Fig 2: Serum N-myc and STAT interactor (NMI) was inversely correlated with prognosis in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) patients. (a) The comparison of serum NMI between HBV-ACLF ameliorated patients (n = 26) and HBV-ACLF non-ameliorated patients (n = 24) at baseline. Horizontal bars indicate the median values and interquartile ranges in each group. (b and c) The kinetic changes of serum NMI in HBV-ACLF ameliorated and non-ameliorated patients during follow-up. (b) ???, Ameliorated. (c) ???, Non-ameliorated. (d) Kaplan–Meier graphs showing the survival probability of two groups of HBV-ACLF patients stratified by serum NMI of 198.5 pg/mL. (e) Area under the receiver operating characteristic curve (AUROC) of serum NMI, Chronic Liver Failure Consortium ACLF score (CLIF-C ACLFs), Model for End-stage Liver Disease (MELD), combination of serum NMI and CLIF-C ACLFs (NMI–CLIF-C ACLFs), or combination of serum NMI and MELD (NMI–MELD) in predicting 3-month mortality of HBV-ACLF patients. , NMI-CLIF-C ACLFs; , NMI-MELD; , CLIF-C ACLFs; , MELD; , NMI; , reference line. ** P < 0.01; *** P < 0.001. [Color figure can be viewed at http://wileyonlinelibrary.com]

Fig 3: NMI is released by activated macrophages. a NMI in the supernatant (Sup) and cytoplasmic fraction of human THP1 cells (ATCC TIB-202™) were analyzed by immunoblotting post S. typhimurium stimulation, using high-mobility group box protein 1 (HMGB1), l-lactate dehydrogenase B 1 (LDHB1), and N-myc as control. ß-actin in the cell and bovine serum albumin (BSA) in the supernatant were used to show equal loading. BSA was shown using commassie blue staining. b NMI in the supernatant (Sup) and cytoplasmic fraction of THP1 cells were analyzed by immunoblotting. The cells were pretreated with (+) or without (-) Brefeldin A for 8 h and stimulated with (+) or without (-) S .typhimurium for 4 h. c NMI released by mouse RAW264.7 cells (ATCC TIB-71™) (2 × 106 cells ml-1) was analyzed by ELISA at different time points post LPS (1 µg ml-1) stimulation. Error bars indicate ± s.e.m. from three biological replicates. d Mice (n = 5 for each group) were intraperitoneally injected with 1 × 104 CFU live S. typhimurium per mouse and the concentration of the NMI in the serum were determined by ELISA. Data are presented as the mean ± s.e.m. **P < 0.01. e, f NMI in the sera of mice (n = 5 for each group) was determined by ELISA 3, 6 or 24 h after intraperitoneal injection of LPS (10 mg kg-1 or 20 mg kg-1) or acetaminophen (APAP) (300 mg kg-1). Data are presented as the mean ± s.e.m. **P < 0.01. g ELISA analysis of NMI in the serum of 24 patients and 8 healthy individuals. Patients succumbed to severe inflammation were labeled with solid cycles. Significance in d and e were tested by unpaired Student’s t-test. Significance in g was tested by Mann–Whitney U-test. **P < 0.01

Fig 4: NMI stimulates macrophages through the TLR4 pathway. a Western blot analysis of mNMI in the BMDM cell lysate after 1 h incubation with recombinant mNMI. The BMDM cells were isolated from Nmi -/- mice and pretreated with macrophage colony-stimulating factor (MCSF). b, c TNF and IL-6 released by BMDMs from WT (C57BL/6) mice, pretreated with bafilomycin A1 (10 nM), TAK-242 (100 nM) or dimethyl sulphoxide (DMSO) for 2 h and stimulated with mNMI (5 µg ml-1) or LPS (100 ng ml-1) for 8 h. d–g TNF and IL-6 levels in the supernatants of BMDMs from WT, Tlr4 -/- and Tlr2 -/- mice were analyzed using ELISA 4 h post activation by different stimulus. h After incubation with mNMI-GFP for 1 h, the percentage of GFP labeled CD11b+F4/80+ cells was determined by Flow cytometric analysis. The cells were isolated from spleen in WT or Tlr4 -/- mice. i NMI in the human THP1 cell (ATCC TIB-202™) lysates interacts with hTLR4. Ni-NTA beads coupled with 2 µg His-hTLR4 and/or His-hMD2 fusion proteins were used as bait. j The luciferase activity of HEK293T cells (ATCC CRL-11268™) are shown after stimulated with 5 µg ml-1 mNMI for 4 h (in the presence or absence of 25 µg ml-1 polymyxin B (PMB)). The cells were pre-transfected with mTLR4-MD2-CD14 and NF-?B promoter with luciferase activity. 100 ng ml-1 LPS was administrated as positive control. In b–e and g, error bars indicate ± s.e.m. from 3 biological replicates. Significance was tested by one-way ANOVA followed by Student–Newman–Keuls test. **P < 0.01

Fig 5: NMI knockout mice have attenuated inflammatory responses. a The survival rate of wild type (WT) (n = 15) and Nmi -/- mice (n = 15) intraperitoneally injected with LPS (50 mg kg-1). *P < 0.05 compared to WT. b, c TNF and IL-6 in the sera of Nmi -/- and WT mice as determined by ELISA 6 or 24 h post LPS-injection. d The survival rate of WT(n = 14) and Nmi -/- mice (n = 14) intraperitoneally injected with LPS (50 µg kg-1) and d-gal (1 g kg-1). **P < 0.01. e, f TNF and IL-6 in the sera of Nmi -/- and WT mice as determined by ELISA 1 h or 3 h after injection of LPS and d-gal. g The survival rate of WT (n = 15) and Nmi -/- mice (n = 15) intraperitoneally injected with acetaminophen (APAP) (600 mg kg-1). Significance in a, d, and g was determined using the log-rank test. **P < 0.01. h, i TNF and IL-6 in the sera of Nmi -/- and WT mice as determined by ELISA 6 or 24 h after injection of APAP. Error bars in b, c, e, f, h, i indicate ± s.e.m. from five individual mice. Significance was tested by unpaired Student’s t-test. *P < 0.05, **P < 0.01