Studied subjects
The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008) as reflected in a priori approval by Ethics Committee of Xi’an Jiaotong University. Informed consent was obtained from the legal guardians of each studied subjects. Thirty-three children under seven years old with Kawasaki disease who were hospitalized in The Children’s Hospital Affiliated to Xi’an Jiaotong University between July 2018 to June 2019 were enrolled in this study. The diagnosis of Kawasaki disease was made in accordance with the Clinical Statements and Guidelines for Kawasaki disease by American Heart Association [1]. The exclusive criteria was the affliction with autoimmune diseases, chronic viral infection, or severe organ failure. Meanwhile, Seventeen healthy children who received regular health examinations during the same period were also enrolled as controls. The baseline characteristics of all studied subjects were listed in Table 1.
CD14+ monocytes purification and naïve CD4+ T cells isolation
Peripheral blood samples were collected from each studied subjects. Peripheral blood mononuclear cells were isolated using Ficoll Plus 1.077 (Solarbio, Beijing, China) by density gradient centrifugation. CD14+ monocytes were purified using human CD14 MicroBeads UltraPure (Miltenyi, Bergisch Gladbach, Germany), while naïve CD4+ T cells were isolated using human Naïve CD4+ T Cell Isolation Kit II (Miltenyi) following manufacturer’s instruction. The purity of enriched cells was more than 95% by flow cytometry determination.
Cell culture
Purified CD14+ monocytes were stimulated with 50ng/ml of recombinant human IL-35 (Peprotech, Rocky Hill, NJ, USA) in the presence of 1×lipopolysaccharide (LPS; eBioscience, Thermo Fisher Scientific, San Diego, USA) for 24 hours. 105 of IL-35 treated CD14+ monocytes were co-cultured in direct or in indirect contact with 105 of autologous naïve CD4+ T cells in the presence of anti-CD3/CD28. In direct contact co-culture, CD14+ monocytes and naïve CD4+ T cells were directly mixed in the 24-well plate. In indirect contact co-culture, CD14+ monocytes were seeded into upper chamber while naïve CD4+ T cells were seeded into lower chamber of the Transwell plate (Corning, Corning, NY, USA). In the last 12 hours of co-culture, phorbol 12-myristate 13-acetate (50ng/ml), ionomycin (1μg/ml), and Brefeldin A (10μg/ml) were added. Moreover, 105 of IL-35 treated CD14+ monocytes were co-cultured with 105 of human umbilical vein endothelial cells (HUVECs) in either direct contact or indirect contact manner. In certain experiments, TNF antagonist etanercept (100μg/ml, Pfizer, England) or granzyme B inhibitor Z-AAD-CMK (100μmol/L, Kamiya Biomed, Washington, USA) was added to the co-culture systems. Cells and supernatants were harvested 48 hours post co-culture.
Enzyme linked immunosorbent assay (ELISA)
Plasma IL-35 concentration was detected using human IL-35 ELISA kit (CUSABIO, Wuhan, Hubei Province, China), and cytokine production in the cultured supernatants was measured using commercial ELISA kits (CUSABIO) following manufacturer’s instruction. Each sample was analyzed in triplicate and the mean value was recorded.
Real-time reverse transcriptional polymerase chain reaction (PCR)
Total RNA was isolated from purified CD14+ monocytes using the TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA). Equal amounts of total RNA from each sample were reverse-transcribed into cDNA using AMV reverse transcription system (Promega, Madison, WI, USA), and real-time PCR was performed using GoTaq qPCR Master Mix (Promega) following manufacturer’s instruction. Real-time PCR primers (IL-12Rβ2 and gp130) assay designed for analysis was purchased from Bio-Rad (Hercules, CA, USA). The primer sequences for Fas ligand (FasL): forward: 5’-ATG TTT CAG CTC TTC CAC CTA CAG AAG GA-3’, reverse: 5’-CAG AGA GAG CTC AGA TAC GTT GAC-3’; TNF-related apoptosis-inducing ligand (TRAIL): forward: 5’-CTG CTG GCA AGT CAA GTG GCA ACT C-3’, reverse: 5’-GTC GCA TCC TGA AAA CTG AAT AGT-3’. GAPDH was applied as standard for data normalization.
Flow cytomerty
Cells were firstly pre-incubated with Cell Activation Cocktail (R&D systems, Minneapolis, MN, USA), and were stained for surface marker with antibodies against CD4 (eBioscience). After washed twice, cells were fixed and permeabilized with intracellular fixation & permeabiliztion buffer (eBioscience), and were then incubated with antibodies against interferon (IFN)-γ and IL-17A (eBioscience) for intracellular staining. BD Bioscience FACS Aria II flow cytometer was used for cell acquisition, and FlowJo V8.6.2 was used for data analysis.
Cell counting kit-8 (CCK-8) assay
Cellular proliferation was measured by CCK-8 method (Beyotimes, Wuhan, Hubei Province, China). Absorbance was measured at wavelength of 450nm.
Cytotoxicity assay
The target HUVECs death was assessed by measurement of lactate dehydrogenase (LDH) expression in the supernatants using LDH Cytotoxicity Assay Kit (Beyotimes) following manufacturer’s instruction. The low-level control was defined as the LDH level in HUVECs cultured alone, while the high-level control was defined as the LDH level in Triton X-100 treated HUVECs. The frequency of target cell death was calculated using the following equation: (experiment value — low-level control)/(high-level control — low-level control) × 100% [21].
Statistical analysis
GraphPad Prism 5 was used for the statistical analyses. Data were presented as mean ± standard deviation. The difference between groups was assessed using Student’s t test, paired t test, or Tukey test. A p value less than 0.05 was considered as significance.