Fatty acid mimetic PBI-4547 restores metabolic homeostasis via GPR84 in mice with non-alcoholic fatty liver disease.

Non-alcoholic Fatty Liver Disease (NAFLD) is the most common form of liver disease and is associated with metabolic dysregulation. Although G protein-coupled receptor 84 (GPR84) has been associated with inflammation, its role in metabolic regulation remains elusive. The aim of our study was to evaluate the potential of PBI-4547 for the treatment of NAFLD and to validate the role of its main target receptor, GPR84. We report that PBI-4547 is a fatty acid mimetic, acting concomitantly as a GPR84 antagonist and GPR40/GPR120 agonist. In a mouse model of diet-induced obesity, PBI-4547 treatment improved metabolic dysregulation, reduced hepatic steatosis, ballooning and NAFLD score. PBI-4547 stimulated fatty acid oxidation and induced gene expression of mitochondrial uncoupling proteins in the liver. Liver metabolomics revealed that PBI-4547 improved metabolic dysregulation induced by a high-fat diet regimen. In Gpr84-/- mice, PBI-4547 treatment failed to improve various key NAFLD-associated parameters, as was observed in wildtype littermates. Taken together, these results highlight a detrimental role for the GPR84 receptor in the context of meta-inflammation and suggest that GPR84 antagonism via PBI-4547 may reflect a novel treatment approach for NAFLD and its related complications.

PBI-4050 via GPR40 activation improves adenine-induced kidney injury in mice.

PBI-4050 (3-pentylbenzenacetic acid sodium salt), a novel first-in-class orally active compound that has completed clinical Phases Ib and II in subjects with chronic kidney disease (CKD) and metabolic syndrome respectively, exerts antifibrotic effects in several organs via a novel mechanism of action, partly through activation of the G protein receptor 40 (GPR40) receptor. Here we evaluate the effects of PBI-4050 in both WT and Gpr40-/- mice on adenine-induced tubulointerstitial injury, anemia and activation of the unfolded protein response (UPR) pathway. Adenine-induced CKD was achieved in 8-week-old C57BL/6 mice fed a diet supplemented with 0.25% adenine. After 1 week, PBI-4050 or vehicle was administered daily by oral-gavage for 3 weeks. Gpr40-/- mice were also subjected to adenine-feeding, with or without PBI-4050 treatment. PBI-4050 improved renal function and urine concentrating ability. Anemia was present in adenine-fed mice, while PBI-4050 blunted these effects and led to significantly higher plasma erythropoietin (EPO) levels. Adenine-induced renal fibrosis, endoplasmic reticulum (ER) stress and apoptosis were significantly decreased by PBI-4050. In parallel, Gpr40-/- mice were more susceptible to adenine-induced fibrosis, renal function impairment, anemia and ER stress compared with WT mice. Importantly, PBI-4050 treatment in Gpr40-/- mice failed to reduce renal injury in this model. Taken together, PBI-4050 prevented adenine-induced renal injury while these beneficial effects were lost upon Gpr40 deletion. These data reinforce PBI-4050's use as a renoprotective therapy and identify GPR40 as a crucial mediator of its beneficial effects.

PBI-4050 Reduces Stellate Cell Activation and Liver Fibrosis through Modulation of Intracellular ATP Levels and the Liver Kinase B1/AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Pathway.

