Of NLRP3 and NLRC4 Inflammasomes

β-arrestin1 Is Critical for the Full Activation of NLRP3 and NLRC4 Inflammasomes Kairui Mao, Shuzhen Chen, Yan Wang, Yan Zeng, Yonglei Ma, Yu Hu, Hong Zhang, Shuhui Sun, Xiaodong Wu, This information is current as Guangxun Meng, Gang Pei and Bing Sun of October 1, 2021. J Immunol 2015; 194:1867-1873; Prepublished online 12 January 2015; doi: 10.4049/jimmunol.1401989 http://www.jimmunol.org/content/194/4/1867 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology b-arrestin1 Is Critical for the Full Activation of NLRP3 and NLRC4 Inﬂammasomes

Kairui Mao,*,1 Shuzhen Chen,*,†,1 Yan Wang,‡,1 Yan Zeng,‡ Yonglei Ma,‡ Yu Hu,* Hong Zhang,‡ Shuhui Sun,x Xiaodong Wu,* Guangxun Meng,‡ Gang Pei,*,{ and Bing Sun*,‡

Inflammasomes are multiprotein complexes that trigger the activation of caspase-1 and the maturation of IL-1b, which are critical for inflammation and control of pathogen infection. Although the function of inflammasomes in immune response and disease development is well studied, the molecular mechanism by which inflammasomes are activated and assembled remains largely unknown. In this study, we found that b-arrestin1, a key regulator of the G protein–coupled receptor signaling pathway, was required for nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain–containing 3 (NLRP3) b and NLR family CARD domain–containing protein 4 (NLRC4) inflammasome–mediated IL-1 production and caspase-1 acti- Downloaded from vation, but it had no effect on absent in melanoma 2 (AIM2) inflammasome activation. Moreover, apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome, which is ASC aggregation mediating caspase-1 activation, was also impaired in b-arrestin1–deficient macrophages upon NLRP3 or NLRC4, but not AIM2 inflammasome activation. Mechanistic study revealed that b-arrestin1 specifically interacted with NLRP3 and NLRC4 and promoted their self-oligomerization. In vivo, in a monosodium urate crystal (MSU)-induced NLRP3-dependent peritonitis model, MSU-induced IL-1b production and neutrophil flux were

significantly reduced in b-arrestin1 knockout mice. Additionally, b-arrestin1 deficiency rescued the weight loss of mice upon log- http://www.jimmunol.org/ phase Salmonella typhimurium infection, with less IL-1b production. Taken together, our results indicate that b-arrestin1 plays a critical role in the assembly and activation of two major canonical inflammasomes, and it may provide a new therapeutic target for inflammatory diseases. The Journal of Immunology, 2015, 194: 1867–1873.

attern recognition receptors recognize a diverse range of cleotide-binding domain and leucine-rich repeat containing (NLR) microbial components and environmental irritants to in- family proteins or absent in melanoma 2–like (AIM2) receptors (ALR) P duce innate immune responses. Among those, some nu- form multiprotein complexes called inflammasomes with the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 (1). Once activated, inflammasomes serve as a platform by guest on October 1, 2021 *State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, for caspase-1 activation and IL-1b maturation as well as pyroptosis Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (2). Canonical inflammasomes, including NLR family pyrin domain– 200031, China; †Department of Microbiology and Immunology, Xiamen Medical College, Xiamen 361008, China; ‡Key Laboratory of Molecular Virology and Im- containing 3 (NLRP3), NLR family CARD domain–containing pro- munology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, tein 4 (NLRC4), and AIM2, once activated by microbial stimuli and Chinese Academy of Sciences, Shanghai 200031, China; xFudan University School of Medicine, Shanghai 200032, China; and {Shanghai Key Laboratory of Signaling endogenous danger signals