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.

Activation of human neutrophils by the anti-inflammatory mediator Esenbeckia leiocarpa leads to atypical apoptosis.

Despite the fact that Esenbeckia leiocarpa, a Brazilian plant, possesses potential anti-inflammatory properties, its effect in neutrophils, key players in inflammation, has never been investigated. In this study, a crude hydroalcoholic extract (CHE) was used to evaluate the potential toxic or agonistic effect of E. leiocarpa in human neutrophils. At a noncytotoxic concentration of 500 µg/mL, CHE increased actin polymerization and cell signaling events, especially p38 MAPK. Its modulatory activity on neutrophil cell apoptosis was investigated by cytology and by flow cytometry and, although CHE increased the apoptotic rate (by cytology) and increased annexin-V binding, it did not, unexpectedly, increase CD16 shedding. CHE increased the degradation of the cytoskeletal proteins gelsolin and paxillin but, surprisingly, not of vimentin. The proapoptotic activity of CHE was reversed by a pan-caspase inhibitor but not by a p38 inhibitor. We conclude that CHE is a novel human neutrophil agonist that induces apoptosis by a caspase-dependent and p38-independent mechanism in an atypical fashion based on its lack of effect on CD16 shedding and vimentin degradation. Since the resolution of inflammation occurs by elimination of apoptotic neutrophils, the ability of CHE to induce neutrophil apoptosis correlates well with its anti-inflammatory properties, as previously reported.

Activation of human neutrophils by Esenbeckia leiocarpa: comparison between the crude hydroalcoholic extract (CHE) and an alkaloid (Alk) fraction.

Esenbeckia leiocarpa, a wide spread native Brazilian tree, was reported recently to possess anti-inflammatory effects in vivo, but the mechanisms involved are still not fully understood and its role in neutrophils is poorly documented. The aim of this study was to compare the effects of a crude hydroalcoholic extract (CHE) and an alkaloid-enriched (Alk) fraction obtained from Esenbeckia leiocarpa bark on human neutrophils by investigating the effect of each fraction alone or in a mixture with classical neutrophil agonists. CHE inhibited intracellular reactive oxygen species (ROS) production but increased the extracellular superoxide (O2-) production, while Alk increased the former and also slightly increased O2- production. We found that CHE and Alk also induced phagocytosis accompanied by Syk activation, adhesion and degranulation. However, neither CHE nor Alk potentiated the effect of classical neutrophil agonists, namely the cytokines GM-CSF for phagocytosis and TNF-α for adhesion or N-formyl-methionyl-leucyl-phenylalanine (fMLP) for degranulation. In addition, based on catalase treatment, CHE and Alk induced neutrophil apoptosis by a hydrogen peroxide (H2O2)-dependent mechanism. Since the elimination of apoptotic neutrophils by professional phagocytes is important for the resolution of inflammation, the ability of CHE and Alk to induce neutrophil apoptosis has to be considered as one possible mechanism associated with the anti-inflammatory activity of these fractions previously reported in vivo.

Modulatory effect of mycophenolate mofetil on carrageenan-induced inflammation in the mouse air pouch model.

UNLABELLED: The treatment of some inflammatory diseases, such as rheumatoid arthritis, remains an important target for studies because some patients are refractory to conventional treatment. Mycophenolate mofetil (MMF), an immunosuppressive drug, has been shown to have a beneficial effect on the therapy of inflammatory and autoimmune diseases. In the present study, we aimed to analyse the anti-inflammatory effect of MMF administered by oral route in the mouse carrageenan-induced air pouch model. RESULTS: MMF significantly inhibited the influx of leukocytes, exudate concentrations (P<0.01), activities of myeloperoxidase (MPO) and adenosine deaminase (ADA), levels of nitrite/nitrate (NO(x)) and inducible nitric oxide synthase (iNOS) mRNA expression, as well as the levels of mRNA expression and proteins of tumor necrosis factor-alpha (TNF-α), Interleukin-beta (IL-1ß) and vascular endothelial growth factor-alpha (VEGF-α) (P<0.05). These results provide evidence that MMF has an important anti-inflammatory effect in reducing the influx of leukocytes and exudate concentrations. These inhibitory effects are correlated with the inhibition of specific pro-inflammatory enzymes (MPO, ADA and iNOS), and the levels of mRNA expression and proteins of TNF-α, IL-1ß and VEGF-α.

