Serum samples from 56 patients with AS(42 men and 14 women) and 43 age- and sex-matched healthy controls were collected with informed consent in accordance to the Declaration of Helsinki principles for research involving human subjects and with approval by the local ethics committee of Nanjing Medical University. AS patients fulfilled the 1984 modified New York criteria for diagnosis of AS without previous systemic treatment[20, 21]. There were neither systemic diseases, autoimmune diseases, active infectious process, known history of bone fractures in the previous 24 months and history of neurological cognitive disease, nor history of osteoporosis among all subject. We tracked the expression of sema4D in 20 patients who were treated with a TNF-α blocker after enrolment at the present study to compare sema4D expression levels before and after anti-TNF-α treatment. Patient serum specimens were stored at −80°C until analysis described below. PBMCs from healthy donors and patients with AS were isolated by discontinuous density-gradient centrifugation, washed twice in sterile phosphate buffered saline (PBS) and resuspended at a concentration of 1×106 cells/ml PBS.
Clinical and laboratory assessment
We recorded the duration of disease, BMI, sex, age, and extraarticular manifestations of AS patients. Disease activity of AS was calculated as CRP and Bath AS disease activity index(BASDAI). BAP (San Diego, California, USA), osteocalcin (Nordic Biosciences, Herlev, Denmark), TRACP-5b and C-terminal cross-linking telopeptide of type I collagen (CTX)(Nordic Biosciences) were evaluated as markers of bone turnover. Serum BAP and TRACP-5b levels were measured by enzyme linked immunosorbent assay. Osteocalcin and CTX were measured by electrochemiluminescence immunoassays (ECLIAs). The intraassay and inter-assay coefficients of variation were below 9% for all parameters. The assay was performed according to the manufacturer’s instructions. Complete blood count and routine biochemical analysis and HLA-B27 were studied.
Serum Sema4D levels and cytokine analysis
The serum concentrations of Sema4D were determined using commercially available ELISA kits (MyBio-Source, San Diego, USA). Assessment was performed according to the manufacturer’s instructions. The levels of soluble IL-17, IL-22 and IL-10 were measured using ELISA kits for each cytokine (R&D Systems, MN, USA). All measurements were performed in triplicate.
CD4+ T Cell Purification and Stimulation
PBMCs were isolated from the blood samples freshly obtained from AS patients and healthy controls by lymphocyte separation medium(San Diego, California, USA), according to the manufacturer’s instruction. Isolation of CD4+ T cells from PBMCs was performed by magnetic cell separation. MACS CD4 microbeads (Miltenyi Biotec, Auburn, CA, USA) were incubated with the PBMCs and applied to a MidiMACS separation column (Miltenyi Biotec, CA, USA)(Miltenyi Biotec), according to the manufacturer’s instructions.
The purity of isolated CD4+ T cells was determined by flow cytometry to be >96% for each population. The purified CD4+ T cells were seeded in 96-well plates at 1×106 cells per well and then stimulated with or without soluble human Sema4D-Fc fusion protein(PeproTech, RockyHill, NJ), Plexin B2 antibody, Plexin B1 antibody or CD72 ligation antibody (BU40; Santa Cruz Biotechnology, USA). To assay in vitro Th17 differentiation, cells were activated for Th17 differentiation with anti-CD3(2μg/ml, plate-bound), anti-CD28 (2μg/ml, soluble). For blocking assays, cells were cocultured with 10ng/ml of Sema4D-Fc and 10 ng/ml of anti-Sema4D antibody or isotype-matched control IgG for 48 hours. Concentrations of human IL-10, IL-22 and IL-17 in culture supernatants were tested by ELISA. At the end of the stimulation period, cells were collected and analyzed by flow cytometry, Quantitative RT-PCR analysis was performed as above description..
Flow Cytometric Analysis
CD4+ T Cells were harvested before and after stimulation. Cell surface markers were stained with the indicated labeled antibodies against indicated cell surface antigens. Cells were prepared in heparinized tubes by Ficoll-Paque density-gradient centrifugation and then analyzed on a FACSCanto (Invitrogen, CA, USA) using FlowJo software(Tree Star) according to the manufacturer’s instructions. The following flow cytometry antibodies were used for the analyzing cell type and cytokine production: PE-CD4, Foxp3-APC, CD25-PE and FITC-IL-17A (BioLegend, California, USA). FITC-, PE- and APC labeled mouse IgG were utilized as isotype controls (Bio-Legend, California, USA).
