Endogenous Danger Signals Research Articles

Palmitic acid induces interleukin-1β secretion via NLRP3 inflammasomes and inflammatory responses through ROS production in human placental cells

Maternal obesity, a major risk factor for adverse pregnancy complications, results in inflammatory cytokine release in the placenta. Levels of free fatty acids are elevated in the plasma of obese human. These fatty acids include obesity-related palmitic acids, which is a major saturated fatty acid, that promotes inflammatory responses. Increasing evidence indicates that nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes mediate inflammatory responses induced by endogenous danger signals. We hypothesized that inflammatory responses associated with gestational obesity cause inflammation. To test this hypothesis, we investigated the effect of palmitic acid on the activation of NLRP3 inflammasomes and inflammatory responses in a human Sw.71 trophoblast cell line. Palmitic acid stimulated caspase-1 activation and markedly increased interleukin (IL)-1β secretion in Sw.71 cells. Treatment with a caspase-1 inhibitor diminished palmitic acid-induced IL-1β release. In addition, NLRP3 and caspase-1 genome editing using a CRISPR/Cas9 system in Sw.71 cells suppressed IL-1β secretion, which was stimulated by palmitic acid. Moreover, palmitic acid stimulated caspase-3 activation and inflammatory cytokine secretion (e.g., IL-6 and IL-8). Palmitic acid-induced cytokine secretion were dependent on caspase-3 activation. In addition, palmitic acid-induced IL-1β, IL-6, and IL-8 secretion was depended on reactive oxygen species (ROS) generation. In conclusion, palmitic acid caused activation of NLRP3 inflammasomes and inflammatory responses, inducing IL-1β, IL-6, and IL-8 secretion, which is associated with ROS generation, in human Sw.71 placental cells. We suggest that obesity-related palmitic acid induces placental inflammation, resulting in association with pregnancy complications.

Tenascin-C drives persistence of organ fibrosis.

The factors responsible for maintaining persistent organ fibrosis in systemic sclerosis (SSc) are not known but emerging evidence implicates toll-like receptors (TLRs) in the pathogenesis of SSc. Here we show the expression, mechanism of action and pathogenic role of endogenous TLR activators in skin from patients with SSc, skin fibroblasts, and in mouse models of organ fibrosis. Levels of tenascin-C are elevated in SSc skin biopsy samples, and serum and SSc fibroblasts, and in fibrotic skin tissues from mice. Exogenous tenascin-C stimulates collagen gene expression and myofibroblast transformation via TLR4 signalling. Mice lacking tenascin-C show attenuation of skin and lung fibrosis, and accelerated fibrosis resolution. These results identify tenascin-C as an endogenous danger signal that is upregulated in SSc and drives TLR4-dependent fibroblast activation, and by its persistence impedes fibrosis resolution. Disrupting this fibrosis amplification loop might be a viable strategy for the treatment of SSc.

Oxidized phosphatidylcholine induces the activation of NLRP3 inflammasome in macrophages.

The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein complex consisting of a receptor, an adaptor protein, and procaspase-1 that induces the secretion of the mature form of IL-1β in response to microbial infection and danger signals. Activation of the NLRP3 inflammasome induced by endogenous danger signal molecules is closely linked to the development and progress of chronic inflammatory diseases. The oxidation of phospholipids occurs upon cellular stress and damage, resulting in the accumulation of oxidized phosphatidylcholines (oxPAPC) such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-phosphocholine (POVPC) at inflammatory sites. In this study, we investigated whether oxidized phosphatidylcholine induces the activation of NLRP3 inflammasome in macrophages, leading to the secretion of IL-1β. POVPC induced the degradation of procaspase-1 to caspase-1(p10), the cleavage of pro-IL-1β to IL-1β, and oligomerization of ASC in primary mouse bone marrow-derived macrophages. POVPC-induced production of caspase-1, and IL-1β was abolished in macrophages derived from NLRP3- or caspase-1-deficient mice. In an air pouch model and a peritonitis model in mice, POVPC injection resulted in the production of caspase-1(p10), IL-1β, and IL-18 in wild-type, but not in NLRP3-deficient, mice. POVPC-induced inflammasome activation was mediated by mitochondrial reactive oxygen species resulting from intracellular Ca2+ signaling and mitochondrial destabilization. Our results demonstrate that endogenously produced oxidized phosphatidylcholines such as POVPC induce the activation of NLRP3 inflammasome, leading to the production of IL-1β in macrophages. The results provide an insight to understand how the oxidized lipids endogenously produced upon cellular stress and tissue damage contribute to the inflammatory reaction at pathologic sites.