Activation of neutrophils by nanoparticles.

The use of nanoparticles (NPs) has increased in the past few years in various fields, including defence, aerospace, electronics, biology, medicine, and so forth. and in applications such as diagnostic technology, bioimaging, and drug/gene delivery. Thus, human exposure to NPs and nanomaterials is unavoidable and will certainly expand in the future resulting in a growing interest in nanotoxicology, the study of toxicity of nanomaterials. A number of studies have reported the effects of NPs in respect to pulmonary inflammation by investigating in vitro activation of pulmonary cells with NPs and in vivo in a variety of models in which neutrophils appear to be the predominant leukocyte cell type in lungs and in bronchoalveolar lavages following inhalation or intratracheal instillation of NPs. Despite the fact that several studies have reported an increased number of neutrophils, the literature dealing with the direct activation of neutrophils by a given NP is poorly documented. This paper will summarize the current literature in this latter area of research and will end with a perspective view in which our laboratory will be involved in the following years.

N-phenylmaleimide derivatives as mimetic agents of the pro-inflammatory process: myeloperoxidase activation.

Myeloperoxidase (MPO) is an important enzyme that catalyzes the reaction between hydrogen peroxide and chloride to generate hypochlorous acid, which oxidizes a range of biomolecules and has been associated with inflammatory diseases. The synthetic compounds N-phenylmaleimide (NFM) and 4-methyl-N-phenylmaleimide (Me-NFM) increased the MPO activity in vitro (of isolated enzyme and in isolated cells after animal treatment) and in vivo assays. MPO-induction may represent a good model system to investigate the molecular and cellular mechanisms of oxidative cell injury induced by activated neutrophils, and the interactions between damaging species involved in the respiratory burst.

Efficacy of tacrolimus in inhibiting inflammation caused by carrageenan in a murine model of air pouch.

BACKGROUND: Tacrolimus (Tac) is a macrolide immunosuppressant drug isolated from Streptomyces tsukubaensis, widely used in organ transplantation. OBJECTIVE: This study examined the effect of tacrolimus administered by oral route (p.o.) on inflammation in mouse subcutaneous air pouch triggered by carrageenan (Cg 1%). METHODS: The air pouch was induced as described by Benincá et al. [Benincá JP, Montanher AB, Zucolotto SM, Schenkel EP, FrödeTS. Anti-inflammatory effects of the Passiflora edulis: forma flavicarpa Degener inhibition of leukocytes, enzymes and pro-inflammatory cytokine levels in the air pouch model, in mice. Food Chem 2007; 104(3); 1097-1105.]. The inflammatory parameters (leukocytes, exudation, myeloperoxidase (MPO) and adenosine-deaminase (ADA) activities, as well as nitrate/nitrate concentrations (NO(x)), interleukin-1 beta (IL-1beta), chemokine to neutrophil (KC) and tumor necrosis factor-alpha (TNF-alpha) levels were analysed 24 h after injection of carrageenan. RESULTS: Tacrolimus, indomethacin and dexamethasone significantly inhibited leukocytes, neutrophils and exudation (P<0.05) when they were administered 0.5 h before inflammation. These drugs, under the same conditions, decreased MPO and ADA activities (P<0.05), NO(x) and IL-1beta levels (P<0.01). Tacrolimus and indomethacin, but not dexamethasone, inhibited KC levels (P<0.01). On the other hand, tacrolimus and dexamethasone, but not indomethacin, decreased TNF-alpha levels (P<0.01). CONCLUSIONS: Results of this study indicate that tacrolimus has an important anti-inflammatory property, showing not only inhibition of pro-inflammatory mediators release, but also inhibition of activated leukocyte infiltration into the site of inflammation. Furthermore, these results showed that most of the anti-inflammatory actions of tacrolimus were similar to those observed in animals treated with either indomethacin or dexamethasone.