NAIP5/NLRC4 Inflammasomes Compounds Inhibit the NLRP1, NLRP3, and Arsenic Trioxide and Other Arsenical

The Journal of Immunology

Arsenic Trioxide and Other Arsenical Compounds Inhibit the NLRP1, NLRP3, and NAIP5/NLRC4 Inﬂammasomes

Nolan K. Maier,* Devorah Crown,* Jie Liu,† Stephen H. Leppla,* and Mahtab Moayeri*

Inflammasomes are large cytoplasmic multiprotein complexes that activate caspase-1 in response to diverse intracellular danger signals. Inflammasome components termed nucleotide-binding oligomerization domain–like receptor (NLR) proteins act as sensors for pathogen-associated molecular patterns, stress, or danger stimuli. We discovered that arsenicals, including arsenic trioxide and sodium arsenite, inhibited activation of the NLRP1, NLRP3, and NAIP5/NLRC4 inflammasomes by their respective activating signals, anthrax lethal toxin, nigericin, and flagellin. These compounds prevented the autoproteolytic activation of caspase-1 and the processing and secretion of IL-1b from macrophages. Inhibition was independent of protein synthesis induction, proteasome-mediated protein breakdown, or kinase signaling pathways. Arsenic trioxide and sodium arsenite did not directly modify or inhibit the activity of preactivated recombinant caspase-1. Rather, they induced a cellular state inhibitory to both the autoproteolytic and substrate cleavage activities of caspase-1, which was reversed by the reactive oxygen species scavenger N-acetylcysteine but not by reducing agents or NO pathway inhibitors. Arsenicals provided protection against NLRP1-dependent anthrax lethal toxin–mediated cell death and prevented NLRP3-dependent neutrophil recruitment in a monosodium urate crystal inflammatory murine peritonitis model. These findings suggest a novel role in inhibition of the innate immune response for arsenical compounds that have been used as therapeutics for a few hundred years. The Journal of Immunology, 2014, 192: 763–770.

