Division of Liver and Pancreas Transplantation, Dumont-UCLA Transplantation Center, Department of Surgery, University of California Los Angeles, Los Angeles, CA
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA
Division of Liver and Pancreas Transplantation, Dumont-UCLA Transplantation Center, Department of Surgery, David Geffen School of Medicine, University of California Los Angeles, 10833 Le Conte Avenue, 77-120 CHS, Los Angeles, CA 90095===
Potential conflict of interest: Nothing to report.
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. (HEPATOLOGY 2013)
Ischemia/reperfusion injury (IRI) in the liver remains a major complication of hemorrhagic shock, liver resection, and transplantation.1 Despite improved preservation and surgical techniques, IRI resulting from donor organ retrieval, cold storage, and warm ischemia during surgery often leads to primary organ nonfunction, predisposes patients to chronic rejection, and contributes to the acute shortage of donor organs available for transplantation. Liver IRI represents an exogenous, antigen-independent inflammatory process that includes Kupffer cell/neutrophil activation and cytokine release followed by hepatocyte and sinusoidal endothelial cell death.1 We and others have documented toll-like receptor 4 (TLR4)–dependent innate immune mechanisms that initiate a liver IRI cascade, transcribe nuclear factor kappa B (NF-κB)–mediated cytokine/chemokine and cell adhesion genes, and lead to the development of local inflammation and apoptosis.2-4
Interleukin-1β (IL-1β), a proinflammatory cytokine produced mainly by macrophages, has many biological functions that are essential to sterile inflammation initiated by endogenous danger signals, such as the up-regulation of endothelial adhesion molecules for the recruitment of innate immune cells5 and the development of an inflammatory phenotype.6 The secretion of IL-1β by inflammatory cells is largely dependent on a multiprotein complex termed the inflammasome, which consists of a nucleotide-binding oligomerization domain–like receptor (NLR) molecule and procaspase-1 and mediates the activation of caspase-1.7-10
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) plays a critical role in the activation of inflammasomes as an adaptor protein that bridges procaspase-1 and inflammasome receptors such as NLR family pyrin domain containing 3 (NLRP3) and absent in melanoma 2.11-13 Indeed, ASC contributes to the immune response through the assembly of inflammasome complexes that activate the downstream effector cysteine protease caspase-1 and result in the generation of active IL-1β and IL-18 from inactive pro–IL-1β and pro–IL-18 precursors.
High mobility group box 1 (HMGB1), an evolutionarily conserved and ubiquitously expressed DNA-binding protein in the nucleus of almost all eukaryotic cells, stabilizes nucleosome formation and facilitates gene transcription, repair, and recombination.14 In addition to its nuclear role, extracellular HMGB1, which is known as one of the key endogenous damage-associated molecular pattern molecules, can activate inflammatory pathways. Indeed, macrophage-derived HMGB1 has been shown to mediate delayed endotoxin lethality and acute lung injury in mice.15-17 HMGB1 can also be released by ischemia-stressed cells,18, 19 and this suggests its role as an endogenous danger signal or alarmin that may engage a diverse receptor repertoire, including TLR2, TLR4, TLR9, and receptor for advanced glycation end products (RAGE), for the initiation of an array of inflammatory responses.3, 20, 21 Although the ASC/caspase-1/IL-1β axis is essential for triggering the inflammation cascade, little is known about its crosstalk with HMGB1. In the present study, we show that ASC mediates caspase-1/IL-1β signaling and promotes HMGB1 to trigger TLR4-driven inflammation. Our results identify a previously unrecognized HMGB1-dependent ASC/caspase-1/IL-1–mediated inflammation response in the mechanism of liver IRI.
Materials and Methods
Male C57BL/6 wild-type (WT) mice (Jackson Laboratory, Bar Harbor, ME) and ASC knockout (KO) mice (bred at the University of California Los Angeles) were used at 8 to 10 weeks of age. The animals were housed in the University of California Los Angeles animal facility under specific pathogen-free conditions, and they received humane care according to the criteria outlined in Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, 1985 revision).
Liver IRI and Treatment.