Hepatic fibrosis is a major cause of morbidity and mortality for which there is currently no effective therapy. We previously showed that 2-(3-pentylphenyl)acetic acid (PBI-4050) is a dual G protein-coupled receptor GPR40 agonist/GPR84 antagonist that exerts antifibrotic, anti-inflammatory, and antiproliferative action. We evaluated PBI-4050 for the treatment of liver fibrosis in vivo and elucidated its mechanism of action on human hepatic stellate cells (HSCs). The antifibrotic effect of PBI-4050 was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation-induced liver fibrosis rodent models. Treatment with PBI-4050 suppressed CCl4-induced serum aspartate aminotransferase levels, inflammatory marker nitric oxide synthase, epithelial to mesenchymal transition transcription factor Snail, and multiple profibrotic factors. PBI-4050 also decreased GPR84 mRNA expression in CCl4-induced injury, while restoring peroxisome proliferator-activated receptor γ (PPARγ) to the control level. Collagen deposition and α-smooth muscle actin (α-SMA) protein levels were also attenuated by PBI-4050 treatment in the bile duct ligation rat model. Transforming growth factor-ß-activated primary HSCs were used to examine the effect of PBI-4050 and its mechanism of action in vitro. PBI-4050 inhibited HSC proliferation by arresting cells in the G0/G1 cycle phase. Subsequent analysis demonstrated that PBI-4050 signals through a reduction of intracellular ATP levels, activation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), and blockade of mammalian target of rapamycin (mTOR), resulting in reduced protein and mRNA levels of α-SMA and connective tissue growth factor and restored PPARγ mRNA expression. Our findings suggest that PBI-4050 may exert antifibrotic activity in the liver through a novel mechanism of action involving modulation of intracellular ATP levels and the LKB1/AMPK/mTOR pathway in stellate cells, and PBI-4050 may be a promising agent for treating liver fibrosis.

A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors: GPR40 and GPR84.

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases.

Silver nanoparticles induce irremediable endoplasmic reticulum stress leading to unfolded protein response dependent apoptosis in breast cancer cells.

Nowadays, silver nanoparticles (AgNP) are widely used in the medical field mainly for their antibacterial properties. Although some studies report a cytotoxic activity of the particles, the mechanisms involved in AgNP-induced cell death remain to be determined. Herein, we report that AgNP of 2 (AgNP2) and 15 nm (AgNP15) induce apoptosis in human MCF-7 and T-47D breast cancer cells. Treatment with AgNP2 and AgNP15 led to accumulation and aggregation of misfolded proteins causing an endoplasmic reticulum (ER) stress and activating the unfolded protein response (UPR). The three main ER sensors, PERK, IRE-1α and ATF-6, were rapidly activated in response to AgNP2 and AgNP15. Although Grp78 levels remained unchanged, AgNP2 and AgNP15 induced upregulation of the transcription factors ATF-4 and GADD153/CHOP. Moreover, the initiating caspase-9 and the effector caspase-7 were activated in response to these NPs. The expression levels of the pro-apoptotic BIM and BAD proteins remained unchanged. In contrast, a downregulation of Mcl-1 and xIAP protein expression as well as a processing of PARP were observed. Pharmacological inhibition of PERK kinase and IRE-1 endonuclease activities, as well as inhibition of ER-stress, partially protected cells from AgNP2- and AgNP15-induced apoptosis. Of note, the non-cancerous MCF-10A cells were more resistant to both AgNP2 and AgNP15 when compared to MCF-7 and T-47D cell lines. Taken together, our results demonstrate that AgNP induce ER stress and can target the UPR-dependent apoptotic pathway in MCF-7 and T-47D, which highlights new potential strategies for the treatment of breast cancers.

Activation of human AML14.3D10 eosinophils by nanoparticles: Modulatory activity on apoptosis and cytokine production.

Eosinophilic inflammation is frequently observed in response to nanoparticle (NP) exposure in airway rodent models of allergies where the number of eosinophils is increased in lungs. Despite this, it is surprising that the potential cytotoxic effect of NP, as well as their direct role on eosinophils is poorly documented. The present study investigated how different NP can alter the biology of the human eosinophilic cell line AML14.3D10. It was found that among NP forms of CeO2, ZnO, TiO2, and nanosilver of 20 nm (AgNP20) or 70 nm (AgNP70) diameters, only ZnO and AgNP20 induced apoptosis. Caspases-7 and -9 were not activated by the tested NP while caspase-3 was activated by AgNP20 only. However, both ZnO and AgNP20 induced cytoskeletal breakdown as evidenced by the cleavage of lamin B1. Using an ELISArray approach for the simultaneous detection of several analytes (cytokines/chemokines), it was found that only ZnO and AgNP20 increased the production of different analytes including the potent pro-inflammatory CXCL8 (IL-8) chemokine. From the data here, we conclude that toxic effects of some NP could be observed in human eosinophil-like cells and that this could be related, at least partially, by induction of apoptosis and production of cytokines and chemokines involved in inflammation. The results of this study also indicate that distinct NP do not activate similarly human eosinophils, since ZnO and AgNP20 induce apoptosis and cytokine production while others such as TiO2, CeO2, and AgNP70 do not.