nflammasomes are large cytoplasmic multiprotein complexes domain–containing protein 4 (NLRC4) inflammasome by direct that form in response to intracellular danger signals. These binding (5, 6). The exact mechanisms by which many disparate I diverse danger signals include pathogen-derived stimuli such signals activate the “promiscuous” NLRP3 inflammasome are un- as bacterial toxins, flagellin, and dsDNA; self-derived molecules known (2). The end result of activation of all inflammasome sensors such as uric acid, amyloid crystals, cholesterol, and ATP; and sig- is the recruitment of caspase-1 to the sensor complex, followed by nals of environmental origin such as aluminum hydroxide, asbestos, its autoproteolytic activation. Activated caspase-1 then rapidly and UV radiation (for reviews, see Refs. 1, 2). The nucleotide- processes the proinflammatory cytokines IL-1b and IL-18 to mature binding oligomerization domain–like receptor leucine-rich repeat forms, allowing their secretion. These cytokines, which are the first proteins (NLRPs), which act as the sensor components of inflam- line of defense for the innate immune response, initiate a cascade of masomes, are activated by several mechanisms. For example, another immunological responses. Inflammasome activation is often thrax lethal toxin (LT), a bipartite toxin made of a receptor binding accompanied by a caspase-1–dependent rapid cell death known as moiety (protective Ag [PA]) and a protease (lethal factor [LF]), pyroptosis (for reviews, see Refs. 1, 2). activates rodent NLRP1 inflammasomes by cleaving them in an N- Not surprisingly, inflammasomes and the innate immune response terminal domain (3, 4). Flagellin activates the NLR family apoptosis play a key role in many infections (7). However, the proinflammatory inhibitory protein 5 (NAIP5)/NLR family caspase-1 recruitment response initiated by inflammasomes has also been implicated in metabolic disorders such as diabetes and inflammatory diseases such as gout and arthritis (8). Furthermore, polymorphisms in the *Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Dis- inflammasome NLR sensors are associated with diseases including eases, National Institutes of Health, Bethesda, MD 20892; and †Center for Molecular vitiligo, rheumatoid arthritis, and Alzheimer’s disease (1). The Medicine, National Heart Lung and Blood Institute, National Institutes of Health, chronic inflammation etiologically associated with numerous can- Bethesda, MD 20892 cers, most notably gastric, hepatic, and colorectal cancers, has also Received for publication May 31, 2013. Accepted for publication November 12, 2013. been linked to activation of these sensors (9). Thus, the role played This work was supported in part by the Intramural Research Program of the National by inflammasome-initiated inflammation in human disease has led Institute of Allergy and Infectious Diseases. to much interest in developing therapeutics targeting inflammasomes Address correspondence and reprint requests to Dr. Mahtab Moayeri, National In- or caspase-1. stitutes of Health Building 33, Room 1W20B, Bethesda, MD 20892. E-mail address: In this study, we show that activation of multiple inflammasomes [email protected] is inhibited by arsenical compounds. Sodium arsenite (NaAsO2) The online version of this article contains supplemental material. and arsenic trioxide (As2O3-Trisenox, a drug with established clinical Abbreviations used in this article: APL, acute promyelocytic leukemia; As2O3, arse- efficacy in treating a number of hematological cancers, including nic trioxide; BMDM, bone marrow–derived macrophage; Boc-D-CMK, Boc-Asp (OBzl)-chloromethylketone; FlaTox, toxin consisting of fusion protein between lethal acute promyelocytic leukemia (APL) and multiple myeloma (10), factor N terminus and flagellin and protective Ag; LF, lethal factor; LFn-Fla, fusion inhibit LT-induced inflammasome-dependent macrophage pyrop- protein between lethal factor N terminus and flagellin; LT, lethal toxin (toxin con- tosis when used at clinically relevant doses. These compounds not sisting of lethal factor and protective Ag); MSU, monosodium urate; NaAsO2, sodium arsenite; NAC, N-acetylcysteine; NAIP5, NLR family apoptosis inhibitory only inhibit NLRP1 inflammasome activation by LT, but also the protein 5; NLR, nucleotide-binding oligomerization domain–like receptor; NLRC4, NAIP5/NLRC4 and NLRP3 inflammasome responses to their effec- NLR family caspase-1 recruitment domain–containing protein 4; NLRP, nucleotide- binding oligomerization domain–like receptor, leucine-rich repeat protein; PA, pro- tors. We found that arsenical compounds inhibit both caspase-1 tective Ag; PML, promyelocytic leukemia protein; ROS, reactive oxygen species. self-activating autoproteolytic activity as well as preactivated www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301434 764 ARSENIC COMPOUNDS INHIBIT THE INFLAMMASOME recombinant caspase-1. The inhibition does not occur through or times (as described in the figure legends). Cells were then treated with direct modification or inhibition of caspase-1 enzymatic function, LT, FlaTox, or medium. Cell viability was assessed by MTT (Sigma-Aldrich) but rather through induction of a cytoplasmic environment in intact as previously described (14). In selected flow cytometry experiments, propidium iodide was used for viability analysis on a BD LSR II flow cells that is inhibitory to its activity. Our findings suggest a novel cytometer (BD Biosciences). role for arsenical compounds as inflammasome inhibitors, with possible off-target utility for treatment of inflammatory conditions, MEK, caspase-1, and IL-1b cleavage as well as a possible explanation of the mechanism for As2O3 ef- RAW264.7 and BALB/cJ BMDM cells were treated with LPS (1 mg/ml) for ficacy in cytokine-dependent hematological cancers. 