HSPA12A attenuates lipopolysaccharide-induced liver injury through inhibiting caspase-11-mediated hepatocyte pyroptosis via PGC-1α-dependent acyloxyacyl hydrolase expression

Liver dysfunction is strongly associated with poor survival of sepsis patients. Cytosolic lipopolysaccharide (LPS) sensing by Caspase-4/5/11 for pyroptosis activation is a major driver of the development of sepsis. Studies in macrophages and endothelial cells have demonstrated that LPS is inactivated by acyloxyacyl hydrolase (AOAH) and leading to desensitizing Caspase-4/5/11 to LPS. However, little is known about the cytosolic LPS-induced pyroptosis in hepatocytes during sepsis. Heat shock protein 12A (HSPA12A) is a novel member of the HSP70 family. Here, we report that LPS increased HSPA12A nuclear translocation in hepatocytes, while knockout of HSPA12A (Hspa12a−/−) in mice promoted LPS-induced acute liver injury. We also noticed that the LPS-induced Caspase-11 activation and its cleavage of gasdermin D (GSDMD) to produce the membrane pore-forming GSDMDNterm (markers of pyroptosis) were greater in livers of Hspa12a−/− mice compared with its wild type controls. Loss- and gain-of-function studies showed that HSPA12A deficiency promoted, whereas HSPA12A overexpression inhibited, cytosolic LPS accumulation, Caspase-11 activation and GSDMDNterm generation in primary hepatocytes following LPS incubation. Notably, LPS-induced AOAH expression was suppressed by HSPA12A deficiency, whereas AOAH overexpression reversed the HSPA12A deficiency-induced promotion of LPS-evoked and Caspase-11-mediated pyroptosis of hepatocytes. In-depth molecular analysis showed that HSPA12A interacted directly with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and increased its nuclear translocation, thereby inducing AOAH expression for cytosolic LPS inactivation, which ultimately leading to inhibition of the Caspase-11 mediated pyroptosis of hepatocytes. Taken together, these findings revealed HSPA12A as a novel player against LPS-induced liver injury by inhibiting cytosolic LPS-induced hepatocyte pyroptosis via PGC-1α-mediated AOAH expression. Therefore, targeting hepatocyte HSPA12A represents a viable strategy for the management of liver injury in sepsis patients.

2 of ALT and AST activities, and hepatocytes were collected for the examination of 22 HSPA12A expression and Caspase-11 activation. 23 24 1.3 Body temperature and animal activities 25 Body temperature in mice was detected at 2, 4 and 6 h following LPS (5 mg/kg) 26 treatment using a rectal probe. Animal activities were also evaluated at 2, 4 and 6 h 27 following LPS (5 mg/kg) treatment according to previous methods 1 . The 28 measurements before LPS treatment served as baseline controls. In brief, the scoring 29 system of activity includes two principal tasks: hunched posture and spontaneous 30 rapid movements interspersed with eating and drinking. The scoring system ranged 31 from 0 to 4, in which 4 (normal) denotes that mice intersperse movement 32 spontaneously and rapidly with eating and drinking but without hunched posture, and 33 0 (severe) is continual hunched posture without movement. The investigator was 34 blinded to the treatment. 1.4 Measurement of Blood pressure, blood gas, blood creatinine (Cr) and urea-37 nitrogen (Urea) Noninvasive blood pressure. 38 Six hours after LPS (5 mg/kg) or NS treatment, mouse systolic blood pressure 39 (SBP) was measured using a non-invasive tail cuff computerized system (ACL-NIBP, 40 Alcott Biotech, China) as described in our previous study 2 . All the measured mice 41 were pre-trained for 5 consecutive days in the pre-warmed tail-cuff device to 42 3 accustom them to the procedure. The investigator was blinded to the treatment.

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Blood gas analysis. Six hours after LPS (5 mg/kg) or NS treatment, mice were 44 anesthetized (1.5% isofluorane), intubated and mechanically ventilated. Arterial blood 45 was drawn from the left ventricle for blood gas measurements, including blood 46 oxygen saturation (SO2), partial pressure of blood oxygen (pO2), and partial pressure        Caspase-11 activation was examined using immunoblotting.

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B. Primary hepatocytes were treated with 500 ng/ml LPS for the indicated durations.

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Caspase-11 activation was examined using immunoblotting.  Culture medium was collected for ALT and AST activity analysis. 121 D. Primary hepatocytes were treated with 500 ng/ml LPS for the indicated durations.

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Culture medium was collected for ALT and AST activity analysis.

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Data are mean ± SD, ** P < 0.01 by Student's two-tailed unpaired t test. n = 5/group.  After incubation with FITC-LPS (500 ng/ml) for 6 h, the primary hepatocytes 161 were immunofluorescence stained for a vesicle marker Flotinllin-1. Hoechst33342 162 was used to counterstain nuclei. The staining was observed using a fluorescence 163 9 microscope. n =3/group. Scale bar = 10 μM. 164 165 Figure S12. LDH activity in culture medium. 166 After incubation with LPS or NS for 6 h, the medium of primary hepatocyte 167 cultures were collected for LDH activity assay. Data are mean ± SD, ** P < 0.01 or * 168 P < 0.05 by two-way ANOVA followed by Tukey's test. n =3/group. Hoechst33342. The staining was observed and quantified using a fluorescence 174 microscope. Data was expressed as FITC fluorescence intensity *10 3 /mm 2 cell area.