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J Immunol. Author manuscript; available in PMC 2017 May 15. Published in final edited form as: J Immunol. 2016 May 15; 196(10): 4331–4337. doi:10.4049/jimmunol.1502340.
The Receptor for Advanced Glycation Endproducts (RAGE) Activates the AIM2 Inflammasome in Acute Pancreatitis Rui Kang1,*, Ruochan Chen1, Min Xie2, Lizhi Cao2, Michael T. Lotze1,3, Daolin Tang1,*, and Herbert J. Zeh III1,* 1
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15219, USA
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2
Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, Hunan 410008, People's Republic of China
3Department
of Immunology, University of Pittsburgh, Pittsburgh, PA 15219, USA
Abstract
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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. Here, we demonstrate that the class III Major Histocompatability Region III receptor for advanced glycation endproducts (RAGE) contributes to AP by modulating inflammasome activation in macrophages. RAGE mediated nucleosome-induced AIM2 (but not NLRP3) inflammasome activation by modulating double-stranded RNA-dependent protein kinase (PKR) phosphorylation in macrophages. Pharmacological and genetic inhibition of the RAGE-PKR pathway attenuated the release of inflammasome-dependent exosomal leaderless cytokines (e.g., IL-1β and HMGB1) in vitro. RAGE or AIM2 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 to guide future development of therapeutic strategies to treat AP.
INTRODUCTION Author Manuscript
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
*
Correspondence should be directed to Dr. Rui Kang ([email protected]), Dr. Daolin Tang ([email protected]), or Dr. Herbert J. Zeh ([email protected]).. CONFLICT OF INTEREST The authors declare no conflicts of interest or financial interests.
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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).
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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 (nDAMPs) that exhibit significant pro-inflammatory 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 antibodies 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).
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Here, we demonstrate that the receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin 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 ceruleininduced AP in experimental animal models. Thus, disruption of nucleosome-RAGE-AIM2 signaling is a potential therapeutic approach for AP therapy.
Materials and Methods Regents
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The antibodies to RAGE and TLR4 were obtained from Abcam (Cambridge, MA, USA). The antibodies to AIM2 and actin were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies to P-PKR and PKR were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). The antibody to NLRP3 was obtained from Adipogen (San Diego, CA, USA). The antibody to Gr-1 was obtained from eBioscience (San Diego, CA). The antibody to F4/80 was obtained from Invitrogen (Grand Island, NY, USA). The antibody to IL-1β was obtained from R&D (Minneapolis, MN, USA). The antibody to caspase-1 was obtained from Adipogen (San Diego, CA, USA). Mouse genomic DNA was obtained from New England BioLabs Inc. (Ipswich, MA, USA). High purity histone protein was obtained from Roche Life Science (Stockholm, Sweden). LPS, ATP, and 2-AP were obtained from InvivoGen (San Diego, CA, USA). Cell culture Mouse peritoneal macrophages (PMs) were isolated from C57BL/6 mice as previously described (11). In brief, mice were injected intraperitoneally with 1.5 mL of 3% Brewer's
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thioglycollate broth for three days. Primary PMs were collected from euthanized animals by peritoneal lavage using 10 mL of ice cold RPMI supplemented with 2% fetal bovine serum (FBS), 1 unit/mL heparin and penicillin/streptomycin. Cells were washed using lavage media without heparin and plated in macrophage culture media of Dulbecco's Modified Eagle's Medium (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 phosphate buffered saline to remove non-adherent cells and leave adherent cells (PMs) in the culture media. The immortalized bone marrow-derived macrophages (iBMDMs) from WT, NLRP3−/−, and AIM2−/− mice were a kind 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% heatinactivated FBS and 100 units of penicillin and 100 μg/ml streptomycin) at 37 °C, 95% humidity, and 5% CO2.
