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Silver Nanoparticles Induce Degradation of the Endoplasmic Reticulum Stress Sensor Activating Transcription Factor-6 Leading to Activation of the NLRP-3 Inflammasome

  • Jean-Christophe Simard
    Correspondence
    To whom correspondence may be addressed: Institut National de la Recherche Scientifique-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Québec H7V1B7, Canada.
    Affiliations
    From the Laboratoire de recherche en inflammation et physiologie des granulocytes, Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Québec H7V1B7, Canada
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  • Francis Vallieres
    Affiliations
    From the Laboratoire de recherche en inflammation et physiologie des granulocytes, Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Québec H7V1B7, Canada
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  • Rafael de Liz
    Affiliations
    From the Laboratoire de recherche en inflammation et physiologie des granulocytes, Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Québec H7V1B7, Canada
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  • Valerie Lavastre
    Affiliations
    From the Laboratoire de recherche en inflammation et physiologie des granulocytes, Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Québec H7V1B7, Canada
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  • Denis Girard
    Correspondence
    To whom correspondence may be addressed: Institut National de la Recherche Scientifique-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Québec H7V1B7, Canada.
    Affiliations
    From the Laboratoire de recherche en inflammation et physiologie des granulocytes, Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Québec H7V1B7, Canada
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Open AccessPublished:January 15, 2015DOI:https://doi.org/10.1074/jbc.M114.610899
      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.

      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.

      Introduction

      Nanoparticles (NP)
      The abbreviations used are: NP
      nanoparticle(s)
      AgNP
      silver nanoparticle(s)
      AgNP15
      silver nanoparticle(s) of 15 nm
      ER
      endoplasmic reticulum
      PERK
      protein kinase RNA-like endoplasmic reticulum kinase
      IRE-1
      inositol-requiring enzyme 1
      ATF-6
      activating transcription factor 6
      SREBP-1
      sterol regulatory element-binding protein-1
      DLS
      dynamic light scattering
      PBMC
      peripheral blood mononuclear cell
      pNA
      p-nitroanilide
      PI
      propidium iodine
      S1P
      Site-1 protease
      S2P
      Site-2 protease.
      are used in many applications and in a variety of sectors, including textile, aerospace, electronics, and medical healthcare. Silver nanoparticles (AgNP) are among the most commonly used NP in nanomedicine, mainly because of their potent antimicrobial properties, increasing the interest to use them for drug delivery (
      • Kim J.S.
      • Kuk E.
      • Yu K.N.
      • Kim J.H.
      • Park S.J.
      • Lee H.J.
      • Kim S.H.
      • Park Y.K.
      • Park Y.H.
      • Hwang C.-Y.
      • Kim Y.K.
      • Lee Y.S.
      • Jeong D.H.
      • Cho M.H.
      Antimicrobial effects of silver nanoparticles.
      ). Indeed, silver ions and nanosilver were shown to be highly toxic for various types of microorganisms, including Pseudomonas spp. and Escherichia spp. (
      • Slawson R.
      • Trevors J.
      • Lee H.
      Silver accumulation and resistance in Pseudomonas stutzeri.
      ,
      • Sondi I.
      • Salopek-Sondi B.
      Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria.
      ). Even if potential exposure of humans to AgNP is already high, it will certainly increase in the becoming years. Because the toxicity of AgNP in humans is not fully understood, it is highly relevant to investigate their mode of action at the cellular and molecular level in humans.
      Endoplasmic reticulum (ER) stress leads to unfolded protein response, a major hallmark of cytotoxicity. To date, three ER stress sensors have been documented: protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE-1), and activating transcription factor 6 (ATF-6). IRE-1 and PERK both contain cytoplasmic kinase domains known to be activated by homodimerization and autophosphorylation in the presence of ER stressors (
      • Bertolotti A.
      • Zhang Y.
      • Hendershot L.M.
      • Harding H.P.
      • Ron D.
      Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.
      ,
      • Liu C.Y.
      • Schröder M.
      • Kaufman R.J.
      Ligand-independent dimerization activates the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum.
      ,
      • Harding H.P.
      • Zhang Y.
      • Ron D.
      Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.
