Severe acute pancreatitis (AP) is responsible for significant human morbidity and mortality worldwide. Currently, no specific treatments for AP exist, primarily due to the lack of a mechanistic understanding of sterile inflammation and the resultant multisystem organ dysfunction, the pathologic response of AP linked to early death. In this study, we demonstrate that the class III major histocompatibility region III receptor for advanced glycation end products (RAGE) contributes to AP by modulating inflammasome activation in macrophages. RAGE mediated nucleosome-induced absent in melanoma 2 (but not NLRP3) inflammasome activation by modulating dsRNA-dependent protein kinase phosphorylation in macrophages. Pharmacological and genetic inhibition of the RAGE–dsRNA-dependent protein kinase pathway attenuated the release of inflammasome-dependent exosomal leaderless cytokines (e.g., IL-1β and high-mobility group box 1) in vitro. RAGE or absent in melanoma 2 depletion in mice limited tissue injury, reduced systemic inflammation, and protected against AP induced by l-arginine or cerulein in experimental animal models. These findings define a novel role for RAGE in the propagation of the innate immune response with activation of the nucleosome-mediated inflammasome and will help guide future development of therapeutic strategies to treat AP.
Acute pancreatitis (AP) is the leading cause of hospitalization in the United States for patients with gastrointestinal disorders, causing significant morbidity and reducing life expectancy (1). Premature activation of digestive enzymes within pancreatic acinar cells is an initiating event that leads to organelle injury and autodigestion of the pancreas (2). A complex cascade of immunological events, including inflammatory mediator production, affects not only the pathogenesis, but also the course of AP (3). Some of these inflammatory mediators are initially released by pancreatic acinar cells, resulting in the recruitment and activation of neutrophils, monocytes, and macrophages, leading to further acinar cell injury. When released, these mediators gain access to the systemic circulation and play a central role in the progression of systemic inflammatory response syndrome and multisystem organ failure (4). The molecular mechanisms linking the progression of local pancreatic damage to systemic inflammation are still poorly understood (5).
Nucleosomes are the repeating subunits of chromatin, consisting of a DNA chain coiled around a core of histone octamer. Besides their nuclear function, histones can also be released into the extracellular space by dead, dying, netting, and injured cells. Extracellular nucleosomes including histones and DNA are nuclear damage-associated molecular pattern molecules that exhibit significant proinflammatory activity in vitro and in vivo (6, 7). We recently demonstrated that the release of nucleosomes via pancreatic injury contributes to the inflammatory response in AP (8). Blocking nucleosome activity by histone-neutralizing Abs significantly promoted survival in an l-arginine–induced experimental animal model of severe AP (8). Thus, it is important to achieve a deeper understanding of the nucleosome-mediated inflammatory signaling pathway (9).
In this study, we demonstrate that the receptor for advanced glycation end products (RAGE), a member of the Ig gene superfamily (10), plays a critical role in mediating the proinflammatory activity of nucleosomes in macrophages. Knockdown or knockout of RAGE in macrophages suppresses nucleosome-induced absent in melanoma 2 (AIM2) inflammasome activation and subsequent proinflammatory mediator release. Targeted ablation of RAGE or AIM2 expression in mice protects against l-arginine– or cerulein-induced AP in experimental animal models. Thus, disruption of nucleosome–RAGE–AIM2 signaling is a potential therapeutic approach for AP therapy.
Materials and Methods
The Abs to RAGE and TLR4 were obtained from Abcam (Cambridge, MA). The Abs to AIM2 and actin were obtained from Cell Signaling Technology (Danvers, MA). The Abs to p–dsRNA-dependent protein kinase (PKR) and PKR were obtained from Santa Cruz Biotechnology (Dallas, TX). The Ab to NLRP3 was obtained from Adipogen (San Diego, CA). The Ab to Gr-1 was obtained from eBioscience (San Diego, CA). The Ab to F4/80 was obtained from Invitrogen (Grand Island, NY). The Ab to IL-1β was obtained from R&D Systems (Minneapolis, MN). The Ab to caspase-1 was obtained from Adipogen. Mouse genomic DNA was obtained from New England BioLabs (Ipswich, MA). High-purity histone protein was obtained from Roche Life Science (Stockholm, Sweden). LPS, ATP, and 2-aminopurine (2-AP) were obtained from InvivoGen (San Diego, CA).
