Patients and tissue specimens
A total of 28 children patients with active IBD who were recruited randomly from the Department of Pediatrics at Ruijin Hospital and Ruijin Hospital North between March 2016 and May 2017. The children included 19 males and 9 females with ages between 1 and 16 years (mean age 8.3±3.25 years). There were 14 cases of ulcerative colitis and 14 cases of Crohn's disease. The diagnosis of IBD was confirmed by 3 physicians on the basis of clinical assessments, endoscopy, pathology, and serology; in addition, the diagnosis was not changed after at least 6 months of follow-up. One block of involved intestinal tissues was collected from each patient during the endoscopic biopsy. Four children who had abdominal pain symptoms but did not have obvious inflammatory changes in the endoscopic and pathological examinations were used as the normal control group, and their intestinal mucosal biopsy tissues were collected. In addition, whole blood specimens were collected from 5 Hp-positive pediatric patients with chronic gastritis confirmed by our department (confirmed by the 13C breath tests, endoscopic examination, and pathology and without other diseases) and 3 healthy volunteers. The detailed features of gastritis patients and healthy volunteers chosen for exosome extraction are presented in Table 1.The blood samples were centrifuged at 3000 × g for 10 min to obtain serum samples and then were stored at -80°C.
Correlation analyses between NLRP12 expression and disease activity
The disease activity of the 28 IBD pediatric patients was evaluated. The Crohn's disease scoring system referenced the Pediatric Crohn’s Disease Activity Index (PCDAI) to score the disease history, laboratory results, changes in body height, and physical examination(14). The reference standard for ulcerative colitis was the Pediatric Ulcerative Colitis Activity Index (PUCAI), and the disease activity scoring was performed based on abdominal pain and stool properties and frequencies (15). The correlation between NLRP12 expression in the intestinal mucosa and the disease activity of IBD pediatric patients was analyzed.
Immunohistochemical detection of NLRP12 expression in intestinal mucosal tissues
The detection procedure was performed using a streptavidin-peroxidase (SP) reagent kit according to the manufacturer’s instructions. The NLRP12 antibody was purchased from Sigma-Aldrich. Result determination: NLRP12-positive cells showed a yellow-brown color in the intestinal epithelial cells or cytoplasm. The blind method was adopted. Five high-power fields in each section were selected under a light microscope to count the percentages of positive cells in each field. The average value was calculated. The classification was performed according to the staining intensity and the number of positive cells. Scoring according to the staining intensity was performed as follows: a light color slightly higher than the background color was 1 point, moderate staining that was significantly higher than the background color was 2 points, and strong staining that was close to a brown color was 3 points. Scoring according to the number of positive cells was performed as follows: a positive cell proportion <10% was 0 points, 10%-30% was 1 point, 31%-50% was 2 points, 51%-75% was 3 points, and >75% was 4 points. A score >4 points obtained via multiplication between these two scores indicated that the patient had positive expression.
Extraction and identification of exosomes
Serum exosomes were extracted using the ExoQuickTM reagent kit (SBI, Palo Alto, CA, USA). The manipulation was performed according to the reagent kit manual. Briefly, 200 μl of serum was mixed with 50 μl of ExoQuick solution and incubated at 4°C overnight. The mixtures were centrifuged at 10 000 g for 30 min at 4°C, and the exosome pellets were resuspended in diluent C (Sigma), resuspended in phosphate-buffered saline (PBS), or lysed immediately for the next experiment. Particle size was measured using a NanoSight LM10 HS-BF instrument (NanoSight Ltd, Salisbury, UK) on the basis of nanoparticle-tracking analysis (NTA). Brieﬂy, exosome pellets resuspended in PBS were diluted to a concentration of 3 μg/μL after protein quantiﬁcation, and then, the exosome suspensions were further diluted 100-fold and analyzed following the manufacturer’s protocol. Transmission electron microscopy (TEM) was performed to detect exosome morphology. Briefly, exosomes suspended in 2% glutaraldehyde were loaded on a copper grid and negatively stained with 3% (w/v) aqueous phosphotungstic acid for 1 min. The grid was then examined using an FEI Tecnai G2 Sprit Twin transmission electron microscope (JEM-1230; Jeol Ltd., Tokyo, Japan). The surface protein markers of exosomes, including CD63 and TSG101, were detected using western blotting. The obtained exosomes were named Exo(Con) and Exo(Hp) to present serum-derived exosomes from healthy volunteers and Hp-positive pediatric patients with chronic gastritis, respectively.
The human intestinal epithelial cell line NCM460 was purchased from INCELL (USA) and cultured in McCoy's 5A culture medium containing 10% fetal bovine serum (FBS), 100 mg/mL penicillin, and 50 μg/mL streptomycin (Gibco, USA) in a 37°C and 5% CO2 cell incubator. The culture medium was replaced with fresh culture medium every 2-3 days. Cells at the logarithmic growth phase were collected, and exosomes were added based on the requirements of the experiment. After siRNA transfection (siRNAs targeting NLRP12 were constructed and synthesized by Genomeditech Shanghai, and transfected into cells using Lipofectamine 2000) and DAPT (N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester) (Sigma-Aldrich, USA) stimulation, the cells were collected for detection.
