DNA: Adding Injury to Insult See Article on Page 348
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repair response is initiated immediately after tissue injury and is needed for a return to tissue homeostasis. An inflammatory response is also initiated after tissue injury and is impressive for its presence after a wide range of insults ranging from trauma, myocardial infarction, to acetaminophen (APAP)-induced hepatocyte death.1 The key features of this response have been identified since ancient times as heat, pain, redness, and swelling, occurring within minutes of tissue injury even in the absence of pathogens. The theoretical framework for explaining the connection between tissue damage and inflammation is based predominantly on the concept of release of damage-associated intracellular molecular pattern molecules (DAMPs) from the damaged cells, resulting in activation of innate immune cells. DAMPs are structurally diverse, ranging from adenosine triphosphate ATP to DNA, which is a ligand for Toll-like receptor 9 (TLR9).2 The clinical significance of the postinjury sterile inflammatory response is that it has repeatedly been shown to increase overall tissue injury, particularly in the liver, which has a robust innate immune system and a large sterile inflammatory response.1 The key demonstration of a role for TLR9 in postinjury inflammatory response was provided for acute injury through APAP and ischemia reperfusion, and this has subsequently been confirmed.3-5 The article by Marques et al. in the current issue provides fascinating new information on broader aspects on the role of DNA and TLR9 in liver inflammation.6 They initially show that, in hepatocytes cultured with APAP, there are nucleic-acid–containing structures in the cytosol and release of DNA into the supernatant. Extracellular Abbreviations: ALI, acute liver injury; APAP, acetaminophen; DAMPs, damage-associated intracellular molecular pattern molecules; HMGB1, high-mobility group box 1; Toll-like receptor 9. Received July 1, 2014; accepted August 21, 2014. Address reprint requests to: Wajahat Z. Mehal, M.D., D.Phil., Section of Digestive Diseases, Yale University, 333 Cedar Street, New Haven, CT 065208019. E-mail: [email protected]; fax: 203-785-7273. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27398 Potential conflict of interest: Nothing to report.
DNA is also demonstrated in vivo after APAP toxicity and is notable for its scale, the fact that it can be detected 6 hours before development of significant necrosis, and also occurs in response to thioacetamideinduced toxicity. These points argue that DNA release is a significant event after a broad range of hepatocyte insults. Neutrophils are an important component of the acute inflammatory response and are also known to extrude DNA in response to mitochondrial and bacterial signals. In APAP-induced injury, the DNA appears to be coming mostly from hepatocytes, and not from neutrophils, which, however, were important for the full necrotic injury response. APAP injury was associated with up-regulation of TLR9 on neutrophils and activation of, predominantly, a nuclear factor kappa B pathway. It was previously demonstrated that a TLR9 antagonist can block APAP injury, and the current article provides evidence for a direct role of extracellular DNA by demonstrating protection through DNASE1. A recent complementary article extends the relevance of these findings to humans.7 Patients with APAP toxicity have elevations in serum DNA, but the clinical significance of this was unknown. Serum levels of mitochondrial and nuclear DNA were, however, higher in patients who died from APAP toxicity than those who survived and were predictive of survival to the same degree as the Model for End-Stage Liver Disease score.7 Collectively, these two studies have a number of important implications. The most obvious is that they complement each other with one providing mechanistic, and the second correlative, clinical data. In addition, approximately 20 DAMPs have been identified and a number of them have been shown to be required for maximal liver injury based on carefully controlled rodent experiments.8 However, these are primarily proof-of-concept experiments with little experimental cross-referencing to evaluate the relative importance of individual DAMPs. This results in a lack of confidence in the clinical relevance of any individual DAMP resulting from, likely, redundancy. These two studies place DNA as an important player in the inflammation that occurs after acute liver injury (ALI) and immediately suggest novel interventions, such as TLR9 antagonists and DNASE1. The identification of elevated levels of DNA in serum of patients with APAP and other acute toxic 35