2 h, with or without drugs (at doses and timing indicated in the figure legends), prior to addition of inflammasome activators (LT, FlaTox, or nigericin, at indicated doses). Cells were then lysed and processed for Materials and Methods Western blotting using primary Abs as previously described (14) in con- Reagents junction with infrared dye–conjugated secondary Abs and visualization with the Odyssey infrared imaging system (LI-COR Biosciences). As2O3 and arsenic (III) chloride were purchased from Alfa Aesar (Ward Hill, MA). Other arsenicals included sodium arsenate (MP Biomedicals, Solon, In vitro caspase-1 assay OH) and arsenic (V) oxide (Strem Chemicals, Newburyport, MA). Cacodylic acid, cycloheximide, actinomycin D, puromycin, buthionine sulfoximine, LPS (50 ng/ml for 8 h, or 1 mg/ml for 2 h) was used to induce IL-1b as N-acetylcysteine (NAC), uric acid, and propidium iodide were from Sigma- a substrate for recombinant caspase-1. Sucrose buffer (250 mM sucrose, Aldrich (St. Louis, MO). Sodium fluoride, sodium orthovanadate, and 10 mM HEPES) lysates of LPS-treated BALB/cJ BMDMs or RAW264.7 NaAsO2 were obtained from Fisher Scientific (Pittsburgh, PA). Staurosporine cells were incubated with 1 U active recombinant mouse caspase-1 per 50 ml was from Biotium (Hayward, CA). Nigericin, anti-Mek1 NT Ab (444942), lysate (MBL International, Woburn, MA) in the presence or absence of G lactacystin, N -monomethyl-L-arginine, and ultrapure LPS were purchased arsenical drug, Boc-Asp(OBzl)-chloromethylketone (Boc-D-CMK; Anas- from Calbiochem (San Diego, CA). Anti-Mek3 NT Ab (sc-959), anti-actin pec, San Jose, CA), or reducing agent for 3 h at 37˚C. In other experiments (sc-1616), and anti-caspase1 p10 Ab (sc-514) were from Santa Cruz Bio- cells were first pretreated with arsenical drugs for 0.5–8 h (as indicated in technology (Santa Cruz, CA). Alexa Fluor 488–conjugated anti-Ly6 Ab was the figure legends) or NAC for 16 h prior to preparation of lysates. Cas- purchased from BioLegend (San Diego, CA). 2-(4-Carboxyphenyl)-4,4,5,5- pase-1–mediated cleavage of IL-1b was analyzed by Western blotting as g tetramethylimidazoline-1-oxyl-3-oxide, S-ethylisothiourea, and N -nitro-L- previously described (14). Because As2O3 as a potent NF-kB inhibitor arginine-methyl ester were obtained from Enzo Life Sciences (Farmingdale, prevents LPS-mediated upregulation of IL-1b when applied before LPS, b NY). Anti–IL-1 Ab (AF-401-NA) was purchased from R&D Systems all LPS priming was performed prior to As2O3 application. (Minneapolis, MN). Secondary Abs used in these studies were anti-goat infrared dye (800CW) (Rockland Immunochemicals, Gilbertsville, PA) and Evaluation of caspase-1 sequestration in a high molecular anti-rabbit infrared dye (800CW) (LI-COR Biosciences, Lincoln, NE). Tris mass complex (carboxyethyl) phosphine hydrochloride was purchased from Affymetrix (Santa Clara, CA). Monosodium urate (MSU) crystals were prepared by Sucrose buffer (250 mM sucrose, 10 mM HEPES) lysates of As2O3-treated crystallization of uric acid as described (11). PA and LF were purified from or heat-shocked (42˚C) RAW264.7 cells were centrifuged at 10,000 3 g Bacillus anthracis as described previously (12). LFn-Fla, a toxin also de- for 10 min at 4˚C. The supernatant and pellet were analyzed for caspase-1 livered by PA, is a fusion of the first 254 aa of LF to full-length flagellin from by Western blotting as previously described (14). Legionella pneumophila (gift of Dr. Russell Vance, University of California at Berkeley, Berkeley, CA) (13). FlaTox is a combination of LFn-Fla and PA. Evaluation of glutathiolated proteins Concentrations of LT correspond to the concentration of each toxin com- m ponent (i.e., 1 mg/ml LT is 1 mg/ml PA plus 1 mg/ml LF). Concentrations of BALB/cJ BMDMs were loaded with 250 M BioGee glutathiolation de- tection reagent (Life Technologies) for 1 h, as described (15). Cells were FlaTox correspond to the concentration of LFn-Fla. The concentration of PA m was always twice that of LFn-Fla in FlaTox experiments (i.e., 1 mg/ml then treated with 50 MAs2O3 for 1 h. Sucrose buffer lysates were pre- FlaTox is 2 mg/ml PA plus 1 mg/ml LFn-Fla). pared and biotin-labeled proteins were precipitated with streptavidin-linked agarose (EMD Millipore, Darmstadt, Germany). Precipitated proteins were Cell culture analyzed for caspase-1 by Western blotting as described (14). RAW264.7 cells and L929 mouse fibroblast cells were grown in DMEM supplemented with 10% FBS, 10 mM HEPES, and 50 mg/ml gentamicin Results (all purchased from Life Technologies, Grand Island, NY). Mouse bone NaAsO2 and As2O3 protect against LT-induced macrophage marrow cells were cultured in complete DMEM (as above) supple- death mented with 30% L929 cell–conditioned supernatant and grown 7–9 d k to allow time for differentiation to bone marrow–derived macrophages A screen of NF- B inhibitors for protection against anthrax LT- (BMDMs). induced macrophage pyroptosis identified NaAsO2 as a potent inhibitor. Both BALB/cJ BMDMs (Fig. 1A) and RAW264.7 cells Animal studies (Supplemental Fig. 1A) were protected by NaAsO2 over a range of All mouse strains used for bone marrow, including mice deficient in pro- concentrations. We investigated other arsenic-containing com- myelocytic leukemia protein (PML) were purchased from The Jackson pounds and found that As2O3, a U.S. Food and Drug Adminis- Laboratory (Bar Harbor, ME). Fischer CDF rats were purchased from tration–approved drug for the treatment of APL, multiple myeloma, Charles River (Wilmington, MA). Rats were given As O (7 mg/kg, i.v.) or 2 3 and other myelodysplastic syndromes also protected against LT in- NaAsO2 (5 mg/kg, i.v.) 30 min prior to LT (12 mg, i.v.) and monitored continuously for malaise or death. For peritonitis studies As2O3 was toxication (Fig. 1A, Supplemental Fig. 1A). Both drugs were non- injected i.p. at 1.25 mg/kg, followed by MSU crystals (i.p., 0.5 mg in 250 ml toxic at protective doses (Fig. 1A, Supplemental Fig. 1A). Longer PBS/mouse). Peritoneal lavages were performed at 2 h with PBS and in- incubation times significantly lowered the protective concentration filtrating cells were counted after hypotonic erythrocyte lysis. For studies on range of As2O3 on BMDMs (Fig. 1B), with doses as low as 3 mM the effects of As2O3 on circulating neutrophils, Ly6 staining of peripheral blood leukocytes was performed followed by flow cytometry analyses on providing 50–60% protection when cells were pretreated for 16 h. a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). All animal In contrast, RAW264.7 cells did not show an added benefit with experiments were performed in strict accordance with guidelines from the longer compound incubation times, requiring a 4-fold higher National Institutes of Health and the Animal Welfare Act, approved by the dose to achieve 60% protection (Supplemental Fig. 1B). An- Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. other derivative, arsenic (III) chloride had similar protection to As2O3, whereas sodium arsenate required a 10-fold higher dose Cytotoxicity assays for protection (Supplemental Fig. 1C). Arsenical compounds RAW264.7 and BALB/cJ BMDM cells were grown in 96-well plates to 90% arsenic (V) oxide and cacodylic acid were not protective (Sup- confluence and pretreated with various drugs or vehicle at a range of doses plemental Fig. 1C). The Journal of Immunology 765