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Western blot Proteins in the cell lysate or supernatants were resolved on 4-12% Criterion XT Bis-Tris gels (Bio-Rad, Hercules, CA, USA) and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated for two hours at 25°C or overnight at 4°C with various primary antibodies. After incubation with peroxidase-conjugated secondary antibodies for one hour at 25°C, the signals were visualized by enhanced or super chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturer's instruction. RNAi
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Specific RAGE-short hairpin RNA (shRNA), TLR2-shRNA, TLR4-shRNA, and controlshRNA were purchased from Sigma. 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, Grand Island, NY, USA) according to the manufacturer's instructions. Experimental animal models of acute pancreatitis
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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 kind 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, USA). For L-arginine-induced pancreatitis, a sterile solution of L-arginine hydrochloride (8%; Sigma) was prepared in normal saline and the pH was adjusted to 7.0. Mice received three hourly intraperitoneal (i.p.) injections of L-arginine (3 g/kg), while controls were administered saline i.p. as a control as described previously (13). For ceruleininduced pancreatitis, mice received seven hourly i.p. injections of 50 μg/kg cerulein (Sigma) in sterile saline, while controls were given saline as described previously (14). ELISA analysis ELISA assays were performed for the measurement of amylase (Abcam), LDH (Abcam), IL-1β (R&D Systems, Minneapolis, MN, USA), TNFα (R&D Systems), and high mobility
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group box 1 (HMGB1) (Sino-Test Corporation, Sagamihara, Japan) in serum and/or cell culture supernatants, and MPO (Abcam) in pancreas tissue homogenates, according to the manufacturer's instructions. Immunofluorescence Tissues were embedded in optimum cutting temperature cryomedium (Sakura, 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). Statistical analysis
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Data are expressed as means ± SEM of three independent experiments. Significance of differences between groups was determined by two-tailed Student's t test or ANOVA LSD 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.
RESULTS Nucleosome release promotes AIM2 inflammasome activation in macrophages
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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 pro-inflammatory mediators including interleukin-1β (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, lipopolysaccharide (LPS)/adenosine triphosphate (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.
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RAGE is required for nucleosome-mediated inflammasome activation in macrophages Toll-like receptors (TLRs) and RAGE are important DAMP 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. J Immunol. Author manuscript; available in PMC 2017 May 15.
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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
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Given that phosphorylation and activation of double-stranded RNA-dependent protein kinase (PKR) is 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-aminopurine (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 of 2-AP itself mildly increased HMGB1 release (Fig. 3D) due to possibility toxicity (21). In addition to IL-1β and HMGB1, 2-AP also partly limited histone/DNAinduced TNFα release (Fig. 3E). Collectively, RAGE-mediated PKR phosphorylation is required for the inflammatory response following nucleosome delivery to macrophages. RAGE depletion protects against acute pancreatitis in experimental animal models
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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 to RAGE−/− mice (Fig. 4A). Histological assessment of pancreatic damage revealed exaggerated acinar cell death, leukocyte infiltration, and interstitial edema in RAGE+/+ mice compared to RAGE−/− mice (Fig. 4B). Immunofluorescent stain 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 myeloperoxidase [MPO] activity, Fig. 4E), pancreatic necrosis (serum lactate dehydrogenase [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 pro-inflammation mediator release in experimental models.
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AIM2 depletion protects against acute pancreatitis in experimental animal models
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Similar to RAGE−/− mice, the AIM2−/− mice were resistant to L-arginine-induced severe AP without death compared to 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.
DISCUSSION
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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. Here, 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 inflammation response to AP.
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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). Here, we demonstrated that AIM2, but not NLRP3, is required for nucleosome-mediated inflammasome activation in macrophages. We also showed here that genetic deletion of AIM2 protects against experimental AP in mice. AIM2 is a member of the hematopoietic interferon-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.
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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 (33). As a newly-identified inflammasome component, PKR can directly bind to NLRP3, NLRP1, AIM2, or NLRC4 by autophosphorylation during inflammasome activation in macrophages (33). Our results indicate that PKR phosphorylation contributes to nucleosome-induced AIM2 inflammasome
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activation and subsequent IL-1β and HMGB1 release. Thus, activation of PKR is implicated in the crosstalk between cell death and inflammasome activation (34).