Mouse peritoneal macrophages (PMs) were isolated from C57BL/6 mice as previously described (11). In brief, mice were injected i.p. with 1.5 ml 3% Brewer’s thioglycollate broth for 3 d. Primary PMs were collected from euthanized animals by peritoneal lavage using 10 ml ice-cold RPMI 1640 supplemented with 2% FBS, 1 U/ml heparin, and penicillin/streptomycin. Cells were washed using lavage media without heparin and plated in macrophage culture media of DMEM supplemented with 10% heat-inactivated FBS and penicillin/streptomycin and incubated at 37°C at 5% CO2 for 2 h. Cultures were washed three times with PBS to remove nonadherent cells and leave adherent cells (PMs) in the culture media. The immortalized bone marrow-derived macrophages (iBMDMs) from wild-type (WT), NLRP3−/−, and AIM2−/− mice were a gift from Dr. Kate Fitzgerald (University of Massachusetts Medical School, Worcester, MA) and Dr. Eicke Latz (University of Bonn, Bonn, Germany). These cells were cultured in DMEM (supplemented with 10% heat-inactivated FBS, 100 U penicillin, and 100 μg/ml streptomycin) at 37°C, 95% humidity, and 5% CO2.
Proteins in the cell lysate or supernatants were resolved on 4–12% Criterion XT Bis-Tris gels (Bio-Rad, Hercules, CA) and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated for 2 h at 25°C or overnight at 4°C with various primary Abs. After incubation with peroxidase-conjugated secondary Abs for 1 h at 25°C, the signals were visualized by enhanced or super chemiluminescence (Pierce, Rockford, IL) according to the manufacturer’s instructions.
Specific RAGE–short hairpin RNA (shRNA), TLR2-shRNA, TLR4-shRNA, and control-shRNA were purchased from Sigma-Aldrich. Cells were seeded in six-well plates at a density of 2 × 106 cells/well to achieve a confluence of 70% overnight. Transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.
Experimental animal models of AP
The protocol for animal use was reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee. RAGE−/− mice (SVEV129 × C57BL/6) were a gift from the late Dr. Angelika Bierhaus (12). TLR4−/− mice (C57BL/6 background) and AIM2−/− mice (C57BL/6 background) were obtained from The Jackson Laboratory (Farmington, CT). For l-arginine–induced pancreatitis, a sterile solution of l-arginine hydrochloride (8%; Sigma-Aldrich) was prepared in normal saline and the pH was adjusted to 7.0. Mice received three hourly i.p. injections of l-arginine (3 g/kg), whereas controls were administered saline i.p. as a control as described previously (13). For cerulein-induced pancreatitis, mice received seven hourly i.p. injections of 50 μg/kg cerulein (Sigma-Aldrich) in sterile saline, whereas controls were given saline as described previously (14).
ELISA assays were performed for the measurement of amylase (Abcam), lactate dehydrogenase (LDH; Abcam), IL-1β (R&D Systems), TNF-α (R&D Systems), and high mobility group box 1 (HMGB1; Shino-Test Corporation, Sagamihara, Japan) in serum and/or cell culture supernatants, as well as myeloperoxidase (MPO; Abcam) in pancreas tissue homogenates, according to the manufacturers’ instructions.
Tissues were embedded in optimum cutting temperature cryomedium (Sakura Finetek, Zoeterwoude, The Netherlands) and cut into 8-μm sections. Sections were subjected to immunofluorescent staining as previously described (15). Nuclear morphology was analyzed with the fluorescent dye Hoechst 33342 (Invitrogen).