undergo self-oligomerization to recruit and Disease Research, School of Life Science and Technology, Tongji University, the adaptor protein ASC. This step leads to self-oligomerization of Shanghai 200092, China ASC into a very large supramolecular structure called the ASC 1K.M., S.C., and Y.W. contributed equally to this work. pyroptosome, which recruits and activates procaspase-1, leading to Received for publication August 5, 2014. Accepted for publication December 5, the processing of pro–IL-1b and pro–IL-18 into the active proin- 2014. flammatory cytokines IL-1b and IL-18, respectively (3–5). Although This work was supported by National 973 Key Project of China Grant it has been well established that activation of inflammasomes plays 2013CB530504, National 863 Project of China Grants 2012AA02A404 and 2012AA020103, National Natural Science Foundation of China Grants 31030029, critical roles in microbial- or danger-associated signal–induced inflam- 31230024, and 81361120409, National Science and Technology Major Projects of matory responses, how inflammatory stimuli promote the forma- China Grants 2012ZX10002-007-003, 2013ZX10004-101-005, and 2013ZX10004- 003-003, and Chinese Academy of Sciences Key Project Grant KJZD-EW-L09-3. tion and activation of the inflammasomes remains largely unclear. b-arrestins are multifunctional proteins that play critical roles in Address correspondence and reprint requests to Dr. Bing Sun and Dr. Gang Pei, State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, the G protein–coupled receptor (GPCR) signaling pathway (6). It Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 has been reported that b-arrestin1 is also involved in autoimmune Yueyang Road, Shanghai 200031, China. E-mail addresses: [email protected] (B.S.) and [email protected] (G.P.) diseases such as experimental autoimmune encephalomyelitis and rheumatoid arthritis (7, 8). On the one hand, b-arrestin1 positively The online version of this article contains supplemental material. + 2 2 regulates CD4 T cell survival and homeostasis by promoting Bcl- Abbreviations used in this article: AIM2, absent in melanoma 2; Arrb1 / , b-arrestin1 deficient; ASC, apoptosis-associated speck-like protein containing 2 expression. On the other hand, b-arrestin1 also regulates Th17 a CARD; BMDC, bone marrow–derived dendritic cell; v-3 FA, omega-3 fatty acid; cell differentiation through scaffolding the interaction of JAK1 LDH, lactate dehydrogenase; MSU, monosodium urate; NLR, nucleotide-binding domain and leucine-rich repeat containing; NLRC4, NLR family CARD domain– and STAT3, thereby promoting STAT3 activation. In the pres- containing protein 4; NLRP3, NLR family pyrin domain–containing 3; NMDA, ent study, we found that in b-arrestin1–deficient peritoneal N-methyl-D-aspartate; poly(dA:dT), poly(deoxyadenylic-thymidylic) acid; WT, macrophages, NLRP3 and NLRC4 inflammasome activation were wild-type. strikingly dampened. In contrast, b-arrestin1 deficiency had Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 no effect on AIM2 inflammasome activation. Moreover, with www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401989 1868 REGULATION OF INFLAMMASOME ACTIVATION BY b-arrestin1 a crosslinking assay and immunofluorescence assay, b-arrestin1 was various combinations of plasmids. At 24 h after the transfection, the cell found to promote NLRP3- and NLRC4-induced ASC pyroptosome lysates were prepared in lysis buffer and incubated with the indicated Ab formation. Further mechanistic study revealed that b-arrestin1 together with protein A/G Plus–agarose immunoprecipitation reagent (Santa Cruz Biotechnology) at 4˚C for 3 h or overnight. After three washes, specifically interacted with NLRP3 and NLRC4 and enhanced their the immunoprecipitates were boiled in SDS sample