ASC/caspase-1/IL-1β signaling triggers inflammatory responses by promoting HMGB1 induction in liver ischemia/reperfusion injury

Abstract

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors, is essential for inducing caspase-1 activation and the consequent secretion of interleukin-1β (IL-1β), which is associated with local inflammation during liver ischemia/reperfusion injury (IRI). However, little is known about the mechanisms by which the ASC/caspase-1/IL-1β axis exerts its function in hepatic IRI. This study was designed to explore the functional roles and molecular mechanisms of ASC/caspase-1/IL-1β signaling in the regulation of inflammatory responses in vitro and in vivo. With a partial lobar liver warm ischemia (90 minutes) model, ASC-deficient and wild-type mice (C57BL/6) were sacrificed at 6 hours of reperfusion. Separate animal cohorts were treated with an anti-IL-1β antibody or control immunoglobulin G (10 mg/kg/day intraperitoneally). We found that ASC deficiency inhibited caspase-1/IL-1β signaling and led to protection against liver ischemia/reperfusion (IR) damage, local enhancement of antiapoptotic functions, and down-regulation of high mobility group box 1 (HMGB1)-mediated, toll-like receptor 4 (TLR4)-driven inflammation. Interestingly, the treatment of ASC-deficient mice with recombinant HMGB1 re-created liver IRI. Moreover, neutralization of IL-1β ameliorated the hepatocellular damage by inhibiting nuclear factor kappa B (NF-κB)/cyclooxygenase 2 signaling in IR-stressed livers. In parallel in vitro studies, the knockout of ASC in lipopolysaccharide-stimulated bone marrow-derived macrophages depressed HMGB1 activity via the p38 mitogen-activated protein kinase pathway and led to the inhibition of TLR4/NF-κB and ultimately the depression of proinflammatory cytokine programs.

Conclusion: ASC-mediated caspase-1/IL-1β signaling promotes HMGB1 to produce a TLR4-dependent inflammatory phenotype and leads to hepatocellular injury. Hence, ASC/caspase-1/IL-1β signaling mediates the inflammatory response by triggering HMGB1 induction in hepatic IRI. Our findings provide a rationale for a novel therapeutic strategy for managing liver injury due to IR.

Figure 1

ASC knockout ameliorates the hepatocellular damage and prevents liver IRI. Mice were subjected to 90min of partial liver warm ischemia, followed by 6h of reperfusion. (A) Hepatocellular function evaluated by sALT (IU/L). Decreased sALT levels in ASC-deficient group as compared with controls. *p<0.01, n=5–6/group. Mean±SD are shown. (B) Suzuki’s histological grading of liver IRI. *p<0.0001, n=5–6/group. Mean±SD are shown. (C) MPO neutrophil activity (U/gm). *p<0.005. n=2–3/group. Mean±SD are shown.

Figure 2

Knockout of ASC signaling reduces HMGB1, TLR4-mediated inflammation, macrophage sequestration and innate cytokine/chemokine programs in IR-stressed livers. (A) Western blot-assisted detection of HMGB1, NF-κB, TLR4, and cleaved caspase-1. β-actin expression serves as an internal control. Representative of three experiments is shown. (B and D) Quantitative RT-PCR-assisted detection of cytokines (HMGB1 [B], TNF-α/IL-12p40 [D]), chemokines (MCP-1/IP-10/CXCL-1 [D]). Data were normalized to HPRT gene expression. *p<0.05, **p<0.01, ***p<0.005, n=3/group. Mean±SD are shown. (C) Representative ischemic liver lobes stained for CD11b (panel a,b,c) and Ly6G (panel d,e,f), magnification ×400, n=3/group.

Figure 3

ASC knockout promotes anti-apoptotic function and reduces IR-induced apoptosis, whereas adjunctive treatment with rHMGB1 recreates liver IRI. (A) Western blot-assisted analysis of Bcl-2, Bcl-xl, and cleaved caspase-3. β-actin was used as an internal control. Representative of three experiments. (B) TUNEL-assisted detection of apoptosis. Representative staining of TUNEL+ apoptotic cells (magnification ×400). n=3/group. (C) Hepatocellular function evaluated by sALT (IU/L). Decreased sALT levels in ASC KO group, compared with WT or ASC-deficient group conditioned with rHMGB1. *p<0.05, **p<0.01, n=3–6/group. Mean±SD are shown. (D) Suzuki’s histological grading of liver IRI. *p<0.0001, n=3–6/group. Mean±SD are shown. (E) Quantitative RT-PCR-assisted detection of TNF-α and IL-1β. Data were normalized to HPRT gene expression. *p<0.05, **p<0.005, n=3/group. Mean±SD are shown.

Figure 4

ASC-mediated caspase-1 signaling induces HMGB1 via MAPKs pathway and NF-κB activation in LPS-stimulated BMMs in vitro. (A) Western blot-assisted analysis of HMGB1, NF-κB, TLR4, and phospho-p38. β-actin was used as an internal control. Representative of three experiments. (B) Quantitative RT-PCR-assisted detection of HMGB1. Data normalized to β-actin gene expression. *p<0.05, n=3/group. Mean±SD are shown. (C) Caspase-1 activity (U). Mean±SD; n=3/group. *p<0.005. (D) ELISA-assisted detection of IL-1β. Mean±SD; n=3/group. *p<0.005. (E) Quantitative RT-PCR-assisted detection of IL-1β and IL-18. Data were normalized to β-actin gene expression. *p<0.005, n=3/group. Mean±SD are shown.

Figure 5

Treatment of WT mice with anti-IL-1β mAb mitigates the hepatocellular damage and liver IRI (6h of reperfusion following 90min of warm ischemia). (A) The sALT levels (IU/L). *p<0.0001, n=5–7/group. Mean±SD are shown. (B) Suzuki’s histological grading of liver IRI. *p<0.0001, n=5–7/group. Mean±SD are shown. (C) MPO neutrophil activity (U/gm). *p<0.05, **p<0.005. n=2–3/group. Mean±SD are shown.

Figure 6

Disruption of IL-1β signaling reduces cytokine/chemokine programs and macrophage traffic in IR-livers. (A) Western blot-assisted expression of NF-κB and COX2; β-actin was used as an internal control. Representative of three experiments are shown. (B) Quantitative RT-PCR-assisted detection of TNF-α, iNOS, and COX2.*p<0.05, **p<0.005, n=3/group. Mean±SD are shown. (C) Left panel: Representative liver sections stained for CD11b (magnification×400). Right panel: Quantification of CD11+ cells/HPF. *p<0.05, **p<0.005, n=3/group.

Figure 7

ASC/Caspase-1/IL-1β - HMGB1 cross talk in the regulation of innate inflammation response in IR-stressed livers.