Cell Death and Damps in Acute Pancreatitis

Cell Death and DAMPs in Acute Pancreatitis

Rui Kang, Michael T Lotze, Herbert J Zeh, Timothy R Billiar, and Daolin Tang

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America

Cell death and inflammation are key pathologic responses of acute pancreatitis (AP), the leading cause of hospital admissions for gastrointestinal disorders. It is becoming increasingly clear that damage-associated molecular pattern molecules (DAMPs) play an important role in the pathogenesis of AP by linking local tissue damage to systemic inflammation syndrome. Endogenous DAMPs released from dead, dying or injured cells initiate and extend sterile inflammation via specific pattern recognition receptors. Inhibition of the release and activity of DAMPs (for example, high mobility group box 1, DNA, histones and adenosine triphosphate) provides significant protection against experimental AP. Moreover, increased serum levels of DAMPs in patients with AP correlate with disease severity. These findings provide novel insight into the mechanism, diagnosis and management of AP. DAMPs might be an attractive therapeutic target in AP. Online address: http://www.molmed.org doi: 10.2119/molmed.2014.00117

INTRODUCTION tion of comorbid disease (4). About one- This triggers downstream signaling and Acute pancreatitis (AP) is inflamma- third of patients with AP develop severe manifests as sterile inflammation tion of the pancreas that can become a necrotizing pancreatitis with persistent (9,12,14). In this review, we outline the fatal disease or lead to severe complica- MOF and a high mortality rate. The main pathogenesis of AP, discuss the relation- tions (1). It is characterized clinically by goals in the clinical management of AP ship between cell death and AP, and abdominal pain and by increased pancre- are adequate fluid resuscitation and the highlight the latest exciting advances in atic enzyme levels in the blood or urine. prevention of MOF (6,7). Both genetic the pathogenic role of DAMPs in AP. Gallstone migration and alcohol abuse and environmental factors affect the de- are the two major risk factors for AP in velopment and severity of pancreatitis PATHOGENESIS OF ACUTE humans (2,3). According to the updated (8). Although the pathogenic mecha- PANCREATITIS Atlanta classification, AP is generally di- nisms remain largely unknown, increas- The pancreas is a dual-purpose gland vided into mild, moderate or severe pan- ing evidence suggests that damage- with exocrine and endocrine functions. creatitis according to the presence or ab- associated molecular pattern molecules The exocrine pancreas produces digestive sence of multiple organ failure (MOF) or (DAMPs) play a central role in the patho- proteases in inactive proenzyme form, local or systemic complications (4). Mild genesis of AP. DAMPs link local tissue namely zymogens. The pancreas is nor- pancreatitis has a good prognosis with damage to systemic inflammation re- mally able to protect itself from zymogen rapid recovery. The late consequences of sponse syndrome (SIRS), which, if severe activation by synthesis of protease in- AP include impaired pancreatic exocrine or sustained, can lead to subsequent hibitors such as pancreatic secretory function and glucose tolerance, diabetes MOF and even death (9,11) (Figure 1). trypsin inhibitor and serine protease in- and development of chronic pancreatitis Most DAMPs are recognized by mem- hibitor (for example, SPINK1/Spink3). By (5). Moderately severe AP is character- brane-bound and cytosolic pattern recog- contrast, pancreatic autodigestion and ized by the presence of transient organ nition receptors (PRRs) expressed by subsequent AP is initiated once these de- failure, local complications or exacerba- both immune and nonimmune cell types. fenses are impaired. In studies of rodent models (for example, repetitive cerulein or L-arginine injections, choline-deficient ethionine supplementation [CDE] diet, Address correspondence to Rui Kang or Daolin Tang, Department of Surgery, University of perfusion of taurocholate into the biliary Pittsburgh, 5117 Centre Ave, Hillman Cancer Center, Pittsburgh, Pennsylvania, 15213. Phone: or pancreatic duct and surgical ligation or 412-623-1211; Fax: 412-623-1212: E-mail: [email protected] or [email protected]. infusion of pancreatic duct models), some Submitted June 11, 2014; Accepted for publication August 4, 2014; Epub essential pathogenic AP events have been (www.molmed.org) ahead of print August 5, 2014. identified (see Figure 1) (15,16). The development of AP involves a complex cascade of events (17), which start with injury or disruption of the pan-