      ). In the case of ATF-6, accumulation of unfolded proteins induces ATF-6 transition to the Golgi, where it is cleaved by two transmembrane proteins, Site-1 and Site-2 proteases (
      • Ye J.
      • Rawson R.B.
      • Komuro R.
      • Chen X.
      • Davé U.P.
      • Prywes R.
      • Brown M.S.
      • Goldstein J.L.
      ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.
      ). ATF-6 cleavage yields a cytoplasmic protein acting as an active transcription factor. Although short-term ER stress events lead to pro-survival transcriptional activities, prolonged ER stress activates the major apoptotic pathways (
      • Yoshida H.
      • Matsui T.
      • Yamamoto A.
      • Okada T.
      • Mori K.
      XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.
      ,
      • Lin J.H.
      • Li H.
      • Yasumura D.
      • Cohen H.R.
      • Zhang C.
      • Panning B.
      • Shokat K.M.
      • Lavail M.M.
      • Walter P.
      IRE1 signaling affects cell fate during the unfolded protein response.
      ). Moreover, ER stress-related events were recently proposed as an early biomarker for nanotoxicological evaluation (
      • Chen R.
      • Huo L.
      • Shi X.
      • Bai R.
      • Zhang Z.
      • Zhao Y.
      • Chang Y.
      • Chen C.
      Endoplasmic reticulum stress induced by zinc oxide nanoparticles is an earlier biomarker for nanotoxicological evaluation.
      ). A few studies have reported ER stress-related events induced by NP in human cell lines and in zebrafish (
      • Chen R.
      • Huo L.
      • Shi X.
      • Bai R.
      • Zhang Z.
      • Zhao Y.
      • Chang Y.
      • Chen C.
      Endoplasmic reticulum stress induced by zinc oxide nanoparticles is an earlier biomarker for nanotoxicological evaluation.
      ,
      • Zhang R.
      • Piao M.J.
      • Kim K.C.
      • Kim A.D.
      • Choi J.Y.
      • Choi J.
      • Hyun J.W.
      Endoplasmic reticulum stress signaling is involved in silver nanoparticles-induced apoptosis.
      ,
      • Christen V.
      • Capelle M.
      • Fent K.
      Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish.
      ).
      Pyroptosis, a type of programmed cell death sharing common features with apoptosis and necrosis, leads to the assembly of the inflammasomes and the formation of large structures called pyroptosomes characterized by aggregation of apoptosis-associated speck-like protein containing a CARD domain (ASC) (
      • Labbé K.
      • Saleh M.
      Pyroptosis: A Caspase-1-dependent programmed cell death and a barrier to infection.
      ). Formation of pyroptosomes allows recruitment and processing of caspase-1 into two active fragments, p10 and p20 (
      • Fernandes-Alnemri T.
      • Wu J.
      • Yu J.W.
      • Datta P.
      • Miller B.
      • Jankowski W.
      • Rosenberg S.
      • Zhang J.
      • Alnemri E.S.
      The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation.
      ). Caspase-1 controls processing and secretion of IL-1β, one of the most potent endogenous pyrogenic molecules. IL-1β is responsible for inflammatory cell infiltration and is known to induce cyclooxygenase and increase expression of adhesion molecules, production of reactive oxygen species, and other inflammatory soluble mediators (
      • Dinarello C.A.
      Immunological and inflammatory functions of the interleukin-1 family.
      ). Secretion of high concentrations of IL-1β is also associated with chronic inflammatory conditions, including rheumatoid arthritis and inflammatory bowel diseases (
      • Dinarello C.A.
      Interleukin-1 in the pathogenesis and treatment of inflammatory diseases.
      ). Interestingly, treatment of some auto-immune diseases with anti-IL-1β antibodies results in significant reduction of disease severity and symptoms. Pyroptosis also leads to the release of cytosolic content via formation of pore in the cellular membrane, thereby increasing the inflammatory process (
      • Franchi L.
      • Eigenbrod T.
      • Muñoz-Planillo R.
      • Nuñez G.
      The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.
      ). Some NP were shown to induce pyroptosis in human cells, namely carbon nanotubes, carbon black NP, and AgNP (
      • Meunier E.
      • Coste A.
      • Olagnier D.
      • Authier H.
      • Lefèvre L.