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RAGE is a member of the immunoglobulin 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 poly-microbial sepsis (12) and DNA-induced lung injury (35). Blockade of RAGE by drugs also attenuates ischemia and reperfusion injury in the liver (36) and heart (37). 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 current 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 (38). TLR9 also contributes to histone activity in liver ischemia and reperfusion injury (39). RAGE plays a major role in regulating the uptake of DNA/HMGB1 in nucleosome complexes (35), and serves to create a ‘zipper’, aligning proinflammatory macrophages and other RAGEexpressing cells to modulate and regulate inflammation in the transition to the adaptive immune response.
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In summary, we demonstrate here 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.
ACKNOWLEDGMENTS We thank Christine Heiner (Departments of Surgery and Anesthesiology, University of Pittsburgh) for her critical reading of the manuscript. Grant support: This work was supported by the National Institutes of Health of USA (R01GM115366 and R01CA160417 to DT; R01 CA181450 to HJZ). This project used University of Pittsburgh Cancer Institute shared resources supported in part by award P30CA047904.
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Author Manuscript Author Manuscript Figure 1. Nucleosomes promote AIM2 inflammasome activation in macrophages
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(A-B) Macrophages (immortalized bone marrow-derived macrophages (iBMDMs) and primary mouse peritoneal macrophages [“PM”]) were treated with histone (300 ng/mL) and/or genomic DNA (500 ng/mL) for 24 hours. The release of IL-1β (A) and HMGB1 (B) in supernatants were assayed using ELISA (n=3, *, p < 0.05 versus untreated group). (C-D) Indicated iBMDMs were treated with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours. The release of IL-1β (C) and HMGB1 (D) in supernatants were assayed using ELISA (n=3, *, p < 0.05 versus WT group). (E) Knockout of NLRP3 suppressed ATP (5 mM, 30min)-induced IL-1β release in LPS-primed NLRP3−/− iBMDMs (n=3, *, p < 0.05 versus WT group). (F) Western blot-analyzed IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) and the precursors of IL-1β (pro-IL-1β) and caspase-1 (pro-Casp-1) in lysates of indicated iBMDMs following treatment with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours.
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Author Manuscript Author Manuscript Figure 2. RAGE is required for nucleosome-mediated inflammasome activation in macrophages
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(A-C) Knockdown of RAGE (but not TLR2 and TLR4) by specific shRNA in iBMDM cells (A) inhibited histone (300 ng/mL)/DNA (500 ng/mL)(“H/D”)-induced IL-1β (B) and HMGB1 (C) release at 24 hours (n=3, *, p < 0.05 versus control shRNA group). (D-E) Peritoneal macrophages (“PM”) were isolated from wild type (WT), RAGE−/−, and TLR4−/− mice and then treated with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours. The release of IL-1β (D) and HMGB1 (E) in supernatants were assayed by ELISA (n=3, *, p < 0.05 versus WT group). (F) Western blot-analyzed IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) and the precursors of IL-1β (pro-IL-1β) and caspase-1 (proCasp-1) in lysates of indicated PMs following treatment with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours.
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Author Manuscript Author Manuscript Figure 3. RAGE-mediated PKR phosphorylation is required for nucleosome-induced inflammasome activation in macrophages
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(A) Peritoneal macrophages (“PM”) were isolated from wild type (WT), RAGE−/−, TLR4−/−, and AIM2−/− mice and then treated with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours. The levels of PKR phosphorylation (P-PKR) and PKR in whole cell extract were assayed using western blot. (B-E) Indicated macrophages were treated with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours in the presence and absence of the PKR inhibitor 2-AP (1 mM). The levels of P-PKR and PKR in whole cell extract were assayed using western blot (B). The release of IL-1β (C), HMGB1 (D), and TNFα (E) in supernatants were assayed using ELISA (n=3, *, p < 0.05 versus H/D treatment group).
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Author Manuscript Author Manuscript Figure 4. RAGE depletion protects against L-arginine-induced acute pancreatitis in experimental models
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(A) RAGE+/+ and RAGE−/− mice received a lethal L-arginine dose (3 g/kg × 3, i.p.). The Kaplan-Meyer method was used to compare differences in survival rates between groups. *, p
J Immunol. Author manuscript; available in PMC 2017 May 15. Published in final edited form as: J Immunol. 2016 May 15; 196(10): 4331–4337. doi:10.4049/jimmunol.1502340.
