Hepatocyte-specific Hmgb1 Deletion

Abstract

High mobility group box 1 (HMGB1) plays a key role in human health and disease. Currently, three different labs in the USA use Cre-lox technology to create mice with a hepatocyte-specific Hmgb1 deletion (HMGB1-HC-KO mice) by backcrossing Hmgb1 ^flox/^flox mice to Alb-Cre mice. This mouse strain has a different phenotype following exposure to several stressors.

We appreciate the interest in our review¹ and the opportunity to clarify theoretical and technical aspects describing the identification and influence of a hepatocyte-specific Hmgb1 deletion. We agree with Huebener et al.'s comments about the efficiency of Hmgb1 knockout in liver.

Currently, 3 different labs in the U.S. use Cre-lox technology to create mice with a hepatocyte-specific Hmgb1 deletion (HMGB1-HC-KO mice) by backcrossing Hmgb1^flox/^flox mice to Alb-Cre mice. This mouse strain has a different phenotype following exposure to several stressors.

Dr. Allan Tsung's lab: “Verification of specificity of HMGB1 KO in HMGB1-HC-KO mice was demonstrated by isolating hepatocytes and analyzing these cells for the presence of HMGB1 mRNA expression using RT-PCR with primers specific for exon 2 of HMGB1…HMGB1 was confirmed by western blotting that was present in both hepatocytes and nonparenchymal cells (NPCs) of control mice, whereas HMGB1-HC-KO mice had HMGB1 expressed only in NPCs.”² Both control and HMGB1-HC-KO mice are born healthy and fertile without any significant liver function abnormalities.² In contrast, HMGB1-HC-KO mice are more sensitive to ischemia/reperfusion injury due to mitochondrial and nuclear injury, indicating an anti-injury function of endogenous HMGB1 in the liver.²

Dr. Robert F. Schwabe's lab: “Quantitative real-time PCR and western blot analysis demonstrated efficient hepatic Hmgb1 deletion, with 90% and 72% reduction of mRNA and protein levels, respectively, and HMGB1 immunohistochemistry confirmed highly efficient deletion in hepatocytes, but not in NPCs.”³ These HMGB1-HC-KO mice have normal mitochondrial function and autophagic response under starvation conditions.³ In contrast, hepatocyte-specific HMGB1 ablation leads to 100% survival following lethal acetaminophen intoxication due to prevention against necrosis-induced sterile inflammation, indicating an injury-promoting function of endogenous HMGB1 in the liver.⁴

Dr. Natalia Nieto's lab: “Cell- and organ-specific Hmgb1 ablation was validated by IHC, which confirmed the absence of HMGB1 mostly in hepatocytes and not in other cells or organs such as the kidney.”⁵ Similarly, in the absence of any treatment, HMGB1-HC-KO mice show normal liver architecture.⁵ In contrast, HMGB1-HC-KO mice are more sensitive to alcohol-induced liver injury due to regulation of fatty acid synthesis and fatty acid β-oxidation, indicating an injury-promoting function of endogenous HMGB1 in the liver.⁵

These findings suggest that HMGB1 plays a dual role in the liver during stress.⁶ HMGB1 expression in the liver not only protects against ischemia/reperfusion injury, but also increases paracetamol toxicity and alcohol injury. As an essential process in maintaining cellular homeostasis and functions, autophagy is implicated in both normal and diseased liver tissue.⁷ Ischemia-reperfusion injury is an important cause of liver damage, occurring during surgical procedures (e.g., hepatic resection and liver transplantation) and hemorrhagic shock. Depending on the context, induction or impairment of autophagy during liver ischemia-reperfusion can either protect against or worsen damage. Induction of autophagy protects against acetaminophen-induced hepatotoxicity by mitophagy and oxidative stress reduction.⁸ An early study showed that autophagy is suppressed in alcoholic liver disease partly because the proteolytic capacity of liver lysosomes can be inhibited by alcohol.⁹ In contrast, recent studies indicate that alcohol can induce autophagy in liver cells in vitro or in vivo.^10,11 The long-term effects and implications of elevated autophagy in the alcohol-exposed liver remain unclear.

HMGB1-dependent autophagy is increasingly implicated in a number of pathophysiologies, including various inflammation-associated diseases and chemotherapy resistance.^1,12 Conditional knockout of HMGB1 in the pancreas,¹³ myeloid cells,¹⁴ and intestinal epithelial cells¹⁵ in mice renders them more sensitive to infection and sterile inflammation partly by promoting nucleosome release and inhibition of autophagy. Loss of HMGB1 in myeloid cells prevents autophagic degradation of inflammasomes.¹⁴ HMGB1 interacts with BECN1 and ATG5 to prevent calpain-mediated cleavage of these proteins, allowing autophagy to proceed.¹⁵ This finding suggests a novel mechanism of the cross-regulation between autophagy and apoptosis during inflammation. The role of HMGB1 in health and disease is not only tissue- and cell-specific,¹⁶ but also structure- and modification-specific.¹⁷ Autophagy activity does not change in a mouse model in which HMGB1-HC-KO mice are crossed to GFP-LC3 transgenic mice under starvation conditions, suggesting that an HMGB1-independent autophagy system exists in the liver.³ Although very useful for in vivo studies, this GFP-LC3 mouse model has a few possible limitations. For example, autophagosomes fuse with lysosomes rapidly, which can reduce the number of GFP-LC3 dots and lead to an underestimation of the autophagy status.¹⁸ Additionally, GFP-LC3 is easily incorporated into intracellular protein aggregates independent of autophagy. This is particularly apparent in mice with conditional knockout of Atg5 and Atg7 in hepatocytes and neurons.^19-21

The different phenotype resulting from Hmgb1 knockout could be derived from some difference(s) in factors/conditions other than the deletion efficiency. Tissue-specific Hmgb1 knockout is desired to avoid off-target effects; however, gene editing and recombination technology is evolving quickly. The liver plays an extremely complex role in metabolism. In-depth studies of HMGB1-dependent and independent autophagy will promote a better understanding of the underlying mechanisms of liver disease. We encourage exchanging HMGB1-HC-KO mice to allow the assessment of multiple functions of HMGB1 in the liver.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Christine Heiner (Department of Surgery, University of Pittsburgh) for her critical reading of the manuscript.

Funding

This work was supported by the National Institutes of Health (R01CA160417 to D.T.), The National Natural Science Foundation-Guangdong Joint Fund (U1132005 and 31171229 to X.S.), and a Science and Information Technology of Guangzhou Key Project (2011Y1-00038 and 20140000000-4,3 to X.S.).