FIGURE 1. NaAsO2 and As2O3 protect murine macrophages from LT-induced pyroptosis without affecting LT translocation or proteolytic activity. (A) BALB/cJ BMDMs were incubated with variable concentrations of NaAsO2 or As2O3 for 15 min before challenge with LT (1 mg/ml). Cell viability was assessed by MTT staining after 1.5 h of toxin treatment. Percentage viability was assessed compared with untreated cells. (B) BALB c/J BMDMs were treated with As2O3 for the indicated times at the indicated concentrations followed by challenge with LT (1 mg/ml). Cell viability was assessed as above. BALB/cJ BMDMs were incubated with (C)

NaAsO2 (75 mM) or (D)As2O3 (50 mM) for 15 min prior to challenge with LT (1 mg/ml) for various periods of time. Western blotting of cell lysates was performed with Abs against the N terminus of MEK1 and MEK3.

NaAsO2 and As2O3 do not inhibit LF translocation or ferent activating danger signals and disparate mechanisms of proteolytic activity activation.

To test whether As2O3 protects against LT toxicity by inhibiting As2O3 does not directly inhibit caspase-1 enzymatic activity cytosolic translocation of LF or its proteolytic activity, the cleavage To determine whether As2O3 inhibits caspase-1 enzymatic activity, of the toxin’s cytoplasmic MEK substrates was monitored in mac- its effect on the in vitro proteolytic function of preactivated purified rophages (16, 17). MEK1 and MEK3 were fully cleaved by 60 min recombinant murine caspase-1 was tested. Pro–IL-1b present in in macrophages treated with LT regardless of whether NaAsO2 lysates of LPS-treated macrophages was used as substrate. As2O3 (Fig. 1C) or As2O3 (Fig. 1D) was added, demonstrating that ar- failed to inhibit pro–IL-1b processing over a wide range of con- senical compounds do not affect LT binding, uptake, translocation, centrations whereas the potent caspase-1 inhibitor Boc-D-CMK or protease activity. Furthermore, the compounds were fully pro- fully blocked processing (Fig. 3A). Similar results were found tective when applied 40 min after LT treatment (Supplemental Fig. with NaAsO2 (Supplemental Fig. 3A). Interestingly, lysates made 1D), a time point when MEK substrates were fully cleaved (Fig. 1C, from cells pretreated with As2O3 at a dose of 7 mM for 8 h (Fig. 1D). These results indicated that protection occurred by targeting late 3B) or 50 mM for 1 h (Fig. 3C) inhibited caspase-1 enzymatic events downstream of MEK cleavage. activity. Furthermore, mixing lysates from As2O3-treated cells with those from untreated cells yielded an intermediate level of caspase- NaAsO2 and As2O3 inhibit multiple inflammasomes 1 inhibition (Fig. 3C). As2O3 at these doses and treatment times LT-mediated macrophage death requires NLRP1-mediated acti- does not upregulate pro–IL-1b or cause IL-1b maturation on its vation of caspase-1 (18), which normally begins at 50–60 min after own (data not shown). These results demonstrate that As2O3-me- toxin treatment (19). Both NaAsO2 (Fig. 2A, left) and As2O3 (Fig. diated inflammasome inhibition does not involve the drug’s direct 2A, right) prevented LT-induced caspase-1 autoproteolysis (upper inhibition of caspase-1 proteolytic activity. Rather, conditions in- b panels) and subsequent IL-1 processing (middle panels) and duced by As2O3 in intact cells are sufficient to inhibit preactivated secretion (lower panels). Furthermore, addition of the compounds recombinant caspase-1. to cells 60 min after LT treatment, at a time when the bulk of the cell’s caspase-1 was processed, did not protect against cell death As2O3 protection is not dependent on caspase-1 sequestration, (Supplemental Fig. 1D). These results indicate that the compounds protein synthesis, proteasome-mediated protein breakdown, or inhibit the LT-induced NLRP1-mediated activation of caspase-1. phosphorylation events To determine whether inhibition by arsenical compounds was spe- We previously showed that heat shock results in the inhibition of cific to the NLRP1 inflammasome, the effect of the compounds on caspase-1 through sequestration in a high molecular mass complex activation of the NLRP3 inflammasome by the ionophore nigericin that can be separated from the cytosol by centrifugation (20). was assessed. As2O3 and NaAsO2 inhibited nigericin-mediated Because arsenical compounds have been shown to upregulate heat caspase-1 processing and IL-1b maturation (Fig. 2B). Further- shock proteins (21, 22), we investigated whether the mechanism more, FlaTox-mediated caspase-1 activation and IL-1b matura- of arsenical inhibition of caspase-1 also involved this high mo- tion, which occurs through the NAIP5/NLRC4 inflammasome, lecular mass complex. Treatment of cells with As2O3 did not was also inhibited by both compounds (Fig. 2C). A slight loss of cause trapping of caspase-1 into a high molecular mass complex inhibition was seen over intoxication times approaching 90 min (Supplemental Fig. 3B). (Fig.2C).As2O3 also protected against the caspase-1–dependent We hypothesized that As2O3-mediated upregulation of cellular cell death induced by FlaTox (Supplemental Fig. 2A). These stress proteins or inhibitory complexes could contribute to caspase- results demonstrate that the compounds inhibit the caspase-1 1 inhibition. Therefore, we tested the effects of the transcription activation induced by inflammasomes that have completely dif- inhibitor actinomycin D and the translation inhibitor puromycin to 766 ARSENIC COMPOUNDS INHIBIT THE INFLAMMASOME