Dendritic Cells Expressing Triggering Receptor Expressed on Myeloid Cells-1 Correlate with Plaque Stability in Symptomatic and Asymptomatic Patients with Carotid Stenosis.

Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory cells, including T-lymphocytes, dendritic cells (DCs) and macrophages that are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. In atherosclerotic tissue the number of DCs increases with the progression of the lesion and produce several inflammatory cytokines and growth factors. Triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role in inflammation. However, relationship of DCs and the role of TREM-1 with the stability of atherosclerotic plaques have not been examined. In this study, we investigated the heterogeneity of the plaque DCs, myeloid (mDC1 and mDC2) and plasmacytoid (pDCs), and examined the expression of TREM-1 and their co-localization with DCs in the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. We found increased expression of HLA-DR, fascin, and TREM-1 and decreased expression of TREM-2 and α-smooth muscle actin in S compared to AS atherosclerotic carotid plaques. Both TREM-1 and fascin were co-localized suggesting increased expression of TREM-1 in plaque DCs of S compared to AS patients. These data were supported by increased mRNA transcripts of TREM-1 and decreased mRNA transcripts of TREM-2 in carotid plaques of S compared to AS patients. There was higher density of both CD1c+ mDC1 and CD141+ mDC2 in the carotid plaques from AS compared to S patients, where as the density of CD303+ pDCs were higher in the carotid plaques of S compared to AS patients. These findings suggest a potential role of pDCs and TREM-1 in atherosclerotic plaque vulnerability. Thus, newer therapies could be developed to selectively block TREM-1 for stabilizing atherosclerotic plaques.

Cholesterol crystals activate the lectin complement pathway via ficolin-2 and MBL - implications for the progression of atherosclerosis

Abstract Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques by inducing inflammation and by functioning as an endogenous danger signal. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. In this study we hypothesized that pattern recognition molecules (PRM) from the lectin pathway bind CC and functions as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC using recombinant proteins, specific inhibitors as well as deficient and normal sera. Binding was assessed by flow cytometry and microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium dependent manner while ficolin-2 binding was calcium independent. No binding was observed for ficolin-1 or ficolin-3. Moreover, we demonstrated that IgM, but not IgG bound to CC and that C1q binding was facilitated by IgM. In conclusion our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.

Acute systemic exposure to silver-based nanoparticles induces hepatotoxicity and NLRP3-dependent inflammation

Nanoparticles (NPs) are increasingly being commercialized for use in biomedicine. NP toxicity following acute or chronic exposure has been described, but mechanistic insight into this process remains incomplete. Recent evidence from in vitro studies suggested a role for NLRP3 in NP cytotoxicity. In this study, we investigated the effect of systemic administration of composite inorganic NP, consisting of Ag:Cu:B (dose range 1–20 mg/kg), on the early acute (4–24 h post-exposure) and late phase response (96 h post-exposure) in normal and NLRP3-deficient mice. Our findings indicate that systemic exposure (≥2 mg/kg) was associated with acute liver injury due to preferential accumulation of NP in this organ and resulted in elevated AST, ALT and LDH levels. Moreover, within 24 h of NP administration, there was a dose-dependent increase in intraperitoneal neutrophil recruitment and upregulation in gene expression of several proinflammatory mediators, including TNF-α, IL-1β and S100A9. Histological analysis of liver tissue revealed evidence of dose-dependent hepatocyte necrosis, increase in sinusoidal Kupffer cells, lobular granulomas and foci of abscess formation which were most pronounced at 24 h following NP administration. NP deposition in the liver led to a significant upregulation in gene expression of S100A9, an endogenous danger signal recognition molecule of phagocytes, IL-1β and IL-6. The extent of proinflammatory cytokine activation and hepatotoxicity was significantly attenuated in mice deficient in the NLRP3 inflammasome, demonstrating the critical role of this innate immune system recognition receptor in the response to NP.