We used a well-established mouse model of partial warm hepatic IRI.2, 22, 23 Separate groups of WT and ASC KO mice were injected with heparin (100 U/kg), and an atraumatic clip was used to interrupt the artery/portal vein blood supply to the left/middle liver lobes. After 90 minutes of ischemia, the clip was removed, and the mice were sacrificed 6 hours after reperfusion. Some of the ASC KO recipients received a single injection of recombinant high mobility group box 1 (rHMGB1; 0.8 mg/kg intraperitoneally; a gift from Dr. A. Tsung, University of Pittsburgh, or Sigma-Aldrich Corp., St. Louis, MO) immediately at reperfusion. Additional WT cohorts were given an antimouse IL-1β monoclonal antibody (mAb; 10 mg/kg intraperitoneally; Novartis, Inc., Basel, Switzerland) or control immunoglobulin G (IgG) 1 day before ischemia. An equivalent of antihuman IL-1β (canakinumab), this mAb binds to mouse IL-1β and neutralizes its activity by blocking its interaction with IL-1 receptors. Sham-operated controls underwent the same procedure but without vascular occlusion.
Hepatocellular Damage Assay.
Serum alanine transaminase (sALT) levels, an indicator of hepatocellular injury, were measured in blood samples with an autoanalyzer (ANTECH Diagnostics, Los Angeles, CA).
Liver Histology and Immunohistochemistry.
Liver paraffin sections (5 μm) were stained with hematoxylin and eosin. The severity of liver IRI was graded blindly with Suzuki's criteria on a scale from 0 to 4.24 No necrosis or congestion/centrilobular ballooning was given a score of 0, whereas severe congestion and >60% lobular necrosis were given a value of 4.
Liver-infiltrating macrophages and neutrophils were detected with a primary mAb against CD11b and lymphocyte antigen 6 complex locus G (Ly6G; BD Biosciences, San Jose, CA), respectively. HMGB1 was detected in hepatocytes and liver-infiltrating macrophages/monocytes with a rabbit anti-HMGB1 antibody (Ab; Cell Signaling Technology, Danvers, MA). The secondary, biotinylated goat antirat IgG or goat antirabbit IgG (Vector, Burlingame, CA) was incubated with immunoperoxidase (ABC kit, Vector). Positive cells were counted blindly in 10 high-power fields (HPFs) per section (×400).
Myeloperoxidase (MPO) Assay.
Neutrophil influx in the liver tissue was assessed with the enzymatic activity of MPO.25 One unit of MPO activity was defined as the quantity of the enzyme degrading 1 μmol of peroxide per minute at 25°C per gram of tissue.
Total RNA was extracted from frozen livers with an RNase mini kit (Qiagen, Valencia, CA); the RNA concentration was determined with a spectrophotometer. RNA (5.0 μg) was reverse-transcribed into complementary DNA. Quantitative polymerase chain reaction was performed with the DNA Engine with the Chromo 4 detector (MJ Research, Waltham, MA). To a final reaction volume of 20 μL, the following were added: 1X SuperMix (Platinum SYBR Green quantitative polymerase chain reaction kit, Invitrogen, Carlsbad, CA), complementary DNA, and 10 μM of each primer. The amplification conditions were 50°C (2 minutes) and 95°C (5 minutes) followed by 40 cycles of 95°C (15 seconds) and 60°C (30 seconds). The primer sequences for the amplification of HMGB1, tumor necrosis factor α (TNF-α), IL-1β, monocyte chemoattractant protein 1 (MCP-1), chemokine (C-X-C motif) ligand 1 (CXCL-1), CXCL-10, IL-18, IL-20p40, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) are shown in Supporting Table 1. Target gene expressions were calculated on the basis of their ratios to the housekeeping gene HPRT.
Apoptosis in formalin-fixed, paraffin-embedded liver sections was detected with a terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining kit (Calbiochem, Gibbstown, NJ).25 Negative controls were prepared through the omission of terminal transferase. Positive controls were generated by a treatment with deoxyribonuclease. TUNEL-positive cells were counted in 10 HPFs per section (×400).
In Vitro Cell Cultures.