The Receptor for Advanced Glycation Endproducts (RAGE) Activates the AIM2 Inflammasome in Acute Pancreatitis Rui Kang1,*, Ruochan Chen1, Min Xie2, Lizhi Cao2, Michael T. Lotze1,3, Daolin Tang1,*, and Herbert J. Zeh III1,* 1
Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15219, USA
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2
Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, Hunan 410008, People's Republic of China
3Department
of Immunology, University of Pittsburgh, Pittsburgh, PA 15219, USA
Abstract
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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. Here, we demonstrate that the class III Major Histocompatability Region III receptor for advanced glycation endproducts (RAGE) contributes to AP by modulating inflammasome activation in macrophages. RAGE mediated nucleosome-induced AIM2 (but not NLRP3) inflammasome activation by modulating double-stranded RNA-dependent protein kinase (PKR) phosphorylation in macrophages. Pharmacological and genetic inhibition of the RAGE-PKR pathway attenuated the release of inflammasome-dependent exosomal leaderless cytokines (e.g., IL-1β and HMGB1) in vitro. RAGE or AIM2 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 to guide future development of therapeutic strategies to treat AP.
INTRODUCTION Author Manuscript
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
*
Correspondence should be directed to Dr. Rui Kang ([email protected]), Dr. Daolin Tang ([email protected]), or Dr. Herbert J. Zeh ([email protected]).. CONFLICT OF INTEREST The authors declare no conflicts of interest or financial interests.
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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).
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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 (nDAMPs) that exhibit significant pro-inflammatory 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 antibodies 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).
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Here, we demonstrate that the receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin 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 ceruleininduced AP in experimental animal models. Thus, disruption of nucleosome-RAGE-AIM2 signaling is a potential therapeutic approach for AP therapy.
Materials and Methods Regents
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The antibodies to RAGE and TLR4 were obtained from Abcam (Cambridge, MA, USA). The antibodies to AIM2 and actin were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies to P-PKR and PKR were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). The antibody to NLRP3 was obtained from Adipogen (San Diego, CA, USA). The antibody to Gr-1 was obtained from eBioscience (San Diego, CA). The antibody to F4/80 was obtained from Invitrogen (Grand Island, NY, USA). The antibody to IL-1β was obtained from R&D (Minneapolis, MN, USA). The antibody to caspase-1 was obtained from Adipogen (San Diego, CA, USA). Mouse genomic DNA was obtained from New England BioLabs Inc. (Ipswich, MA, USA). High purity histone protein was obtained from Roche Life Science (Stockholm, Sweden). LPS, ATP, and 2-AP were obtained from InvivoGen (San Diego, CA, USA). Cell culture Mouse peritoneal macrophages (PMs) were isolated from C57BL/6 mice as previously described (11). In brief, mice were injected intraperitoneally with 1.5 mL of 3% Brewer's
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thioglycollate broth for three days. Primary PMs were collected from euthanized animals by peritoneal lavage using 10 mL of ice cold RPMI supplemented with 2% fetal bovine serum (FBS), 1 unit/mL heparin and penicillin/streptomycin. Cells were washed using lavage media without heparin and plated in macrophage culture media of Dulbecco's Modified Eagle's Medium (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 phosphate buffered saline to remove non-adherent cells and leave adherent cells (PMs) in the culture media. The immortalized bone marrow-derived macrophages (iBMDMs) from WT, NLRP3−/−, and AIM2−/− mice were a kind 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% heatinactivated FBS and 100 units of penicillin and 100 μg/ml streptomycin) at 37 °C, 95% humidity, and 5% CO2.