Toll-like Receptor 2 Regulates Nerve Growth Factor Synthesis via NF-κB Signaling in Human Intervertebral Disc Cells

Introduction Intervertebral disc degeneration is a leading cause of chronic low back pain (LBP), but how degeneration contributes to LBP is poorly understood. Nerve growth factor (NGF) levels increase during disc degeneration and evidence suggests NGF promotes disc innervation, neuronal sensitization and low back pain, making NGF a possible therapeutic target. However, current anti-NGF therapeutics have limited efficacy and safety concerns. Mechanisms leading to increased NGF in the disc are poorly understood and the molecular signaling mechanisms regulating NGF in inflammatory mediator rich environments, such as a degenerating disc, are unknown. During degeneration, proteoglycans and extra-cellular matrix (ECM) proteins are degraded and fragmented. ECM fragments can act as endogenous danger signal ligands for toll-like receptors (TLR). TLR activation induces cytokine and chemokine expression, and could thus regulate expression of inflammatory mediators, such as IL-1β or TNFα, during disc degeneration. NGF is often increased in environments with elevated levels of inflammatory mediators. Therefore, we hypothesized that TLR activation in the disc induces NGF expression. Materials and Methods Non-degenerate human intervertebral discs from organ donors without a history of low back pain were collected for cell isolation. NP and AF tissues were separated and enzymatically digested. Cells were treated with IL-1β (control), peptidoglycan (PGN, TLR2 agonist) and lipopolysaccharide (LPS, TLR4 agonist). Neutralizing antibodies against TLR2 were used to prevent TLR2 activation. Activated cell-signaling pathways following IL-1β and PGN treatment were evaluated by western blot and immunofluorescence. Small molecule inhibitors blocking p38 MAPK (SB203580) and NF-κB (BMS-345541) activity were added to cell cultures in combination with IL-1β and PGN. NGF gene expression was evaluated by qRT-PCR after 6, 12 and 24 hours, and NGF protein levels were examined by western blot and ELISA after 48 hours. Results TLR 2 activation significantly increased NGF gene expression in NP and AF cells following 6, 12 and 24 hours of treatment, while TLR 4 activation had little effect on NGF expression. TLR 2 activation significantly increased NGF protein secretion after 48 hours in NP and AF cells while TLR 4 activation did not increase NGF protein levels. TLR 2 neutralization with antibodies showed that TLR 2 is required for PGN induced NGF expression. TLR 2 activation of the p38 MAPK, ERK1/2, JNK and NF-κB signaling pathways was analyzed using phosphorylation specific antibodies. TLR activation increased p38 MAPK, ERK1/2 and p65 (NF- κB pathway) phosphorylation in disc cells compared with untreated cells. Furthermore, immunofluorescence found that TLR activation induces p65 translation to the nucleus, indicative of NF-κB signaling. Inhibiting NF-κB signaling decreased TLR 2 induced NGF expression. Conclusion This study found that TLR 2 activation directly regulates NGF expression via NF-κB signaling in human intervertebral disc cells. These findings represent a novel regulatory mechanism of NGF in the IVD. In the central nervous system NF-κB distinct signaling pathways regulate NGF. Therefore, these finding may provide therapeutic strategies to target NGF and low back pain without affecting the central nervous system.

Inhibition of IL-1R1/MyD88 signalling promotes mesenchymal stem cell-driven tissue regeneration.