Bone marrow–derived macrophages (BMMs) were generated as described.23 Cells (1 × 106/well) were cultured for 7 days, and this was followed by incubation with lipopolysaccharide (LPS; 100 ng/mL) for 6 hours or rHMGB1 (1 μg/mL) for 24 hours (both from Sigma-Aldrich Corp.).
Proteins (30 μg per sample) from livers or cell cultures were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). Polyclonal rabbit antimouse cleaved caspase-3, B cell lymphoma 2 (Bcl-2), B cell lymphoma extra large (Bcl-xL), HMGB1, COX2, phospho-p38 mitogen-activated protein kinase (MAPK), β-actin (Cell Signaling Technology, Danvers, MA), TLR4 (IMGENEX, San Diego, CA), NF-κB, and polyclonal goat antimouse cleaved caspase-1 (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Relative protein quantities were determined with a densitometer and were expressed in absorbance units.
Caspase-1 Enzymatic Activity Assay.
Caspase-1 enzymatic activity was determined with a colorimetric assay kit (R&D System, Minneapolis, MN). Briefly, BMMs were cultured with recombinant TNF-α (100 ng/mL) for 24 hours, and cellular protein was extracted with a cold protein lysis buffer. The cell lysate (50 μL) was added to 50 μL of a caspase-1 reaction buffer in a 96-well, flat-bottom microplate. Each sample was then added to a 200 mM caspase-1 substrate (WEHD-pNA), and this was followed by 2 hours of incubation at 37°C. The enzymatic activity of caspase-1 was measured on an enzyme-linked immunosorbent assay (ELISA) reader at the wavelength of 405 nm.
A mouse ELISA kit was used to measure IL-1β levels in BMM culture supernatants (eBioscience, San Diego, CA).
Data are expressed as means and standard deviations. Differences between experimental groups were analyzed with a Student t test. All differences were considered statistically significant at P < 0.05.
Ab, antibody; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; Bax, B cell lymphoma 2–associated X protein; Bcl-2, B cell lymphoma 2; Bcl-xL, B cell lymphoma extra large; BMM, bone marrow–derived macrophage; COX2, cyclooxygenase 2; CXCL, chemokine (C-X-C motif) ligand; ELISA, enzyme-linked immunosorbent assay; HMGB1, high mobility group box 1; HPF, high-power field; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IgG, immunoglobulin G; IL, interleukin; iNOS, inducible nitric oxide synthase; IR, ischemia/reperfusion; IRAK, interleukin-1 receptor-associated kinase; IRI, ischemia/reperfusion injury; KO, knockout; LBP, lipopolysaccharide binding protein; LPS, lipopolysaccharide; Ly6G, lymphocyte antigen 6 complex locus G; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein 1; MD-2, myeloid differentiation 2; MPO, myeloperoxidase; mRNA, messenger RNA; MyD88, myeloid differentiation protein 88; NALP3, NACHT, LRR and PYD, domains–containing protein 3; NF-κB, nuclear factor kappa B; NLR, nucleotide-binding oligomerization domain–like receptor; NLRP3, NLR family pyrin domain containing 3; qRT-PCR, quantitative real-time polymerase chain reaction; RAGE, receptor for advanced glycation end products; rHMGB1, recombinant high mobility group box 1; sALT, serum alanine aminotransferase; TIRAP, toll-interleukin 1 receptor domain containing adaptor protein; TLR, toll-like receptor; TNF-α, tumor necrosis factor α; TRAF6, tumor necrosis factor receptor–associated factor 6, E3 ubiquitin protein ligase; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WT, wild type.
Disruption of ASC Signaling Ameliorates Liver IRI.