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[NF-κB], mitogen-activated protein kinase [MAPK], signal transducer and activator of transcription 3 [STAT3] and inflammasome). Of these, NF-κB appears to the main inflammatory pathway in pancreatitis, although conflicting reports have been published (27–30). Recruitment and activated innate immune cells at the site of injury lead to further acinar cell injury and increase the levels of circulating DAMPs. It is important to note that neutrophil depletion markedly reduces tissue damage severity in AP, suggesting a critical role for tissue infiltration of neutrophils in AP (31). DAMPs ultimately are the mediators of the systemic inflammatory response and cause further pancreatic damage, as well as MOF. In particular, acute lung injury and adult respiratory distress syndrome are the leading causes of death in patients with AP (6). More recently, myeloid NF-κB activation was found to contribute to IL-6 synthesis and IL-6 transsignaling, which promotes pancreatitis-associated lung injury and lethality (32). A better under- Figure 1. Emerging role of DAMPs in acute pancreatitis. A number of rodent models have standing of the relationship between local proven useful for studying the pathogenic mechanisms of acute pancreatitis. Increasing tissue injury and systemic inflammation evidence indicates that DAMPs such as HMGB1, DNA, HSPs, histones and ATP play a cen- syndrome in the development of AP may tral role in the pathogenesis of acute pancreatitis because DAMPs link local tissue dam- provide more targeted therapeutic op- age and death to systemic inflammation response syndrome, which leads to subsequent tions (see Figure 1). MOF and even death.

CELL DEATH AND ACUTE PANCREATITIS creatic acini, which then permits the leak- reticulum, can cause mitochondrial cal- The severity of experimental AP corre- age of active pancreatic enzymes includ- cium overload, which leads to enhanced lates with the extent and type of cell in- ing amylolytic, lipolytic and proteolytic generation of mitochondrial ROS and mi- jury and death. Although multiple forms enzymes that destroy local tissues. This tochondrial membrane permeabilization. of cell death exist in physiological and results in edema, vascular damage, hem- Mitochondria dysfunction-mediated ox- pathological conditions (33), necrosis and orrhage and cell death (18,19). In addition idative injury results in endoplasmic apoptosis are the most widely studied to oxidative stress (20) and calcium over- reticulum stress, lysosomal damage and types in both clinical and experimental load (21), hypotension (22) and low aci- the release of proteases (for example, AP (34,35). Necrotic cells are capable of nar pH (23) contribute to these initiation cathepsin and trypsin) to degrade cytoso- activating proinflammatory and im- processes. After initial production of ac- lic proteins that cause pancreatic acinar munostimulatory responses by releasing tive pancreatic enzymes, local cell death cell death (25,26). Dead, dying and in- DAMPs and other molecules, whereas and systemic inflammation ensue. Mito- jured pancreatic acinar cells release intra- apoptosis is usually considered immuno- chondria, the energy factories of cells, cellular contents, including DAMPs (for logically silent because the cytoplasmic regulate pancreatic cell death through example, high mobility group box 1 content is packaged in apoptotic bodies control of the production of adenosine [HMGB1], DNA, histones and ATP), and these membrane-bound cell frag- triphosphate (ATP) and reactive oxygen which in turn promote infiltration of vari- ments are rapidly taken up and de- species (ROS), as well as calcium (24). ous immune cells (for example, neu- graded by phagocytes or autophagy (36). Dysfunction of mitochondrial calcium trophils, monocytes and macrophages) However, excessive apoptotic cells can uptake and efflux, including elevation of and activation of inflammatory signaling activate the inflammatory and immune cytosolic calcium from the endoplasmic pathways (for example, nuclear factor-κB responses by releasing nuclear DAMPs

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served in necrosis, whereas pyknosis and karyorrhexis occur in apoptotic cells. A number of observations have indicated that necrosis is the major type of pancreatic acinar cell death (34,41). Al- though some data clearly implicates that the release of DAMPs from necrosis is responsible for the inflammatory response, much less is known about the signaling mechanisms responsible for this function. A good example of such a mechanism is derived from the study of poly (ADP-ribose) polymerase (PARP) in necrosis. PARP-1 activation during DNA damage induces energy failure by de- pleting NAD+; the cell consumes ATP to replete the NAD+ level, ultimately resulting in energy failure and DAMP release in necrosis (42). Genetic or pharmacologic blockade of PARP inhibits necrosis and demonstrates a reduced pancreatitis response (43). In addition to the passive ATP depletion-mediated mechanisms, several active mechanisms (for example, oxidative stress, calcium overload, mitochondrial permeability transition pore opening and cathepsin release) also participate in the regulation Figure 2. Release and activity of DAMPs in acute pancreatitis. Various pathologic insults of the necrotic process in AP (34,44,45). cause mitochondrial dysfunction and endoplasmic reticulum stress in pancreatic acinar The actual mechanisms of necrosis in- cells, which leads to activation of multiple pathologic signals such as oxidative stress and volved in the development of sequential calcium overload. These signals are common triggers of cell death (for example, necrosis, cellular and organ response in AP need apoptosis, necroptosis, pyroptosis and autophagic cell death) through different regulator further investigation. or effector proteins as indicated. In contrast to the intracellular physiological role of DAMPs, extracellular DAMPs from cell death are important mediators of local and sys- Apoptosis temic inflammatory responses through different receptors (for example, TLRs, NLRs, RAGE and P2X7). Apoptosis is a process of programmed cell death that generally includes a cell death receptor-dependent (extrinsic) (nDAMPs) and mitochondrial DAMPs stimuli, whereas autolysis is caused by pathway and a mitochondria-dependent (mitDAMPs) (10,37–39). In addition to cell self-digestion from its own enzymes (intrinsic) pathway (46). Morphological necrosis and apoptosis, additional path- (40). Morphological features of necrotic features of apoptotic cells include cell ways (for example, necroptosis, pyropto- cells include swelling of the cell, cell shrinkage, membrane blebbing, chro- sis and autophagy) regulate DAMP re- membrane rupture and the release of in- matin condensing, DNA fragmentation lease and affect AP progression (Figure 2). tracellular contents, including proteins and apoptotic body formation. The cas- Collectively, these findings contribute and nonproteins (for example, DNA and pases are central initiators and effectors greatly to our understanding of the im- RNA). Nuclear morphological changes of apoptosis, although the existence of a munologic choices made during cell resulting from breakdown of DNA show caspase-independent pathway promotes death (12,14). The roles of different types three types: pyknosis (nucleus shrinks apoptosis (47). Caspase-8 mediates the of cell death in AP are discussed below. and the chromatin condenses into a solid cell death receptor-dependent pathway, basophilic mass), karyorrhexis (nucleus whereas caspase-9 triggers the mitochon- Necrosis shrinks further) and karyolysis (DNA is dria-dependent apoptotic pathway. Both Necrosis is a process of cell self- completely digested and none of the nu- caspase-8 and -9 can activate caspase-3 to destruction from external or internal cleus is visible). Karyolysis usually is ob- cleave specific groups of substrate pro-

