Construction of recombinant IL-35 expressing E. coli/IL-35
pET-28a(+) vector was obtained from Cwbiotech, China. The sequence of IL-35 was a generous gift from Prof. Jiyu Ju (Weifang Medical University, Shandong). The IL-35 segment was gained using EcoI/XhoI double digestion and subcloned into pET-28a(+) vector linearized to construct pET-28a(+)-IL35 recombinant plasmid, which was transformed into E. coli strain BL21(DE3) to get E. coli/IL-35. The same strain carrying empty-load vector (pET-28a(+)) was used as E. coli/0. The expression of the exogenous protein was induced by the addition of 1 mM Isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h at 37 °C post cultured overnight in LB-medium. Then cells were harvested by centrifugation at 7000×g for 10 min at 4 °C, and the obtained pellets were assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to confirm the expression of IL-35 by anti-IL-12A (Abcam, China).
The entire experiment was approved by the Animal Care and Use Committee of Tianjin Medical University (China) according to the Chinese Council on Animal Care guidelines. The male SPF BALB/c mice (Aoyide Co., Tianjin, China) aged 6–8 weeks and weighing 18 ± 2 g. They were housed in comfortable cages at Animal Care Facility of Tianjin General Surgery Institute and allowed to acclimatize to standard lighting, and temperature conditions with food and water freely available before the experiment were performed.
The mice were randomly divided into four groups (n = 10 mice per group) as follows: normal group, DSS group, DSS + E. coli0 group, DSS + E. coli/IL-35 group. DSS-induced colitis model was established in mice according to Kihara et al. . Colitis was induced in 30 mice by free access to 3% DSS solution for 5 days followed by additional 5 days with drinking water without DSS. Mice in DSS group were given 0.2 mL PBS once a day through an oral-gastric tube during the last 5 days. In the DSS + E. coli/IL-35 group, mice were treated with 0.2 mL of PBS suspensions (1 × 1010 CFU/mouse/day) of E. coli/IL-35 induced with 1 mM IPTG for 4 h, while those were given the same amount of PBS containing E. coli0 as control in DSS + E. coli0 group. All mice were sacrificed by cervical dislocation and examined as described below after fast for 6 h.
Assessment of inflammation severity
The degree of inflammation in mice was comprehensively assessed by daily disease activity index (DAI) and length of the colon. Briefly, DAI was adopted based on the scoring system of Murthy et al. , which represents the sum of scores for weight loss, stool consistency and rectal bleeding divided by three. The colon (from the ileocecal junction to the anus) was collected and the length of it was measured. Then the colon was cutted longitudinally and washed by cold saline, and the colonic contents were removed.
Colon samples were cleaned with saline, fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 5 μm sections. Specimens were dewaxed, hydrated and stained with standard hematoxylin and eosin (H&E) to examine pathological changes in a blinded fashion. The colitis score was used to determine the extent of the inflammation based on a previously published grading system , which according to the criteria (a) inflammation severity: 0 (none), 1 (slight), 2 (moderate), 3 (severe); (b) depth of injury: 0 (none), 1 (mucosal), 2 (mucosal and submucosal), 3 (transmural); (c) cryp damage: 0 (none), 1 (basal 1/3 damage), 2 (basal 2/3 damage), 3 (crypt lost, only surface epithelium intact), 4 (entire crypt and epithelium lost); (d) percent involvement: 1 (1–25%), 2 (26–50%), 3 (51–75%), 4 (76–100%). All evaluations were performed by observers unaware of the treatment groups.
To quantitate the inflammatory cell infiltration, sections were stained with specific antibodies (Abcam, China). Immunohistochemical staining was performed by using anti-Ly6G and CD3 antibodies to detect intracolonic cellular infiltration of neutrophils and CD3+ T cells. Endogenous peroxides of colon specimens were blocked with 3% H2O2 followed by deparaffination and rehydration, and antigen retrieval was processed by heating in the microwave. The primary antibody was at a dilution of 1:100. Sections only incubated with secondary antibodies were used as negative control. After enclosed by 5% bovine serum albumin (BSA), the specimens were stained according to the instructions of Strept Avidin–Biotin Complex (SABC) kit. Stained sections were photographed using an Olympus inverted microscope (Olympus Imaging America, Center Valley, PA).
Fluorescence-activated cell sorting (FACS) analysis
FACS analysis was used, as previously described , to determine the subpopulation of CD4+CD25+Foxp3+Tregs and CD4+IL-17A+ Th17 in spleen and mesenteric lymph nodes (MLN). Briefly, splenic and MLN single-cell suspensions were prepared with the final concentration of 1 × 107/mL before immunofluorescent staining. After the lymphocytes were gated from the whole population, the CD4+ cells were gated by anti-mouse CD4 antibody for both Tregs and Th17 cells. Then, for the Tregs, the population of both positive staining of anti-mouse CD25 and intracellular Foxp3 antibodies in CD4+ cells were calculated. And for the Th17 cells, the population of intracellular IL-17A+ cells stained with anti-mouse IL-17A antibody were calculated. In addition, the isotype controls of both Tregs and Th17 cells had been done at the same time. All fluorescent-labeled antibodies were purchased from either eBioscience (eBioscience, San Diego, CA) or BioLegend (BioLegend, San Diego, CA).
Real-time PCR analysis of inflammation-related genes of colon
The transcriptional gene levels of IL-6, IL-10, IL-35 and IL-12 in colon tissues were determined by quantitative real-time PCR instrument. Total RNA was extracted from colon tissue using TRIzol reagent, and two μg RNA was reverse transcribed into cDNA with the QuantiTect Reverse Transcription Kit (Qiagen) using random hexamers. Quantitative real-time PCR was conducted using the TaqMan gene expression assay with a LightCycler 1.5. GAPDH served as an internal control. The sequences of primers used for analysis were designed as follows: GADPH, sense 5′-AGGTCGGTGTGAACGGATTTG-3′, antisense 5′-TGTAGACCATGTAGTTGAGGTCA-3′; IL-12a, sense 5′-GACCTGGACCCTGAGATTGTGAA-3′, antisense 5′-GGTCCCTGTGCAGCACGTTA-3′; IL-10, sense 5′-AGAAGCATGGCCCAGAAATCA-3′, antisense 5′-GGCCTTGTAGACACCTTGGT -3′; IL-6, sense 5′-CCACTTCACAAGTCGGAGGCTTA-3′, antisense 5′-GCAAGTGCATCATCGTTGTTCATAC-3′; EBI3, 5′-GTTCTCCACGGTGCCCTAC-3′, antisense 5′-CGGCTTGATGATTCGCTC-3′; IL-12p40, sense 5′-CCTGTGACACGCCTGAAGAAGATG-3′, antisense 5′-CTTGTGGAGCAGCAGATGTGAGTG-3′.
Enzyme-linked immunosorbent assay (ELISA)
Serum was prepared and subjected to ELISA to determine levels of IL-10, IL-35 and IL-6 using ELISA kits (Biolgend, http://www.biolegend.com/) according to the protocol provided by the manufacturer. ELISA was performed in triplicate for each sample.
SPSS 17.0 (SPSS Inc., Chicago, USA) was used for the statistical analysis. The enumeration data were performed as mean ± standard deviation (SD). One-way ANOVA was used for multiple-group comparisons and with proper post hoc analysis. A significant difference was defined as p < 0.05.