We analyzed the hepatocellular function in mouse livers subjected to 90 minutes of warm ischemia followed by 6 hours of reperfusion. As shown in Fig. 1A, sALT levels were decreased in ASC KO mice versus WT controls (12,506.8 ± 12,717 versus 32,812 ± 5133 IU/L, P < 0.01). These data correlated with Suzuki's grading of histological liver ischemia/reperfusion (IR) damage. Indeed, ASC-deficient mice showed minimal sinusoidal congestion and vacuolization without edema or necrosis (Suzuki's score = 1.4 ± 0.6; Fig. 1B). Similar findings were recorded for ASC-deficient livers subjected to 90 minutes of warm ischemia only (Suzuki's score = 1.2 ± 0.4; Supporting Fig. 2A,B). In contrast, ASC-proficient (WT) livers revealed moderate to severe edema and extensive hepatocellular necrosis at 6 hours of reperfusion (Suzuki's score = 3.7 ± 0.5, P < 0.0001; Fig. 1B). The liver MPO activity, an index of neutrophil accumulation, was suppressed in ASC KO mice versus WT controls (0.32 ± 0.076 versus 4.1 ± 0.2 U/g, P < 0.005; Fig. 1C).
ASC Deficiency Inhibits HMGB1 in Liver IRI.
As shown in Fig. 2A, western blot–assisted expression of HMGB1 (2.0-2.2 AU), NF-κB (2.6-2.8 AU), TLR4 (1.7-1.9 AU), and cleaved caspase-1 proteins (1.5-1.7 AU) was consistently increased in WT livers versus ASC-deficient livers (0.8-1.0, 1.0-1.2, 0.2-0.4, and 0.5-0.7 AU, respectively). We further analyzed messenger RNA (mRNA)–level coding for HMGB1 by qRT-PCR (Fig. 2B). ASC KO mice showed decreased hepatic expression of HMGB1 versus controls (P < 0.05). In contrast to ASC-proficient (WT) controls, the baseline HMGB1 levels were decreased in ASC-deficient mice, as evidenced both in vitro (BMM cultures) and in vivo (normal livers; Supporting Fig. 4A,B). To determine whether ASC signaling may have influenced macrophage and neutrophil trafficking patterns, we performed immunohistochemical staining in IR-stressed livers. ASC-deficient livers were devoid of both CD11b+ macrophages (Fig. 2Ca-c) and Ly6G+ neutrophils (Fig. 2Cd-f) in comparison with WT controls (7.4 ± 4.4 versus 38.1 ± 18.6 CD11b+ macrophages per HPF, P < 0.005; 10.7 ± 5.6 versus 42.5 ± 18.5 LY6G+ neutrophils per HPF, P < 0.0001). In agreement with these immunostaining data, the qRT-PCR–assisted detection of mRNA coding for TNF-α/IL-12p40 (P < 0.01), CXCL-10/MCP-1 (P < 0.05), and CXCL-1 (P < 0.0005) was reduced in ASC-deficient livers versus WT controls (Fig. 2D).
ASC Deficiency Promotes Antiapoptotic Function and Decreases Apoptosis in IR-Stressed Livers.
To determine whether ASC affects IR-induced apoptosis, we performed western blots to detect antiapoptotic genes. The expression of Bcl-2 and Bcl-xL was up-regulated in ASC KO livers (1.8-2.0 and 1.5-1.7 AU; Fig. 3A) versus WT livers (0.2-0.4 AU). Moreover, ASC deficiency inhibited the expression of cleaved caspase-3 (0.3-0.5 AU) in comparison with controls (1.9-2.1 AU). In agreement with the western analysis, the frequency of TUNEL+ cells per HPF in the ischemic liver lobes was diminished in ASC KO mice versus their WT counterparts [7.9 ± 15.22 (Fig. 3Bc) versus 75.4 ± 15.12 TUNEL+ cells per HPF (Fig. 3Bb), P < 0.001].
Exogenous rHMGB1 Re-Creates Liver IRI in ASC-Deficient Mice.