Tissue injury and the healing response lead to the release of endogenous danger signals including Toll-like receptor (TLR) and interleukin-1 receptor, type 1 (IL-1R1) ligands, which modulate the immune microenvironment. Because TLRs and IL-1R1 have been shown to influence the repair process of various tissues, we explored their role during bone regeneration, seeking to design regenerative strategies integrating a control of their signalling. Here we show that IL-1R1/MyD88 signalling negatively regulates bone regeneration, in the mouse. Furthermore, IL-1β which is released at the bone injury site, inhibits the regenerative capacities of mesenchymal stem cells (MSCs). Mechanistically, IL-1R1/MyD88 signalling impairs MSC proliferation, migration and differentiation by inhibiting the Akt/GSK-3β/β-catenin pathway. Lastly, as a proof of concept, we engineer a MSC delivery system integrating inhibitors of IL-1R1/MyD88 signalling. Using this strategy, we considerably improve MSC-based bone regeneration in the mouse, demonstrating that this approach may be useful in regenerative medicine applications.

Translocation of Endogenous Danger Signal HMGB1 From Nucleus to Membrane Microvesicles in Macrophages.

High mobility group box 1 (HMGB1) is a nuclear protein that can be released from activated or dead cells. Extracellular HMGB1 can serve as a "danger signal" and novel cytokine that mediates sterile inflammation. In addition to its soluble form, extracellular HMGB1 can also be carried by membrane microvesicles. However, the cellular mechanisms responsible for nuclear HMGB1 translocation to the plasma membrane and release onto membrane microvesicles have not been investigated. Tobacco smoking is a major cause of sterile inflammation in many diseases. Smoking also increases blood levels of HMGB1. In this study, we found that exposure of macrophages to tobacco smoke extract (TSE) stimulated HMGB1 expression, redistribution, and release into the extracellular milieu both as a soluble molecule and, surprisingly, as a microvesicle-associated form (TSE-MV). Inhibition of chromosome region maintenance-1 (CRM1), a nuclear exporter, attenuated TSE-induced HMGB1 redistribution from the nucleus to the cytoplasm, and then its release on TSE-MVs. Our study demonstrates a novel mechanism for the translocation of nuclear HMGB1 to the plasma membrane, and then its release in a microvesicle-associated form. J. Cell. Physiol. 231: 2319-2326, 2016. © 2016 Wiley Periodicals, Inc.

The purinergic receptor P2X7 role in control of Dengue virus-2 infection and cytokine/chemokine production in infected human monocytes

Purinergic signaling has a crucial role in intracellular pathogen elimination. The P2X7 purinergic receptor (P2X7R), once activated by ATP, leads to pro-inflammatory responses including reactive oxygen species production. ATP can be released by injured cells, as endogenous danger signals. Dengue fever may evolve to a severe disease, leading to hypovolemic shock and coagulation dysfunctions as a result of a cytokine storm. Our aim was to evaluate the role of P2X7R activation during Dengue virus (DENV) infection. Extracellular ATP inhibited viral load in pretreated monocytes, as measured by NS1 secretion and by decrease in DENV+ P2X7+ cell frequencies, suggesting that P2X7R is involved in the antiviral response. Nitric oxide (NO) has anti-DENV properties and is decreased after DENV infection. NO production after ATP stimulation is abrogated by KN62 treatment, a specific P2X7R inhibitor, indicating that P2X7R likely is acting in the virus containment process. Additionally, TNF, CXCL8, CCL2 and CXCL10 factors that are associated with dengue severity were modulated by the P2X7R activation. We conclude that P2X7R is directly involved in the modulation of the antiviral and inflammatory process that occurs during DENV infection in vitro, and may have an important role in patient recovery in a first moment.

Correcting the NLRP3 inflammasome deficiency in macrophages from autoimmune NZB mice with exon skipping antisense oligonucleotides.

