Redox-dependent regulation of hepatocyte absent in melanoma 2 inflammasome activation in sterile liver injury in mice

Abstract

Sterile liver inflammation, such as liver ischemia-reperfusion, hemorrhagic shock after trauma, and drug-induced liver injury, is initiated and regulated by endogenous mediators including DNA and reactive oxygen species. Here, we identify a mechanism for redox-mediated regulation of absent in melanoma 2 (AIM2) inflammasome activation in hepatocytes after redox stress in mice, which occurs through interaction with cytosolic high mobility group box 1 (HMGB1). We show that in liver during hemorrhagic shock in mice and in hepatocytes after hypoxia with reoxygenation, cytosolic HMGB1 associates with AIM2 and is required for activation of caspase-1 in response to cytosolic DNA. Activation of caspase-1 through AIM2 leads to subsequent hepatoprotective responses such as autophagy. HMGB1 binds to AIM2 at a non-DNA-binding site on the hematopoietic interferon-inducible nuclear antigen domain of AIM2 to facilitate inflammasome and caspase-1 activation in hepatocytes. Furthermore, binding of HMGB1 to AIM2 is stronger with fully reduced all-thiol HMGB1 than with partially oxidized disulfide-HMGB1, and binding strength corresponds to caspase-1 activation. These data suggest that HMGB1 redox status regulates AIM2 inflammasome activation.

Conclusion: These findings suggest a novel and important mechanism for regulation of AIM2 inflammasome activation in hepatocytes during redox stress and may suggest broader implications for how this and other inflammasomes are activated and how their activation is regulated during cell stress, as well as the mechanisms of inflammasome regulation in nonimmune cell types. (Hepatology 2017;65:253-268).

Figure 1. AIM2-mediated caspase-1 activation in hepatocytes is protective during hypoxia-reoxygenation and hemorrhagic shock

(A) Plasma ALT in Control/AIM2^−/− mice after HS/R (n=3/gp control; n=7/gp HS/R; **P<0.01; *P<0.05); (B) Fold increase of cytoplasmic dsDNA from livers of control mice (no HS/R) or mice after HS/R (n=3–5/gp; **P<0.01); (C) Annexin V/PI flow cytometry dot plots for WT/AIM2^−/− hepatocytes after normoxia or 6h hypoxia/1h reoxygenation; (D) Relative caspase-1 activity (percentage of normoxic levels) in WT/AIM2^−/− hepatocytes after normoxia or 6h hypoxia/1h reoxygenation (^#P<0.05, normoxia vs. hypoxia-reoxygenation; **P<0.01, WT vs AIM2^−/−); (E) Immunoprecipitation (IP) of ASC in WT hepatocytes after 6h hypoxia/1h reoxygenation, or 3h poly(dA:dT) (1/2μg) and immunoblotting for AIM2; (F) Relative mitochondrial DNA (mtDNA) copy number (compared with normoxic levels – dotted line); data representative of at least 3 separate repeats.

Figure 2. Redox-mediated caspase-1 activation is dependent on HMGB1

(A) Western blot of caspase-1 in liver lysates from WT/HC-HMGB1^−/− mice after HS+4.5h of resuscitation (res);1 mouse per lane; (B) Relative caspase-1 activity in liver lysates from WT/HC-HMGB1^−/− mice after HS+4.5h of resuscitation. Data are shown as percentage of WT control (Ctrl) livers (mean±s.d. n=3/gp control; n=5/gp HS+4.5res; **P<0.01); (C) Plasma ALT in mice after HS/R (n=2/gp control;n=6–7/gp HS+4.5res; *P<0.05); (D) Annexin V/PI flow cytometry dot plots for WT/HC-HMGB1^−/− hepatocytes after normoxia or 6h hypoxia/1h reoxygenation (H-R); (E) Immunoprecipitation (IP) of ASC in WT/HC-HMGB1^−/− hepatocytes after 6h hypoxia/1h reoxygenation or poly(dA:dT) for 3h followed by immunoblot for AIM2 or ASC; data representative of at least 3 separate repeats.

