A total of 903 sepsis patients and 1,150 healthy individuals from three regions of China were recruited in this study between February 2013 and June 2018. There were 370 patients and 400 healthy controls included in our prior cohorts . The patients were enrolled in the study if they met the following inclusion criteria: 1) the diagnosis of sepsis, severe sepsis, or septic shock was established by ICU senior attending physicians according to the International Sepsis Definitions Conference ; 2) the patients were older than 18 years of age and belonged to the Chinese Han population; and 3) the patients had a probability of survival greater than 24 hours. Patients were excluded from this study if they had diabetes, malignant tumors, human immunodeficiency virus, or autoimmune diseases or were pregnant, readmitted to a hospital or receiving immunosuppressive therapy, corticosteroid therapy or chemoradiotherapy. The sepsis, severe sepsis or septic shock is the initial situation of the disease in the patients. The healthy controls, which were enrolled at the health examination center at each hospital, had no history of sepsis, no recent acute illness and no chronic illness, such as autoimmune diseases, diabetes, cancer and major cardiac, renal, hepatic, and endocrinological disorders. The healthy controls also belonged to the Chinese Han population, and their healthy status was determined by reviewing the medical examination reports and questioning the participants. The age, sex, source of infection, cause of infection, pathogenic bacteria, and Acute Physiology and Chronic Health Evaluation (APACHE) II results of each patient were documented, and blood samples were collected within 12 hours after the diagnosis of sepsis, severe sepsis, or septic shock was established. The survival of all the patients was observed for a 28-day period. The participants’ confidentiality was preserved according to the guidelines for studies of human subjects. This study was approved by the Ethics Committees of the participating hospitals, and written informed consent was signed by all enrolled subjects or their families prior to enrollment.
DNA isolation and genotyping
Genomic DNA was isolated from Peripheral blood mononuclear cells (PBMCs) using the TIANamp Blood DNA Kit (TianGen Biotech, Beijing, China) according to the vendor’s recommendations. The ADAM17 -172A>G polymorphism was genotyped using the ABI PRISM® SNaPshot™ Multiplex Kit (Applied Biosystems, Carlsbad, CA, USA). The sequences of the forward and reverse primers were 5'-GGCCTAGCCCCTCAATCCTCTT-3' and 5'-TTTTTTTGGTAACGCCACCTG -CCTTC-3', respectively. The obtained data were analyzed using Gene Mapper 4.1 (Applied Biosystems, Foster City, CA, USA).
Mononuclear cell isolation and cell culture
PBMCs were isolated from the blood samples collected from sepsis patients and healthy individuals by density gradient centrifugation using Lymphoprep™ (Axis-Shield PoCAS, Oslo, Norway). The PBMCs from the 18 healthy individuals were randomly selected for in vitro LPS stimulation experiments. Mouse leukemia cells of monocyte macrophage (RAW264.7 cells), human umbilical vascular endothelial cells (HUVECs) and human embryonic kidney cells (HEK293T) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown at 37 °C with 5% CO2 in DMEM or RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA) and penicillin/streptomycin (Sigma-Aldrich, USA).
Plasmid or virus construction
Plasmids for the overexpression of human pCMV-EGR1 were obtained from Longqian Biotech (Shanghai, China). Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommended protocol. The EGR1 adenovirus packages used for the overexpression or silencing experiments with HUVECs were designed and synthesized by Shanghai Genechem Co., Ltd. (China). The expression of EGR1 infection was detected by Western blotting, quantitative real-time PCR (qRT-PCR) and fluorescence.
C57BL/6 mice (8-12 weeks, males and females in half) were purchased from Guangdong Laboratory Animal Center (Guangzhou, China). The mice used for the experiments in this study were bred and maintained under conventional housing conditions in our animal facility. The animal experimental protocols were reviewed and approved by the Ethics Committee of Affiliated Hospital of Guangdong Medical University, China. EGR1 antisense oligonucleotide (As-ODN) (5’-TACCGTCGCCGGTTC-3’) was constructed to inhibit EGR1 by targeting the corresponding DNA sequence. The EGR1 sense ODN (S-ODN) (5’-TCGTGCCGCTGCCAT-3’) was used as a negative control . All ODNs were synthesized by Takara Biomedical Technology Co., Ltd. The mice were intraperitoneally injected with LPS (Escherichia coli O55:B5, Sigma-Aldrich, USA) at a dose of 10 mg/kg to establish the endotoxemia model. Blood was collected from the mice and centrifuged for measurement of the serum concentrations of TNF-α, IL-6 and IL-1β using each specific enzyme-linked immunosorbent assay (ELISA) kits. Lung and liver tissues were collected for the detection of ADAM17 and EGR1 mRNA expression. For the survival experiments, the mice were intraperitoneally injected with LPS (20 mg/kg) and monitored for 72 hours.