      • Dardenne C.
      • Bernad J.
      • Béraud M.
      • Flahaut E.
      • Pipy B.
      Double-walled carbon nanotubes trigger IL-1β release in human monocytes through Nlrp3 inflammasome activation.
      ,
      • Yang E.J.
      • Kim S.
      • Kim J.S.
      • Choi I.H.
      Inflammasome formation and IL-1β release by human blood monocytes in response to silver nanoparticles.
      ,
      • Reisetter A.C.
      • Stebounova L.V.
      • Baltrusaitis J.
      • Powers L.
      • Gupta A.
      • Grassian V.H.
      • Monick M.M.
      Induction of inflammasome-dependent pyroptosis by carbon black nanoparticles.
      ). Therefore, studying the impact of several distinct NP in the regulation of the inflammasome has become highly relevant for investigating their toxicity.
      In this study, we show that low concentrations of silver nanoparticles of 15 nm (AgNP15) induced ER stress response but did not led to cell death, whereas higher concentrations resulted in atypical ER stress response associated with ATF-6 degradation and pyroptotic cell death through NLRP-3 inflammasome activation. Our data suggest a link between these two processes.

      DISCUSSION

      AgNP are gaining increasing attention as the utilization of these NP is becoming more and more important in a plethora of medical products (
      • Nowack B.
      • Krug H.F.
      • Height M.
      120 years of nanosilver history: implications for policy makers.
      ) and, therefore, increasing potential human exposure, raising important toxicological considerations. In this study, we provide new evidences for a potential cytotoxic role of AgNP15, as demonstrated by activation of ER stress events accompanied by the activation of the NLRP-3 inflammasome and pyroptosis. Different types of nanoparticles were shown to induce ER stress response, including zinc oxide (
      • Chen R.
      • Huo L.
      • Shi X.
      • Bai R.
      • Zhang Z.
      • Zhao Y.
      • Chang Y.
      • Chen C.
      Endoplasmic reticulum stress induced by zinc oxide nanoparticles is an earlier biomarker for nanotoxicological evaluation.
      ). However, only a few studies reported that AgNP can induce ER stress events in zebrafish and in human Chang liver cells (
      • Zhang R.
      • Piao M.J.
      • Kim K.C.
      • Kim A.D.
      • Choi J.Y.
      • Choi J.
      • Hyun J.W.
      Endoplasmic reticulum stress signaling is involved in silver nanoparticles-induced apoptosis.
      ,
      • Christen V.
      • Capelle M.
      • Fent K.
      Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish.
      ).
      Accumulation of unfolded proteins in the ER leads to transition of ATF-6, where it is processed by S1P and S2P (
      • Ye J.
      • Rawson R.B.
      • Komuro R.
      • Chen X.
      • Davé U.P.
      • Prywes R.
      • Brown M.S.
      • Goldstein J.L.
      ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.
      ). The results of the present study indicate that 25 μg/ml AgNP15 induce rapid processing of ATF-6 (within 1 h) in human monocytes, an effect not observed at lower concentrations. Interestingly, activation of the NLRP-3 inflammasome was also induced only in response to stimulation with 25 μg/ml AgNP15, suggesting that ATF-6 could act as a molecular switch to induce the activation of this inflammasome. Indeed, inhibition of S2P (and S1P, to a lesser extent) blocked not only ATF-6 processing, but also activation of the inflammasome. Our results are in agreement with others reporting that inhibition of S2P was more efficient than S1P to block processing of ATF-6 (
      • Guan M.
      • Fousek K.
      • Chow W.A.
      Nelfinavir inhibits regulated intramembrane proteolysis of sterol regulatory element binding protein-1 and activating transcription factor 6 in castration-resistant prostate cancer.
      ,
      • Guan M.
      • Fousek K.
      • Jiang C.
      • Guo S.
      • Synold T.
      • Xi B.
      • Shih C.C.
      • Chow W.A.
      Nelfinavir induces liposarcoma apoptosis through inhibition of regulated intramembrane proteolysis of SREBP-1 and ATF6.
      ). Interestingly, a recent study demonstrated that the classical ER stressor, tunicamycin, is sufficient for the induction of IL-1β, suggesting a possible link between ER stress and the inflammasome (
      • Kim S.