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teins to induce apoptosis. The Bcl-2 fam- Autophagy Perhaps more importantly, the expres- ily is divided into antiapoptosis (for ex- Autophagy, an evolutionarily con- sion of vacuole membrane protein 1 ample, Bcl-2, Bcl-XL, Bcl-w and MCL-1) served process, can deliver endogenous (VMP1) is increased in AP and mediates and proapoptosis (for example, Bax, Bak, cytoplasmic constituents (for example, “zymophagy,” a selectively autophagic Box, Bad, Bim, Bid, PUMA and NOXA) damaged organelles and unused pro- pathway to remove zymogen granules, proteins that are majorly responsible for teins) and exogenous pathogens to lyso- and prevents pancreatic acinar cell death regulating the intrinsic pathway by con- somes for degradation (59). Autophagy by interaction with Beclin1, an essential trolling mitochondrial outer-membrane has three different forms: microau- regulator of autophagy and apoptosis permeabilization (MOMP) (48). When tophagy, chaperone-mediated autophagy (71–74). Mitochondrial injury and lipid MOMP occurs, several mitochondrial and macroautophagy. Macroautophagy abnormalities have been associated with proteins (for example, cytochrome c and (hereafter called autophagy) is the well- pancreatitis (66). The functional interac- second mitochondria-derived activator of studied dynamic membrane process that tion of zymophagy and other selective caspases [Smac/DIABLO]) are released is regulated by the family of autophagy- autophagic pathways including mi- into the cytosol to induce the formation related proteins (ATGs). The major mor- tophagy and lipophagy in AP have been of apoptosome (49,50). phological features of autophagy involve insufficiently explored. Lysosomes can Cerulein, L-arginine, ethanol, the formation and maturation of au- be involved in various cellular transport lipopolysaccharide (LPS), platelet-acti- tophagic vacuoles, including processes, including autophagy. Lysoso- vating factor, and cytokines (for exam- phagophores, autophagosomes and au- mal dysfunction from deficiency of ple, tumor necrosis factor [TNF]-α and tolysosomes. Hydrolases (cathepsins L Gnptab enzyme and lysosomal mem- interleukin [IL]-1β) have been shown to and B) from lysosomes contribute to the brane protein (LAMP)-2 can lead to au- induce apoptosis in AP. Besides caspase degradation of substrates in the autophagic flux impairment in AP and Bcl-2 family proteins, other compo- tolysosome, which produce amino acids (67,69,70). Loss of LAMP-2 impairs au- nents of the death machinery (for exam- and ATP for protein synthesis and en- tophagosome-lysosome fusion and pro- ple, X-linked inhibitor of apoptosis and ergy metabolism. Autophagy generally motes necrosis and inflammation, which p53) are involved in the regulation of ex- exerts a prosurvival function during is associated with HMGB1 release in AP trinsic or intrinsic apoptosis in AP. stress, whereas excessive autophagy may (70). Interestingly, autophagosomes are Given that apoptosis generally limits the contribute to cell death and DAMP re- increased significantly in trypsin in- inflammatory cascade, the apoptosis lease (60,61). DAMP-induced autophagy hibitor SPINK1/Spink3-deficient pancre- level of acinar cells is inversely related is involved in the regulation of inflam- atic acinar cells (75). However, it is un- to AP severity (35,51). Thus, induction of mation and the immune response clear how SPINK1 regulates autophagy apoptosis in pancreatic acinar cells ex- (14,62,63), while autophagy participates flux and zymophagy. Collectively, these erts a protective effect (52,53), whereas in the secretion, release and degradation studies suggest that autophagy has both suppression of apoptosis by caspase in- of DAMPs in a context-dependent man- positive and negative impacts on pancre- hibitors increases the severity of AP (54). ner (64). atitis (Figure 3). Caspases not only mediate apoptosis but Autophagy dysfunction has been im- also protect cells against necrosis by plicated in the pathogenesis of human Necroptosis cleavage and inactivation of PARP or diseases, including AP (65,66). One early Necroptosis is a process of programmed trypsin (55). Perhaps paradoxically, in- feature of AP is the accumulation of large necrotic cell death (76). Morphological tracellular heat shock proteins (HSPs) autophagic vacuoles in pancreatic acinar features of necroptosis are similar to that protect pancreatic acinar cells partly by cells (67,68). Autophagic flux is impaired of necrosis and various types of au- inhibition of apoptosis (56,57), suggest- in AP, which mediates acinar cell vacuole tophagic vesicles are frequently observed ing a pathogenic role of apoptosis in AP formation, trypsinogen activation, cell (76). Stimulation of death receptors upon (58). Subsequently, researchers also ob- death and the inflammatory response ligation by TNF-α, FasL and TNF-related served that pancreatic acinar cells un- (for example, inflammasome activation) apoptosis-inducing ligand (TRAIL) dergoing apoptosis can release histones, (67,69,70), suggesting a protective role of under apoptosis-deficient conditions, in DNA and HMGB1, which facilitate pan- autophagy in AP. In contrast to the above particular with pan-caspase inhibitor creatic injury and the inflammatory re- concept, ATG5-mediated autophagosome Z-VAD-FMK or inhibition of caspase-8, sponse (10,11). Whether induction or information in acinar cells is not blocked in may induce necroptosis. Receptor-inter- hibition of apoptosis would be beneficial pancreatitis, and inhibition of autophagy acting protein (RIP) kinases play a cen- in a clinical setting remains unproven. In from ATG5-deficient mice improves tral role in the regulation of necroptosis. addition, the current pharmacologic cerulein-induced AP with reduced The RIP kinases contain seven members agents that target apoptosis largely lack trypsinogen activation, suggesting that that facilitate NF-κB activation and specificity. autophagy is a harmful response (67,68). death-inducing processes. As caspase-8

