Jean-Christophe Simard

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-1β. 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-1β secretion. S100A8- and S100A9-mediated activation of NF-κB, the NLR family, pyrin domain-containing 3 (NLRP3) protein, and pro-IL-1β 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.

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 Crohns 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.

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.

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

Background: Some nanoparticles are known to induce endoplasmic reticulum (ER) stress and lead to cell death. Results: Silver nanoparticles induce ATF-6 degradation, leading to activation of the NLRP-3 inflammasome and pyroptosis. Conclusion: ATF-6 is an important target to silver nanoparticles. Significance: Our results provide a new link between ER stress and 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-1β 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-1β production. The absence of caspase-4 expression resulted in a significant reduction of pro-IL-1β. 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-1β, 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.

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. Copyright

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.

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-1β 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.

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-1β, 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-1β. 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.

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.

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

Abstract 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.