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Western blot Proteins in the cell lysate or supernatants were resolved on 4-12% Criterion XT Bis-Tris gels (Bio-Rad, Hercules, CA, USA) and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated for two hours at 25°C or overnight at 4°C with various primary antibodies. After incubation with peroxidase-conjugated secondary antibodies for one hour at 25°C, the signals were visualized by enhanced or super chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturer's instruction. RNAi
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Specific RAGE-short hairpin RNA (shRNA), TLR2-shRNA, TLR4-shRNA, and controlshRNA were purchased from Sigma. 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, Grand Island, NY, USA) according to the manufacturer's instructions. Experimental animal models of acute pancreatitis
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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 kind 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, USA). For L-arginine-induced pancreatitis, a sterile solution of L-arginine hydrochloride (8%; Sigma) was prepared in normal saline and the pH was adjusted to 7.0. Mice received three hourly intraperitoneal (i.p.) injections of L-arginine (3 g/kg), while controls were administered saline i.p. as a control as described previously (13). For ceruleininduced pancreatitis, mice received seven hourly i.p. injections of 50 μg/kg cerulein (Sigma) in sterile saline, while controls were given saline as described previously (14). ELISA analysis ELISA assays were performed for the measurement of amylase (Abcam), LDH (Abcam), IL-1β (R&D Systems, Minneapolis, MN, USA), TNFα (R&D Systems), and high mobility
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group box 1 (HMGB1) (Sino-Test Corporation, Sagamihara, Japan) in serum and/or cell culture supernatants, and MPO (Abcam) in pancreas tissue homogenates, according to the manufacturer's instructions. Immunofluorescence Tissues were embedded in optimum cutting temperature cryomedium (Sakura, 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). Statistical analysis
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Data are expressed as means ± SEM of three independent experiments. Significance of differences between groups was determined by two-tailed Student's t test or ANOVA LSD 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.
RESULTS Nucleosome release promotes AIM2 inflammasome activation in macrophages
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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 pro-inflammatory mediators including interleukin-1β (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, lipopolysaccharide (LPS)/adenosine triphosphate (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.
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RAGE is required for nucleosome-mediated inflammasome activation in macrophages Toll-like receptors (TLRs) and RAGE are important DAMP 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. J Immunol. Author manuscript; available in PMC 2017 May 15.
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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
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Given that phosphorylation and activation of double-stranded RNA-dependent protein kinase (PKR) is 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-aminopurine (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 of 2-AP itself mildly increased HMGB1 release (Fig. 3D) due to possibility toxicity (21). In addition to IL-1β and HMGB1, 2-AP also partly limited histone/DNAinduced TNFα release (Fig. 3E). Collectively, RAGE-mediated PKR phosphorylation is required for the inflammatory response following nucleosome delivery to macrophages. RAGE depletion protects against acute pancreatitis in experimental animal models
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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 to RAGE−/− mice (Fig. 4A). Histological assessment of pancreatic damage revealed exaggerated acinar cell death, leukocyte infiltration, and interstitial edema in RAGE+/+ mice compared to RAGE−/− mice (Fig. 4B). Immunofluorescent stain 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 myeloperoxidase [MPO] activity, Fig. 4E), pancreatic necrosis (serum lactate dehydrogenase [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 pro-inflammation mediator release in experimental models.
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AIM2 depletion protects against acute pancreatitis in experimental animal models
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Similar to RAGE−/− mice, the AIM2−/− mice were resistant to L-arginine-induced severe AP without death compared to 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.
DISCUSSION
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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. Here, 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 inflammation response to AP.
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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). Here, we demonstrated that AIM2, but not NLRP3, is required for nucleosome-mediated inflammasome activation in macrophages. We also showed here that genetic deletion of AIM2 protects against experimental AP in mice. AIM2 is a member of the hematopoietic interferon-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.
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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 (33). As a newly-identified inflammasome component, PKR can directly bind to NLRP3, NLRP1, AIM2, or NLRC4 by autophosphorylation during inflammasome activation in macrophages (33). Our results indicate that PKR phosphorylation contributes to nucleosome-induced AIM2 inflammasome
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activation and subsequent IL-1β and HMGB1 release. Thus, activation of PKR is implicated in the crosstalk between cell death and inflammasome activation (34).