Silver nanoparticles of 70 nm and 20 nm affect differently the biology of human neutrophils.

The influence of size of nanoparticles (NP), especially in regard to pulmonary toxicity, has been widely investigated. In general, NP with smaller diameters are more pro-inflammatory in vivo, at least in terms of neutrophil influx. Nevertheless, the influence of size of NP on polymorphonuclear neutrophil (PMN) cell biology is poorly documented. In the study here, it was decided to determine if AgNP with a diameter of 70 nm (AgNP70) will alter the biology of human PMN similarly to AgNP20 previously reported to induce apoptosis and inhibit de novo protein synthesis. The results here indicated that, in contrast to AgNP20, AgNP70 delayed PMN apoptosis. However, both AgNP20 and AgNP70 inhibited de novo protein synthesis. Both forms of AgNP did not significantly increase reactive oxygen species (ROS) production, but AgNP20 significantly increased the cell production of the CXCL8 chemokine (IL-8). In addition, AgNP20, but not AgNP70, induced the release of albumin and matrix metalloproteinase-9 (MMP-9/gelatinase B) into culture supernatants. Consistent with this latter observation, gelatinase activity was increased by AgNP20, as assessed by zymography. From these outcomes, it is concluded that two NP with different initial diameters can possess similar - as well as distinct - biological properties in modulating human PMN functions. These outcomes are testimony to the complexity of the modes of action of NP at the cellular level.

Gold nanoparticles induce apoptosis, endoplasmic reticulum stress events and cleavage of cytoskeletal proteins in human neutrophils.

Gold nanoparticles (AuNPs) are promising candidates for developing nanomedicines, for the treatment of different disorders, including inflammatory diseases. However, how AuNPs could alter the biology of human neutrophils, key player cells in inflammation, is a poorly documented area of research. Here we found that, although AuNP of 20 nm (AuNP20) could be internalized in cytosolic vacuoles but that AuNP70 were localized at the cell membrane, both induced apoptosis similarly by a caspase-dependent mechanism. AuNPs induced degradation of the cytoskeletal proteins vimentin, lamin B1 and gelsolin, but, unexpectedly, did not increase their cell surface expression. Consequent with caspase-4 processing, AuNPs were found to activate endoplasmic reticulum (ER)-stress, as evidenced by activation of the three ER sensors, IRE1 (inositol-requiring protein-1), ATF-6 (activating transcription factor-6) and PERK (protein kinase RNA (PKR)-like ER kinase). AuNPs are novel human neutrophil proapoptotic agents indicating that they are toxic to these cells. However, the fact that they do not induce cell surface expression of cytoskeletal proteins could decrease potential adverse effects and toxicity of AuNPs by limiting, for example, the production of autoantibody against cytoskeleton components.

Prolonged cultures of unstimulated human neutrophils lead to the apparition and persistence of rest-in-plate structures (RIPs) recognized by professional phagocytes in vitro and in vivo.