FIGURE 2. NaAsO2 and As2O3 prevent inﬂammasome- mediated caspase-1 activation in macrophages. BALB/cJ BMDMs were primed with LPS (1 mg/ml,2h),thenpretreatedwith

NaAsO2 (75 mM) or As2O3 (50 mM) for 15 min, and then treated with (A)LT(1mg/ml), (B) nigericin (50 mM), or (C)FlaTox (1 mg/ml) for various periods of time. Western blotting of culture supernatants for secreted IL-1b (bottom panel)oroflysatesfor the p10 subunit of caspase-1 and intracellular forms of IL-1b (upper two panels) was performed with appropriate Abs.

determine whether protein synthesis was required for As2O3 pro- (Fig. 4B, Supplemental Fig. 2B), indicating that As2O3 did not tection. Pretreatment of macrophages with a range of concen- manifest its effects through proteasome-mediated breakdown of trations of puromycin or actinomycin D (Fig. 4A) did not reverse a protein or proteins. As2O3 protection, indicating that protein synthesis is not necessary Finally, neither phosphatase inhibitors sodium fluoride and so- for As2O3-mediated inflammasome inhibition. These results were dium vanadate nor the kinase inhibitor staurosporine reversed the supported by our previous finding that NaAsO2 and As2O3 could inhibition of caspase-1 enzymatic activity in lysates of As2O3-treated protect cells even when applied 40 min after LT intoxication, cells (Supplemental Fig. 3C). Similarly, pretreatment of cells with making a requirement for new protein synthesis unlikely. Similar phosphatase or kinase inhibitors had no effect on As2O3-based results were found with NaAsO2 and with use of the translation protection from LT-mediated pyroptosis (data not shown). These inhibitor cycloheximide (Supplemental Fig. 4A). results indicate that it is unlikely that As2O3-based protection Because proteasome inhibition prevents activation of the NLRP1 involves phosphorylation signaling pathways. inflammasome (19, 23), we tested the role of protein breakdown induced by the arsenical compounds in the inhibition of the As2O3 protection requires ROS but does not involve the NO NAIP5/NLRC4 inflammasome. Unlike NLRP1 activation, which pathway requires proteasome activity, FlaTox activation of the NAIP5/ As2O3 and NaAsO2 have been shown to directly induce ROS, and NLRC4 inflammasome is not impacted by proteasome inhibition some of their effects occur through the modification of ROS- (Fig. 4B, Supplemental Fig. 2B). We found that treatment of cells controlling cellular enzymes (24). We used the potent anti-oxidant with the proteasome inhibitor lactacystin did not reverse As2O3 and ROS scavenger NAC to determine whether the protective protection against FlaTox or As2O3 inhibition of IL-1b processing effects of As2O3 require induction of an altered oxidative state in The Journal of Immunology 767

As2O3 inhibition of inﬂammasome activation is independent of promyelocytic leukemia protein degradation

As2O3 is known to catalyze the degradation of PML (25). A recent report described inhibition of the NLRP3 inflammasome by As2O3 (26) and attributed this inhibition to the drug’s degradation of PML, which was suggested to be essential to NLRP3 inflammasome activation. Evidence for the breakdown of PML as the mechanism of NLRP3 inflammasome inhibition by As2O3, however, was not directly shown in those studies. To determine whether PML degradation is the mechanism of As2O3-mediated inflammasome inhibition, we assessed the effect of As2O3 on inflammasome activation in BMDMs deficient in PML. As2O3 inhibited IL-1b maturation in response to NLRP3 (Fig. 5A) and NAIP5/NLRC4 (Fig. 5B) stimuli in PML-deficient macrophages. The effect of LT-induced NLRP1 activation was not assessed in the knockout mice that are on the LT-nonresponsive C57BL/6 background (18). These results demonstrate that PML degradation is not required for As2O3- mediated inflammasome inhibition.