Data are expressed as mean ± SEM of three independent experiments. Significance of differences between groups was determined by a two-tailed Student t test or ANOVA least significant difference test. The Kaplan–Meier method was used to compare the differences in mortality rates between groups. A p value <0.05 was considered statistically significant.
Nucleosome release promotes AIM2 inflammasome activation in macrophages
Our previous study demonstrated that nucleosome release following pancreatic injury promotes the recruitment and activation of macrophages in AP (8). Inflammasomes are important signaling platforms that activate highly proinflammatory mediators, including IL-1β, IL-18, and HMGB1 (16, 17). To determine whether nucleosome release promotes inflammasome activation in macrophages, we treated the iBMDMs and primary PMs with exogenous histone and genomic DNA. There was a significant effect of combining histone and DNA in promotion of IL-1β (Fig. 1A) and HMGB1 (Fig. 1B) release, suggesting that nucleosomes promote inflammasome activation in macrophages. The NLRP3 inflammasome mediates activation of sterile inflammatory pathways in AP (18). However, histone/DNA-induced IL-1β (Fig. 1C) or HMGB1 (Fig. 1D) release was not affected in NLRP3−/− iBMDMs. As a positive control, LPS/ATP-induced IL-1β release (Fig. 1E) was diminished in NLRP3−/− iBMDMs. In contrast, histone/DNA-induced IL-1β (Fig. 1C) and HMGB1 (Fig. 1D) release was suppressed in AIM2−/− iBMDMs. Similarly, Western blot analysis demonstrated reduced extracellular levels of IL-1β and cleaved caspase-1 (p20) in the culture supernatants of AIM2−/− iBMDMs, but not NLRP3−/− iBMDMs (Fig. 1F). These findings suggest that nucleosome selectively promotes AIM2 inflammasome activation in macrophages.
RAGE is required for nucleosome-mediated inflammasome activation in macrophages
TLRs and RAGE are important damage-associated molecular pattern receptors in infection and sterile inflammation. To further determine which receptor is required for histone/DNA-induced inflammasome activation, we transfected iBMDMs with specific shRNA targeting RAGE, TLR2, or TLR4, respectively. We achieved a >80% reduction in the protein expression of RAGE, TLR2, or TLR4 in iBMDMs (Fig. 2A). Importantly, knockdown of RAGE, but not TLR2 or TLR4, significantly attenuated histone/DNA-induced IL-1β (Fig. 2B) and HMGB1 (Fig. 2C) release in macrophages. Similarly, histone/DNA-induced release of IL-1β (Fig. 2D) and HMGB1 (Fig. 2E) were significantly reduced in PMs from RAGE−/− mice, but not TLR4−/− mice. Moreover, Western blot analysis demonstrated reduced extracellular levels of IL-1β and cleaved caspase-1 (p20) in the culture supernatants of RAGE−/− PMs following histone/DNA treatment (Fig. 2F). These findings indicate that nucleosomes selectively promote inflammasome activation in a RAGE-dependent fashion.
RAGE-mediated PKR phosphorylation is required for nucleosome-induced inflammasome activation in macrophages
Given that phosphorylation and activation of PKR are required for inflammasome-dependent IL-1β and HMGB1 release in macrophages (19), we next analyzed whether RAGE promotes nucleosome-mediated inflammasome activation by regulating PKR phosphorylation. The level of PKR phosphorylation was significantly increased following histone/DNA treatment (Fig. 3A). In contrast, knockout of RAGE, but not TLR4 and AIM2, attenuated histone/DNA-induced PKR phosphorylation in isolated PMs (Fig. 3A). To explore whether PKR is required for nucleosome-mediated inflammasome activation, we treated iBMDMs and PMs with 2-AP, a potent PKR inhibitor (20). 2-AP inhibited histone/DNA-induced PKR phosphorylation (Fig. 3B) and subsequent IL-1β (Fig. 3C) and HMGB1 (Fig. 3D) release in iBMDMs and PMs. Treatment with 2-AP itself mildly increased HMGB1 release (Fig. 3D) owing to possibile toxicity (21). In addition to IL-1β and HMGB1, 2-AP also partly limited histone/DNA-induced TNF-α release (Fig. 3E). Collectively, RAGE-mediated PKR phosphorylation is required for the inflammatory response following nucleosome delivery to macrophages.