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both apoptosis and necroptosis by com- bined Z-VAD-FMK and necrostatin-1 treatment exacerbates cerulein-induced AP (84). These data highlight the impor- tance of regulatory switches between different forms of cell death that influence the development of AP.

Pyroptosis The term pyroptosis was originally coined to describe peculiar bacterial infection-induced death in macrophages (85) and it is now a form of cell death in immune cells mediated by inflammasome in response to immune stimuli (for example, PAMPs and DAMPs) (86). Apart from extracellular ATP, HMGB1 and histone also can induce pyroptosis in immune cells and further augment the infection or sterile inflammation (87,88). For example, RAGE-dependent uptake of HMGB1 by macrophages leads to caspase-1-mediated pyroptosis in the devel- Figure 3. The yin and yang of autophagy in acute pancreatitis. On one hand, VMP1- opment of inflammation, whereas TLR9 Beclin1 complex-mediated zymophagy, a selective autophagy, prevents pancreatic aci- is required for histone-mediated pyrop- nar cell death through degradation of zymogen. On the other hand, deficiency of tosis during liver injury (87–89). Pyropto- LAMP2, Gnptab and SPINK1 cause impaired autophagy flux and increased autophago- sis shares morphological features with some accumulation, which mediates vacuole formation in pancreatic acinar cells, intra- both apoptosis and necroptosis, includ- cellular activation of trypsinogen, cell death and the inflammatory response. By contrast, ing cytoplasmic swelling, DNA fragmen- deficiency of ATG5 decreases autophagosome formation, which reduces intracellular ac- tation and pore formation. The formation tivation of trypsinogen and pancreatic damage in AP. Thus, autophagy plays a dual role in the regulation of AP. of pyroptotic DNA fragmentation is independent of caspase-activated DNase. In contrast to apoptosis, pyroptosis has a substrates, binding of RIP1/RIPK-1 to inhibitor enables selective inhibition of proinflammatory nature because of the RIP3/RIPK-3 forms a phosphorylation necroptosis and underscores its potential release of cell contents after rapid plasma complex to assemble the necrosome (77). contribution to diseases (79). membrane permeabilization or the secre- Cellular FLICE inhibitory protein (cFLIP) The expression of RIP3 protein is in- tion of proinflammatory cytokines (for is a homolog of caspase-8. In the pres- creased in AP. Notably, RIP3 knockout example, IL-1β, IL-18, IL-33 and HMGB1) ence of the long isoform of cFLIP mice exhibit significantly decreased after caspase-1 activation (86,90). Inflam-