Inflammasomes are molecular complexes activated by infection and cellular stress, leading to caspase-1 activation and subsequent interleukin-1β (IL-1β) processing and cell death. The autoimmune NZB mouse strain does not express NLRP3, a key inflammasome initiator mediating responses to a wide variety of stimuli including endogenous danger signals, environmental irritants and a range of bacterial, fungal and viral pathogens. We have previously identified an intronic point mutation in the Nlrp3 gene from NZB mice that generates a splice acceptor site. This leads to inclusion of a pseudoexon that introduces an early termination codon and is proposed to be the cause of NLRP3 inflammasome deficiency in NZB cells. Here we have used exon skipping antisense oligonucleotides (AONs) to prevent aberrant splicing of Nlrp3 in NZB macrophages, and this restored both NLRP3 protein expression and NLRP3 inflammasome activity. Thus, the single point mutation leading to aberrant splicing is the sole cause of NLRP3 inflammasome deficiency in NZB macrophages. The NZB mouse provides a model for addressing a splicing defect in macrophages and could be used to further investigate AON design and delivery of AONs to macrophages in vivo.

Assessing Extracellular ATP as Danger Signal In Vivo: The pmeLuc System.

Inflammation is the key pathophysiological response triggered by noxious agents in multicellular organisms. Central to inflammation is detection of exogenous or endogenous danger signals by immune cells. Extracellular ATP is a ubiquitous danger signal released during septic or sterile inflammation. The development of reliable techniques to measure extracellular ATP in vivo has become an urgent need in inflammation studies after the discovery that the most potent plasma membrane receptor responsible for NLRP3 inflammasome activation is an ATP-activated receptor, P2RX7. Here we describe an easy bioluminescence technique for the measurement of extracellular ATP in vivo.

Allergens and Activation of the Toll-Like Receptor Response.

Pattern recognition receptors (PRRs) provide a crucial function in the detection of exogenous and endogenous danger signals. The Toll-like receptors (TLRs) were the first family of PRRs to be discovered and have been extensively studied since. Whilst TLRs remain the best characterized family of PRRs there is still much to be learnt about their mode of activation and the mechanisms of signal transduction they employ. Much of our understanding of these processes has been gathered through the use of cell based signaling assays utilizing specific gene-reporters or cytokine secretion based readouts. More recently it has become apparent that the repertoire of ligands recognized by these receptors may be wider than originally assumed and that their activation may be sensitized, or at least modulated by the presence of common household allergens such as the cat dander protein Fel d 1, or the house dust mite allergen Der p 2. In this chapter we provide an overview of the cell culture and stimulation processes required to study TLR signaling in HEK293 based assays and in bone marrow-derived macrophages.

High Glucose and Lipopolysaccharide Prime NLRP3 Inflammasome via ROS/TXNIP Pathway in Mesangial Cells.

While inflammation is considered a central component in the development in diabetic nephropathy, the mechanism remains unclear. The NLRP3 inflammasome acts as both a sensor and a regulator of the inflammatory response. The NLRP3 inflammasome responds to exogenous and endogenous danger signals, resulting in cleavage of procaspase-1 and activation of cytokines IL-1β, IL-18, and IL-33, ultimately triggering an inflammatory cascade reaction. This study observed the expression of NLRP3 inflammasome signaling stimulated by high glucose, lipopolysaccharide, and reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine in glomerular mesangial cells, aiming to elucidate the mechanism by which the NLRP3 inflammasome signaling pathway may contribute to diabetic nephropathy. We found that the expression of thioredoxin-interacting protein (TXNIP), NLRP3, and IL-1β was observed by immunohistochemistry in vivo. Simultaneously, the mRNA and protein levels of TXNIP, NLRP3, procaspase-1, and IL-1β were significantly induced by high glucose concentration and lipopolysaccharide in a dose-dependent and time-dependent manner in vitro. This induction by both high glucose and lipopolysaccharide was significantly inhibited by N-acetyl-L-cysteine. Our results firstly reveal that high glucose and lipopolysaccharide activate ROS/TXNIP/ NLRP3/IL-1β inflammasome signaling in glomerular mesangial cells, suggesting a mechanism by which inflammation may contribute to the development of diabetic nephropathy.

Dietary fatty acid composition is sensed by the NLRP3 inflammasome: omega-3 fatty acid (DHA) prevents NLRP3 activation in human macrophages.

