For the analysis of Th17 cells, PBMCs were suspended at a density 1 × 106 cells/ml in complete culture medium (RPMI 1640 supplemented with 100 U/ml penicilin and 100 ug/mL streptomycin, 2 mM glutamine and with 10% heat-inactivated fetal calf serum, Gibco, USA). The cell suspension was transferred to each well of 24-well plates. Cultures were stimulated with phorbol myristate acetate (PMA, 50 ng/mL) plus ionomycin (1 uM, all from Alexis Biochemicals, San Diago, CA), in the presence of monensin (500 ng/mL, from eBioscience, San Diago, CA) in an incubator at 37 °C under a 5% CO2 environment. After 5 h of culture, the contents of the well were transferred to 15-mL sterile tubes. The cells were than centrifuged at 1500 rpm for 5 min. For the analysis of Tregs, PBMCs were aliquoted into tubes for further staining38.
Surface and intracellular staining
Cells were aliquoted into tubes and washed once in phosphate-buffered saline (PBS). For Th17 analysis, the cells were incubated with fluorescein isothiocyanate (FITC) anti-human CD4 monoclonal antibody (Ab) at 4 °C for 20 min. For Treg analysis, the cells were incubated with FITC anti-human CD4 and phycoerythrin (PE) anti-human CD25. After the surface staining, the cells were stained with fluorescein phycoerythrin (PE) anti-human IL-17A for Th17 detection or PC5 anti-human FoxP3 for Treg detection after fixation and permeabilization according to the manufacturer’s instructions. Isotype controls were performed to enable correct compensation and confirm Ab specificity. All of the Abs were from eBioscience, San Diego, CA. Flow cytometry analysis was performed using Flow Jo software.
Plasma Cytokine and Chemokine Determinations
Plasma concentrations of 18 cytokines and chemokines of including IFN-γ (sensitivity is 0.2 pg/ml), IL-5 (0.3 pg/ml), IL-6 (0.4 pg/ml), IL-10 (0.1 pg/ml), IL-12 (0.04 pg/ml), IL-17A (0.1 pg/ml), IL-17F (0.1 pg/ml), IL-21 (0.6 pg/ml), IL-22 (8.2 pg/ml), IL-23 (0.9 pg/ml), IL-27 (5.1 pg/ml), Eotaxin (1.4 pg/ml), CCL2 (0.6 pg/ml), CCL3 (1.1 pg/ml), CCL4 (4.7 pg/ml), RANTES (0.2 pg/ml), CXCL1 (2.8 pg/ml), CXCL8 (1.2 pg/ml), CXCL10 (0.3 pg/ml), CXCL12α (20.5 pg/ml) were detected by using Luminex bead-based multiplex assay (eBiosciences, San Diego, CA, USA) under close compliance with manufacturer’s guidelines.
Quantitative Real-time PCR Analysis of Toll like receptors, cytokines and transcription factors
Measurement of mRNA expression of TLRs, cytokines and transcription factors, namely TLR2 and TLR4, IL-17 and IL-23, and associated transcription factors relevant to the study, T-bet (for Th1 cells), GATA3 (for Th2 cells), RORγτ (for Th17 cells), and FoxP3 (for Treg cells), was performed using SYBR Green System on PBMCs for all AE patients and HC subjects, as well as on liver samples of patients from the MAAE group (who were operated on).
Total RNA was obtained using TRIzol (Invitrogen) according to the manufacturer’s instructions and quantified by A260 absorbance. 2 μg of RNA was reverse-transcribed to cDNA, and used as a template for Real-time PCR. qRT-PCRs were conducted with the SYBR Green PCR premix following the manufacturer’s protocols (Invitrogen, CA, USA) with primers purchased from Sangon, Shanghai (see Supplementary Table S2). Measurements were performed using SYBR Green program on i-Q 5.0 Real-time PCR system (BioRad, Foster City, CA, USA). The relative amounts of PCR products were determined using the relative standard curve method and GAPDH was used as an internal control. The 2−ΔΔCt method was used to determine the specific Ct value of each target gene.
Immunohistochemistry of IL-17A and IL-23
Liver tissue samples were prepared for immunohistochemistry, and a semi-quantitative method was applied for analyzing IL-17A and IL-23 immunostaining. Two senior pathologists blinded to each other’s results read the sections; scores considered both staining intensity and the percentage of cells stained at a specific range of intensity, as described previously39.
All continuous variables were expressed as median and interquartile range (IQR) and categorical variables as number and percentage. All AE patients were compared with HC subjects and MAAE with MIAE patients. In the liver, measurements performed in LA were compared with those performed in DA. We used Mann-Whitney rank-sum test to detect the differences between groups and Spearman correlation analysis to test correlation between two continuous variables. All tests were two-sided and a probability value of p ≤ 0.05 was considered to be statistically significant. Evaluation of the diagnostic values for IL-5, IL-23 and their combination was made using Receiver Operative Curves (ROC). Areas under curves (AUC) and sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) were calculated and compared between groups. Statistical analysis of the data was conducted with the Statistical Package for Social Sciences (SPSS), version 17.0.