RAGE depletion protects against AP in experimental animal models
Severe AP was induced with i.p. injection of l-arginine as previously described (22). The RAGE+/+ mice were substantially more susceptible to AP with significantly higher mortality rates compared with RAGE−/− mice (Fig. 4A). Histological assessment of pancreatic damage revealed exaggerated acinar cell death, leukocyte infiltration, and interstitial edema in RAGE+/+ mice compared with RAGE−/− mice (Fig. 4B). Immunofluorescent staining of F4/80 (a macrophage marker) and Gr-1 (a neutrophil marker) confirmed that the leukocyte infiltration was reduced in RAGE+/+ mice (Fig. 4C). The level of serum amylase, consistent with AP, was also significantly lower in RAGE−/− mice (Fig. 4D). Consistently, pancreatic neutrophil recruitment (as assessed by levels of pancreatic MPO activity, Fig. 4E), pancreatic necrosis (serum LDH activity, Fig. 4F), and circulating levels of IL-1β (Fig. 4G) and HMGB1 (Fig. 4H) were significantly lower in the RAGE−/− mice as well. Severe AP is often associated with acute lung injury, a significant cause of morbidity and mortality in this disease. Knockout of RAGE in mice also limited lung injury in animals with AP (Fig. 4B). Similarly, cerulein-induced mild AP (no animal death) was also associated with increased acinar cell injury (Fig. 5A), infiltration of macrophages/neutrophils (Fig. 5B), serum amylase levels (Fig. 5C), pancreatic MPO activity (Fig. 5D), serum LDH activity (Fig. 5E), and circulating levels of IL-1β (Fig. 5F) and HMGB1 (Fig. 5G) in RAGE+/+ mice when compared with RAGE−/− mice. Collectively, these findings support the notion that RAGE depletion protects against AP with decreased tissue injury and proinflammatory mediator release in experimental models.
AIM2 depletion protects against AP in experimental animal models
Similar to RAGE−/− mice, the AIM2−/− mice were resistant to l-arginine–induced severe AP without death compared with AIM2+/+ mice (Fig. 6A). Moreover, the acinar cell injury (Fig. 6B), infiltration of macrophages/neutrophils (Fig. 6C), serum amylase levels (Fig. 6D), pancreatic MPO activity (Fig. 6E), serum LDH activity (Fig. 6F), and circulating levels of IL-1β (Fig. 6G) and HMGB1 (Fig. 6H) were significantly decreased in AIM2−/− mice compared with AIM2−/− mice.
Macrophages are critical inflammatory cells involved in the pathophysiology of AP following acinar cell injury (23). Acinar cell death products such as nucleosomes are thought to play an essential role in driving macrophage activation and the systemic inflammatory response in the setting of AP (8). Circulating nucleosomes, complexes of DNA and histones, are common prognostic markers of disease in the setting of infection as well as sterile inflammation. In the present study, we demonstrated that RAGE contributes to nucleosome-mediated macrophage activation by regulating PKR-dependent AIM2 inflammasome activation. Thus, RAGE-dependent AIM2 inflammasome activation links local cell death, nucleosome release, and the systemic inflammatory response to AP.