As2O3 reduces inflammatory cell infiltrate in a murine peritonitis model The anti-inflammatory properties of As O were tested in a murine A 2 3 FIGURE 3. As2O3 inhibits caspase-1 through indirect mechanisms. ( ) peritoneal inflammation model. As O caused up to a 2-fold re- m 2 3 Sucrose lysates from BALB/cJ BMDMs pretreated with LPS (1 g/ml, 2 h) duction in the number of cells recruited to the peritoneum when were incubated (37˚C, 3 h) with active recombinant caspase-1 (1 U/50 ml) in administered i.p. (Fig. 6A) or i.v. (data not shown) 30 min prior to the presence or absence of a range of As2O3 concentrations or positive control caspase-1 inhibitor Boc-D-CMK (400 mM). IL-1b cleavage was MSU crystals. This decrease was not due to As2O3-mediated death monitored by Western blot. (B) BALB/cJ BMDMs pretreated with LPS (50 of peripheral neutrophils, because i.v. injection of As2O3 did not ng/ml, 8 h) were incubated with 7 mMAs2O3 for 8 h followed by sucrose cause a significant decrease in the number of neutrophils in cir- lysis. Lysates were mixed with active recombinant caspase-1 (1 U/50 ml) and culation (data not shown). These results demonstrate that As2O3 incubated at 37˚C for 3 h. IL-1b cleavage was assessed by Western blot. (C) can also inhibit the NLRP3 inflammasome in vivo. RAW264.7 cells were LPS treated (1 mg/ml, 2 h), followed by As2O3 NaAsO2 and As2O3 extend survival time in LT-treated rats treatment (50 mM, 1 h). Sucrose lysates from As2O3-pretreated cells were mixed in various ratios with sucrose lysates from untreated cells. Recombi- LT induces rapid death of rats in40–100minthroughanunknown nant caspase-1 was added and IL-1b cleavage was monitored as above. NLRP1-dependent process (27). Pretreatment of toxin-sensitive Fischer rats with As2O3 or NaAsO2 prior to LT challenge extended cells. Pretreatment of RAW264.7 cells with NAC, in a dose range mean time to death (Fig. 6B), demonstrating the ability of these drugs where this ROS scavenger did not impact NLRP1 inflammasome to alter the outcome of NLRP1/caspase-1 activation in vivo. activation, reversed the protective effects of As2O3 on LT-treated macrophages (Fig. 4C). NAC treatment of BALB/cJ BMDMs also Discussion restored IL-1b processing in response to LT treatment (Fig. 4D). In this work we show that arsenical compounds inhibit activation NAC treatment also reversed the caspase-1 inhibitory ability of of caspase-1 and IL-1b processing by the NLRP1, NLRP3, and lysates made from As2O3-treated cells (Fig. 4E). NAIP5/NLRC4 inflammasome sensors. This inhibition of caspase-1 It has been shown that a generally oxidative cellular environment autoproteolytic activation and downstream cytokine processing was can lead to inhibition of caspase-1 through reversible glutathiolation not due to inhibition of the uptake or activity of the inflammasome- of catalytic cysteine residues (15), and we hypothesized that arsenic activating stimuli/toxins. It was also not a result of direct modifi- treatment may create such an oxidative environment. However, cation or inactivation of caspase-1 enzymatic activity. Instead, treatment with the ROS generator buthionine sulfoximine or with inhibition occurred through induction of a cellular state that did not hydrogen peroxide (Supplemental Fig. 4B) did not protect cells from involve protein synthesis, proteasome function, or phosphorylation LT-induced pyroptosis. Additionally, spiking lysates from As2O3- events and that was inhibitory to both autoproteolytic processing of treated cells with the reducing agents DTT (Fig. 4F) or Tris (car- caspase-1 and its enzymatic activity toward its cytokine substrates. boxyethyl) phosphine hydrochloride (data not shown) did not reverse A potent ROS scavenger, NAC, could reverse the protection pro- the inhibitory effect of arsenic treatment on caspase-1 enzymatic vided by the arsenical compounds, which have been previously activity. Use of the biotin-labeled glutathione analog BioGee cou- shown to be potent inducers of ROS (24). Our findings suggest that pled with immunoprecipitation of caspase-1 from As2O3-treated arsenical compounds may be considered as a treatment in many cells, indicated that caspase-1 was not glutathiolated in response to conditions that involve inflammasome activation and uncontrolled arsenic treatment (data not shown). Nitrosylation of caspase-1 was proinflammatory responses, including gout, arthritis, and various also eliminated as an inhibitory modification. Pretreatment of cells inherited disorders (28). with various inhibitors of NOS or with a scavenger of NO did not Arsenic has been used therapeutically for .2000 y in traditional reverse As2O3 protection (Supplemental Fig. 4C). Chinese medicine and in Western medicine since the time of These results indicate that specific ROS changes induced by Hippocrates. Its extensive use in the 18th and 19th centuries ex- As2O3 (but not general increases in ROS) are required to inhibit panded to treatment of eczema, ulcers, malaria, plague, asthma, inflammasome activation. This also suggests that an ROS scav- psoriasis, anemia, arthritis, epilepsy, Hodgkin’s disease, and leu- enger such as NAC can have both activating and inhibitory effects kemia (10, 29). Fowler’s solution, an alkaline solution of white on inflammasome activation. arsenic, became a foundation of 19th century pharmacopeia and 768 ARSENIC COMPOUNDS INHIBIT THE INFLAMMASOME

FIGURE 4. Protein synthesis or breakdown is not required for protection but can be reversed by ROS scavengers. (A) RAW264.7 cells were treated with variable concentrations of puromycin or actinomycin D for 1 h before incubation with

As2O3 (50 mM, 15 min). Cells were then challenged with LT (1 mg/ml) and cell viability was assessed at 2 h. (B) BALB/cJ BMDMs were primed with 1 mg/ml LPS for 1.5 h and pretreated with 20 mM lactacystin for 1 h, followed by As2O3 (50 mM, 15 min). Cells were then treated with FlaTox (1 mg/ml, 1 h). Western blotting was performed with Abs against IL- 1b.(C) RAW264.7 cells were pretreated for 16 h with variable concentrations of NAC, or (inset) with 25 mM NAC followed by As2O3 (50 mM, 15 min) and LT (1 mg/ml, 2 h). Cell viability was assessed by MTT staining and determined relative to untreated cells. Alternatively, cell death was evaluated by propidium iodide staining (inset). (D) BALB/cJ BMDMs were pretreated overnight with 25 mM NAC and primed with 1 mg/ ml LPS for 2 h, followed ﬁrst by As2O3 (50 mM, 15 min) and then by LT (1 mg/ml, 2 h). Western blotting was performed with Abs against IL-1b.(E) RAW264.7 cells were pretreated for 16 h with 25 mM NAC. Cells were then treated and processed as in Fig. 3C. (F) RAW264.7 cells were LPS treated

(1 mg/ml, 2 h) followed by As2O3 treatment (50 mm, 1 h). Sucrose lysates were spiked with varying concentrations of DTT. Recombinant caspase-1 was added and IL-1b cleavage was monitored as above.

remained in use until 1950. Pharmacology texts in 1880 described was that arsenic compounds could induce binding of an endoge- the curative properties of arsenic as “almost magical,” and no nous caspase-1 inhibitory protein or cofactor within the cell. Heat other medication was said to remedy such an extensive assortment shock inhibits caspase-1 through sequestration of the enzyme into of ailments (30–32). Arsenic has a wide range of effects on cel- a high molecular mass complex with an unknown binding partner lular proteins and pathways. It has the ability to directly bind to (20). Because arsenic treatment is known to induce a heat shock– thiol groups on proteins, potentially altering their function. A like cellular state (21, 22), we tested whether a similar large number of cellular proteins have been identified that are modified molecular complex was formed following treatment with As2O3. by arsenic, including the catalytic subunit of IkB kinase, resulting Arsenical treatment did not induce sequestration of caspase-1 into in inhibition of the NF-kB pathway and dampening inflammatory a high molecular mass complex. Arsenic treatment could also responses at the transcriptional level (33, 34). Modification of the result in signaling events that lead to phosphorylation of various antioxidant enzymes glutathione peroxidase and thioredoxin re- cellular proteins involved in interactions with or inhibition of ductase (35, 36) results in potent ROS induction by arsenical caspase-1. We found that pan-kinase and phosphatase inhibitors compounds. In this study we present a new effect of arsenical did not impact As2O3 effects on caspase-1 activity. compounds as inhibitors of IL-1b processing and secretion. Arsenic is a potent inducer of ROS (24). The antioxidant NAC Caspase-1 is the IL-1b–activating/cleaving enzyme responsible reversed the protective effects of As2O3 and restored caspase-1 for the first line of cytokine responses by resident innate immune activity and IL-1b release, indicating that ROS generation could cells at all tissue sites. We found that As2O3 and NaAsO2 inhibit play a role in the protective effects of the drug. Paradoxically, inflammasome-induced caspase-1 activation. The enzymatic ac- some investigators have found that ROS production can be an tivity of caspase-1, a cysteine protease, is dependent on catalytic activation signal for the NLRP3 inflammasome (37) and that ROS cysteine residues that may be vulnerable to arsenic modification. causes the induction of NLRP3 expression (38). In the present Because arsenic compounds did not inhibit caspase-1 proteolytic study we demonstrate that a known potent inducer of ROS can activity in cell lysates, this possibility was eliminated. Lysates also inhibit NLRP3 inflammasome activation. Our findings sup- prepared from cells pretreated with As2O3, however, inhibited port the hypothesis that ROS production is but one of a combi- cleavage of IL-1b by recombinant caspase-1, indicating that nation of cellular signals necessary for NLRP3 activation (39) and a cellular condition inhibitory to its activity was induced in cells. that certain ROS events can inhibit activation (40). Importantly, Because generation of the autoproteolytic fragment p10 requires general increases in ROS do not inhibit all inflammasomes, as the recruitment of procaspase-1 to the NLR platform, we hypothe- pro-oxidants buthionine sulfoximine and hydrogen peroxide did sized that this recruitment was possibly inhibited. One hypothesis not protect cells from LT- or caspase-1–induced death. Therefore,