Polymorphonuclear neutrophil cells (PMNs) are known to spontaneously undergo apoptosis and then eliminated by professional phagocytes to prevent inflammation, a process called efferocytosis. However, when efferocytosis is impaired, PMNs will fall into secondary necrosis. Whether this state can persist for a certain period of time is unclear, since most of the studies investigating secondary necrosis are performed within 24h following induction by a proapoptotic agent. In this study, freshly isolated human PMNs were incubated without addition of exogenous agents in order to force them to undergo apoptosis and then secondary necrosis, an ideal experimental condition to study the behavior of secondary necrotic PMNs in absence of efferocytosis. By monitoring PMN cell morphology over time, we observed that an increasing proportion of cells harbored a ghost-like phenotype. Because these cellular remnants persist in plates for several days, we introduce here the terminology RIPs for 'rest-in-plate' structure. Heating of freshly isolated PMNs for 5min did not lead to the apparition of RIPs over time. In vivo administration of 7-days old RIPs in the murine air pouch model induced a slight inflammation resorbed within 24h. PKH26-stained RIPs were found to be ingested by professional phagocytes in vitro and in vivo in the murine air pouch and peritonitis models. Therefore, aged-PMNs have the potential to become RIPs in absence of efficient efferocytosis. Fortunately RIPs are recognized by professional phagocytes and, therefore, the concept of resolution of inflammation based on elimination of apoptotic and secondary necrotic PMNs could also be applied to RIPs.

Silver nanoparticles rapidly induce atypical human neutrophil cell death by a process involving inflammatory caspases and reactive oxygen species and induce neutrophil extracellular traps release upon cell adhesion.

Inflammation is one of the major toxic effects reported in response to in vitro or in vivo nanoparticle (NP) exposure. Among engineered NPs, silver nanoparticles (AgNPs) are very attractive for the development of therapeutic strategies, especially because of their antimicrobial properties. In humans, neutrophils, key players in inflammation, are the most abundant blood leukocytes that spontaneously undergo apoptosis, a central cell death mechanism regulating inflammation. The aim of this study was to evaluate the effect of AgNPs on neutrophil apoptosis. Transmission electronic microscopy reveals that AgNPs rapidly penetrate inside neutrophils. AgNPs induced atypical cell death where the cell volume increased and the cell surface expression of CD16 remained unaltered unlike apoptotic neutrophils where cell shrinkage and loss of CD16 are typically observed. The AgNP-induced atypical cell death is distinct from necrosis and reversed by a pancaspase inhibitor or by inhibitors of the inflammatory caspase-1 and caspase-4. In addition, AgNPs induced IL-1ß production inhibited by caspase-1 and caspase-4 inhibitors and also induced caspase-1 activity. Reactive oxygen species (ROS) production was increased by AgNPs and the atypical cell death was inhibited by the antioxidant n-acetylcysteine. Under similar experimental conditions, adhesion of neutrophils leads to neutrophil extracellular trap (NET) release induced by AgNPs. However, this process was not reversed by caspase inhibitors. We conclude that AgNPs rapidly induced an atypical cell death in neutrophils by a mechanism involving caspase-1, -4 and ROS. However, in adherent neutrophils, AgNPs induced NET release and, therefore, are novel agents able to trigger NET release.

Silver nanoparticles induce degradation of the endoplasmic reticulum stress sensor activating transcription factor-6 leading to activation of the NLRP-3 inflammasome.

In the past decade, the increasing amount of nanoparticles (NP) and nanomaterials used in multiple applications led the scientific community to investigate the potential toxicity of NP. Many studies highlighted the cytotoxic effects of various NP, including titanium dioxide, zinc oxide, and silver nanoparticles (AgNP). In a few studies, endoplasmic reticulum (ER) stress was found to be associated with NP cytotoxicity leading to apoptosis in different cell types. In this study, we report for the first time that silver nanoparticles of 15 nm (AgNP15), depending on the concentration, induced different signature ER stress markers in human THP-1 monocytes leading to a rapid ER stress response with degradation of the ATF-6 sensor. Also, AgNP15 induced pyroptosis and activation of the NLRP-3 inflammasome as demonstrated by the processing and increased activity of caspase-1 and secretion of IL-1ß and ASC (apoptosis-associated speck-like protein containing a CARD domain) pyroptosome formation. Transfection of THP-1 cells with siRNA targeting NLRP-3 decreased the AgNP15-induced IL-1ß production. The absence of caspase-4 expression resulted in a significant reduction of pro-IL-1ß. However, caspase-1 activity was significantly higher in caspase-4-deficient cells when compared with WT cells. Inhibition of AgNP15-induced ATF-6 degradation with Site-2 protease inhibitors completely blocked the effect of AgNP15 on pyroptosis and secretion of IL-1ß, indicating that ATF-6 is crucial for the induction of this type of cell death. We conclude that AgNP15 induce degradation of the ER stress sensor ATF-6, leading to activation of the NLRP-3 inflammasome regulated by caspase-4 in human monocytes.