36
GARCIA-MARTINEZ AND MEHAL
injuries raises a number of issues. It places liver injury in the growing area of the science and biology of extracellular DNA, which has implications for pathogenesis, diagnostics, and therapy. For pathogenesis, it is likely that it is not just the amount of DNA, but also its qualitative characteristics that will be important. These characteristics will range from modifications, such as methylation, to tertiary structure. Hypomethylated DNA is well known to be a stronger ligand for TLR9 than methylated DNA, and other structural modifications also result in changes in ability to activate TLR9.9 Changes in TLR9 ligand activity are similarly induced by cellular proteins that are bound to DNA. For example, high-mobility group box 1 (HMGB1) significantly increases the ability of DNA to activate TLR9. Qualitative changes, particularly changes in methylation, have been reported in circulating DNA of tumor origin and are more likely associated with actively transcribed regions, which are less tightly packed.10 Such an association opens up the possibility that the transcriptional state of the cell may affect the inflammatory activity of DNA released. The increasing confidence of a role for extracellular DNA in liver injury has the added value of explaining the end-organ damage that is frequently observed during ALI. Extracellular DNA may be acting as a DAMP and inducing lung and liver injury. This could be further investigated by testing the ability of serum from patients with APAP toxicity to activate immune cells in vitro and seeing whether this correlates with end-organ damage. Diagnostically, a particular DNA profile may be associated with liver diseases of different etiologies and provide an additional level of information. Therapeutically, this opens up for testing TLR9 antagonists that are in clinical development for treating Lupus, as well as DNA-binding polymers that can scavenge DNA and prevent binding to receptors.11 Antibody-mediated therapy to components of known DNA-binding proteins, such as HMGB1, offer additional options. It is interesting to note that the highly stable structure of DNA makes it well suited to survive the myriad of insults that can kill a cell, allowing DNA to remain intact to function as a DAMP.
HEPATOLOGY, January 2015
With the explosion of information on new molecules, DNA can sometimes seem to be a wellinvestigated molecule that has nothing new to reveal. As these studies show, this is not the case, and this original rock star of biology still has some undiscovered functions.
IRMA GARCIA-MARTINEZ, PH.D. WAJAHAT Z. MEHAL, M.D., D.PHIL.
Section of Digestive Diseases Yale University New Haven, CT
References 1. Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology 2012;143:1158-1172. 2. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 2010;464:104-107. 3. Imaeda AB, Watanabe A, Sohail MA, Mahmood S, Mohamadnejad M, Sutterwala FS, et al. Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest 2009; 119:305-314. 4. Bamboat ZM, Balachandran VP, Ocuin LM, Obaid H, Plitas G, DeMatteo RP. Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury. HEPATOLOGY 2010;51:621-632. 5. Gill R, Ruan X, Menzel CL, Namkoong S, Loughran P, Hackam DJ, Billiar TR. Systemic inflammation and liver injury following hemorrhagic shock and peripheral tissue trauma involve functional TLR9 signaling on bone marrow-derived cells and parenchymal cells. Shock 2011;35:164-170. 6. Marques PE, Oliveira AG, Pereira RV, David BA, Gomides LF, Saraiva AM, et al. Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice. HEPATOLOGY 2015;61:348-360. 7. McGill MR, Staggs VS, Sharpe MR, Lee WM, Jaeschke H; Acute Liver Failure Study Group. Serum mitochondrial biomarkers and damageassociated molecular patterns are higher in acetaminophen overdose patients with poor outcome. HEPATOLOGY 2014;60:1336-1345. 8. Mehal WZ. Who is the scientist in bio-medical research, the author or the reviewer? Front Pharmacol 2014;5:50. 9. Wang Z, Zheng Y, Hou C, Yang L, Li X, Lin J, et al. DNA methylation impairs TLR9 induced Foxp3 expression by attenuating IRF-7 binding activity in fulminant type 1 diabetes. J Autoimmun 2013;41:50-59. 10. Rykova EY, Morozkin ES, Ponomaryova AA, Loseva EM, Zaporozhchenko IA, Cherdyntseva NV, Vlassov VV, et al. Cell-free and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content. Expert Opin Biol Ther 2012;12(Suppl 1):S141-S153. 11. Hennessy EJ, Parker AE, O’Neill LA. Targeting Toll-like receptors: emerging therapeutics? Nat Rev Drug Discov 2010;9:293-307.