FIGURE 5. PML degradation is not the mechanism of 2/2 As2O3-mediated inﬂammasome inhibition. PML BMDMs were primed with LPS (1 mg/ml, 2 h), then pretreated with

As2O3 (50 mM) for 15 min, and then treated with (A) nigericin (50 mM) or (B) FlaTox (1 mg/ml) for various periods of time. Western blotting of lysates for intracellular forms of IL-1b was performed with appropriate Abs. The Journal of Immunology 769

refractory multiple myeloma (42). Trials of myelodysplastic syn- drome patients treated with As2O3 alone or in combination with thalidomide have demonstrated hematological improvement and increases in progression-free and overall survival compared with control patients (43). A number of mechanisms have been pro- posed to explain the drug’s efficacy in treatment of these cancers, including apoptosis, degradation of PML, induction of oxidative damage, and inhibition of angiogenesis and NF-kB signaling (for a review, see Ref. 44). We hypothesize that the ability of As2O3 to b FIGURE 6. NaAsO2 and As2O3 inhibit inflammasomes in vivo. (A) inhibit caspase-1, and thus IL-1 inflammatory signaling, plays Inflammatory cell recruitment was assessed after 2 h in the peritoneum of a major role in its anti-cancer effects. Many cancers cells, including BALB/cJ mice injected i.p. with MSU (0.5 mg/250 ml/mouse). Mice had multiple myeloma, breast cancer, and advanced melanoma cells, been injected with either PBS (i.p.) or As2O3 (1.25 mg/kg, i.p.). Error bars actively secrete IL-1b, providing a proliferative advantage through represent SEM; n = 5 animals/group; p = 0.0291. (B) Fischer rats pre- either autocrine or paracrine signaling (45–47). For example, IL-1b treated with 7 mg/kg As2O3 i.v. (n = 7) or 5 mg/kg NaAsO2 i.v. (n =7) secreted by myeloma cells has been shown to have paracrine effects were challenged with LT (12 mg, i.v.). Times to death (one symbol rep- on bone marrow stromal cells, inducing them to produce the IL-6 resenting one animal) were compared with control animals that did not b receive drug treatment (n = 5); p values comparing each treatment group to that is required for a proliferative cancer environment (45). IL-1 controls are ,0.0075. signaling has also been shown to play a role in tumor angiogenesis (48). As2O3 is already known to potently inhibit NF-kBsignaling (33, 34), and thus this compound obstructs inflammation at two site-specific ROS regulation may be an important factor in ar- distinct steps: NF-kB–dependent upregulation of proinflammatory senical compound–mediated effects. cytokines, such as IL-1b, as well as maturation and release of the Reversible modification of redox-sensitive and catalytically nec- cytokine through inhibition of caspase-1 activation as shown in essary cysteine residues on caspase-1 under conditions of high ROS the present study. Thus, As2O3-mediated disruption of IL-1b have been observed (15). Therefore, we hypothesized that redox- maturation may partially account for the successful treatment of dependent protein modifications could be the mechanism of arsenic- multiple myeloma patients with As2O3 in recent clinical trials. The based inhibition of caspase-1. Inhibitors of NOS or a NO scavenger, varied effects of As2O3 on cellular processes and its continued use however, did not alter the effects of As2O3. Similarly, various re- as a therapeutic necessitate further study of its molecular mecha- ducing agents did not alter arsenic-based inhibition of caspase-1. nisms of action. Finally, we did not find glutathiolated caspase-1 in arsenic-treated Because of the roles that inflammasomes and caspase-1–mediated cells. Therefore, we found no evidence of redox-based caspase-1 inflammatory signaling play in various other human malignancies modifications due to arsenic treatment. ROS increases can induce (8), the identification of inflammasome-inhibiting compounds is of expression of numerous proteins, and it is known that arsenicals clinical importance (49). We suggest that As2O3 and other arsenical induces various cellular stress responses such as expression of heat compounds can also be used as potent anti-inflammatories for shock proteins and the tumor suppressor protein p53 (24). We found treatment of localized diseases, such as in topical treatments for that inhibitors of protein synthesis did not alter NaAsO2 or As2O3 skin conditions. These drugs have been used to treat animal protection against inflammasome activation, indicating that de novo models of asthma, coronary restenosis, colitis, and lupus (50–53). protein synthesis was not required for arsenical compound effects. Gout, a condition in which uric acid crystals in joints activate the Another effect of As2O3 is the breakdown of different cellular NLRP3 inflammasome to cause painful inflammation (54), is an proteins, such as PML (25). While this manuscript was in prep- example of a condition that could benefit from a caspase-1 in- aration, Lo et al. (26) described inhibition of the NLRP3 inflam- hibitory treatment. We demonstrated a 2-fold reduction in MSU- masome by As2O3 and attributed this inhibition to degradation of induced cell recruitment upon treatment with As2O3, comparable PML, which they suggested to be essential to NLRP3 inflamma- to what was seen with allopurinol in other studies (55). One can some formation. We demonstrate that PML breakdown is not the imagine other possibilities for use of these compounds as anti- mechanism of As2O3-mediated NLRP3 inflammasome inhibition. inflammatories in diseases where caspase-1 activation plays a Furthermore, we show that the drug’s inhibitory effect is not prominent role. specific to the NLRP3 inflammasome, as we found potent inhi- Our studies demonstrate that arsenical compounds, including the bition of the NLRP1 and NAIP5/NLRC4 inflammasomes, which U.S. Food and Drug Administration–approved anti-cancer thera- presumably do not require PML. As2O3 was effective at inhibiting peutic Trisenox, are potent inhibitors of caspase-1 and the innate IL-1b processing in PML-deficient macrophages in response to immune response and thus may have potential in treatment for in- both NLRP3- and NAIP5/NLRC4-activating stimuli. Moreover, flammatory disorders. The application of the drug’s anti-inflammatory we demonstrate that proteasome activity, which catalyzes the actions in various inflammasome-related diseases is an area of breakdown of PML by As2O3 (25), is not necessary for inflam- future study by our laboratory. masome inhibition by this drug. Instead, our results show that in addition to preventing the autoproteolytic activation of caspase-1 in intact cells, the drug can inhibit activity of preactivated recombi- Acknowledgments nant caspase-1. We thank Dr. Russell Vance for providing the LFn-Fla protein. We thank As O is a U.S. Food and Drug Administration–approved drug the Flow Cytometry Section of the National Institute of Allergy and Infec- 2 3 tious Diseases Research Technologies Branch for use of facilities and for (under brand name Trisenox) used for treatment of refractory expertise. APL, multiple myeloma, and other lymphoma conditions. Single- agent therapy with As2O3 or combination therapy with all-trans retinoic acid leads to complete remission in .80% of newly di- Disclosures agnosed APL patients (41). Recent clinical trials have demon- N.K.M., M.M., and S.H.L. are inventors on U.S. provisional patent appli- strated modest efficacy of As2O3 alone in treating advanced or cation no. 61/784138 filed March 14, 2013. 770 ARSENIC COMPOUNDS INHIBIT THE INFLAMMASOME