The Nod-like receptor protein 3 (NLRP3) inflammasome is considered to be a pivotal host platform responsible for sensing of exogenous and endogenous danger signals, including those generated as a result of metabolic dysregulation, and for the subsequent, IL-1β-mediated orchestration of inflammatory and innate immunity responses. In this way, although the molecular link between diet-induced obesity and inflammasome activation is still unclear, free fatty acids (FFA) have been proposed as a triggering event. We report that dietary fatty acid (FA) composition is sensed by the NLRP3 inflammasome in human macrophages. For this purpose, we have analysed three roles of FA supplementation: as a priming signal for ATP-activated macrophages, in determining where the administration of dietary FAs interferes with LPS-mediated inflammasome activation and by inducing inflammasome activation per se. In this study, we confirm that saturated (SFAs) activated the NLRP3 inflammasome and stimulated the secretion of the IL-1β cytokine, while PUFAs were mainly inhibitors. Moreover, in general, DHA (n-3 PUFA) was more effective in preventing inflammasome activation than arachidonic acid (n-6 PUFA).

Suppression of NLRP3 inflammasome by γ-tocotrienol ameliorates type 2 diabetes

The Nod-like receptor 3 (NLRP3) inflammasome is an intracellular sensor that sets off the innate immune system in response to microbial-derived and endogenous metabolic danger signals. We previously reported that γ-tocotrienol (γT3) attenuated adipose tissue inflammation and insulin resistance in diet-induced obesity, but the underlying mechanism remained elusive. Here, we investigated the effects of γT3 on NLRP3 inflammasome activation and attendant consequences on type 2 diabetes. γT3 repressed inflammasome activation, caspase-1 cleavage, and interleukin (IL) 1β secretion in murine macrophages, implicating the inhibition of NLRP3 inflammasome in the anti-inflammatory and antipyroptotic properties of γT3. Furthermore, supplementation of leptin-receptor KO mice with γT3 attenuated immune cell infiltration into adipose tissue, decreased circulating IL-18 levels, preserved pancreatic β-cells, and improved insulin sensitivity. Mechanistically, γT3 regulated the NLRP3 inflammasome via a two-pronged mechanism: 1) the induction of A20/TNF-α interacting protein 3 leading to the inhibition of the TNF receptor-associated factor 6/nuclear factor κB pathway and 2) the activation of AMP-activated protein kinase/autophagy axis leading to the attenuation of caspase-1 cleavage. Collectively, we demonstrated, for the first time, that γT3 inhibits the NLRP3 inflammasome thereby delaying the progression of type 2 diabetes. This study also provides an insight into the novel therapeutic values of γT3 for treating NLRP3 inflammasome-associated chronic diseases.

Seprafilm Mediated Immune Response Mimic Danger Signal: A Pilot Study in Ovarian Cancer Cases

Danger signal was first proposed in 1994. In response to danger signal, dendritic cells, as the director of the T-cell immune system, would be matured and further enhance T-cell response. Hyaluronan had been reported as one of endogenous danger signals. Because of Seprafilm (most used for preventing post-operation adhesion) is also an hyaluronate-based agent, so we choose Seprafilm as our “candidate” of emerging danger signal to increasing T-cell response. We perform a pilot study in 10 ovarian cancer patients to compare the immune risk profiles between cases using Seprafilm (5 cases) and not using Seprafilm (5 cases). We found that there is no statistical difference on the immune risk profiles between two groups. However, a trend of increased CD4 in Seprafilm group was noted, indicating that Seprafilm might be an optional agent mimic danger signal. The potential value of Seprafilm to augment host immunosurveillance to improve survival rate in cancer patients is unknown, further study is needed to clarify the possibility of clinical applications.