(namely cFLIPL), caspase-8 fails to medi- necroptosis and MOF in experimental AP masome is a multiprotein oligomer that ate apoptosis, but can cause cleavage of (80,81). These observations suggests that is assembled in the cytoplasm by cytoso- both RIP1 and RIP3, which inhibits necroptosis mediates AP development. lic NOD-like receptors (NLRs). A well- necroptosis. By contrast, inhibition of Inhibition of necroptosis by gene deletion studied NLR inflammasome is the caspase-8 expression or activity by of RIP3 or using necrostatin-1 diminishes NLRP3 inflammasome, which recognizes knockout of caspase-8 or its interactor DAMP release and protects mice from various agonists. The NLRP3 inflamma- FAS-associated death domain protein lethal sepsis (82). RIP-mediated necroptosis some activation requires the priming (FADD) results in increased RIP1 stabil- also is implicated in sterile inflammation step, in which DAMPs such as HMGB1 ity that induces necroptosis. The mixed from trauma and ischemia-reperfusion in- or HMGB1/DNA complex might induce lineage kinase domain-like protein jury (79), which is accompanied by high the expression of NLRP3 by signaling (MLKL) is the interacting target of RIP3 levels of DAMP release. These studies through TLR4 and TLR9, respectively and a component of necrosome to in- suggest that inhibition of necroptosis (91,92). In addition to NLRs, non-NLR duce necroptosis (78). The discovery of blocks DAMP release and prevents sterile inflammasomes such as absent in mela- necrostatin-1 as a RIP1-specific kinase inflammation (83). Intriguingly, blocking noma 2 (AIM2) inflammasome have the

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ability to detect foreign dsDNA. RAGE is DAMPs are nDAMPs, including strated to regulate cell survival, prolifer- required for both HMGB1/DNA com- HMGB1, histones and nDNA. Other ation, differentiation and death in both plex-induced AIM2 inflammasome activ- PAMPs include S100 proteins, HSPs, immune and nonimmune cells. The pre- ity and autophagy, although upregulated complement, ATP and uric acid. Mito- cise structures and affinities of these lig- autophagy subsequently limits inflam- chondria are now recognized not only as and-receptor interactions remain to be masome activity (93). The regulatory central players in cell death but also as fully characterized. mechanisms of inflammasome activation an important source of DAMPs. mit- are extremely complex (94). Activation of DAMPs, including mitDNA, N-formyl HMGB1 AND ACUTE PANCREATITIS double-stranded RNA-dependent pro- peptides, transcription factor A (TFAM, a HMGB1 is a highly conserved protein tein kinase (PKR) is implicated in the mitochondrial HMGB1 homologue) and with location-dependent functions. In the crosstalk between inflammasomes, ROS, play emerging roles in inflamma- nucleus, HMGB1 as a DNA chaperone DAMP release and cell death (95). tion by the activation of neutrophils, regulates nucleosome structure, genomic TLR9 plays a fundamental role in monocytes and macrophages (96). stability and a number of DNA-associated DNA recognition and activation of innate DAMP sensing can be further classified events. Loss of HMGB1 in cells increases immunity. Both activation of TLR9 and as extracellular or intracellular depend- DNA damage and apoptosis and inhibits the NLRP3 inflammasome contribute to ing on whether the DAMP is released autophagy during stress (98,99). Once re- pancreatic acinar cell death and sterile from the stressed cell. Intracellular leased during death, HMGB1 acts as a inflammation in AP (10). Thus, genetic DAMP sensing involves detection by in- DAMP, regulating the inflammatory and deletion of NLRP3, caspase-1 and TLR9 tracellular receptors in the same cell that immune responses (100,101). Ample evi- in mice protects against cerulein-induced produced the DAMP (14). dence also shows that HMGB1 can act as pancreatitis (10). The potential mecha- A number of receptors (for example, an intracellular DAMP and drive activa- nism responsible for these phenomena is TLRs, NLRs, RIG-I-like receptors (RLRs), tion of intracellular receptors through nu- involved in increased mitochondrial the receptor for advanced glycation end cleic acid recognition. Caspase-3/7 and DNA (mitDNA) and nuclear DNA products [RAGE], and P2X7) have been PARP-1 are required for HMGB1 release (nDNA) release during apoptosis, which reported to mediate DAMP activity in in apoptosis and necrosis, (42,102 respec- activates TLR9 as well as NLRP3 inflam- different cell types, including immune tively), whereas ATG5 and PKR are re- masome pathways (10,38,39). Taken to- and nonimmune cells. TLRs play a cen- quired for HMGB1 release in autophagy gether, interplay between pyroptosis and tral role in innate immunity. TLR-1, -2, and pyroptosis, (103,104 respectively). DNA-sensing pathway is involved in the -4, -5 and -6 are located on the cell sur- RAGE and TLRs are positive receptors sterile inflammatory response. face, whereas TLR-3, -7, -8 and -9 are ex- mediating the proinflammatory activity pressed on the endosomal and lysosomal of HMGB1, whereas CD24 and TIM3 are DAMPs AND THEIR RECEPTORS compartment. NLRs and RLRs act as in- negative receptors limiting HMGB1 activ- Why is the immune system so con- tracellular surveillance molecules and ity in inflammation and tumor immunity cerned with cell death? The current no- RAGE is a member of the immunoglobu- (105). The redox status of HMGB1 also tion is that DAMPs released or exposed lin gene superfamily. RAGE also is pres- regulates its activity (106). In particular, from dying or dead cells contribute to in- ent in the mitochondria and regulates HMGB1 in reduced all-thiol form pro- flammatory and immune responses to re- mitochondrial respiration (97). Posttrans- motes cell migration, whereas HMGB1 in move dead cells and initiate tissue healing lational modification (for example, oxi- disulfide form induces cytokine produc- (12,13). Failure of this control mechanism dation and proteolysis) of DAMPs affects tion (106). By contrast, oxidized HMGB1 can lead to uncontrolled inflammation their receptor binding ability and activ- loses the above immune activities. More- and serious diseases such as sepsis, arthri- ity. In addition to receptors, endocytosis over, reduced HMGB1 induces au- tis, atherosclerosis, lupus and cancer. By also mediates DAMP activity in immune tophagy, whereas oxidized HMGB1 in- contrast, pathogen-associated molecular and cancer cells (88,97). A fine-tuned duces caspase-dependent apoptosis (107). patterns (PAMPs) are foreign danger sig- mechanism of these actions in disease However, the redox status within tissue nals and can induce immune cells to ac- needs further exploration. in disease is a dynamic process and the tively secrete DAMPs by various nonclas- Once released, extracellular DAMPs extracellular activity of HMGB1 may sical pathways, which indicates cross-talk induce the activation of multiple inflam- change accordingly (108). between DAMPs and PAMPs in the regu- matory pathways that lead to the pro- HMGB1 play an important role in lation of innate immune responses (14). duction and release of inflammatory cy- human health and disease (109). The A growing number of endogenous tokines, interferons, chemokines and cell serum levels of HMGB1 are significantly substances from multiple subcellular adhesion molecules. Apart from their elevated and correlate with the severity compartments are being identified as po- main effects on immunity, DAMPs and of AP (110). HMGB1 seems to act as an tential DAMPs. The most prominent their receptors also have been demon- important cytokine mediator in the