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RAGE is a member of the immunoglobulin 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 poly-microbial sepsis (12) and DNA-induced lung injury (35). Blockade of RAGE by drugs also attenuates ischemia and reperfusion injury in the liver (36) and heart (37). 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 current 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 (38). TLR9 also contributes to histone activity in liver ischemia and reperfusion injury (39). RAGE plays a major role in regulating the uptake of DNA/HMGB1 in nucleosome complexes (35), and serves to create a ‘zipper’, aligning proinflammatory macrophages and other RAGEexpressing cells to modulate and regulate inflammation in the transition to the adaptive immune response.
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In summary, we demonstrate here 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.
ACKNOWLEDGMENTS We thank Christine Heiner (Departments of Surgery and Anesthesiology, University of Pittsburgh) for her critical reading of the manuscript. Grant support: This work was supported by the National Institutes of Health of USA (R01GM115366 and R01CA160417 to DT; R01 CA181450 to HJZ). This project used University of Pittsburgh Cancer Institute shared resources supported in part by award P30CA047904.
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Author Manuscript Author Manuscript Figure 1. Nucleosomes promote AIM2 inflammasome activation in macrophages
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(A-B) Macrophages (immortalized bone marrow-derived macrophages (iBMDMs) and primary mouse peritoneal macrophages [“PM”]) were treated with histone (300 ng/mL) and/or genomic DNA (500 ng/mL) for 24 hours. The release of IL-1β (A) and HMGB1 (B) in supernatants were assayed using ELISA (n=3, *, p < 0.05 versus untreated group). (C-D) Indicated iBMDMs were treated with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours. The release of IL-1β (C) and HMGB1 (D) in supernatants were assayed using ELISA (n=3, *, p < 0.05 versus WT group). (E) Knockout of NLRP3 suppressed ATP (5 mM, 30min)-induced IL-1β release in LPS-primed NLRP3−/− iBMDMs (n=3, *, p < 0.05 versus WT group). (F) Western blot-analyzed IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) and the precursors of IL-1β (pro-IL-1β) and caspase-1 (pro-Casp-1) in lysates of indicated iBMDMs following treatment with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours.
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Author Manuscript Author Manuscript Figure 2. RAGE is required for nucleosome-mediated inflammasome activation in macrophages
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(A-C) Knockdown of RAGE (but not TLR2 and TLR4) by specific shRNA in iBMDM cells (A) inhibited histone (300 ng/mL)/DNA (500 ng/mL)(“H/D”)-induced IL-1β (B) and HMGB1 (C) release at 24 hours (n=3, *, p < 0.05 versus control shRNA group). (D-E) Peritoneal macrophages (“PM”) were isolated from wild type (WT), RAGE−/−, and TLR4−/− mice and then treated with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours. The release of IL-1β (D) and HMGB1 (E) in supernatants were assayed by ELISA (n=3, *, p < 0.05 versus WT group). (F) Western blot-analyzed IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) and the precursors of IL-1β (pro-IL-1β) and caspase-1 (proCasp-1) in lysates of indicated PMs following treatment with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours.
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Author Manuscript Author Manuscript Figure 3. RAGE-mediated PKR phosphorylation is required for nucleosome-induced inflammasome activation in macrophages
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(A) Peritoneal macrophages (“PM”) were isolated from wild type (WT), RAGE−/−, TLR4−/−, and AIM2−/− mice and then treated with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours. The levels of PKR phosphorylation (P-PKR) and PKR in whole cell extract were assayed using western blot. (B-E) Indicated macrophages were treated with histone (300 ng/mL)/DNA (500 ng/mL) (“H/D”) for 24 hours in the presence and absence of the PKR inhibitor 2-AP (1 mM). The levels of P-PKR and PKR in whole cell extract were assayed using western blot (B). The release of IL-1β (C), HMGB1 (D), and TNFα (E) in supernatants were assayed using ELISA (n=3, *, p < 0.05 versus H/D treatment group).
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Author Manuscript Author Manuscript Figure 4. RAGE depletion protects against L-arginine-induced acute pancreatitis in experimental models
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(A) RAGE+/+ and RAGE−/− mice received a lethal L-arginine dose (3 g/kg × 3, i.p.). The Kaplan-Meyer method was used to compare differences in survival rates between groups. *, p