      • Joe Y.
      • Jeong S.O.
      • Zheng M.
      • Back S.H.
      • Park S.W.
      • Ryter S.W.
      • Chung H.T.
      Endoplasmic reticulum stress is sufficient for the induction of IL-1β production via activation of the NF-κB and inflammasome pathways.
      ). Moreover, tunicamycin was previously shown to induce ATF-6 degradation (
      • Ye J.
      • Rawson R.B.
      • Komuro R.
      • Chen X.
      • Davé U.P.
      • Prywes R.
      • Brown M.S.
      • Goldstein J.L.
      ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.
      ). Another study also demonstrates that inhibition of ER stress by sodium 4-phenylbutyrate results in a significant reduction in IL-1β levels, supporting the role of ER stress in IL-1β production (
      • Kim H.J.
      • Jeong J.S.
      • Kim S.R.
      • Park S.Y.
      • Chae H.J.
      • Lee Y.C.
      Inhibition of endoplasmic reticulum stress alleviates lipopolysaccharide-induced lung inflammation through modulation of NF-κB/HIF-1α signaling pathway.
      ). However, to the best of our knowledge, the role of ATF-6 in the activation of the NLRP-3 inflammasome had never been investigated before.
      The inflammasomes are multiprotein complexes activated upon cytosolic perturbations such as cellular infection (
      • Franchi L.
      • Eigenbrod T.
      • Muñoz-Planillo R.
      • Nuñez G.
      The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.
      ,
      • Schroder K.
      • Tschopp J.
      The inflammasomes.
      ). This complex mediates the activation of the inflammatory caspase-1, which is critical for the secretion of mature IL-1β and formation of pores at the cell membrane (
      • Fink S.L.
      • Cookson B.T.
      Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages.
      ). Multiple inflammasomes have been characterized including NLRC-4, AIM-2, and NLRP-3. Among them, NLRP-3 is the one that has been more studied and is known to be activated upon various types of stimuli, including asbestos, silica, uric acid, and ATP (
      • Martinon F.
      • Pétrilli V.
      • Mayor A.
      • Tardivel A.
      • Tschopp J.
      Gout-associated uric acid crystals activate the NALP3 inflammasome.
      ,
      • Dostert C.
      • Pétrilli V.
      • Van Bruggen R.
      • Steele C.
      • Mossman B.T.
      • Tschopp J.
      Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica.
      ,
      • Kahlenberg J.M.
      • Lundberg K.C.
      • Kertesy S.B.
      • Qu Y.
      • Dubyak G.R.
      Potentiation of caspase-1 activation by the P2X7 receptor is dependent on TLR signals and requires NF-κB-driven protein synthesis.
      ). Conversely to the NLRC-4 or the AIM-2, the NLRP-3 inflammasome is controlled by a priming step that requires de novo protein translation (
      • Bauernfeind F.G.
      • Horvath G.
      • Stutz A.
      • Alnemri E.S.
      • MacDonald K.
      • Speert D.
      • Fernandes-Alnemri T.
      • Wu J.
      • Monks B.G.
      • Fitzgerald K.A.
      • Hornung V.
      • Latz E.
      Cutting edge: NF-κB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression.
      ). Although carbon nanotubes and carbon black have also been reported to activate this inflammasome in THP-1 and murine bone marrow-derived cells (
      • Reisetter A.C.
      • Stebounova L.V.
      • Baltrusaitis J.
      • Powers L.
      • Gupta A.
      • Grassian V.H.
      • Monick M.M.
      Induction of inflammasome-dependent pyroptosis by carbon black nanoparticles.
      ,
      • Yang M.
      • Flavin K.
      • Kopf I.
      • Radics G.
      • Hearnden C.H.
      • McManus G.J.
      • Moran B.
      • Villalta-Cerdas A.
      • Echegoyen L.A.
      • Giordani S.
      • Lavelle E.C.
      Functionalization of carbon nanoparticles modulates inflammatory cell recruitment and NLRP3 inflammasome activation.