qRT-PCR and Western blot analysis
Total RNA from the PBMCs, cells or tissues was extracted using the TRIzol reagent (Invitrogen), and complementary DNA (cDNA) was synthesized from 1 μg of total pure RNA using the PrimeScriptTM RT reagent kit with cDNA Eraser (Takara) in accordance to the manufacturer’s recommended protocol. The primer sequences used in this study were designed by Sangon Biotech (see Additional file 1). The protein concentrations were determined using the BCA protein assay kit (Thermo Fisher Scientific, USA). The following rabbit polyclonal antibodies were used: anti-ADAM17 (diluted at 1:1000, Abcam, UK), anti-EGR1 (1:1000, Abcam, UK), anti-SP1 (1:1000, Abcam, UK), anti-AP2α (1:500, Abcam, UK), and anti-β-actin antibody (1:2000, CST, USA).
The concentrations of IL-1β, IL-6, and TNF-α in the supernatants obtained from cell culture medium or animal plasma were measured using each specific ELISA kits according to the manufacturer’s instructions. The following ELISA kits were used: TNF-α, IL-6, and IL-1β (Boster Biological Technology, USA).
Flow cytometric analysis
Cell apoptosis was detected by flow cytometry using the Annexin V-PE/7-AAD Apoptosis Detection Kit (YEASEN Biotechnology, Shanghai, China) following the manufacturer's instructions. The cells were harvested, washed twice with ice-cold PBS, and resuspended to a density of 1×106 cells/mL in binding buffer. The cells were mixed with 5 μL of Annexin V-PE and 10 μL of 7-AAD and incubated at room temperature for 15 minutes. The cells were subsequently mixed with 400 μL of binding buffer and detected by flow cytometry.
In order to predict the possible transcription factors that might bind to the promoter region of the ADAM17 gene, we submitted a 200-bp sequence around the -172 position in the ADAM17 gene promoter to the following three transcription factor prediction websites: http://tfbind.hgc.jp/, http://jaspar.genereg.net/cgi-bin/jaspar_db.pl, and http://gene regulation. com/pub/programs/alibaba2/index.html. The final set of transcription factors was obtained by combining the results obtained using these three software packages (see Additional file 2).
Luciferase plasmid constructs and luciferase reporter assay
The human ADAM17 promoter sequence carrying rs12692386 (A or G allele at position -172) was cloned into pGL3 luciferase reporter vectors (Promega, Madison, WI, USA). The primers F-5'-CTAGCAAAATAGGCTGTCC-3' and R-5'-GCCGGGCCTTTCTTTATG-3' were used to obtain the promoter gene fragment of ADAM17 by PCR amplification, and the pGL3-ADAM17-A/G plasmids were ultimately obtained. HEK293T cells were cotransfected with ADAM17 promoter reporter vectors and the transcription factor-overexpressing plasmid by Lipofectamine 2000 (Invitrogen, USA). The level of luciferase activity was assayed using the dual-luciferase reporter assay system (Promega, USA). Firefly luciferase activity was normalized to Renilla luciferase activity.
Chromatin immunoprecipitation (ChIP)-qPCR
ChIP assays were performed the standard protocol with some modifications. ChIP coupled with quantitative PCR was used to investigate the protein-DNA interactions at binding sites in the ADAM17 gene. Briefly, chromatin was cross-linked with transcription factors, sheared into fragments (100–750 bp) by sonication and immunoprecipitated using antibody against EGR1 (CST, USA). The recovered DNA was analyzed by PCR using primers flanking the putative transcription factor-binding sites as indicated. The primers used for PCR of the ChIP fragments were as follows: F-5’-CGTGGGCGGGGCAAG-3’ and R-5’-GCAGGTGGCGTTACCAA -AGG-3’. The PCR program was 95 °C for 30 seconds followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 30 seconds.
The association between ADAM17 polymorphism and sepsis was determined using the Chi-squared test or Fisher’s exact test, and the sample sizes were based on our previous experience. The Benjamin-Hochberg procedure for multiple testing correction was used to analyze the false discovery rate, and P<0.05 after Benjamin-Hochberg correction for multiple testing was considered significant. The survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Analysis of clinical variables such as sex were analyzed by the Chi-squared test. Image J software was used for densitometry quantification of the Western blot bands. The values are shown as the mean ± standard deviation (SD). The expression of ADAM17 and EGR1, and cytokine levels in the independent groups were compared using the non-parametric Mann-Whitney U test (two-tailed). The statistical analyses were performed using SPSS 19.0 (IBM, NY, USA) or GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA), and P value less than 0.05 was considered statistically significant. Additional details are provided in the figure legends, where appropriate.