homology to mice genes were synthesized by GenePharma (Shanghai, China).
The CTSS expression plasmid was constructed as follows. Briefly, the restriction sites of XhoI and BamHI were inserted into the ends of the CTSS open reading frame based on primers designed from the cDNA sequences of CTSS stored in the NCBI database. The specific primers were upstream primer 5′-TAAGATGGCTGTTTTGGA TG and downstream primer 5′-TTCTTTTCCCAGATGAGACGC. Purified PCR products were ligated with a pMD18-T vector using T4 DNA ligase (Takara), and the constructed pMD18-T-CTSS plasmid was confirmed by sequencing. The pcDNA3.1 and pMD18-T-CTSS vectors were purified simultaneously with BamHI and XhoI and ligated with recombinant pcDNA3.1-CTSS to obtain the recombinant pcDNA3.1-CTSS. The DNA of pcDNA3.1-CTSS was confirmed by sequencing. Cells were transfected using Lipofectamine™ RNAiMAX Transfection Reagent (13778075, Invitrogen) following manufacturer’s protocols.
Enzyme-linked immunosorbent assay (ELISA)
Patients and mice plasma samples were assayed for CTSS, IL-1β, IL-10, and TNF-α levels using an ELISA kit, following the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA isolation was performed using Trizol reagent according to the manufacturer’s instructions and the One-Step SYBR PrimeScript PLUS RT-RNA PCR Kit (Takara) was selected for RT-PCR analysis. The TaqMan Universal Master Mix II (Thermo, USA) was used for cDNA generation in reverse transcription. Gene relative expression was normalized to β-actin using 2−∆∆Ct. The experiments were performed in triplicate. Primers used in the study can be found in Table 1.
Western blot analysis
Western blot analysis was performed using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The blots were incubated with primary antibodies overnight at 4°C. Following three washes, membranes were then incubated with secondary antibody overnight at 4°C. Signals were visualized with ECL and exposed using a ChemiDoc XRS imaging system (BioRad, Hercules, CA, USA).
Flow cytometry analysis
Flow cytometry was used for the detection of polarization in macrophage phenotypes. Macrophage stimulated by PA (500 μM) for 24 h, were washed with pre-cooling buffer (1% bovine serum albumin in PBS containing 0.01% NaN3, Thermo Fisher) and incubated with 10% mouse serum for 20 min. Then, the cells were incubated with the LIVE/DEAD Fixable Dead Cell Stain Kit (Thermo Fisher), CD206-APC (Invitrogen), and CD11C-PE (BD) at the manufacturer’s recommended dilution for 40 min at 4°C. The samples were then washed, centrifuged at 1,500 × g for 5 min and re-suspended in PBS, and then analyzed with a FACS Canto II system and BD FACS DIVA software (BD).
Dual-luciferase activity assay
PCR was performed to obtain the CTSS UTR target site. A luciferase reporter with CTSS UTR and a putative IRF5 binding site was established in a pMiRreport vector. Macrophage were co-transfected with a recombinant and mutant recombinant CTSS promoter with the IRF5 binding site using Lipofectamine 3000. Luciferase reporter assays were performed using a dual-luciferase assay system (Promega).
Electrophoretic mobility shift assay (EMSA)
EMSA was used to verify the interaction between IRF5 and the CTSS gene. The specific promoter fragments of CTSS containing the GCAAAC-box and mutated GCAAAC-box were synthesized as biotin end-labeled and unlabeled oligonucleotides. The unlabeled GCAAAC-box oligonucleotides served as a competitor. The anti-IRF5 antibody was used for supershift identification. The assay was performed using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher) according to the manufacturer’s instructions.
Isolation and identification of exosomes
ExoQuick-TC (SBI) and total exosomal isolation were performed according to the manufacturer's instructions. Briefly, the collected cell culture medium was centrifuged at 2000 × g for 30 min and the supernatant was collected. One-fifth of the ExoQuick-TC exosomal precipitate or half of the total exosomal isolate was added to the supernatant and the suspension was incubated overnight at 4°C. The suspensions were centrifuged at 1,500 × g for 30 min for ExoQuick-TC or at 10,000 × g for 60 min for total exo-some isolation. They were re-suspended in PBS. Prior to further experiments, exosomal marker proteins were identified. The exosomes were re-suspended in Trizol for RNA analyses; or in lysis buffer (8 M urea/2.5% SDS, 5 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 mM phenylmethylsulphonyl fluoride) for protein analyses or used for transmission electron microscopy.
Scanning electron microscopy
Fixed specimens were placed on a 400-mesh carbon/Formvar coated grid at optimal concentration and allowed to absorb into the formvar for 1 min. For immunogold staining, the raster was placed in blocking buffer for 1 h in the blocking/permeation step. Without rinsing, the raster was immediately added to the primary antibody dilution (1:300 anti-CD9 ab92726, Abcam and anti-GPC1 PIPA528055, Thermo Fisher) overnight at 4°C. As a control, some grids were not exposed to the primary antibody. The next day, the grids were washed with PBS and then floated in 10 nm gold particles (Aurion, Hatfield, PA, USA) ligated with appropriate secondary antibodies and incubated at room temperature for 2 h. They were then rinsed with PBS and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 min. The grids were rinsed in PBS and distilled water, dried, and stained with uranyl acetate. Samples were photographed with a Tecnai Bio Twin transmission electron microscope (FEI, Hillsboro, OR, USA) and images were taken with an AMT CCD camera (Advanced Microscopy Technology, Danvers, MA, USA).
Hematoxylin and eosin (HE) staining
The pancreas was dissected along the pancreatic duct and a 0.5 cm × 0.5 cm specimen was fixed in 4% paraformaldehyde solution. After embedding in paraffin, the samples were cut into 4-μm-thick sections and stained with HE. Then, the sections were observed under an optical microscope and photographed.
Pancreatic acinar cells were cultured in 24-well plates. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and then washed three times in PBS containing 0.1% Tween-20 to permeabilize cells. Cells were then blocked with 5% BSA for 30 min in PBS. Primary antibodies were incubated at 1:100 in PBS with 1% BSA and 0.05% Tween-20 at 4°C overnight. Secondary antibodies were incubated at room temperature for 2 h. Images were taken with a Zeiss Axiovert 40 CFL.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to stain the mice pancreas sections (Roche, Shanghai, China). Pancreatic acinar cells positive for TUNEL were counted in 20 fields with ×200 magnification.
Pancreatic acinar cell viability was tested using CCK8 assay according to the manufacturer’s instructions. After transfection and treatment, cells were incubated in six-well plates and 10 μL cell counting kit-8 solutions were added to each well. Cells were incubated for 2 h at 37°C, and absorbance was measured at 450 nm with a spectrophotometer.
To conduct immunohistochemical analyses, samples were blocked in normal goat serum with 5% BSA in TBS for 1 h at room temperature. The sections were incubated with primary antibody at a dilution of 1:400 overnight at 4 °C and then washed with PBS three times. After incubation with secondary antibodies (16 h), the sections were subjected to a DAB reaction. The sections were photographed using a digitalized microscope camera (Nikon, Tokyo, Japan).
Transcription site prediction
Sequences were submitted to the JASPAR database (http://jaspar.genereg.net/) for transcription site prediction.
The data are presented as the means ± standard error of the mean (SEM). Tukey’s test or Student’s t-test for unpaired results was used to evaluate the differences among more than three groups or between two groups, respectively. Differences were considered significant for values of p < 0.05.