      ), the effect of AgNP on the inflammasome has never been reported. Herein, we show a cytotoxic and inflammatory role of AgNP15 as evidenced by induction of pyroptotic cell death and IL-1β secretion. Indeed, AgNP15-treated cells harbored the characteristics of pyroptotic cell death: increased processing and activity of caspase-1, pyroptosome formation, loss of membrane integrity, and secretion of IL-1β. The experiments on THP-1, primary monocytes, and macrophages also provide new mechanisms of action of the regulation of the NLRP-3 inflammasome. For instance, the basal expression level of the different components of this pathway were found to be slightly different in THP-1, primary monocytes, and macrophages. This resulted in a significant variation in the amount of mature IL-1β secreted within the extracellular space. Indeed, AgNP15-treated primary monocytes secreted significant amount of IL-1β without the priming of LPS. Of note, we could detect a significant expression level of NLRP-3 and pro-IL-1β in resting monocytes, possibly explaining the secretion of IL-1β in AgNP15-treated cells.
      Secretion of IL-1β is associated with acute inflammation and pathogenesis of multiple chronic inflammatory diseases (
      • Dinarello C.A.
      Interleukin-1 in the pathogenesis and treatment of inflammatory diseases.
      ), fitting well with previous studies indicating that nanosilver particles possess some toxic and proinflammatory effects (

      M. Kanapathipillai, A. Brock, D. E. Ingber (2014) Nanoparticle targeting of anti-cancer drugs that alter intracellular signaling or influence the tumor microenvironment Adv. Drug Deliv. Rev 10.1016/j.addr.2014.05.005

      • Lohcharoenkal W.
      • Wang L.
      • Chen Y.C.
      • Rojanasakul Y.
      Protein nanoparticles as drug delivery carriers for cancer therapy.
      ,
      • Chen X.
      • Schluesener H.J.
      Nanosilver: a nanoproduct in medical application.
      • Kulthong K.
      • Srisung S.
      • Boonpavanitchakul K.
      • Kangwansupamonkon W.
      • Maniratanachote R.
      Determination of silver nanoparticle release from antibacterial fabrics into artificial sweat.
      ). However, in a subacute murine inhalation model, some authors reported that AgNP induce minimal lung toxicity or inflammation (
      • Stebounova L.V.
      • Adamcakova-Dodd A.
      • Kim J.S.
      • Park H.
      • O'Shaughnessy P.T.
      • Grassian V.H.
      • Thorne P.S.
      Nanosilver induces minimal lung toxicity or inflammation in a subacute murine inhalation model.
      ). These observations explain why some authors are interested in trying to answer whether AgNP are allies or adversaries (
      • Bartłomiejczyk T.
      • Lankoff A.
      • Kruszewski M.
      • Szumiel I.
      Silver nanoparticles: allies or adversaries?.
      ). Therefore, the results of the present study are in line with the need to further study the mechanisms of the action of AgNP on mammalian cells at the molecular level, to assure a safe application of AgNP.
      Although we provide new evidences in the toxicity of AgNP, the exact mechanisms underlying the effects of AgNP15 still remain to be clarified. Even if we clearly observed AgNP15 inside cells, particularly close to or in the nucleus, we cannot definitely conclude that AgNP have to penetrate cells to exert biological effects. Based on the internalization inhibition experiments, we may speculate that internalization of AgNP15 might be dispensable for its effects on IL-1β secretion. Moreover, the effect of AgNP15 was clearly dependent on caspase-1 as inhibition of this protein resulted in abolition of IL-1β secretion. Furthermore, although we observed ATF-6 degradation in response to AgNP15, it cannot be ruled out that silver ions are somewhat involved. However, it is important to specify that we have used a commercial source of AgNP, and we have used them as is, as they are probably used in different applications. Our results also indicate that the processing of ATF-6 and the appearance of the cleaved fragment correlate with the increased expression of caspase-1 p20, caspase-1 activity, IL-1β secretion, and the percentage of PI-positive cells, supporting inflammasome activation. Through inhibition of ATF-6 processing, we clearly see a reduction in caspase-1 activity, IL-1β secretion, and the percentage of PI-positive cells, suggesting that ATF-6 degradation is an event related to activation of the NLRP-3 inflammasome and pyroptotic cell death. It is noteworthy that this effect was clearly different from apoptosis cell death.