buffer for 10 min self-oligomerization. In vivo, b-arrestin1 was involved in a mono- and analyzed by immunoblot. sodium urate (MSU)–induced peritonitis model by promoting IL-1b For interaction of NLRs and b-arrestin1, NLRP3, NLRC4, and NLRP12 production and neutrophil influx. During Salmonella typhimurium were cloned into p3xFLAG-CMV-10 expression vector (Sigma-Aldrich) to express 3xFLAG-NLRs. 3xFLAG peptide (Sigma-Aldrich) was used infection, which causes severe inflammation through NLRC4 according to the manufacturer’s recommendations. In short, after 24 h inflammasome activation, b-arrestin1 deficiency played a protective transfection, the cell lysates were prepared in lysis buffer and incubated role and modulated IL-1b production as well. Our results suggest with anti-FLAG M2 affinity gel at 4˚C overnight. The gel was washed and the positive role of b-arrestin1inregulatingNLRP3andNLRC4 eluted with 100 mg/ml 3xFLAG peptide for .3 h at 4˚C, and the super- inflammasomes, which may be a potential target in the treatment of natant free of gel was analyzed by immunoblot. b For endogenous coimmunoprecipitation experiments, different stimu- IL-1 –associated autoinflammatory diseases. lated BMDCs were then prepared in lysis buffer and incubated with 0.5 ml anti-NLRP3 or rabbit IgG. The subsequent procedures were performed as Materials and Methods described above. Mice and reagents Triton X-100 insoluble fraction was generated as previously described (11). Then 1% Nonidet P-40 buffer containing EDTA-free protease in- C57BL/6 mice were obtained from Shanghai Laboratory Animal Center hibitor “mixture” and phosphatase inhibitor mixture (Nacalai Tesque) were 2 2 (Chinese Academy of Sciences). b-arrestin1–deficient (Arrb1 / ) mice on used to dissolve the insoluble part at 4˚C for .1 h. After centrifugation at a C57BL/6 background were provided by Robert J. Lefkowitz (Duke 5000 3 g for 10 min at 4˚C, the supernatant was collected and applied for University Medical Center, Durham, NC). All mice were maintained in immunoprecipitation assay as described above. Downloaded from pathogen-free conditions. Animal care and use were in accordance with the guidelines of the Institute of Biochemistry and Cell Biology, Chinese ASC pyroptosome detection Academy of Sciences. ASC pyroptosomes were detected as previously reported (12). Macrophages All reagents were from Sigma-Aldrich unless stated otherwise. ATP was were seeded in six-well plates (2 3 106 cells per well) and treated with purchased from Roche. Anti-NLRP3 Ab was obtained from Alexis (804-880- different stimuli. The cells were centrifuged and resuspended in 500 ml C100). Anti–b-arrestin1 Ab was purchased from Abcam. Anti–caspase-1 Ab buffer containing 20 mM HEPES-KOH (pH 7.5), 150 mM KCl, 1%

was obtained from Santa Cruz Biotechnology. Anti-ASC Ab was produced http://www.jimmunol.org/ Nonidet P-40, 0.1 mM PMSF, and protease inhibitor mixture, followed by by immunization of rabbits with ASC expressed by Escherichia coli. Anti- shearing 10 times through a 21-gauge needle. The cell lysates were then NLRC4 Ab was purchased from Millipore. Anti-P2X7R Ab was purchased centrifuged at 5000 3 g for 10 min at 4˚C, and the pellets were washed from Abcam. MSU was prepared as previously reported (9). S. typhimurium twice with PBS and resuspended in 500 ml PBS. The resuspended pellets was a gift from Weihuan Fang (Zhejiang University, Zhejiang, China). were cross-linked with disuccinimidyl suberate (4 mM) for 30 min and Flagellin of S. typhimurium was provided by Feng Shao (National Institute of pelleted at 5000 3 g for 10 min. The cross-linked pellets were lysed in 30 Biological Sciences, Beijing, China). IL-1b and IL-6 ELISA kits were ob- ml SDS sample buffer, separated using 12% SDS-PAGE, and immuno- tained from R&D Systems. Lactate dehydrogenase (LDH) release was as- blotted using anti-mouse ASC Abs. sessed in cell-free medium at indicated times following the manufacturer’s instructions (cytotoxicity detection kit; Roche Diagnostics). Anti-FLAG M2 Confocal microscopy afﬁnity gel and 3xFLAG peptide were purchased from Sigma-Aldrich. Cells Peritoneal macrophages were plated overnight on coverslips. After stim- by guest on October 1, 2021 ulation as described above, the cells were washed and ﬁxed with 4% PFA in