Activation of the inflammasome contributes to the pathogenesis of inflammatory diseases, including cancer, diabetes, inflammatory bowel disease, rheumatoid arthritis, atherosclerosis, sepsis, and pancreatitis (24). NLRP3 inflammasome-mediated IL-1β production from infiltrating macrophages within the pancreas can lead to the death of pancreatic β cells and subsequent diabetes (25). Moreover, reactive oxygen species production during apoptosis or mitophagy deficiency in pancreatic β cells also accelerates NLRP3 inflammasome activation and IL-1β production, as well as the expression of chemotactic factors (26–28). Genetic deletion of NLRP3 protects against experimental AP and chronic obesity-induced pancreatic damage (18). In this study, we demonstrated that AIM2, but not NLRP3, is required for nucleosome-mediated inflammasome activation in macrophages. We also showed in the present study that genetic deletion of AIM2 protects against experimental AP in mice. AIM2 is a member of the hematopoietic IFN-inducible nuclear protein HIN-200 family and functions as a cytosolic dsDNA sensor, which, when activated, promotes the assembly of an inflammasome (29, 30). These findings have enhanced our understanding of the molecular mechanisms by which individual inflammasomes are activated by specific signaling mechanisms in AP.
The regulatory mechanisms of inflammasome activation are extremely complex and facilitate a balanced inflammasome-mediated immune response in disease (31). Early studies show that PKR is a serine/threonine protein kinase that is activated by autophosphorylation after binding to dsRNA (32). Phosphorylation of PKR can be observed in response to several activators of the inflammasome (19). As a newly identified inflammasome component, PKR can directly bind to NLRP3, NLRP1, AIM2, or NLRC4 by autophosphorylation during inflammasome activation in macrophages (19). Our results indicate that PKR phosphorylation contributes to nucleosome-induced AIM2 inflammasome activation and subsequent IL-1β and HMGB1 release. Thus, activation of PKR is implicated in the crosstalk between cell death and inflammasome activation (33).
RAGE is a member of the Ig superfamily of cell surface receptors that recognize multiple ligands, including AGE, S100, HMGB1, DNA, and RNA (10). RAGE plays a critical role in infection and sterile inflammation. For example, RAGE−/− mice are resistant to polymicrobial sepsis (12) and DNA-induced lung injury (34). Blockade of RAGE by drugs also attenuates ischemia and reperfusion injury in the liver (35) and heart (36). However, the role of RAGE in the pathogenesis of pancreatic disease remains poorly defined. We previously demonstrated that loss of RAGE inhibits pancreatic cancer development (15). Our present study indicates that RAGE promotes the development of pancreatitis partly through mediating nucleosome-induced AIM2 inflammasome activation and proinflammatory mediator release in macrophages. Moreover, mice deficient in RAGE are unable to mount a typical inflammatory response in experimental AP. TLR9 plays a fundamental role in CpG genomic DNA recognition and activation of innate immunity. TLR9 regulates acinar cell death with sterile inflammation in AP (18). The interplay between RAGE and TLR9 regulates HMGB1–DNA complex activity (37). TLR9 also contributes to histone activity in liver ischemia and reperfusion injury (38). RAGE plays a major role in regulating the uptake of DNA/HMGB1 in nucleosome complexes (34) and serves to create a “zipper,” aligning proinflammatory macrophages and other RAGE-expressing cells to modulate and regulate inflammation in the transition to the adaptive immune response.
In summary, we demonstrate in this study that RAGE plays a critical role not only in pancreatic injury, but also in inflammasome activation and subsequent proinflammatory mediator release by macrophages during AP. We also demonstrated that nucleosome-mediated PKR phosphorylation is important for AIM2 inflammasome activation in macrophages. Overproduction of inflammasome-related cytokines (e.g., IL-1β and HMGB1) is associated with the development of AP. Thus, targeting the RAGE-dependent AIM2 inflammasome pathway may be a potential therapeutic approach to AP.
We thank Christine Heiner (Departments of Surgery and Anesthesiology, University of Pittsburgh) for critical reading of the manuscript.
This work was supported by National Institutes of Health Grants R01 GM115366, R01 CA160417 (both to D.T.), and R01 CA181450 (to H.J.Z.). This project used University of Pittsburgh Cancer Institute shared resources supported in part by National Institutes of Health Grant P30CA047904.
Abbreviations used in this article:
absent in melanoma 2
high-mobility group box 1
immortalized bone marrow–derived macrophage
dsRNA-dependent protein kinase
receptor for advanced glycation end products
short hairpin RNA
The authors have no financial conflicts of interest.