Figure 3. HMGB1 binds AIM2 to facilitate inflammasome activation after redox stress in hepatocytes

(A) IP of AIM2 followed by immunoblotting for ASC or HMGB1 in cell lysates from WT/AIM2^−/− hepatocytes after 6h hypoxia/1h reoxygenation (H-R) or poly(dA:dT) for 3h; (B) IP of HMGB1 followed by immunoblotting for AIM2 in cell lysates from WT/HC-HMGB1^−/− hepatocytes after 6h hypoxia/1h reoxygenation (H-R) or poly(dA:dT) for 3h (left); (C) Quantification of immunoprecipitation results from 3 separate repeats (right; **P<0.01; ND, not detectable); (D) Immunoprecipitation (IP) of Myc, followed by IB for FLAG at 24h after transfection of HC-HMGB1−/− hepatocytes with myc-HMGB1 and FLAG-tagged AIM2 after normoxia or 6h hypoxia/1h reoxygenation (H-R); data representative of at least 3 separate repeats; (E) Confocal immunofluorescence of liver (scale bar=5μm) in WT mouse after HS+4.5h resuscitation. AIM2 (green), HMGB1 (red), DAPI nuclear stain (blue), actin (white). Colocalization of AIM2 and HMGB1 indicated by arrows (left); (F) Quantification of numbers of punctae per hepatocyte of HMGB1, AIM2 and HMGB1/AIM2 colocalization as measured by confocal immunofluorescent staining (right; n=10/gp);

Figure 4. HMGB1 binds to AIM2 at HIN domain

(A) Western blot (WB) for caspase-1 in WT/AIM2^−/− hepatocytes after transfection with 1μg of either nuclear DNA (nuDNA) or mitochondrial DNA (mtDNA) or lipofectamine control (no DNA); (B) Immunoprecipitation (IP) of FLAG, followed by WB for Myc at 24h after transfection of HEK293 cells with myc-HMGB1 and FLAG-tagged AIM2 or FLAG-tagged AIM2-HIN domain (HIN). (C) Immunoprecipitation (IP) of Myc, followed by WB for Flag at 24h after transfection of HEK293 cells with myc-HMGB1 and FLAG-tagged AIM2 or FLAG-tagged AIM2-HIN domain (HIN). Data shown are representative of 3 independent experiments.

Figure 5. His79/Ser80 on AIM2-HIN domain are important for binding of HMGB1

(A) Molecular docking analysis of AIM2-HIN (4JBM) and HMGB1 (2YRQ) showing putative interaction site at H65,S66 (corresponding to His79/Ser80 on full length sequence in protein data base) on AIM2-HIN with N-terminus of HMGB1; (B) Ten HIN family sequences were aligned with ClustalW and displayed using WebLogo, with symbol heights corresponding to relative amino acid frequency. The arrowhead shows the position of the amino acid mutation (H79/S80) used to create FLAG-HIN mutant; (C) IP of Myc followed by Western blot (WB) of Flag in cell lysates from HEK293A cells overexpressing My-HMGB1, Flag-HIN, Flag-HIN(H65K), Flag-HIN(T35K) or Flag-HIN(H65KS66K); (D) WB for capase-1 in cell lysates from AIM2^−/− hepatocytes overexpressing Flag-HIN, Flag-HIN(H65K), Flag-HIN(T35K) or Flag-HIN(H65KS66K); data representative of at least 3 separate repeats.

Figure 6. Redox status of HMGB1 in livers during hemorrhagic shock

(A) Representative spectra of whole protein ESI-MS of HMGB1 isoforms isolated from liver tissue of control, (B) mice treated with HS+1.5res and (C) HS+4.5res. Molecular weights and a schematic representation of each isoform are indicated on each spectra when required. Specific HMGB1 post translational modifications (redox and acetyl) had been confirmed by LC-MS/MS following enzymatic digestion of the whole protein isoform isolate. Data are representative of at least three individual mice per group. (D) Quantification of HMGB1 post translational modifications by whole protein ESI-MS compared to authentic synthetic standards in liver from control and HS/R treated mice (n=3 mice per group performed in duplicate). Data are expressed as fold change to control mice.

Figure 7. Redox status of HMGB1 affects its binding to AIM2

(A) WB for caspase-1 in cell lysates from peritoneal macrophages (MΦ) +/− LPS (10ng/mL) or HC-HMGB1^−/− hepatocytes +/− overexpression of Myc-HMGB1, Myc-HMGB1(C23S) or Myc-HMGB1(C45) after 6h hypoxia/1h reoxygenation (H-R) (B) Immunoprecipitation of Flag or (C) Myc in cell lysates from HEK293A cells transfected with Flag-AIM2 and Myc-HMGB1(C23S) or Myc-HMGB1(C45S) for 24h and treated without H₂O₂ (B) or with 0.5mM H₂O₂ (C) for 6h followed by western blot (IB) for Myc or Flag. Data shown are representative of 3 independent experiments.