Exercise Training Decreases Hepatic Injury and Metastases Through Changes in Immune Response to Liver Ischemia/Reperfusion in Mice.

BACKGROUND AND AIMS: Liver ischemia/reperfusion injury (IRI) induces local and systemic inflammation in which neutrophil extracellular traps (NETs) are major drivers. IRI markedly augments metastatic growth, which is consistent with the notion that the liver IRI can serve as a premetastatic niche. Exercise training (ExT) confers a sustainable protection, reducing IRI in some animal models, and has been associated with improved survival in patients with cancer; however, the impact of ExT on liver IRI or development of hepatic metastases is unknown. APPROACH AND RESULTS: Mice were randomized into exercise (ExT) and sedentary groups before liver IRI and tumor injection. Computerized dynamic network analysis of 20 inflammatory mediators was used to dissect the sequence of mediator interactions after ischemia/reperfusion (I/R) that induce injury. ExT mice showed a significant decrease in hepatic IRI and tissue necrosis. This coincided with disassembly of complex networks among inflammatory mediators seen in sedentary mice. Neutrophil infiltration and NET formation were decreased in the ExT group, which suppressed the expression of liver endothelial cell adhesion molecules. Concurrently, ExT mice revealed a distinct population of infiltrating macrophages expressing M2 phenotypic genes. In a metastatic model, fewer metastases were present 3 weeks after I/R in the ExT mice, a finding that correlated with a marked increase in tumor-suppressing T cells within the tumor microenvironment. CONCLUSIONS: ExT preconditioning mitigates the inflammatory response to liver IRI, protecting the liver from injury and metastases. In light of these findings, potential may exist for the reduction of liver premetastatic niches induced by liver IRI through the use of ExT as a nonpharmacologic therapy before curative surgical approaches.

Neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive, inflammatory form of fatty liver disease. It is the most rapidly rising risk factor for the development of hepatocellular carcinoma (HCC), which can arise in NASH with or without cirrhosis. The inflammatory signals promoting the progression of NASH to HCC remain largely unknown. The propensity of neutrophils to expel decondensed chromatin embedded with inflammatory proteins, known as neutrophil extracellular traps (NETs), has been shown to be important in chronic inflammatory conditions and in cancer progression. In this study, we asked whether NET formation occurs in NASH and contributes to the progression of HCC. We found elevated levels of a NET marker in serum of patients with NASH. In livers from STAM mice (NASH induced by neonatal streptozotocin and high-fat diet), early neutrophil infiltration and NET formation were seen, followed by an influx of monocyte-derived macrophages, production of inflammatory cytokines, and progression of HCC. Inhibiting NET formation, through treatment with deoxyribonuclease (DNase) or using mice knocked out for peptidyl arginine deaminase type IV (PAD4-/- ), did not affect the development of a fatty liver but altered the consequent pattern of liver inflammation, which ultimately resulted in decreased tumor growth. Mechanistically, we found that commonly elevated free fatty acids stimulate NET formation in vitro. CONCLUSION: Our findings implicate NETs in the protumorigenic inflammatory environment in NASH, suggesting that their elimination may reduce the progression of liver cancer in NASH. (Hepatology 2018).

Hypoxia mediates mitochondrial biogenesis in hepatocellular carcinoma to promote tumor growth through HMGB1 and TLR9 interaction.

The ability of cancer cells to survive and grow under hypoxic conditions has been known for decades, but the mechanisms remain poorly understood. Under certain conditions, cancer cells undergo changes in their bioenergetic profile to favor mitochondrial respiration by activating the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and up-regulating mitochondrial biogenesis. In this study, we hypothesized that augmented mitochondrial biogenesis plays a critical role for cancer cells to survive hypoxia. Consistent with this hypothesis, both hypoxic human hepatocellular carcinoma (HCC) tumors and HCC cell lines subjected to hypoxia increase mitochondrial biogenesis. Silencing of PGC-1α in hypoxic HCC cell lines halts their proliferation. Mechanistic investigations in vitro indicated that intracellular high mobility group box 1 (HMGB1) protein, a nuclear protein overexpressed in HCC, is essential for the process. Silencing of HMGB1 in hypoxic HCC cell lines resulted in a significant decrease in PGC-1α activation and mitochondrial biogenesis. Without HMGB1, hypoxic HCC cells had significantly reduced adenosine triphosphate production, decreased cellular proliferation, and increased apoptosis. In a diethylnitrosamine-induced murine model of HCC, genetic blocking of HMGB1 in hypoxic tumors resulted in a significant decrease in tumor growth. Tumors lacking HMGB1 had a significant reduction in mitochondrial biogenesis and a significant increase in mitochondrial dysfunction. Further in vitro mechanistic experiments indicated that during hypoxia HMGB1 translocates from the nucleus to the cytoplasm and binds to cytoplasmic Toll-like receptor-9. This binding leads to activation of p38 and subsequent phosphorylation of PGC-1α, with resultant up-regulation of mitochondrial biogenesis. CONCLUSION: Taken together, our findings suggest that during hypoxia HMGB1 up-regulates mitochondrial biogenesis in HCC cancer cells, promoting tumor survival and proliferation. (Hepatology 2017;66:182-197).

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

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).

Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury.

UNLABELLED: Innate immunity plays a crucial role in the response to sterile inflammation such as liver ischemia/reperfusion (I/R) injury. The initiation of liver I/R injury results in the release of damage-associated molecular patterns, which trigger an innate immune and inflammatory cascade through pattern recognition receptors. Neutrophils are recruited to the liver after I/R and contribute to organ damage and innate immune and inflammatory responses. Formation of neutrophil extracellular traps (NETs) has been recently found in response to various stimuli. However, the role of NETs during liver I/R injury remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo. This was associated with increased NET markers, serum level of myeloperoxidase-DNA complexes, and tissue level of citrullinated-histone H3 compared to control mice. Treatment with peptidyl-arginine-deiminase 4 inhibitor or DNase I significantly protected hepatocytes and reduced inflammation after liver I/R as evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. In vitro, NETs increase hepatocyte death and induce Kupffer cells to release proinflammatory cytokines. Damage-associated molecular patterns, such as High Mobility Group Box 1 and histones, released by injured hepatocytes stimulate NET formation through Toll-like receptor (TLR4)- and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout or TLR9 knockout neutrophils confers significant protection from liver I/R injury with a significant decrease in NET formation. In addition, we found inhibition of NET formation by the peptidyl-arginine-deiminase 4 inhibitor and that DNase I reduces High Mobility Group Box 1 and histone-mediated liver I/R injury. CONCLUSION: Damage-associated molecular patterns released during liver I/R promote NET formation through the TLR signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R.

Delayed inhaled carbon monoxide mediates the regression of established neointimal lesions.

OBJECTIVE: Intimal hyperplasia (IH) contributes to the failure of vascular interventions. While many investigational therapies inhibit the development of IH in animal models, few of these potential therapies can reverse established lesions. Inhaled carbon monoxide (CO) dramatically inhibits IH in both rats and pigs when given perioperatively. It also prevented the development of pulmonary arterial hypertension in rodents. Interestingly, CO could reverse pulmonary artery structural changes and right heart hemodynamic changes when administered after the establishment of pulmonary hypertension. Thus, we hypothesize that inhaled CO may mediate the regression of established neointimal lesions. METHODS: Rats underwent carotid artery balloon angioplasty injury. Carotid arteries were collected at 2 and 4 weeks after injury for morphometric analysis of the neointima. Another group was treated with inhaled CO (250 parts per million) for 1 hour daily from week 2 until week 4. Additional rats were sacrificed 3 days after initiating CO treatment, and the carotid arteries were examined for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end-labeling, proliferation by Ki67 staining, and autophagy by microtubule-associated protein light chain 3 I/II staining. RESULTS: At 2 weeks following injury, sizable neointimal lesions had developed (intimal/media = 0.92 ± 0.22). By 4 weeks, lesion size remained stable (0.80 ± 0.09). Delayed inhaled CO treatment greatly reduced neointimal lesion size vs the 2- and 4-week control mice (0.38 ± 0.05; P < .05). Arteries from the CO-treated rats exhibited significantly reduced apoptosis compared with control vessels (3.18% ± 1.94% vs 16.26% ± 5.91%; P = .036). Proliferation was also dramatically reduced in the CO-treated animals (2.98 ± 1.55 vs 10.37 ± 2.80; P = .036). No difference in autophagy between control and CO-treated rats was detected. CONCLUSIONS: Delayed administration of inhaled CO reduced established neointimal lesion size. This effect was mediated by the antiproliferative effect of CO on medial and intimal smooth muscle cells without increases in arterial wall apoptosis or autophagy. Future studies will examine additional time points to determine if there is temporal variation in the rates of apoptosis and autophagy.

Hepatocyte-specific high-mobility group box 1 deletion worsens the injury in liver ischemia/reperfusion: a role for intracellular high-mobility group box 1 in cellular protection.

UNLABELLED: High-mobility group box 1 (HMGB1) is an abundant chromatin-associated nuclear protein and released into the extracellular milieu during liver ischemia-reperfusion (I/R), signaling activation of proinflammatory cascades. Because the intracellular function of HMGB1 during sterile inflammation of I/R is currently unknown, we sought to determine the role of intracellular HMGB1 in hepatocytes after liver I/R. When hepatocyte-specific HMGB1 knockout (HMGB1-HC-KO) and control mice were subjected to a nonlethal warm liver I/R, it was found that HMGB1-HC-KO mice had significantly greater hepatocellular injury after I/R, compared to control mice. Additionally, there was significantly greater DNA damage and decreased chromatin accessibility to repair with lack of HMGB1. Furthermore, lack of hepatocyte HMGB1 led to excessive poly(ADP-ribose)polymerase 1 activation, exhausting nicotinamide adenine dinucleotide and adenosine triphosphate stores, exacerbating mitochondrial instability and damage, and, consequently, leading to increased cell death. We found that this was also associated with significantly more oxidative stress (OS) in HMGB1-HC-KO mice, compared to control. Increased nuclear instability led to a resultant increase in the release of histones with subsequently more inflammatory cytokine production and organ damage through activation of Toll-like receptor 9. CONCLUSION: The lack of HMGB1 within hepatocytes leads to increased susceptibility to cellular death after OS conditions.