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pathogenesis of severe AP and has a erties, HSPs easily complex with other necrotic cells are the primary source of wider therapeutic window. Early block- molecules including PAMPs; several circulating DNA during AP. ade or delayed therapeutic delivery (for studies demonstrate that the previously Different types of nucleic acid recep- example, ethyl pyruvate [111], A box reported cytokine activity of HSP was tors and sensing molecules have been [112] and anti-HMGB1 antibody [113]) due to contaminating bacterial products identified (133). TLR9 is critical for the targeting HMGB1 limits development including LPS and flagellin (121,122). recognition of CpG DNA. The expression and associated MOF in experimental AP. This supports the argument against the of TLR9 in the pancreas is increased in Antioxidants (for example, pyrrolidine role of HSPs as DAMPs. AP (138) and genetic deletion of TLR9 dithiocarbamate 114) and anticoagulants The expression of inducible HSPs (for protects mice from AP (10). TLR9 also (for example, antithrombin III [115] and example, HSP27, HSP60, HSP70 and mediates the innate immune response to danaparoid sodium [116]) also inhibit HSP90) is upregulated in experimental nDNA, mitDNA, histone and HMGB1 in HMGB1 release and therefore protect AP (123,124). Preconditioning of animals AP and other sterile injuries (10,89). The against injury in AP. By contrast, intracel- with either a thermal or chemical stressor interaction between RAGE and TLR9 is lular HMGB1 protects against AP partly induces HSP expression, which in turn required for HMGB1-nDNA complex through limiting nDAMP (histones and protects against AP (56,57). Knockout of and TFAM-mitDNA complex-mediated DNA) release and subsequent inflamma- heat shock factor protein 1 in mice in- immune responses (139,140). RAGE also tory cell recruitment and activation (11). hibits HSP synthesis and develops more has the ability to directly recognize DNA These observations indicate the dual role severe AP induced by cerulein (125). (141). A study indicates that besides of HMGB1 in the regulation of AP. HSP27, HSP60 and HSP70 are direct ef- RAGE and TLR9, TLR2 is required for HMGB1 function is not only complicated fectors in mediating this protective effect the HMGB1-nucleosome complex- in the pancreas, but also in other tis- against AP by regulation of NF-κB sig- mediated immune response in macro - sues/cells according to studies of naling (30), intrapancreatic trypsinogen phages and dendritic cells (142). These HMGB1 knockout or knockin mice (117). activation (56,126), calcium overload findings improve our understanding of (125), actin cytoskeleton (127), apoptosis how DNA-protein complexes trigger in- HSPS AND ACUTE PANCREATITIS (128) and autophagy (129). Although nate immune responses in human dis- HSPs are conserved stress proteins and studies of the role of extracellular HSP in eases, including AP. their expression is upregulated in acti- AP are limited, one study shows that ad- vated cells responding to heat shock, in- ministration of recombinant HSP70 in HISTONE AND ACUTE PANCREATITIS fection, hypoxia and other stressors. The mice aggravates cerulein-induced AP in Histones are basic components of major function of HSP is assisting with a TLR4-dependent manner (130). Serum chromosome structural units, namely the folding and unfolding of newly HSP27 is increased in patients with pan- nucleosomes. They are divided into core translated proteins during stress. Accord- creatitis and pancreatic cancer (131), histones (H2A, H2B, H3 and H4) and ing to their molecular size, mammalian highlighting its role as a potential bio- link histones (H1/H5). In the nucleus, HSPs are categorized into the following marker in these diseases. histones and their posttranslational groups: HSP100, HSP90, HSP70, HSP60, modifications regulate chromosome HSP40 and the small HSPs (for example, DNA AND ACUTE PANCREATITIS structure and function (143). In addition HSP25/27, αB-crystallin and HSP22). DNA is the hereditary material that to their nuclear function, emerging Overexpression of intracellular HSPs in- carries genetic information. According to studies indicate that histones as well as hibits apoptosis and inflammatory cy- the endosymbiotic theory, nDNA and nucleosomes can be released into the ex- tokine production (for example, TNF-α, mtDNA have separate evolutionary ori- tracellular space upon infection (for ex- IL-1β and HMGB1) following cellular gins. mtDNA evolved from the circular ample, sepsis) (144), sterile inflamma- stresses. HSP70 is directly involved in genomes of bacteria when an early eu- tion (for example, trauma, chaperone-mediated autophagy (118). karyotic cell engulfed a prokaryotic cell ischemia-reperfusion injury and pancre- HSP70 and HSP27 confer protection in (132). DNA is a potent activator of innate atitis) (11,89,145) and death (for exam- cellular stress by enhancing autophagy immunity by activation of type I inter- ple, apoptosis, necrosis and NETosis) and mitophagy (119,120). Some HSPs feron, inflammasome, and NF-κB path- (146). NETosis is a unique form of cell such as HSP70 and HSP60 are rapidly re- ways (133). Monitoring levels of circulat- death that is first observed in neu- leased following nonprogrammed cell ing DNA has been proposed as a useful trophils upon microbial infection (147). death (12,13). Extracellular HSPs induce tool in the noninvasive diagnosis of Neutrophils have the ability to release immune cell activation and the proin- human diseases. Serum DNA level is ele- chromosomal components including hi- flammatory response through binding to vated significantly in patients with se- stones to capture and kill microbes PRRs, including TLR2, TLR4 and CD91. vere AP and may be an early predictor of (147). Once released, histones exhibit Due to their molecular chaperone prop- severity in AP (134–137). Apoptotic or DAMP activity with significant induc-