A
repair response is initiated immediately after tissue injury and is needed for a return to tissue homeostasis. An inflammatory response is also initiated after tissue injury and is impressive for its presence after a wide range of insults ranging from trauma, myocardial infarction, to acetaminophen (APAP)-induced hepatocyte death.1 The key features of this response have been identified since ancient times as heat, pain, redness, and swelling, occurring within minutes of tissue injury even in the absence of pathogens. The theoretical framework for explaining the connection between tissue damage and inflammation is based predominantly on the concept of release of damage-associated intracellular molecular pattern molecules (DAMPs) from the damaged cells, resulting in activation of innate immune cells. DAMPs are structurally diverse, ranging from adenosine triphosphate ATP to DNA, which is a ligand for Toll-like receptor 9 (TLR9).2 The clinical significance of the postinjury sterile inflammatory response is that it has repeatedly been shown to increase overall tissue injury, particularly in the liver, which has a robust innate immune system and a large sterile inflammatory response.1 The key demonstration of a role for TLR9 in postinjury inflammatory response was provided for acute injury through APAP and ischemia reperfusion, and this has subsequently been confirmed.3-5 The article by Marques et al. in the current issue provides fascinating new information on broader aspects on the role of DNA and TLR9 in liver inflammation.6 They initially show that, in hepatocytes cultured with APAP, there are nucleic-acid–containing structures in the cytosol and release of DNA into the supernatant. Extracellular Abbreviations: ALI, acute liver injury; APAP, acetaminophen; DAMPs, damage-associated intracellular molecular pattern molecules; HMGB1, high-mobility group box 1; Toll-like receptor 9. Received July 1, 2014; accepted August 21, 2014. Address reprint requests to: Wajahat Z. Mehal, M.D., D.Phil., Section of Digestive Diseases, Yale University, 333 Cedar Street, New Haven, CT 065208019. E-mail: [email protected]; fax: 203-785-7273. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27398 Potential conflict of interest: Nothing to report.
DNA is also demonstrated in vivo after APAP toxicity and is notable for its scale, the fact that it can be detected 6 hours before development of significant necrosis, and also occurs in response to thioacetamideinduced toxicity. These points argue that DNA release is a significant event after a broad range of hepatocyte insults. Neutrophils are an important component of the acute inflammatory response and are also known to extrude DNA in response to mitochondrial and bacterial signals. In APAP-induced injury, the DNA appears to be coming mostly from hepatocytes, and not from neutrophils, which, however, were important for the full necrotic injury response. APAP injury was associated with up-regulation of TLR9 on neutrophils and activation of, predominantly, a nuclear factor kappa B pathway. It was previously demonstrated that a TLR9 antagonist can block APAP injury, and the current article provides evidence for a direct role of extracellular DNA by demonstrating protection through DNASE1. A recent complementary article extends the relevance of these findings to humans.7 Patients with APAP toxicity have elevations in serum DNA, but the clinical significance of this was unknown. Serum levels of mitochondrial and nuclear DNA were, however, higher in patients who died from APAP toxicity than those who survived and were predictive of survival to the same degree as the Model for End-Stage Liver Disease score.7 Collectively, these two studies have a number of important implications. The most obvious is that they complement each other with one providing mechanistic, and the second correlative, clinical data. In addition, approximately 20 DAMPs have been identified and a number of them have been shown to be required for maximal liver injury based on carefully controlled rodent experiments.8 However, these are primarily proof-of-concept experiments with little experimental cross-referencing to evaluate the relative importance of individual DAMPs. This results in a lack of confidence in the clinical relevance of any individual DAMP resulting from, likely, redundancy. These two studies place DNA as an important player in the inflammation that occurs after acute liver injury (ALI) and immediately suggest novel interventions, such as TLR9 antagonists and DNASE1. The identification of elevated levels of DNA in serum of patients with APAP and other acute toxic 35