      Caspase-4 belongs to the human inflammatory caspase family along with caspase-1, caspase-5, and caspase-12 (
      • McIlwain D.R.
      • Berger T.
      • Mak T.W.
      Caspase functions in cell death and disease.
      ). The involvement of caspase-4 in ER stress has been previously established (
      • Binet F.
      • Chiasson S.
      • Girard D.
      Evidence that endoplasmic reticulum (ER) stress and caspase-4 activation occur in human neutrophils.
      ,
      • Hitomi J.
      • Katayama T.
      • Eguchi Y.
      • Kudo T.
      • Taniguchi M.
      • Koyama Y.
      • Manabe T.
      • Yamagishi S.
      • Bando Y.
      • Imaizumi K.
      • Tsujimoto Y.
      • Tohyama M.
      Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death.
      ) but has also been recently challenged (
      • Binet F.
      • Chiasson S.
      • Girard D.
      Arsenic trioxide induces endoplasmic reticulum stress-related events in neutrophils.
      ,
      • Obeng E.A.
      • Boise L.H.
      Caspase-12 and caspase-4 are not required for caspase-dependent endoplasmic reticulum stress-induced apoptosis.
      ). Nevertheless, to date, only a few studies reported the potential involvement of caspase-4 in the activation of the inflammasomes (
      • Sollberger G.
      • Strittmatter G.E.
      • Kistowska M.
      • French L.E.
      • Beer H.D.
      Caspase-4 is required for activation of inflammasomes.
      ,
      • Kajiwara Y.
      • Schiff T.
      • Voloudakis G.
      • Gama Sosa M.A.
      • Elder G.
      • Bozdagi O.
      • Buxbaum J.D.
      A critical role for human caspase-4 in endotoxin sensitivity.
      ). Our results indicate that caspase-4 is indeed required for the priming of the inflammasome, as the absence of this caspase resulted in a severe impairment of pro-IL-1β synthesis. However, the absence of caspase-4 also resulted in a significant increase in the activity of active caspase-1 and the proportion of PI-positive and pyroptosome-positive cells. These results suggest that caspase-4 would therefore regulate negatively the NLRP-3 inflammasome activation but would play an important role in its priming step. It is noteworthy that, in caspase-4-deficient THP-1 cells, the NF-κB pathway is markedly affected because caspase-4 was previously found to interact with TRAF-6, agreeing with the lack of pro-IL-1β in these cells (
      • Lakshmanan U.
      • Porter A.G.
      Caspase-4 interacts with TNF receptor-associated factor 6 and mediates lipopolysaccharide-induced NF-κB-dependent production of IL-8 and CC chemokine ligand 4 (macrophage-inflammatory protein-1).
      ). In another study, IL-1β production was found to be dependent on NF-κB in THP-1 cells (
      • Yallapu M.M.
      • Gupta B.K.
      • Jaggi M.
      • Chauhan S.C.
      Fabrication of curcumin encapsulated PLGA nanoparticles for improved therapeutic effects in metastatic cancer cells.
      ). Together with the fact that priming of the inflammasomes is dependent of NF-κB activation and that caspase-4-deficient cells have reduced NF-κB transduction, this information is in agreement with our observation on the impairment of pro-IL-1β in caspase-4-deficient cells. Because we made our observations at a concentration of 25 μg/ml AgNP15, and not at 1–10 μg/ml, it would be of interest in the future to establish whether the same would be true for AgNP with a different starting diameter because this variable is highly important in the induction of different biological effects previously reported for cell toxicity of AgNP in bacteria, yeast, algae, crustaceans, and mammalian cells in vitro (
      • Ivask A.
      • Kurvet I.
      • Kasemets K.
      • Blinova I.
      • Aruoja V.
      • Suppi S.
      • Vija H.
      • Käkinen A.
      • Titma T.
      • Heinlaan M.
      • Visnapuu M.
      • Koller D.
      • Kisand V.
      • Kahru A.
      Size-dependent toxicity of silver nanoparticles to bacteria, yeast, algae, crustaceans and Mammalian cells in vitro.
      ). Because of the great potential and interest that AgNP represent for developing future therapies based on drug delivery, further studies need to be conducted to limit undesired potential effects of AgNP.

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