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Nanoparticles Activate the NLR Pyrin Domain Containing 3 (Nlrp3)

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Nanoparticles Activate the NLR Pyrin Domain Containing 3 (Nlrp3) Nanoparticles activate the NLR pyrin domain containing 3(Nlrp3)inflammasome and cause pulmonary inflammation through release of IL-1α and IL-1β Amir S. Yazdia, Greta Guardaa, Nicolas Riteaub, Stefan K. Drexlera, Aubry Tardivela, Isabelle Couillinb, and Jürg Tschoppa,1 aDepartment of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland; and bLaboratory of Molecular Immunology and Embryology, University of Orleans and Centre National de la Recherche Scientifique, F-45071 Orleans, France Edited by Marc Feldmann, Imperial College London, London, United Kingdom, and approved October 1, 2010 (received for review June 13, 2010) Nanoparticles are increasingly used in various fields, including somes (12). To date four inflammasomes are functionally char- biomedicine and electronics. One application utilizes the opacifying acterized: the Nlrp1 (NLR pyrin domain containing 1, Nalp1), effect of nano-TiO2, which is frequently used as pigment in cosmet- Nlrp3 (NLR pyrin domain containing 3, Nalp3), Nlrc4 (NLR con- ics. Although TiO2 is believed to be biologically inert, an emerging taining a caspase recruitment domain 4, Ipaf), and AIM2 (absent literature reports increased incidence of respiratory diseases in peo- in melanoma) inflammasomes. The best-characterized inflam- ple exposed to TiO2. Here, we show that nano-TiO2 and nano-SiO2, masome consists of three main components, the Nod-like re- but not nano-ZnO, activate the NLR pyrin domain containing 3 ceptor (NLR)-family protein, Nlrp3, procaspase-1, and the ASC (Nlrp3) inflammasome, leading to IL-1β release and in addition, in- (apoptosis speck-like protein containing a CARD) adapter, which duce the regulated release of IL-1α. Unlike other particulate Nlrp3 bridges interactions between the former proteins (12). Following fl agonists, nano-TiO2–dependent-Nlrp3 activity does not require cy- autoactivation via in ammasome assembly, caspase-1 cleaves toskeleton-dependent phagocytosis and induces IL-1α/β secretion IL-1β, whose biologically active form is then secreted. β in nonphagocytic keratinocytes. Inhalation of nano-TiO2 provokes The potent actions of mature IL-1 dictate that its synthesis and lung inflammation which is strongly suppressed in IL-1R– and IL-1α– secretion are tightly regulated. A first signal, such as Toll-like – β deficient mice. Thus, the inflammation caused by nano-TiO2 in vivo receptor activation, triggers synthesis of pro IL-1 by transcrip- is largely caused by the biological effect of IL-1α. The current use of tional induction, whereas a second stimulus leads to inflamma- – nano-TiO2 may present a health hazard due to its capacity to induce some oligomerization, caspase-1 autoactivation, and caspase-1 IL-1R signaling, a situation reminiscent of inflammation provoked dependent cleavage and release of the biologically active, mature by asbestos exposure. IL-1β. This second signal, by which an active inflammasome complex is formed, is triggered by an ever-expanding group of caspase-1 | titanium dioxide | silica dioxide chemically and biologically unrelated danger-associated molecu- lar patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) (13). Whereas Nlrp3 inflammasome-dependent in- anoparticles are single particles with a diameter less than flammation promotes pathogen clearance in a number of in- N100 nm (1). Nanomaterials are increasingly used for manu- fection models, such as Candida albicans (14) and Klebsiella facturing industrial items, such as antibacterial surface coatings pneumonia (15), the Nlrp3 inflammasome can also initiate path- and semiconductors. The most frequently used nanoparticles are ological inflammatory reactions in response to endogenous or inorganic metal oxides such as silica dioxide (SiO2) and titanium exogenous danger signals such as ATP (16), monosodium urate dioxide (TiO2), due to their large surface-to-volume ratio (2). In crystals (MSU) (17), UVB irradiation (18), and the environ- particular, nano-TiO2 is frequently used as white pigment in many mental pollutants, silica and asbestos (19, 20). Although data everyday products such as white paint, paper, and plastics, and suggest that potassium efflux (13), cathepsin B (19), and/or the their use is especially prevalent in cosmetics (3). generation of reactive oxygen species (ROS) (20) may modulate μ Although m-sized and larger TiO2 particles are believed to inflammasome activation, the exact molecular mechanisms me- be biologically inert (4), an augmented frequency of respiratory diating inflammasome activation remain elusive (21). diseases in workers exposed to TiO2 was reported in the late 1980s (5). Supporting this observation, several recent studies demon- Results strated that pulmonary uptake of TiO2 nanoparticles worsened Nanoparticles Activate the Nlrp3 Inflammasome in Myeloid Cells and respiratory diseases such as pulmonary fibrosis or lipopolysac- Lead to IL-1β Secretion in Primary Human Keratinocytes. To deter- IMMUNOLOGY fl charide (LPS)-induced lung in ammation in