36
GARCIA-MARTINEZ AND MEHAL
injuries raises a number of issues. It places liver injury in the growing area of the science and biology of extracellular DNA, which has implications for pathogenesis, diagnostics, and therapy. For pathogenesis, it is likely that it is not just the amount of DNA, but also its qualitative characteristics that will be important. These characteristics will range from modifications, such as methylation, to tertiary structure. Hypomethylated DNA is well known to be a stronger ligand for TLR9 than methylated DNA, and other structural modifications also result in changes in ability to activate TLR9.9 Changes in TLR9 ligand activity are similarly induced by cellular proteins that are bound to DNA. For example, high-mobility group box 1 (HMGB1) significantly increases the ability of DNA to activate TLR9. Qualitative changes, particularly changes in methylation, have been reported in circulating DNA of tumor origin and are more likely associated with actively transcribed regions, which are less tightly packed.10 Such an association opens up the possibility that the transcriptional state of the cell may affect the inflammatory activity of DNA released. The increasing confidence of a role for extracellular DNA in liver injury has the added value of explaining the end-organ damage that is frequently observed during ALI. Extracellular DNA may be acting as a DAMP and inducing lung and liver injury. This could be further investigated by testing the ability of serum from patients with APAP toxicity to activate immune cells in vitro and seeing whether this correlates with end-organ damage. Diagnostically, a particular DNA profile may be associated with liver diseases of different etiologies and provide an additional level of information. Therapeutically, this opens up for testing TLR9 antagonists that are in clinical development for treating Lupus, as well as DNA-binding polymers that can scavenge DNA and prevent binding to receptors.11 Antibody-mediated therapy to components of known DNA-binding proteins, such as HMGB1, offer additional options. It is interesting to note that the highly stable structure of DNA makes it well suited to survive the myriad of insults that can kill a cell, allowing DNA to remain intact to function as a DAMP.
HEPATOLOGY, January 2015
With the explosion of information on new molecules, DNA can sometimes seem to be a wellinvestigated molecule that has nothing new to reveal. As these studies show, this is not the case, and this original rock star of biology still has some undiscovered functions.
IRMA GARCIA-MARTINEZ, PH.D. WAJAHAT Z. MEHAL, M.D., D.PHIL.
Section of Digestive Diseases Yale University New Haven, CT
References 1. Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology 2012;143:1158-1172. 2. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 2010;464:104-107. 3. Imaeda AB, Watanabe A, Sohail MA, Mahmood S, Mohamadnejad M, Sutterwala FS, et al. Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest 2009; 119:305-314. 4. Bamboat ZM, Balachandran VP, Ocuin LM, Obaid H, Plitas G, DeMatteo RP. Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury. HEPATOLOGY 2010;51:621-632. 5. Gill R, Ruan X, Menzel CL, Namkoong S, Loughran P, Hackam DJ, Billiar TR. Systemic inflammation and liver injury following hemorrhagic shock and peripheral tissue trauma involve functional TLR9 signaling on bone marrow-derived cells and parenchymal cells. Shock 2011;35:164-170. 6. Marques PE, Oliveira AG, Pereira RV, David BA, Gomides LF, Saraiva AM, et al. Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice. HEPATOLOGY 2015;61:348-360. 7. McGill MR, Staggs VS, Sharpe MR, Lee WM, Jaeschke H; Acute Liver Failure Study Group. Serum mitochondrial biomarkers and damageassociated molecular patterns are higher in acetaminophen overdose patients with poor outcome. HEPATOLOGY 2014;60:1336-1345. 8. Mehal WZ. Who is the scientist in bio-medical research, the author or the reviewer? Front Pharmacol 2014;5:50. 9. Wang Z, Zheng Y, Hou C, Yang L, Li X, Lin J, et al. DNA methylation impairs TLR9 induced Foxp3 expression by attenuating IRF-7 binding activity in fulminant type 1 diabetes. J Autoimmun 2013;41:50-59. 10. Rykova EY, Morozkin ES, Ponomaryova AA, Loseva EM, Zaporozhchenko IA, Cherdyntseva NV, Vlassov VV, et al. Cell-free and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content. Expert Opin Biol Ther 2012;12(Suppl 1):S141-S153. 11. Hennessy EJ, Parker AE, O’Neill LA. Targeting Toll-like receptors: emerging therapeutics? Nat Rev Drug Discov 2010;9:293-307.