Human S100A9 potentiates IL-8 production in response to GM-CSF or fMLP via activation of a different set of transcription factors in neutrophils.

Inflammation is highly regulated by various agents. Unexpectedly, we report here that the damage-associated molecular pattern S100A9 protein, a potent neutrophil activator and inducer of cytokine production in monocytes, is not a direct activator of cytokine production in human neutrophils. However, S100A9 primed IL-8 production in fMLP- and GM-CSF-stimulated neutrophiles via NF-κB and CREB-1, and NF-κB, STAT3 and STAT5, respectively. Pharmacological inhibition confirmed the importance of these transcription factors by significantly decreasing IL-8 production. This is the first time that a different set of transcription factors are shown to be involved in S100A9-primed neutrophils in response to proinflammatory agonist.

Interaction between silver nanoparticles of 20 nm (AgNP20 ) and human neutrophils: induction of apoptosis and inhibition of de novo protein synthesis by AgNP20 aggregates.

Cytotoxic and proinflammatory properties of silver nanoparticles (AgNPs) have been reported in few studies but the direct interaction between AgNPs and neutrophils, which play a key role in inflammation, has never been documented. Here, we examined the role of AgNPs with a starting size of 20 nm (AgNP20 ) in human neutrophils. Using dynamic light scattering for the characterization of NPs suspended under identical conditions to those used for in vitro experiments, we found that, at 10 µg ml(-1) , 92% of AgNP20 possess a diameter of 17.1 nm but, at 100 µg ml(-1) , a tri-modal size distribution with large aggregates was observed (> 500 nm). Neutrophil cell size increased when treated with AgNP20 and transmission electronic microscopy experiments revealed that AgNP20 can rapidly interact with the cell membrane, penetrate neutrophils, localize in vacuole-like structures, and be randomly distributed in the cytosol after 24 h. Treatment with 100 µg ml(-1) AgNP20 for 24 h (but not 10 µg ml(-1) ) increased the neutrophil apoptotic rate and inhibited de novo protein synthesis. We conclude that AgNP20 induced apoptosis and can act as potent inhibitors of de novo protein synthesis at 100, but not 10 µg ml(-1) in human neutrophils.

Curcumin inhibits agent-induced human neutrophil functions in vitro and lipopolysaccharide-induced neutrophilic infiltration in vivo.

Curcumin, extracted from the rhizome of Curcuma longa, is known to possess anti-inflammatory activities. Despite the fact that neutrophils are key player cells in inflammation, the role of curcumin on neutrophil cell biology is not well documented and, in particular, how curcumin can alter primed neutrophils is unknown. In addition, the effect of curcumin on agent-induced neutrophilic inflammation is not well documented. Here, we demonstrated that curcumin inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)- or lipopolysaccharide (LPS)-induced suppression of human neutrophil apoptosis. In addition, we found that curcumin reversed the ability of phorbol myristate acetate (PMA) to induce reactive oxygen species as assessed by flow cytometry using the CM-H2DCF-DA probe. Using an antibody array approach, curcumin was found to inhibit LPS-induced cytokine production, including MIP-1α, MIP-1ß, IL-6, IL-8 (CXCL-8) and GRO-α. The inhibitory effect of curcumin on IL-8 production was confirmed by ELISA. Using both an electrophoretic mobility shift assay and a TransFactor p50 NF-κB ELISA, we demonstrated that curcumin inhibited LPS-induced NF-κB activation. In vivo, using the murine air pouch model of acute inflammation, we demonstrated that intraperitoneal administration of curcumin inhibited LPS-induced neutrophilic infiltration in vivo. As assessed by a murine antibody array approach, curcumin was found to decrease the local production of several cytokines/chemokines induced by LPS, including, but not limit to, MIP-1α and MIP-1ß. We conclude that curcumin possesses potent modulatory activities on primed or agent-induced human neutrophils in vitro and that it possesses important anti-inflammatory activities in vivo by inhibiting LPS-induced neutrophilic inflammation.