For the proliferation assay, isolated CD4+ T cells were labeled with the Cell TraceTM CFSE Cell Proliferation Kit (Invitrogen, CA, USA) at a final concentration of 4μM. CFSE-labeled CD4+ T cells were incubated under the described conditions. 1×106 million of CFSE-labeled T cells, were seeded into a flat 96-well plate. Soluble anti-sema4D (see above), soluble anti-CD72 (Biolegend, San Diego, CA, USA), or matched isotypes were added as indicated. T cell proliferation was recorded after 3 days and 5 days, based on CFSE dilution using flow cytometry.
Western blot assay
Cells were collected after induction and cell lysate was prepared from 1 × 107 cells. The proteins were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Sigma, USA). The membrane was blocked in 5% bovine serum albumin or nonfat dry milk in Tris-buffered saline for 2 h at room temperature, then incubated with mouse primary antibodies overnight at 4℃. The membrane was washed and incubated with primary antibodies against CYP1A1 or β-actin (all from Cell Signaling Technology, Danvers, MA, USA), and horseradish peroxidase-conjugated secondary antibodies were added. Then, the bands were detected using ECL luminescent reagents (Pierce, USA). GAPDH was used as a loading control.
RNA extraction and real-time quantitative PCR (qPCR)
To detect levels of IL-17A, ROR-γt, Foxp3, and GAPDH mRNA expression, total RNA from human PBMCs and CD4+ T cells was extracted using the QIAGEN RNeasy Mini Kit (QIAGEN, Hilden, Germany), and complementary DNA (cDNA) was synthesized using a Super-Script II cDNA synthesis kit (Invitrogen, USA) according to the manufacturer’s protocols. Quantitative RTPCR analysis was performed using the QuantiFastTM SYBR Green PCR Kit (QIAGEN, Hilden, Germany) with an ABI 7500 instrument (Applied Biosystems, CA, USA). The primer sequences were as follows: IL-17, forward, 5’-CGGACTGTGATGGTCAACCTGA-3’,reverse,5’-GCACTTTGCCTCCCAGATCACA-3’; FoxP3,forward,5’-GGCACAATGTCTCCTCCAGAGA-3’,reverse,5’-CAGATGAAGCCTTGGTCAGTGC-3’;ROR-γt,forward,5’-CAGAATGACCA-GATTGTGCTT-3’,reverse,5’-TCCATGCCACCGTATTTGC-3’;AhR,forward,5’-CAAATCAGAGACTGGCAGGA-3’,reverse,5’-AGAAGACCAAGGCATCTGCT-3’;CYP1A1,forward,5’-GTTCTTGGAGCTTCCCCGAT-3’,reverse,5’-CTGACACGAAGGCTGGAAGT-3’,andGAPDH,forward,5’-GTCTCCTCTGACTTCAACAGCG-3’,reverse,5’-ACCACCCTGTTGCTGTAGCCAA-3’. All reactions were carried out in triplicate in the same plate.
Cell culture and Luciferase assay
The EL-4 cells were cultured at 37℃ and 5%CO2 in RPMI1640(Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum. The EL-4 cells were plated in 96-well plates (1×106 cells per well), and the cells in each well were co-transfected with a vector, pGL3 [luc2P/XRE/Hygro] , containing a xenobiotic responsive elements (XRE) that drives transcription of the luciferase reporter geneluc 2P (Photinuspyralis) was used. Fresh culture media were added to the cells along with FICZ(300nM), Sema4D(10ng/ml), anti-PlexinB1(5μg/ml), anti-PlexinB2(5μg/ml), anti-CD72(5μg/ml) or CH233191(30μM), either alone or in combination, and incubated for 12h. The cells were washed and lysed, and the supernatants were collected from the lysed cell preparations. The luciferase activity was measured by a luciferase assay system (Promega, Madison, WI, USA) and a multimode reader according to the manufacturer’s instructions.
Ethoxyresorufin-O-deethylase (EROD) activity
The EL-4 cells were plated into 6-well plates at a density of 1 × 106 cells/mL, and stimulated with Sema4D, anti-PlexinB1, anti-PlexinB2, anti-CD72, CH223191, and FICZ either alone or in combination, and incubated for 24h, and supernatant was collected. Then, CYP1A1 activity was measured by using the EROD enzyme assay, as previously described. Fluorescence intensity was detected by using a FL600 plate reader (Biotek, Winooski, VT, USA), with excitation at 530 nm and emission at 590 nm.
Statistical significance was calculated using SPSS 20.0. Nonparametric Mann-Whitney U tests were used to compare 2 groups, and comparisons between 3 groups were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test. The correlation analysis was performed using the Pearson correlation test. For all statistical analyses, p values less than 0.05 were considered significant.