HEK293T cells were cultured in humidified 5% CO2 at 37˚C in DMEM PBS for 15 min, permeabilized with Triton X-100, and blocked with 1% supplemented with 10% (v/v) FBS, penicillin (100 U/ml), and strepto- BSA in PBS for 30 min. After incubation with FITC-conjugated anti-ASC mycin (100 U/ml). Lipofectamine 2000 (Invitrogen) was used for trans- Ab and anti-NLRP3 Ab for 2 h, cells were washed and incubated with Alexa fection of HEK293T cells. Fluor 561 goat anti-mouse Ab (BD Biosciences) for 1 h. Finally, the cells In preparation for the isolation of peritoneal macrophages, mice were were stained with DAPI. The confocal microscopy analyses were performed i.p. injected with 1 ml 4% thioglycollate, and peritoneal exudate cells using a Leica TCS SP2. were isolated from the peritoneal cavity 4 d postinjection. The cells were then incubated at 37˚C for 6 h and washed three times with HBSS. The In vivo peritonitis model remaining adherent cells were used as the peritoneal macrophages de- m scribed in the experiments. Mice were injected i.p. with 1 mg MSU in 200 l PBS; control mice re- Bone marrow–derived dendritic cells (BMDCs) were prepared as follows: ceived PBS. At 6 h after injection, mice were sacrificed and peritoneal cells bone marrow cells were flushed from the femurs and tibias of C57BL/6 and were obtained with 10 ml PBS with 1% (v/v) FBS, followed by centri- Arrb12/2 mice and depleted of red cells with ammonium chloride. Cells fugation and total cell counting. Subsequently, cells were stained for were cultured at 1 3 106 cells per well in 24-well plates in DMEM sup- neutrophil surface marker GR-1 and detected using flow cytometry. The number of neutrophils was calculated as total cells multiplied by the plemented with 20 ng/ml murine GM-CSF (PeproTech). Fresh medium was + changed every 2 d. On day 7, cells were collected for further experiments. percentage of GR-1 cells. The number of peritoneal macrophages was calculated as total cells multiplied by the percentage of CD11b and F4/80 In vitro stimulation of macrophages double-positive cells. For IL-1b and IL-6 measurements, 600 ml PBS with 1% (v/v) FBS was used and the supernatant IL-1b and IL-6 of peritoneal Unless otherwise indicated, the macrophages were primed with 200 ng/ml lavage fluid were determined. LPS from E. coli 0111:B4 (Sigma-Aldrich) for 4 h before stimulation with 5 mM ATP for 30 min, 20 mM nigericin for 30 min, 250 mg/ml MSU for Animal infections 3 h, poly(deoxyadenylic-thymidylic) acid [poly(dA:dT)] for 6 h, flagellin for 6h,orlog-phaseS. typhimurium strain SL1344 for 2 h. Poly(dA:dT) was For S. typhimurium infection, mice were starved for 12 h and infected i.p. transfected with Lipofectamine 2000 (Invitrogen) according to the manu- with 13 102 CFU log-phase bacteria (prepared as described above) in 100 ml facturer’s recommendations. S. typhimurium was grown overnight (16–18 h) sterile PBS, after which food and water were supplied. At day 5 postinfec- in Luria–Bertani broth at 37˚C and inoculated at 1:50 in fresh Luria–Bertani tion, peritoneal fluid was collected for detection of IL-1b and IL-6. Spleen broth, followed by another 2.5–3 h culture, when the bacteria came into its and liver were harvested, homogenized in PBS, and the bacteria loads were log phase. The bacteria were diluted in fresh macrophage