To clarify the function of HMGB1 in the ASC-mediated inflammatory response, we administered rHMGB1 to ASC KO mice immediately at reperfusion after 90 minutes of warm ischemia. As shown in Fig. 3C, an rHMGB1 infusion increased sALT levels at 6 hours of reperfusion in comparison with untreated ASC-deficient mice (29,354.3 ± 2971 versus 12,506.8 ± 12,717 IU/L, P < 0.05). These data correlated with Suzuki's grading of histological liver damage (Fig. 3D). Hence, unlike ASC-deficient but otherwise untreated livers (Suzuki's score = 1.5 ± 0.7), those conditioned with adjunctive rHMGB1 revealed moderate to severe edema, sinusoidal congestion, and hepatocellular necrosis (Suzuki's score = 3.6 ± 0.51, P < 0.0001). A similar effect was displayed in rHMBG1-treated WT livers subjected to 90 minutes of ischemia and 6 hours of reperfusion (Supporting Fig. 5A,B). In contrast, rHMGB1 did not affect well-preserved histological architecture in sham controls. Furthermore, adjunctive rHMGB1 significantly increased the expression of mRNA coding for TNF-α (P < 0.05) and IL-1β (P < 0.005) in ASC KO livers versus otherwise untreated ASC-deficient livers (Fig. 3E). These findings were confirmed in vitro: the addition of rHMGB1 to either WT or ASC-deficient BMM cultures increased cleaved caspase-1/TLR4 and IL-1β expression (Supporting Fig. 3A,B).
ASC Promotes HMGB1/TLR4-Mediated Inflammation.
To further elucidate the molecular mechanisms of the HMGB1- and ASC-mediated inflammatory response in liver IRI, we used an LPS-stimulated BMM cell culture system. As shown in Fig. 4A, western blot–assisted expression of HMGB1 was reduced in ASC-deficient BMMs (0.9-1.2 AU) versus WT controls (2.0-2.2 AU). In contrast to LPS-stimulated WT BMMs (2.1-2.3 AU), the expression of TLR4 (0.2-0.4 AU) and NF-κB (0.3-0.5 AU) was decreased in ASC-deficient BMMs. Moreover, LPS-stimulated ASC-deficient BMMs showed reduced expression of phospho-p38 MAPK (0.1-0.2 AU) versus WT BMMs (2.7-2.9 AU). Next, we analyzed HMGB1 gene expression with qRT-PCR (Fig. 4B). The mRNA-level coding for HMGB1 was decreased in LPS-stimulated BMMs of ASC KO mice versus LPS-stimulated WT cells (P < 0.05). These findings were confirmed by decreased caspase-1 activity in ASC-deficient BMMs but not WT BMMs (0.51 ± 0.357 versus 3.55 ± 0.19 U, P < 0.005; Fig. 4C). To investigate the role of ASC/caspase-1/IL-1–mediated inflammatory responses, we analyzed the production of IL-1β in BMMs by ELISA. As shown in Fig. 4D, LPS-stimulated ASC-deficient BMMs revealed decreased IL-1β levels in comparison with WT BMMs (186.5 ± 108.7 versus 1722.7 ± 125.9 pg/mL, P < 0.005). Furthermore, our qRT-PCR results showed that IL-1β and IL-18 decreased in LPS-stimulated, ASC-deficient BMMs versus WT BMMs (P < 0.005; Fig. 4E).
Neutralization of IL-1β Mitigates Liver IRI in WT Mice.
Having demonstrated that ASC/caspase-1/IL-1β contributes to the IR inflammation response, we next investigated the role of IL-1β by using a neutralizing anti–IL-1β mAb in our model. The disruption of IL-1β signaling alleviated IR liver damage, as evidenced by diminished sALT levels (11,300 ± 4595.5 versus 33,626 ± 5156.6 and 32,617 ± 3859.4 IU/L, P < 0.0001; Fig. 5A) and well-preserved liver histology (Suzuki's score = 1.1 ± 0.5; Supporting Fig. 1 and Fig. 5B). In contrast, livers in phosphate-buffered saline and IgG groups revealed moderate to severe edema (Suzuki's score = 3.6 ± 0.5) and extensive hepatocellular necrosis (Suzuki's score = 3.7 ± 0.48, P < 0.0001; Fig. 5B). In agreement with these data, MPO activity was suppressed in the anti–IL-1β mAb–treated group versus the phosphate-buffered saline and IgG controls [0.34 ± 0.1 versus 3.13 ± 0.72 (P < 0.05) and 3.08 ± 0.11 U/g (P < 0.005); Fig. 5C].
