In total, 323 subjects were recruited from outpatients or inpatients of the First Affiliated Hospital of Zhejiang University between October 2016 and December 2017. These subjects were divided into four groups: (i) HB-ACLF: 135 CHB patients with ACLF diagnosed by the criteria of the Asian-Pacific Association for the Study of the Liver (APASL)—i.e., “acute hepatic insult manifesting as jaundice (≥ 5 mg/dL) and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease.” ; (ii) acute decompensated cirrhosis (AD): 30 AD patients were characterized by cirrhosis complicated with ascites, hepatic encephalopathy and upper gastrointestinal bleeding. Cirrhosis was defined by previous endoscopy, liver biopsy, radiological evidence, or clinical manifestation of liver decompensation. (iii) chronic hepatitis B (CHB): 128 age-/sex-matched patients with stable chronic hepatitis B were included. Chronic hepatitis B was diagnosed by serum HBsAg positive for more than six months together with histology or imaging or laboratorial or clinical evidence of cirrhosis or liver fibrosis or long-term liver inflammation; (iv) healthy controls (HC): 180 age-/sex-matched healthy subjects were used as controls. Multi-organ failure was diagnosed with two or more extrahepatic organ failures happen according to the chronic liver failure sequential organ failure assessment (SOFA) score. Exclusion criterions were as follows: age younger than 18 years, human immunodeficiency virus infection, pregnancy, immunotherapy, cancer, and a history of autoimmune diseases. Written consent was obtained from each subject or their nominated next of kin if the participants could not provide informed consent. This study was performed according to the principles of the Helsinki Declaration and was approved by the Ethic Committee of the First Affiliated Hospital of Zhejiang University. The baseline characteristics of the patients are shown in (Additional file 1: Table S1). The model for end-stage liver disease (MELD) score and CLIF-consortium organ failure (CLIF-C) score were calculated to assess the severity of the disease.
CD3-percp-cy5.5 (Catalogue #45-0036-42), CD8-APC (Catalogue #17-0086-42), CD8-FITC (Catalogue #11-0086-42), CD14-APC (Catalogue #17-0149-42), CD56-FITC (Catalogue #4278380), CD16-PerCP-eFluor™710 (Catalogue #46-0168-42), CD11b-PerCP-eFluor™710 (Catalogue #46-0110-80), IFN-γ-PE (Catalogue #12-7319-42), TLR2-FITC (Catalogue #11-9922-41) and Mouse IgG1-PE (Catalogue #12-4714-81) were all bought from eBioscience. HLA-DR-PE (Catalogue #555812), IL-10-APC (Catalogue #554707), CXCR3-APC (Catalogue #550967) and IFN-γ-FITC (Catalogue #561053), Rat IgG2a-APC (Catalogue #554690) and mouse IgG-APC (Catalogue #5065947) were purchased from BD Biosciences. CD16-FITC (Catalogue #302006), HLA-DR-APC (Catalogue #307609), CD3-FITC (Catalogue #317306) and TLR4-APC (Catalogue #312815) were purchased from Biolegend. EP2-PE (Catalogue #10477) and Rabbit IgG-PE (Catalogue D5-1610) were purchased from Cayman Chemical.
Isolation of peripheral blood mononuclear cells
Whole-blood samples were collected within 24 h after admission. After centrifugation, the plasma was collected and stored immediately at − 80 °C. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density gradient centrifugation (GE healthcare, Pittsburgh, USA).