vivo (6, 7). The mine the possible IL-1–stimulatory effect of metallic nanoparticles, particle size of TiO2 was a critical factor in triggering this pul- the human macrophage cell line THP1, was stimulated with various monary inflammatory response (8, 9). nanoparticles for 6 h. In contrast to ZnO (15 nm), both TiO2 (ana- Cytokines of the interleukin-1 family, such as interleukin-1α tase, 20 nm or rutile, 80 nm) and SiO2 (15 nm) induced caspase-1 (IL-1α), interleukin-1β (IL-1β), and interleukin-18 (IL-18) are cleavage and IL-1β secretion (Fig. 1A). Because the organ primarily potent mediators of innate immunity. They are produced as exposed to everyday metallic nanoparticles is the skin, we also stim- proforms, which are further processed upon activation. Pathways ulated primary human keratinocytes with nanoparticles (Fig. 1B). leading to IL-1α processing, which is active both as pro–IL-1α and as cleaved fragment, are yet to be characterized in detail, but were 2+ reported to involve the Ca -dependent protease calpain (10). Author contributions: A.S.Y., I.C., and J.T. designed research; A.S.Y., G.G., N.R., S.K.D., Besides its function as an autocrine growth factor, IL-1α is known A.T., and I.C. performed research; A.S.Y., G.G., N.R., S.K.D., A.T., and I.C. analyzed data; to drive sterile inflammation, in particular in response to dying and A.S.Y. and J.T. wrote the paper. cells (11). Compared with IL-1α processing and activation, the The authors declare no conflict of interest. mechanisms mediating pro–IL-1β and pro–IL-18 maturation are This article is a PNAS Direct Submission. better characterized. Pro-IL–1β and pro-IL–18 are cleaved by the 1To whom correspondence should be addressed. E-mail: [email protected]. protease caspase-1, that itself is autoactivated through caspase-1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. clustering within large multiprotein complexes called inflamma- 1073/pnas.1008155107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1008155107 PNAS | November 9, 2010 | vol. 107 | no. 45 | 19449–19454 Downloaded by guest on September 30, 2021 Similar to macrophages, human keratinocytes secreted mature IL-1β intracellular ROS have been proposed to be criticial for Nlrp3 in a dose-dependent manner following exposure to nano-TiO2 and inflammasome activation in response to “classic” Nlrp3 agonists. nano-SiO2 but not carbon nanotubes, another nanoparticle, which is To further characterize the activating capacity of nanoparticles in used primarily in electronic and material science. this context, we examined whether Nlrp3 inflammasome activity To examine whether mouse and human cells behaved similarly, was suppressed by the ATP-sensitive potassium channel inhibitor, mouse bone marrow-derived dendritic cells (BMDC) (Fig. 1C) glibenclamide or the chemical ROS scavenger (2R,4R)-4- and bone marrow-derived macrophages (BMDM) (Fig. S1) were aminopyrrolidine-2, 4-dicarboxylate (APDC). As for “classic” treated with nano-TiO2 and nano-SiO2. Nano-TiO2 and nano- Nlrp3 agonists such as MSU, cellular pretreatment with gliben- SiO2 were potent stimuli for IL-1β secretion in both murine my- clamide or APDC diminished inflammasome-dependent IL-1β eloid cell types, whereas release of inflammasome-independent secretion triggered by nanoparticles (Fig. S3A). fl cytokines such as TNF were minimally affected by nano-TiO2 Although the above results suggested the current in amma- stimulation as compared with the TLR9 agonist CpG DNA, which some activation scheme proposed for other particles, we found an induced TNF secretion without activating inflammasome-de- important difference between nano-TiO2 and known particulate pendent IL-1β secretion (Fig. S2). activators such as MSU, alum, hemozoin, and μm-sized SiO2 (20). As particulate danger signals (e.g., asbestos, MSU, and alum) Whereas these above-mentioned particulates can activate the trigger secretion of mature IL-1β via the Nlrp3 inflammasome, we Nlrp3 inflammasome in myeloid cells, they do not trigger activa- hypothesized that the Nlrp3 inflammasome may also mediate tion in keratinocytes (Fig. 2 A and B). This is likely to reflect the low responses to nano-TiO2 and nano-SiO2. To test this hypothesis, capacity of keratinocytes to phagocytose particles. In contrast, we treated human and mouse macrophages in which Nlrp3 small diameter (15 nm) but not large diameter (1.5 μm) SiO2 could inflammasome components were knocked down or knocked out, activate the inflammasome in keratinocytes (Fig. 2B), whereas both μ fl respectively, with nano-TiO2 or nano-SiO2. THP1 cells stably nano-sized and m-sized SiO2 activated the Nlrp3 in amma- infected with shRNA directed against caspase-1 or Nlrp3 (Fig. 1A) some in myeloid cells (Fig. 2A), suggesting that the inflammasome- − − − − or BMDCs from ASC / or Nlrp3 / mice (Fig. 1C) did not se- activating signal was triggered without the need for particle phago- crete IL-1β in response to nano-TiO2 or nano-SiO2, indicating cytosis. In support of this, disruption of actin-mediated phagocytosis that the Nlrp3 inflammasome mediated responses to these ago- by cytochalasin D was unable to suppress nanoparticle-induced nists.
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