Neutralization of IL-1β Inhibits NF-κB/COX2 in IR-Stressed Livers.
To investigate the mechanism by which an anti–IL-1β mAb treatment may exert anti-inflammatory effects, we analyzed the expression of NF-κB, COX2, and inflammatory mediators in IR livers. As shown in Fig. 6A, the anti–IL-1β Ab depressed western blot–assisted expression of NF-κB (0.4-0.6 AU) and COX2 proteins (0.1-0.2 AU) in comparison with WT (2.6-2.8 and 1.4-1.6 AU, respectively) and IgG controls (2.0-2.2 and 1.6-1.8 AU, respectively). Moreover, treatment with anti–IL-1β mAb decreased mRNA expression coding for COX2 (P < 0.05), iNOS (P < 0.005), and TNF-α (P < 0.005) versus controls (Fig. 6B). Neutralization of IL-1β reduced the frequency of CD11b+ macrophages sequestered in ischemic liver lobes versus WT and IgG-treated controls [23.1 ± 6.44 versus 38.1 ± 18.64 (P < 0.05) and 40.8 ± 12.91 cells per HPF (P < 0.005); Fig. 6C].
The adaptor protein ASC contributes to immune responses through activation of cysteine protease caspase-1–dependent IL-1β.26 Although under normal conditions ASC-associated inflammasomes are autorepressed, they become activated by a wide range of pathogen stimuli, including oxidative stress, ischemia, and damage signals. As an endogenous danger signal or alarmin, HMGB1, released from activated macrophages/necrotic cells, may bind immune receptors, including TLRs and RAGE, to trigger immune responses.21 This study has identified the essential role of HMGB1 in ASC/caspase-1/IL-1β–dependent inflammatory ASC KO responses in hepatic IRI. Indeed, global decreased sALT levels, depressed local macrophage/neutrophil sequestration, reduced hepatocellular apoptosis, and mitigated proinflammatory cytokine/chemokine programs in IR-stressed livers. Moreover, ASC deficiency diminished the induction of HMGB1 and alleviated IR-triggered liver damage through negative regulation of TLR4.
The molecular mechanisms of ASC/caspase-1/IL-1β signaling for programming an inflammatory phenotype might involve the activation of multiple intercellular pathways. We found that disruption of ASC inhibited HMGB1/TLR4 expression and led to decreased induction of inflammatory mediators; this suggests that ASC/caspase-1/IL-1β plays an important role in triggering local inflammation. In fact, the adaptor ASC was initially believed to exert its effects by bridging the interaction between NLRs and caspase-1 in inflammasome complexes.27 The activation of ASC within inflammasomes leads to the maturation of caspase-1 and the processing of its IL-1β and IL-18 substrates. Our in vitro data demonstrate that ASC deficiency decreased caspase-1 activity and IL-1β/IL-18 production in LPS-stimulated BMMs, and this implies a role for ASC in caspase-1/IL-1β–mediated inflammation.
Although the ASC/caspase-1/IL-1β axis is essential for the initiation of an inflammatory response, the molecular pathways involved in crosstalk with HMGB1 have not been elucidated. Our data demonstrate that the treatment of ASC KO mice with rHMGB1 increased IR-induced hepatocellular damage, whereas the disruption of ASC without exogenous rHMGB1 prevented hepatic inflammatory development. These results are consistent with the ability of endogenous HMGB1 to promote liver IR damage19 and suggest that HMGB1 might have a distinct role during ASC/IL-1β–mediated inflammation in hepatic IRI.