S100A8 and S100A9 induce cytokine expression and regulate the NLRP3 inflammasome via ROS-dependent activation of NF-κB(1.).

S100A8 and S100A9 are cytoplasmic proteins expressed by phagocytes. High concentrations of these proteins have been correlated with various inflammatory conditions, including autoimmune diseases such as rheumatoid arthritis and Crohn's disease, as well as autoinflammatory diseases. In the present study, we examined the effects of S100A8 and S100A9 on the secretion of cytokines and chemokines from PBMCs. S100A8 and S100A9 induced the secretion of cytokines such as IL-6, IL-8, and IL-1ß. This secretion was associated with the activation and translocation of the transcription factor NF-κB. Inhibition studies using antisense RNA and the pharmacological agent BAY-117082 confirmed the involvement of NF-κB in IL-6, IL-8, and IL-1ß secretion. S100A8- and S100A9-mediated activation of NF-κB, the NLR family, pyrin domain-containing 3 (NLRP3) protein, and pro-IL-1ß expression was dependent on the generation of reactive oxygen species. This effect was synergistically enhanced by ATP, a known inflammasome activator. These results suggest that S100A8 and S100A9 enhance the inflammatory response by inducing cytokine secretion of PBMCs.

Damage-associated molecular pattern S100A9 increases bactericidal activity of human neutrophils by enhancing phagocytosis.

The damage-associated molecular-pattern S100A9 is found at inflammatory sites in infections and various autoimmune diseases. It is released at very high concentrations in the extracellular milieu by activated neutrophils and monocytes in response to various agents. This proinflammatory protein is found in infected mucosae and tissue abscesses where it acts notably as a potent neutrophil activator. In this study, we examined the role of S100A9 in the control of infections. S100A9 was found to increase human neutrophil bactericidal activity toward Escherichia coli. Although S100A9 induced the accumulation of reactive oxygen species over time through the activation of NADPH oxidase, its antimicrobial activity was mediated mainly by enhancing the efficiency of neutrophil phagocytosis. Interestingly, S100A9 did not act by increasing cell surface expression of CD16, CD32, or CD64 in neutrophils, indicating that its biological effect in FcR-mediated phagocytosis is independent of upregulation of FcγR levels. However, S100A9-induced phagocytic activity required the phosphorylation of Erk1/2, Akt, and Syk. Taken together, our results demonstrate that S100A9 stimulates neutrophil microbicidal activity by promoting phagocytosis.

Induction of neutrophil degranulation by S100A9 via a MAPK-dependent mechanism.

S100A9 is a proinflammatory protein, expressed abundantly in the cytosol of neutrophils and monocytes. High extracellular S100A9 concentrations have been correlated with chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease, as well as with phagocyte extravasation. This study tested the hypothesis that S100A9 induces degranulation in human neutrophils. S100A9 was found to up-regulate the surface expression of CD35 and CD66b, proteins contained in secretory vesicles and specific/gelatinase granules, respectively. In addition, gelatinase and albumin, stored, respectively, in specific/gelatinase granules and secretory vesicles, were detected in the supernatants of neutrophils stimulated with S100A9. In contrast, stimulation with S100A9 had no effect on CD63 expression or MPO secretion, two proteins contained in azurophilic granules. S100A9 induced the phosphorylation of the MAPKs, ERK1/2, p38, and JNK. Inhibition of p38 and JNK but not ERK1/2, with specific inhibitors (SB203580, JNKII, and PD98059, respectively), blocked neutrophil degranulation induced by S100A9. Taken together, these results support the hypothesis and clearly indicate that S100A9 induces the degranulation of secretory and specific/gelatinase granules but not of azurophilic granules in a process involving p38 and JNK and further support its classification as a DAMP.