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required for the induction of apoptosis (33). Further, ATP can be passively released into the extracellular space during cell death, which mediates the immune response by P2X7 receptor (152). Several mechanisms have been reported to regulate passive ATP release. For example, caspase and pannexin 1 regulate ATP release in apoptosis (153). In addition, active secretion of ATP has been identified in immune and cancer cells. The underlying mechanisms of ATP secretion include ion channel-mediated conductive release as well as autophagy- mediated exocytosis of ATP-enriched vesicles (154–156). Extracellular ATP regulates multiple cell processes, including cell migration, metabolism, inflammation, immunity, differentiation, Figure 4. Role of intracellular and extracellular HMGB1 in acute pancreatitis. Under normal apoptosis and autophagy (151). How- conditions, HMGB1 is located in the nucleus of pancreatic acinar cells to regulate nucle- ever, the signaling mechanism of these osome stability. However, under conditions of increased pathologic insult, increased oxida- ATP-mediated activities remains to be tive stress and calcium overload leads to HMGB1 release. Loss of intracellular HMGB1 in elucidated. pancreatic acinar cells accelerates DNA damage, cell death and nDAMP (for example, In AP, extracellular ATP binds to its re- histone) release. Subsequently, extracellular histone promotes immune cell (for example, ceptor P2X7 and results in NLRP3 in- macrophage and neutrophil) recruitment and activation, which leads to HMGB1 release. Consequently, circulating HMGB1 levels are increased and function as a mediator of flammasome assembly, caspase-1 activa- β lethal systemic inflammation in acute pancreatitis. tion and IL-1 secretion (10). Inhibition of P2X7 through genetic deletion, or treatment with P2X7 antagonists (for ex- tion of proinflammatory and toxic re- recruit and activate immune cells such as ample, A-438079), limits pancreatic in- sponses in vivo and in vitro (144,145). macrophages to secrete HMGB1 into the jury and the inflammatory response in Activation of TLRs (for example, TLR2, circulation of CH mice. Moreover, anti- an experimental animal AP model (10). TLR4 and TLR9) and NLRP3 inflamma- H3 antibody and anti-HMGB1 antibody In another study, mitochondria-mediated some mediate the activity of histones protect against AP in CH mice (11). intracellular ATP depletion was found to (87,89,145,148). Dynamic changes in cir- These findings suggest interplay be- be able to cause necrosis in AP (157). In- culating levels of histones as well as nu- tween intracellular and extracellular terestingly, the P2X7 receptor not only cleosomes serve as potential biomarkers nDAMPs in the pathogenesis of AP (see mediates extracellular ATP activity, but and novel therapeutic targets in human Figure 4). also regulates ATP release in several cells diseases (149,150). (158). Thus, it seems likely that the ATP- Our recent study indicates that oxida- ATP AND ACUTE PANCREATITIS P2X7 signaling pathway is a novel regu- tive stress-mediated DNA damage is re- ATP, the major energy currency mole- lating mechanism for AP. sponsible for histone release in AP (11). cule of various cellular processes, is nor- This process is controlled by intracellular mally present in the cell cytoplasm and CONCLUDING REMARKS AND FUTURE HMGB1 (Figure 4). Loss of intracellular is used as a substrate for protein kinase PERSPECTIVES HMGB1 in pancreatic tissue leads to activation (151). Most ATP in mam- It has been widely accepted that in- local oxidative injury, which facilitates malian cells is generated within the mi- flammation and cell death are key patho- nuclear catastrophe and proinflamma- tochondria. The ability of autophagy to logic responses of AP and determine dis- tory nucleosomal (histone and DNA) re- defend cells against various stressors is ease severity. Mitochondria-mediated cell lease (11). Consistently, antioxidant N- obtained partly through the generation death is essential for the initiation of AP. acetyl-L-cysteine protects against AP in of substrates to maintain ATP produc- As mentioned above, imbalanced produc- pancreatic HMGB1 conditional knockout tion. Intracellular ATP levels determine tion of mitochondrial calcium, ATP and mice (termed CH mice). Once released, cell death fate (33). ATP depletion leads ROS are the most common causes of cel- extracellular histones (H3 and H4) can to necrosis, whereas ATP production is lular injury and death. Various signaling