Measurement of plasma cytokines and PGE2
The plasma cytokine levels of ACLF patients were measured using a multiplex panel (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. PGE2 was measured using the Prostaglandin E Metabolites ELISA kit (Cayman Chemical, Michigan, USA). Both measurements were performed according to the manufacturer’s instructions. The detection limits were as follows:IL-1β, 17.3 pg/ml; IL-1rα, 15 pg/ml; IL-2, 16.5 pg/ml; IL-4, 11 pg/ml; IL-5, 27 pg/ml; IL-6, 24.8 pg/ml; IL-7, 11 pg/ml; IL-8, 14.5 pg/ml; IL-9: 21 pg/ml; IL-10: 33.3 pg/ml; IL-12: 15.5 pg/ml; IL-13: 13.5 pg/ml; IL-5; 57.3 pg/ml; IL-17: 24.8 pg/ml; eotaxin, 17 pg/ml; FGF basic, 14 pg/ml; G-CSF, 36.3 pg/ml; GM-CSF, 16 pg/ml; IFN-γ, 15.5 pg/ml; IP-10, 14.5 pg/ml; MCP-1, 18.5 pg/ml; MIP-1α, 8.8 pg/ml; PDGF-bb, 32.5 pg/ml; MIP-1β, 31.5 pg/ml; RANTES, 15.8 pg/ml; TNF-α, 12.5 pg/ml; VEGF, 109.3 pg/ml; PGE2, 2 pg/ml.
Measurement of cell surface markers
Isolated PBMCs or 100 μl of heparin-treated peripheral whole blood was incubated with antibodies for 15 min at room temperature. After incubation, whole-blood samples were lysed with FACS lysing solution and were washed with phosphate-buffered saline before analysis using an Accuri C6 cytometer (Accuri, BD). The isotype IgG was used as the control.
To explore factors driving EP2 expression, PBMC from healthy controls were incubated for 7 days with a range of IP-10 and MIP-1β and assessed for EP2 expression.
Gating strategies of flow cytometry for immune subsets are introduced in Figure S1 (Additional file 2): CD8+T cells (CD3+/CD8+), CD4+T cells (CD3+/CD8−), NK (CD3−/CD56+), NKT (CD3+/CD56+), monocytes (CD14+), neutrophils (CD16+).
Intracellular cytokine assays
In vitro, PBMCs (2 × 105/well) were stimulated with lipopolysaccharide (LPS) (100 ng/ml; Sigma-Aldrich, St. Louis, MO) for 72 h in the presence or absence of AH6809 (150 μM; Cayman Chemical) or DMSO (0.1%) in 1640 RPMI medium (Invitrogen, Oslo, Norway) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 U/ml of penicillin, and 100 U/ml of streptomycin (Invitrogen) at 37 °C in 5% CO2. For the last 6 h, monensin (1.7 μg/ml; Biolegend) was added to the medium to inhibit cytokine secretion, and the cells were re-stimulated with LPS (100 ng/ml). Next, the cells were incubated with fixation/permeabilization solution (BD biosciences) at 4 °C for 20 min. After incubation, the cells were washed, stained with IFN-γ-PE and IL-10-APC for 15 min at room temperature and analyzed by flow cytometry.
Measurement of supernatant cytokines
In vitro, PBMCs (2 × 105/well) were stimulated with LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO) for 72 h in the presence or absence of AH6809 (150 μM) or butaprost (50 μM; Sigma) or DMSO in 10% FBS RPMI medium. Next, the supernatants were collected and stored at − 80 °C immediately. Cytokines in the supernatants were measured using a multiplex panel (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. The limitations of detection have been mentioned above.
One hundred microliters of whole blood were incubated with AF488-conjugated Escherichia coli (K-12 strain) bio-particles (Molecular probe, Eugene, OR, USA) in 96-well plates and was analyzed by FACS according to previously described protocols . For blocking experiments, the blood was preincubated with AH6809 or DMSO for 1 h at 37 °C in 5% CO2.
Oxidative burst assays
One hundred microliters of whole blood samples were incubated with or without heat-inactivated E. coli (8.04*107cfu/ml) or PMA (50 ng/ml) in 96-well plates for 30 min. Next, the cells were assessed for oxidative burst using an ROS assay kit (Genecopoeia, MD, USA) and were analyzed by FACS according to the manufacturer’s protocol. For blocking experiments, blood was pre-incubated with AH6809 or DMSO for 1 h at 37 °C in 5% CO2.
The data are shown as the mean ± standard deviation (SD), mean ± standard error of the mean (SEM), median (range) or number (percentage). For comparisons between two independent groups, the Mann–Whitney U test was used. Comparisons between paired groups were performed by the Wilcoxon signed-rank test. Correlations between variables were calculated by the Spearman’s rank correlation test. P < 0.05 at two sides was considered statistically significant. All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software).