2.1. PI-IBS ptients
35 patients (inpatients and outpatients) were enrolled, diagnosed in accordance with the diagnostic criteria of IBS (Rome III,2006) 【22】 from January 2018 to December 2019 at Hainan General Hospital . The patients’ age ranged from 18 to 50 years old, with the mean age of 34.5 years, including 18 female and 17 male. The patients had at least once intestinal infection history but did not receive standard and enough antibiotics treatment. All the patients had no any organic disorders and reported abdominal discomfort-associated abnormal defecation when enrolled. 33 healthy volunteers were assigned into the control group. The research protocol in the current study was permitted by Ethnic Committee of Hainan General Hospital. The written informed consent was obtained from all patients and volunteers.
The patients were checked by colonoscopy to exclude obvious organic diseases. 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 【7】. 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.
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 FACSas described previously【7, 9】.
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.
2.5. Statistics analysis
Data were analyzed using Student’s t-test (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).