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molecules and proteins have related or ACKNOWLEDGMENTS acute pancreatitis in mice. Gastroenterology. overlapping actions in the regulation of We apologize to the researchers who 146:1097–107. cell death. This regulation not only modi- were not referenced due to space limita- 12. Zitvogel L, Kepp O, Kroemer G. (2010) Decoding cell death signals in inflammation and immunity. fies the magnitude of the death response tions. We thank Christine Heiner (Depart- Cell. 140:798–804. but also results in switching between dif- ment of Surgery, University of Pittsburgh, 13. Bianchi ME. (2007) DAMPs, PAMPs and alarmins: ferent types of death. Such complex Pittsburgh, PA, USA) for her critical read- all we need to know about danger. J. Leukoc. Biol. death events then progress to a systemic ing of the manuscript. This work was 81:1–5. inflammatory response that is mediated supported by the National Institutes of 14. Tang D, et al. (2012) PAMPs and DAMPs: signal 0s that spur autophagy and immunity. Immunol. by the release of various intracellular Health (R01CA160417 to D Tang; Rev. 249:158–75. content including DAMPs. DAMPs are R01CA181450 to HJ Zeh/MT Lotze) 15. Lerch MM, Gorelick FS. (2013) Models of acute and not only passively released during cell and a 2013 Pancreatic Cancer Action chronic pancreatitis. Gastroenterology. 144:1180–93. death and tissue injury, but also actively Network-AACR Career Development 16. Saluja AK, Dudeja V. (2013) Relevance of animal secreted by activated immune cells. Award (grant number 13-20-25-TANG). models of pancreatic cancer and pancreatitis to human disease. Gastroenterology. 144:1194–8. DAMPs have multiple forms and their The work supporting the findings re- 17. Sah RP, Garg P, Saluja AK. (2012) Pathogenic activities vary greatly depending on type viewed in this manuscript was aided by mechanisms of acute pancreatitis. Curr. Opin. of death, posttranslational modification, core support from the University of Gastroenterol. 28:507–15. and their receptors. In particular, Pittsburgh Cancer Institute (NIH grant 18. Pandol SJ, Saluja AK, Imrie CW, Banks PA. (2007) HMGB1, one of the best-characterized P30CA047904). Acute pancreatitis: bench to the bedside. Gas- DAMPs, has a unique role in AP accord- troenterology. 132:1127–51. 19. Dawra R, et al. (2011) Intra-acinar trypsinogen ac- DISCLOSURES ing to experimental and clinical studies. tivation mediates early stages of pancreatic in- It is also worth mentioning that intra- The authors declare that they have no jury but not inflammation in mice with acute cellular and extracellular DAMPs may competing interests as defined by Molecu- pancreatitis. Gastroenterology. 141:2210–7. have different mechanisms of action in lar Medicine, or other interests that might 20. Tsai K, et al. (1998) Oxidative stress: an important the pathogenesis of AP (see Figure 4). 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