2.1. PI-IBS ptients
From January 2018 to December 2019, thirty-five IBS patients including inpatients and outpatients were recruited for the study at Hainan General Hospital. The inpatients met the criteria that having abdominal pain, diarrhea, or abdominal pain accompanied by changes in stool characteristics. No obvious biochemical or pathological abnormalities were observed after multiple examinations, including colonoscopy. All hospitalized patients excluded other organic diseases and were clearly diagnosed with irritable bowel syndrome. All 35 patients met the Rome III dignosis criteria for IBS and were positively confirmed onset after an episode of acute gastroenteritis with diarrhea and/or vomiting in addition to a series of examinations such as microbiology test, hence, meeting the definition of PI-IBS 【26】. Moreover, microscopic colitis and other inflammation GI diseases such as celiac disease were excluded by histological screening of mucosal biopsies throughout the entire colon, obtained by colonoscopy prior to inclusion according to established diagnostic criteria. The exclusive criteria for patients were as follows: 1) experienced major abomenial surgery. 2) evidence of metabolic, gastrointestinal, cardiovascular, psychological or malignant disease. 3) gastrointestinal organic disease including peptic ulcer, Crohn’s disease, ulcerative colitis and pancreatitis. 4) pregnancy or lactating. 5) patients who are taking nonsteroidal anti-inflammatory drugs, steroids, or antibiotics. The patients’ age ranged from 17 to 53 years old, with the mean age of 33.9 years, including 19 female and 16 male. In this study, 33 voluntary healthy controls were recruited by advertisement and had regular examination including colonoscopy to exclude any inflammatory diseases. None of them had GI complaints, chronic pain conditions, infectious or inflammatory disorders such as rheumatoid arthritis, psychiatric illnesses, or were taking pharmaceutical agents. The age of health controls ranged from 19 to 55 years old, with the mean age of 32.9 years, including 15 female and 18 male. The peripheral blood and colonic tissue were collected through colon biopsy and preserved in -80°C freezer for further examination. In addition, some tissue was processed into frozen sections. Some tissue was smashed into powder within ultrosonic disintegrator and the supernatant was preserved under -80°C freezer for further examination.
2.2. Morphological analysis
The colonic tissue was collected by colonoscopy biopsy and processed into ultra-thin frozen sections by liquid nitrogen quick-frozen slicing. The sections were treated by immunofluorescence histochemical staining. The primary antibody was florescence- conjugated rabbit anti-mouse anti-δ1 TCR or δ2 TCR monoclonal antibodies. The stained tissue sections were scanned under Laser scanning confocal microscope (Olympus FV10i,Olympus,Tokyo, Japan). The intensity of fluorescence was calculated automatically using the image analysis software.
2.3. Enrichment of Vδ1 γδ T cells
The enrichment of T cells were conducted as previously described by Cheng et, al 【10】. Briefly, 5ml peripheral blood of PI-IBS patients was collected and the total T cells were isolated by centrifuging with lymphocyte separation medium Ficoll (Sigma-Aldrich, ST. Louis, MO, USA). The total T cells were stained with anti humanVδ1-PE and anti humanVδ2-APC (BD Biosciences, Franklin Lakes, NJ, USA) and the proportion of Vδ1 and Vδ2 γδ T cells was measured by FACS. In conclusion, the proportion of Vδ1 T cells were all increased in 35 PI-IBS patients compared with control group.
The total T cells were stained with FITC conjugated anti-δ1 TCR mAb (100ul antibodies per 1´108 cells, incubated at 4°C for 30 min), followed by stained with microbeads conjugated anti-FITC mAb (100ul antibodies per 1´108 cells, incubated at 4°C for 30 min). The Vδ1 γδT cells were positively selected by MACS(Miltenyi Biotec GmbH, Cologne, NRW, Germany). For further purification of Vδ1 γδ T cells, the residual αβ+ T cells were depleted using PE-conjugated anti-αβTCR antibody with anti-PE microbeads.
2.4. Functional Evaluation of Vδ1 γδ T cells
The enriched Vδ1 γδ T cells were incubated in the presence of IL-23 (10 ng/ml) under 37°C, 5%CO2 for 48 hours ,followed by functional evaluation including proliferation, activation and cytokine producing capability.
Proliferation
Cell Counting Kit -8 (CCK-8) Assay was used to evaluate the enriched Vδ1
γδ T cells’ proliferation. Briefly, the cells were seeded at 4×103 cells/well in 96-well plates, then incubated at 37 °C for 24 h in a total volume of 100 μl medium. The cells were added with 10μl CCK8/well and cultured at 37°C,5% for 8 hours. The absorbance value at 450nm was measured on a Thermomax Microplate Reader (Menlo Park, CA, U.S.A). The proliferation response was expressed as the OD value of mean±standard deviation (SD) of triplicate determinations.
Activation
Vδ1γδT cells were stained with PE conjugated anti-CD69 mAb or anti-CD62L mAb (100ul antibodies per 1´108 cells, incubated with IL-23 and TLR4 at 4°C for 30 min) . The expression of CD69 and CD62L on Vδ1γδT cells was determined by FACS as described previously【10, 13】.
Production of proinflammatory cytokines
The concentration of IFNγ and IL-17 in the supernatants from the cultured Vd1 γδ T cells and the colonic tissue were measured by ELISA in accordance with the manufacture’s instruction. Briefly, the supernatant from cultured cells and tissues were removed and replaced with fresh media (RPMI-1640) and were then returned to standard culture conditions for 24hours. Subsequently, the IL-17 protein of cell supernatant was analyzed by IL-17 high-sensitivity (0.25–16pg/mL sensitivity range) ELISA kit (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s protocol.
2.5. Statistics analysis
Experimental data were analyzed with Kolmogorov-Smirnov test by SPSS software to explore whether data complied with a normal distribution. The result of K-S analysis indicated a normality distribution of all data. The unpaired Student’s t-test was used to compare differences between two groups (SPSS 19.0 software). Data were expressed as the mean ± standard error of the mean. Values in the same row with different superscripts are significant (P < 0.05), while values with same superscripts are not significant (P > 0.05).