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)
3
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 (The abbreviations used are: NP
nanoparticle(s)
AgNPsilver nanoparticle(s)
AgNP15silver nanoparticle(s) of 15 nm
ERendoplasmic reticulum
PERKprotein kinase RNA-like endoplasmic reticulum kinase
IRE-1inositol-requiring enzyme 1
ATF-6activating transcription factor 6
SREBP-1sterol regulatory element-binding protein-1
DLSdynamic light scattering
PBMCperipheral blood mononuclear cell
pNAp-nitroanilide
PIpropidium iodine
S1PSite-1 protease
S2PSite-2 protease.
1
). Indeed, silver ions and nanosilver were shown to be highly toxic for various types of microorganisms, including Pseudomonas spp. and Escherichia spp. (2
, 3
). 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 (
4
, 5
, 6
). 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 (7
). 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 (8
, 9
). Moreover, ER stress-related events were recently proposed as an early biomarker for nanotoxicological evaluation (10
). A few studies have reported ER stress-related events induced by NP in human cell lines and in zebrafish (10
, 11
, 12
).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) (
13
). Formation of pyroptosomes allows recruitment and processing of caspase-1 into two active fragments, p10 and p20 (14
). 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 (15
). Secretion of high concentrations of IL-1β is also associated with chronic inflammatory conditions, including rheumatoid arthritis and inflammatory bowel diseases (16
). 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 (17
). Some NP were shown to induce pyroptosis in human cells, namely carbon nanotubes, carbon black NP, and AgNP (18
, 19
, 20
). 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 (
36
) 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 (10
). However, only a few studies reported that AgNP can induce ER stress events in zebrafish and in human Chang liver cells (11
, 12
).Accumulation of unfolded proteins in the ER leads to transition of ATF-6, where it is processed by S1P and S2P (
7
). 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 (38
, 39
). 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 (40
). Moreover, tunicamycin was previously shown to induce ATF-6 degradation (7
). 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 (41
). 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 (
17
, 32
). 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 (42
). 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 (43
, 44
, 45
). 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 (35
). Although carbon nanotubes and carbon black have also been reported to activate this inflammasome in THP-1 and murine bone marrow-derived cells (- 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.
J. Immunol. 2009; 183: 787-791
20
, 46
), 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 (
16
), fitting well with previous studies indicating that nanosilver particles possess some toxic and proinflammatory effects (47.
48
, 49
50
). However, in a subacute murine inhalation model, some authors reported that AgNP induce minimal lung toxicity or inflammation (51
). These observations explain why some authors are interested in trying to answer whether AgNP are allies or adversaries (52
). 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 (
53
). The involvement of caspase-4 in ER stress has been previously established (25
, 27
) but has also been recently challenged (34
, 54
). Nevertheless, to date, only a few studies reported the potential involvement of caspase-4 in the activation of the inflammasomes (30
, 55
). 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 (22
). In another study, IL-1β production was found to be dependent on NF-κB in THP-1 cells (37
). 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 (26
). 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.REFERENCES
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Article Info
Publication History
Published online: January 15, 2015
Accepted:
December 3,
2014
Received:
September 9,
2014
Identification
Copyright
© 2015 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.
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