As an intracellular protein, HMGB1 translocates to the nucleus, where it binds DNA to regulate gene transcription.28 However, extracellular HMGB1 has been shown to act as a cytokine mediator in response to inflammatory stimuli due to infection,15 whereas HMGB1 promotes TLR4-mediated inflammation in hepatic IRI.3, 4 Both hepatocytes and infiltrating macrophages/monocytes (including Kupffer cells) did express HMGB1 in IR-stressed WT and ASC-deficient livers. However, HMGB1 levels selectively increased in the ischemic WT liver infiltrate but not in the ASC-deficient liver infiltrate (Supporting Fig. 1A-D). In agreement with our findings, enhanced HMGB1 activated TLR4/NF-κB and led to increased macrophage sequestration along with expression of proinflammatory cytokines (TNF-α/IL-12p40) and chemokines (MCP-1/CXCL-10). Using a well-controlled in vitro culture system, we found that ASC deficiency decreased mRNA and protein HMGB1 expression in LPS-stimulated BMMs and that TLR4 and NF-κB expression was diminished in ASC-deficient BMMs. Furthermore, rHMGB1 increased cleaved caspase-1 and IL-1β levels in BMM cultures (Supporting Fig. 3A,B), and this suggests that HMGB1 was at least in part responsible for the activation of caspase-1/IL-1β signaling in IR-stressed livers. Interestingly, ASC deficiency resulted in inhibition of phosphorylated p38 MAPK. In agreement with the essential role of MAPKs in IL-1β–mediated inflammation,29 our data imply that HMGB1 induction in the ASC/caspase-1/IL-1β–mediated inflammation cascade in hepatic IRI is p38 MAPK–dependent.
IL-1β is an important cytokine that targets inflammatory injury due to hepatic IR. IL-1β can express its biological activity only from pro–IL-1β to mature IL-1β through proteolytic cleavage by the protease caspase-130 and induce the release of HMGB1 from monocytes and macrophages.15 Moreover, IL-1β can form complexes with HMGB1 to enhance immune responses.31, 32 Our results demonstrate that IL-1β blockade reduced IR-induced hepatocellular damage and improved liver function. Blocking IL-1β decreased the expression of NF-κB and COX2. Indeed, COX2 overexpression has been associated with hypoxia/ischemia and inflammatory chronic diseases,33 whereas COX2 inhibition has improved liver transplant function.34 It is plausible that IL-1β stimulates COX2 production through NF-κB in IR-stressed livers. Our findings support recent clinical data on the efficacy of antihuman IL-1β (canakinumab) therapy in type 2 diabetes35 and autoimmune inflammatory disorders caused by mutations in the NLRP3 nucleotide-binding domain, such as cryopyrin-associated periodic syndromes36 and Schnitzler syndrome.37
ASC, originally identified as a protein that mediates apoptosis in human leukemia cells,38 interacts with B cell lymphoma 2–associated X protein (Bax) to induce apoptosis via the p53-Bax pathway.39 In addition, HMGB1 release can occur during the process of apoptotic cell death.40 Our data demonstrate that ASC knockdown decreased HMGB1 and caspase-3 but increased antiapoptotic Bcl-2/Bcl-xL expression. These findings were further supported by the increased frequency of apoptotic cells in WT ischemic livers, whereas ASC deficiency markedly decreased hepatocellular apoptosis. Hence, because ASC-mediated apoptosis is essential to the mechanism of hepatic IRI, blocking ASC-mediated signaling may represent a novel strategy for regulating apoptotic pathways in the ischemia-stressed liver.
Figure 7 depicts molecular mechanisms by which the ASC/caspase-1/IL-1β-HMGB1 axis may regulate the liver IRI immune cascade. ASC contributes to inflammatory responses through the activation of inflammasomes, which in turn activate caspase-1 and catalyze pro–IL-1β/pro–IL-18 into mature IL-1β/IL-18. IL-18 is closely related to and shares a similar dimensional structure with IL-1β. ASC/caspase-1/IL-1 promotes HMGB1 induction through the activation of p38 MAPK, which triggers TLR4 and NF-κB to program proinflammatory mediators. In addition, HMGB1 might provide a positive feedback mechanism to regulate caspase-1 activation. ASC/caspase-1–mediated elaboration of IL-1β and COX2 downstream are required for inflammatory development in the course of hepatic IRI.
In conclusion, ASC/caspase-1/IL-1β signaling promotes HMGB1 induction to facilitate a TLR4-dependent inflammatory phenotype leading to IR hepatocellular damage. By identifying HMGB1 as a novel mediator in ASC/caspase-1/IL-1β–triggered inflammation, our findings provide a rationale for refined therapeutic strategies against liver IRI.