NCM460 cell and exosome protein samples were obtained using RIPA lysis buffer. The concentrations of samples were determined using the bicinchoninic acid (BCA) method. Polyacrylamide gels were prepared, and samples were separated using electrophoresis. Proteins were transferred onto a membrane and blocked. The primary antibody was added and incubated at 4°C overnight. Then, the membrane was incubated with the secondary antibody at room temperature for 1 h. The blots were exposed to X-ray films and photographed. The grey density value of the target band was analyzed using the Image J system to reflect the protein expression level. The experiment was repeated 3 times. The following primary antibodies were used: NLRP12 (1:1000, Sigma-Aldrich, USA), anti-CD63 (1:1000, Abcam, USA), TSG101 (1:1000, Abcam, USA), calnexin (1:1000, Sigma-Aldrich, USA), GADPH (1:1000, Sangon Biotech, China), NLRP3 (1:1000, Sigma-Aldrich, USA), pro-caspase-1 (1:1000, Abcam, USA), Notch1 (anti-Notch1 intracellular domain, NICD,1:1000, Abcam, USA), HES-1 (1:500, OriGene Technologies, USA), HES-5 (1:1000, Abcam, USA), HEY-1 (1:1000, Proteintech Group, USA), and MCP-1 (1:1000, Abcam, USA).
Real-time quantitative PCR (qRT-PCR)
Total RNA was extracted from the NCM460 cells and mouse colon tissues using TRIzol (Invitrogen). The extracted total RNA (2 µg) was reverse transcribed into cDNA using a PrimeScript RT MasterMix reagent kit (TaKaRa). Next, qRT-PCR was performed using a SYBR Green Supermix reagent kit (TaKaRa) in a LightCycler 480 system (Roche). The Ct value was obtained from the PCR reaction curve to calculate the relative expression level. GADPH was used as the internal control. ΔCt was the difference in expression between the target gene and the internal control gene. The expression levels of the genes were expressed using 2-ΔCt. All experiments were repeated at least 3 times. The primer sequences are shown in Table 2.
Enzyme-linked immunosorbent assay (ELISA)
The cell culture supernatant was collected from NCM460 cells transfected with the NLRP12 siRNA. The IL-1β secretion level was detected using an ELISA kit(Anogen, Missassauga, ON,Canada), and fecal Lipocalin-2 (LCN-2) levels were also quantified using mouse Lipocalin-2 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. After dilution of the standards and the sample loading, incubation, and washing steps, the absorbance value of each well was measured at a wavelength of 450 nm using a Softmax Pro5 microplate reader. In addition, one blank control was used.
Establishment of a colitis model and exosome intervention
A dextran sulfate sodium salt (DSS, molecular mass 36-50 kDa, MP Biomedicals, France)-induced mouse colitis model was established. All mice were normally housed for 1 week before the experiments to observe changes in eating, drinking, activities, and body weight. A total of 28 C57BL/6 mice were randomly divided into 4 groups (n=7 mice/group) as follows: normal control group (Con group), model group (DSS group), Exo(Con) intervention group (DSS+Exo(Con) group), and Exo(Hp) intervention group (DSS+Exo(Hp) group). For 9 d, the control group received normal saline for drinking and the model group received a 5% DSS solution for drinking. Based on their consumption of the 5% DSS solution, the intervention group received an intraperitoneal injection of exosomes (50 µg/animal) at model establishment and every 24 h. During model establishment, changes in body weight, stool properties, and blood in the stools of the mice were observed every day. Disease activity index (DAI) scoring was performed based on the above indicators. For fecal sample collection, mice were placed in clean polycarbonate cages without bedding until they defecated.Fecal samples were reconstituted in PBS containing 0.1% Tween 20 at a concentration of 100 mg feces/ ml and vortexed for 30 min to yield a homogenous suspension. Mice were sacrificed 9 d after model establishment. The mouse colons and spleens were collected to evaluate the colon lengths and spleen sizes. Colon tissues at approximately 2 cm from the ileocecum were isolated and fixed in 4% formalin for histopathological examinations and scoring. In addition, some tissues were cryopreserved at -80°C for subsequent RNA extraction and qRT-PCR detection.
Detection of changes in the expression of inflammasome-associated genes, signaling pathways, and inflammatory cytokines using PCR array and antibody array technology
After exosome intervention in NCM460 cells for 24 h, cell supernatants and cell samples were collected. Total RNA was extracted from the cell samples using TRIzol and reverse transcribed into cDNA. Changes in inflammasome-associated genes and signaling pathways were detected using PCR array technology. The PCR ARRAY of Human Inflammasomes and the Human Signal Transduction Pathway Finder PCR Array were purchased from BioTNT (Shanghai, China). In addition, changes in inflammatory cytokines in the cell supernatants were detected using inflammatory cytokine antibody array technology. The Human Inflammation Array was purchased from Raybiotech.
Measurement data are all expressed as the mean±SEM. Data input and plotting were performed using GraphPad Prism 5.0 or EXCEL 2007. The statistical significance of the differences was calculated by performing Student’s t-test or one-way analysis of variance (anova). The enumeration data were evaluated by performing χ2 test or Fisher’s exact test as appropriate. P<0.05 was the standard for the statistical examinations. P<0.05 indicates significant differences, and P>0.05 indicates without significant differences.