The Role of HMGB1 in the Pathogenesis of Type 2 Diabetes

Significance. With an alarming increase in recent years, diabetes mellitus has become a global challenge. Despite advances in treatment of diabetes mellitus, currently, medications available are unable to control the progression of diabetes and its complications. Growing evidence suggests that inflammation is an important pathogenic mediator in the development of diabetes mellitus. The perspectives including suggestions for new therapies involving the shift from metabolic stress to inflammation should be taken into account. Critical Issues. High-mobility group box 1 (HMGB1), a nonhistone nuclear protein regulating gene expression, was rediscovered as an endogenous danger signal molecule to trigger inflammatory responses when released into extracellular milieu in the late 1990s. Given the similarities of inflammatory response in the development of T2D, we will discuss the potential implication of HMGB1 in the pathogenesis of T2D. Importantly, we will summarize and renovate the role of HMGB1 and HMGB1-mediated inflammatory pathways in adipose tissue inflammation, insulin resistance, and islet dysfunction. Future Directions. HMGB1 and its downstream receptors RAGE and TLRs may serve as potential antidiabetic targets. Current and forthcoming projects in this territory will pave the way for prospective approaches targeting the center of HMGB1-mediated inflammation to improve T2D and its complications.

A Novel Function for P2Y2 in Myeloid Recipient-Derived Cells during Graft-versus-Host Disease.

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. During the initiation phase of acute GvHD, endogenous danger signals such as ATP are released and inform the innate immune system via activation of the purinergic receptor P2X7 that a noninfectious damage has occurred. A second ATP-activated purinergic receptor involved in inflammatory diseases is P2Y2. In this study, we used P2y2(-/-) mice to test the role of this receptor in GvHD. P2y2(-/-) recipients experienced reduced GvHD-related mortality, IL-6 levels, enterocyte apoptosis, and histopathology scores. Chimeric mice with P2y2 deficiency restricted to hematopoietic tissues survived longer after GvHD induction than did wild-type mice. P2y2 deficiency of the recipient was connected to lower levels of myeloperoxidase in the intestinal tract of mice developing GvHD and a reduced myeloid cell signature. Selective deficiency of P2Y2 in inflammatory monocytes decreased GvHD severity. Mechanistically, P2y2(-/-) inflammatory monocytes displayed defective ERK activation and reactive oxygen species production. Compatible with a role of P2Y2 in human GvHD, the frequency of P2Y2(+) cells in inflamed GvHD lesions correlated with histopathological GvHD severity. Our findings indicate a novel function for P2Y2 in ATP-activated recipient myeloid cells during GvHD, which could be exploited when targeting danger signals to prevent GvHD.

Necrosis-Induced Sterile Inflammation Mediated by Interleukin-1α in Retinal Pigment Epithelial Cells.

Endogenous danger signals released from necrotic cells contribute to retinal inflammation. We have now investigated the effects of necrotic cell extracts prepared from ARPE-19 human retinal pigment epithelial cells (ANCE) on the release of proinflammatory cytokines and chemokines by healthy ARPE-19 cells. ANCE were prepared by subjection of ARPE-19 cells to freeze-thaw cycles. The release of various cytokines and chemokines from ARPE-19 cells was measured with a multiplex assay system or enzyme-linked immunosorbent assays. The expression of interleukin (IL)–1α and the phosphorylation and degradation of the endogenous nuclear factor–κB (NF-κB) inhibitor IκB-α were examined by immunoblot analysis. Among the various cytokines and chemokines examined, we found that ANCE markedly stimulated the release of the proinflammatory cytokine IL-6 and the chemokines IL-8 and monocyte chemoattractant protein (MCP)–1 by ARPE-19 cells. ANCE-induced IL-6, IL-8, and MCP-1 release was inhibited by IL-1 receptor antagonist and by an IKK2 inhibitor (a blocker of NF-κB signaling) in a concentration-dependent manner, but was not affected by a pan-caspase inhibitor (Z-VAD-FMK). Recombinant IL-1α also induced the secretion of IL-6, IL-8, and MCP-1 from ARPE-19 cells, and IL-1α was detected in ANCE. Furthermore, ANCE induced the phosphorylation and degradation of IκB-α in ARPE-19 cells. Our findings thus suggest that IL-1α is an important danger signal that is released from necrotic retinal pigment epithelial cells and triggers proinflammatory cytokine and chemokine secretion from intact cells in a manner dependent on NF-κB signaling. IL-1α is therefore a potential therapeutic target for amelioration of sterile inflammation in the retina.