References 27. Newman, Z. L., M. P. Printz, S. Liu, D. Crown, L. Breen, S. Miller-Randolph, P. Flodman, S. H. Leppla, and M. Moayeri. 2010. Susceptibility to anthrax lethal 1. Martinon, F., A. Mayor, and J. Tschopp. 2009. The inflammasomes: guardians of toxin-induced rat death is controlled by a single chromosome 10 locus that the body. Annu. Rev. Immunol. 27: 229–265. includes rNlrp1. PLoS Pathog. 6: e1000906. 2. Lamkanfi, M., and V. M. Dixit. 2012. Inflammasomes and their roles in health 28. Dinarello, C. A. 2011. A clinical perspective of IL-1b as the gatekeeper of in- and disease. Annu. Rev. Cell Dev. Biol. 28: 137–161. flammation. Eur. J. Immunol. 41: 1203–1217. 3. Hellmich, K. A., J. L. Levinsohn, R. Fattah, Z. L. Newman, N. Maier, I. Sastalla, 29. Waxman, S., and K. C. Anderson. 2001. History of the development of arsenic S. Liu, S. H. Leppla, and M. Moayeri. 2012. Anthrax lethal factor cleaves mouse derivatives in cancer therapy. Oncologist 6(Suppl. 2): 3–10. Nlrp1b in both toxin-sensitive and toxin-resistant macrophages. PLoS ONE 7: e49741. 30. Doyle, D. 2009. Notoriety to respectability: a short history of arsenic prior to its 4. Levinsohn, J. L., Z. L. Newman, K. A. Hellmich, R. Fattah, M. A. Getz, S. Liu, present day use in haematology. Br. J. Haematol. 145: 309–317. I. Sastalla, S. H. Leppla, and M. Moayeri. 2012. Anthrax lethal factor cleavage 31. Zhu, J., Z. Chen, V. Lallemand-Breitenbach, and H. de The´. 2002. How acute of Nlrp1 is required for activation of the inflammasome. PLoS Pathog. 8: promyelocytic leukaemia revived arsenic. Nat. Rev. Cancer 2: 705–713. e1002638. 32. Aronson, S. M. 1994. Arsenic and old myths. R. I. Med. 77: 233–234. 5. Kofoed, E. M., and R. E. Vance. 2011. Innate immune recognition of bacterial 33. Kapahi, P., T. Takahashi, G. Natoli, S. R. Adams, Y. Chen, R. Y. Tsien, and ligands by NAIPs determines inflammasome specificity. Nature 477: 592–595. M. Karin. 2000. Inhibition of NF-kB activation by arsenite through reaction with 6. Zhao, Y., J. Yang, J. Shi, Y.-N. Gong, Q. Lu, H. Xu, L. Liu, and F. Shao. 2011. a critical cysteine in the activation loop of IkB kinase. J. Biol. Chem. 275: The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion 36062–36066. apparatus. Nature 477: 596–600. 34. Roussel, R. R., and A. Barchowsky. 2000. Arsenic inhibits NF-kB-mediated 7. Franchi, L., R. Munõz-Planillo, and G. Nuń˜ez. 2012. Sensing and reacting to gene transcription by blocking IkB kinase activity and IkBa phosphorylation microbes through the inflammasomes. Nat. Immunol. 13: 325–332. and degradation. Arch. Biochem. Biophys. 377: 204–212. b 8. Hoffman, H. M., and A. A. Wanderer. 2010. Inflammasome and IL-1 -mediated 35. Lu, J., E.-H. Chew, and A. Holmgren. 2007. Targeting thioredoxin reductase is disorders. Curr. Allergy Asthma Rep. 10: 229–235. a basis for cancer therapy by arsenic trioxide. Proc. Natl. Acad. Sci. USA 104: 9. Zitvogel, L., O. Kepp, L. Galluzzi, and G. Kroemer. 2012. Inflammasomes in 12288–12293. carcinogenesis and anticancer immune responses. Nat. Immunol. 13: 343–351. 36. Styblo, M., S. V. Serves, W. R. Cullen, and D. J. Thomas. 1997. Comparative 10. Emadi, A., and S. D. Gore. 2010. Arsenic trioxide: an old drug rediscovered. inhibition of yeast glutathione reductase by arsenicals and arsenothiols. Chem. Blood Rev. 24: 191–199. Res. Toxicol. 10: 27–33. 11. Martin, W. J., M. Walton, and J. Harper. 2009. Resident macrophages initiating 37. Martinon, F. 2010. Signaling by ROS drives inflammasome activation. Eur. J. and driving inflammation in a monosodium urate monohydrate crystal-induced Immunol. 40: 616–619. murine peritoneal model of acute gout. Arthritis Rheum. 60: 281–289. 38. Bauernfeind, F., E. Bartok, A. Rieger, L. Franchi, G. Nuń˜ez, and V. Hornung. 12. Park, S., and S. H. Leppla. 2000. Optimized production and purification of 2011. Cutting edge: reactive oxygen species inhibitors block priming, but not Bacillus anthracis lethal factor. Protein Expr. Purif. 18: 293–302. activation, of the NLRP3 inflammasome. J. Immunol. 187: 613–617. 13. von Moltke, J., N. J. Trinidad, M. Moayeri, A. F. Kintzer, S. B. Wang, N. van 39. Gross, O., C. J. Thomas, G. Guarda, and J. Tschopp. 2011. The inflammasome: Rooijen, C. R. Brown, B. A. Krantz, S. H. Leppla, K. Gronert, and R. E. Vance. an integrated view. Immunol. Rev. 243: 136–151. 2012. Rapid induction of inflammatory lipid mediators by the inflammasome 40. Rubartelli, A. 2012. Redox control of NLRP3 inflammasome activation in health in vivo. Nature 490: 107–111. and disease. J. Leukoc. Biol. 92: 951–958. 14. Newman, Z. L., N. Sirianni, C. Mawhinney, M. S. Lee, S. H. Leppla, 41. Lengfelder, E., W.-K. Hofmann, and D. Nowak. 2012. Impact of arsenic trioxide M. Moayeri, and L. M. Johansen. 2011. Auranofin protects against anthrax lethal in the treatment of acute promyelocytic leukemia. Leukemia 26: 433–442. toxin-induced activation of the Nlrp1b inflammasome. Antimicrob. Agents 42. Ro¨llig, C., and T. Illmer. 2009. The efficacy of arsenic trioxide for the treatment Chemother. 55: 1028–1035. of relapsed and refractory multiple myeloma: a systematic review. Cancer Treat. 15. Meissner, F., K. Molawi, and A. Zychlinsky. 2008. Superoxide dismutase 1 Rev. 35: 425–430. regulates caspase-1 and endotoxic shock. Nat. Immunol. 9: 866–872. 43. Wei, W., F. Zhou, Y. Zhang, L. Guo, H. Shi, and J. Hou. 2012. A combination of 16. Duesbery, N. S., C. P. Webb, S. H. Leppla, V. M. Gordon, K. R. Klimpel, thalidomide and arsenic trioxide is effective and well tolerated in patients with T. D. Copeland, N. G. Ahn, M. K. Oskarsson, K. Fukasawa, K. D. Paull, and myelodysplastic syndromes. Leuk. Res. 36: 715–719. G. F. Vande Woude. 1998. Proteolytic inactivation of MAP-kinase-kinase by 44. Carney, D. A. 2008. Arsenic trioxide mechanisms of action: looking beyond anthrax lethal factor. Science 280: 734–737. acute promyelocytic leukemia. Leuk. Lymphoma 49: 1846–1851. 17. Vitale, G., L. Bernardi, G. Napolitani, M. Mock, and C. Montecucco. 2000. 45. Lust, J. A., and K. A. Donovan. 1999. The role of interleukin-1b in the patho- Susceptibility of mitogen-activated protein kinase kinase family members to genesis of multiple myeloma. Hematol. Oncol. Clin. North Am. 13: 1117–1125. proteolysis by anthrax lethal factor. Biochem. J. 352: 739–745. 46. Dunn, J. H., L. Z. Ellis, and M. Fujita. 2012. Inflammasomes as molecular 18. Boyden, E. D., and W. F. Dietrich. 2006. Nalp1b controls mouse macrophage mediators of inflammation and cancer: potential role in melanoma. Cancer Lett. susceptibility to anthrax lethal toxin. Nat. Genet. 38: 240–244. 314: 24–33. 19. Wickliffe, K. E., S. H. Leppla, and M. Moayeri. 2008. Anthrax lethal toxin- 47. Goldberg, J. E., and K. L. Schwertfeger. 2010. Proinflammatory cytokines in induced inflammasome formation and caspase-1 activation are late events de- breast cancer: mechanisms of action and potential targets for therapeutics. Curr. pendent on ion fluxes and the proteasome. Cell. Microbiol. 10: 332–343. Drug Targets 11: 1133–1146. 20. Levin, T. C., K. E. Wickliffe, S. H. Leppla, and M. Moayeri. 2008. Heat shock 48. Dinarello, C. A. 2010. Why not treat human cancer with interleukin-1 blockade? inhibits caspase-1 activity while also preventing its inflammasome-mediated Cancer Metastasis Rev. 29: 317–329. activation by anthrax lethal toxin. Cell. Microbiol. 10: 2434–2446. 49. Lo´pez-Castejoń, G., and P. Pelegrıń. 2012. Current status of inflammasome 21. Bhagat, L., V. P. Singh, R. K. Dawra, and A. K. Saluja. 2008. Sodium arsenite blockers as anti-inflammatory drugs. Expert Opin. Investig. Drugs 21: 995–1007. induces heat shock protein 70 expression and protects against secretagogue- 50. Bobe´, P., D. Bonardelle, K. Benihoud, P. Opolon, and M. K. Chelbi-Alix. 2006. induced trypsinogen and NF-kB activation. J. Cell. Physiol. 215: 37–46. Arsenic trioxide: a promising novel therapeutic agent for lymphoproliferative 22. Kim, Y.-H., E.-J. Park, S. T. Han, J.-W. Park, and T. K. Kwon. 2005. Arsenic and autoimmune syndromes in MRL/lpr mice. Blood 108: 3967–3975. trioxide induces Hsp70 expression via reactive oxygen species and JNK pathway 51. Shen, L., F. Gong, W. Tian, W. Li, F. Zhang, J. Qian, A. Sun, Y. Zou, W. Yang, in MDA231 cells. Life Sci. 77: 2783–2793. and J. Ge. 2013. Anti-inflammatory effects of arsenic trioxide eluting stents in 23. Muehlbauer, S. M., H. Lima, Jr., D. L. Goldman, L. S. Jacobson, J. Rivera, a porcine coronary model. Biomed. Res. Int. 2013: 937936. M. F. Goldberg, M. A. Palladino, A. Casadevall, and J. Brojatsch. 2010. Pro- 52. Singer, M., G. Trugnan, and M. K. Chelbi-Alix. 2011. Arsenic trioxide reduces teasome inhibitors prevent caspase-1-mediated disease in rodents challenged 2,4,6-trinitrobenzene sulfonic acid-induced murine colitis via nuclear factor-kB with anthrax lethal toxin. Am. J. Pathol. 177: 735–743. down-regulation and caspase-3 activation. Innate Immun 17: 365–374. 24. Flora, S. J. S. 2011. Arsenic-induced oxidative stress and its reversibility. Free 53. Xu, Z. P., J. M. Huo, Y. L. Sang, J. Kang, and X. Li. 2012. Effects of arsenic Radic. Biol. Med. 51: 257–281. trioxide (As2O3) on airway remodeling in a murine model of bronchial asthma. 25. Lallemand-Breitenbach, V., J. Zhu, Z. Chen, and H. de The´. 2012. Curing APL Can. J. Physiol. Pharmacol. 90: 1576–1584. through PML/RARA degradation by As2O3. Trends Mol. Med. 18: 36–42. 54. Kingsbury, S. R., P. G. Conaghan, and M. F. McDermott. 2011. The role of the 26. Lo, Y.-H., Y.-W. Huang, Y.-H. Wu, C.-S. Tsai, Y.-C. Lin, S.-T. Mo, W.-C. Kuo, NLRP3 inflammasome in gout. J Inflamm Res 4: 39–49. Y.-T. Chuang, S.-T. Jiang, H.-M. Shih, and M.-Z. Lai. 2013. Selective inhibition 55. Martinon, F., V. Pe´trilli, A. Mayor, A. Tardivel, and J. Tschopp. 2006. Gout- of the NLRP3 inflammasome by targeting to promyelocytic leukemia protein in associated uric acid crystals activate the NALP3 inflammasome. Nature 440: mouse and human. Blood 121: 3185–3194. 237–241.