Fig 2: Expression of TFPI2 and CLIP1 in human fatty liver after CS.a Macroscopic view of the human liver; HE staining, ORO staining, and IHC staining of TFPI2 and CLIP1. b–d Western blotting and statistical analyses of the TFPI2 and CLIP1 proteins in human liver tissue. e Analysis of the necrotic area via human liver histology. f Analysis of the ORO-positive area in human liver tissue. g, h Quantitative analysis of IHC staining of TFPI2 and CLIP1 in human liver tissue. n = 5 per group; the data are the mean ± SD; ns, not significant; *P < 0.05; and **P < 0.01. i Mechanistic scheme. In a rat model of fatty liver, severe steatosis, cold ischemia, and subsequent reperfusion led to the accumulation of ROS in hepatocytes, with the released HMGB1 activating the TLR4/NF-κB inflammatory pathway in ECs. Additionally, R24 of the KD1 domain of TFPI2 directly binds to CLIP1 in ECs, inhibiting the ubiquitination and degradation of TIRAP by CLIP1, thereby activating the TLR4/NF-κB-mediated inflammatory response, resulting in severe IRI. HOPE effectively ameliorates the entire pathological process.

Fig 3: BIG1 deficiency reduced PIP2 production and prohibited the binding of TIRAP to the plasma membrane of macrophages induced by LPS.a WT and BIG1−/− BMDMs were treated with or without LPS (100 ng/ml) for 30 min. The cell lysates were subjected to immunoblotting with the indicated antibodies. b The membrane-bounded and cytosolic proteins from WT and BIG1−/− BMDMs stimulated with LPS (100 ng/ml) for 30 min were measured by immunoblotting with the indicated antibodies. c WT and BIG1−/− BMDMs were treated with or without LPS (100 ng/ml) for 30 min, followed by confocal imaging analysis with TIRAP antibody. Scale bars, 20 μm. d WT and BIG1−/− BMDMs were treated with LPS (100 ng/ml) for 30 min, followed by dot blot with the indicated antibodies (up panel). The activity of PIP5K was measured by luminescence assay kit (down panel). e The cell surface proteins from WT and BIG1−/− BMDMs treated with LPS (100 ng/ml) for 30 min, were measured by dot blot and immunoblotting with the indicated antibodies, respectively. f WT and BIG1−/− BMDMs were treated with or without LPS (100 ng/ml) for 30 min, followed by confocal imaging analysis with PIP2 antibody. Scale bars, 20 μm. g PIP2 fluorescent intensities from more than 50 individual cells were quantified in f. h WT and BIG1−/− BMDMs were treated with or without LPS (100 ng/ml) for 30 min, then cellular PIP2 levels from extracted acidic lipids were measured by using the PIP2 mass ELISA kit. i, j BIG1−/− BMDMs transfected with active mutant ARF3 (Q71L) or vector were treated with or without LPS (100 ng/ml) for 30 min, followed by dot blot and Western blot with the indicated antibodies (i) or measuring the activity of PIP5K (j). k THP-1-derived macrophages transfected with negative control siRNA or si-BIG1 were treated with or without LPS (100 ng/ml) for 30 min. The membrane-bounded and cytosolic proteins were isolated, followed by dot blotting with the indicated antibodies. Data show pooled technical replicates from three independent experiments (d, g, h, j). All immunoblot data are representative of three independent experiments with similar results. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (one way ANOVA in d, g, h, j).

Fig 4: Confocal immunofluorescence of integrins and inflammatory response proteins with/without Ti treatment. (A-C) Localization of CD18, CD11b and CD11a after Ti co-culture. (D and E) Confocal images of Myd88 and TIRAP localizations. Non-linear adjustments. (F) Statistical analysis was performed among multiple marks comparing fluorescence ratio. Green represents anti-IgA staining. Scale bar, 10 µm. *P<0.05. Ti, titanium; TIRAP, TIR-domain-containing adapter protein.

Fig 5: According to proteomic data, the most immune-related proteins were activated 12 h after infection. (A) Detection of TLR activation by relative mRNA level. TLR2 shows highlight expression in all TLRs; TLR2 was selected for subsequent investigation. (B) To investigate the pathway by which TLR2 exerts functionality, qPCR was performed on critical proteins downstream of TLR2; TRIF, TIRAP, and TRAM were not upregulated. (C) Two pathways downstream of TLR2 were verified by western blotting. The expression levels of PI3K-AKT and NF-κB pathway proteins were significantly upregulated; however, the MyD88 pathway, which is usually considered a TLR2 junction protein, was not activated. (D) JEV infection promoted the upregulation of inflammatory factors (TNF-α, CCL3, IL-1beta, and iNOS), thus indicating an inflammatory response. All samples were tested in three parallel groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.