medium and added determined as previously described (13). to the LPS-priming macrophages at different multiplicities of infection. Cells were cultured at 37˚C for 2 h before analysis. Statistical analysis Immunoprecipitation and immunoblot analysis Data are presented as the means 6 SD from three independent experiments. Statistical comparisons between different treatments were performed by an Immunoprecipitation and immunoblot analysis were performed as de- unpaired Student t test, where p , 0.05 was considered statistically sig- scribed previously (10). In short, HEK293T cells were transfected with nificant and p , 0.01 was highly significant. The Journal of Immunology 1869

Results b-arrestin1 regulates NLRP3 inflammasome activation b-arrestin1 was reported to play an important role in autoimmune diseases, such as experimental autoimmune encephalomyelitis and rheumatoid arthritis (7, 8); however, the molecular mechanism remains poorly studied. Given that Th17 cells play a central role in autoimmune disease (14, 15) and IL-1, IL-6, and IL-23, produced mainly by dendritic cells and macrophages, are essential for the induction of Th17 cells (16–18), we prepared BMDCs from the wild-type (WT) and Arrb12/2 mice and stimulated these cells with LPS for different lengths of time. As shown in Supplemental Fig. 1, b-arrestin1 deficiency resulted in a dramatic decrease of IL-1b production at 48 h of stimulation, but not IL-6, TNF-a, and IL-23 production. It has been reported that IL-1b secretion in- volves two sequential processes: the activation of NF-kB, which contributes to the expression of pro–IL-1b and NLRP3 (19), and the second signal, the assembly of inflammasomes to generate active caspase-1 for processing pro–IL-1b (9, 20, 21). The secretion of IL-1b by LPS-stimulated BMDCs at a later time point may be Downloaded from due to the generation of endogenous danger signal, such as ATP, from dead dendritic cells. To investigate whether b-arrestin1isinvolvedinNLRP3 inflammasome activation, we stimulated LPS-primed WT and b 2/2 FIGURE 2. -arrestin1 is required for NLRC4 but not AIM2 inflamma- Arrb1 peritoneal macrophages with ATP, nigericin, or MSU. some–mediated IL-1b production and caspase-1 activation. (A) Production b 2/2 Inconsistent with the findings in BMDCs, IL-1 production, but of IL-1b by LPS-primed peritoneal macrophages from WT mice or Arrb1 http://www.jimmunol.org/ 2/2 not IL-6, was markedly decreased in Arrb1 macrophages mice left untreated (Mock) or infected with various dilutions of S. typhi- (Fig. 1A, 1B). Reduced generation of caspase-1 active form p10 murium for 2 h. (B) Immunoblot analysis of IL-1b p17 and caspase-1 p10 in 2 2 was also observed in Arrb1 / macrophages in all stimuli tested supernatants and pro–IL-1b, procaspase-1, b-arrestin1, and b-actin in (Fig. 1C). It was interesting that b-arrestin1 could also be detected lysates of peritoneal macrophages treated as in (A). (C) Production of IL-1b 2/2 in the supernatant upon NLRP3 inflammasome activation by LPS-primed peritoneal macrophages from WT mice or Arrb1 mice D (Supplemental Fig. 2A). Because b-arrestin1 deficiency had no left untreated (Mock) or incubated with various dosages flagellin for 6 h. ( ) b effect on the LPS-induced NLRP3 and pro–IL-1b production Immunoblot analysis of IL-1 p17 and caspase-1 p10 in supernatants and pro–IL-1b, procaspase-1, b-arrestin1, and b-actin in lysates of peritoneal (Supplemental Fig. 2B), the diminished caspase-1 activation in macrophages treated as in (C). (E) Production of IL-1b by LPS-primed b by guest on October 1, 2021 -arrestin1–deficient macrophages was not the result of the peritoneal macrophages from WT mice or Arrb12/2 mice transfected with availability of NLRP3. Given that arrestins were reported to fa- various dosages of poly(dA:dT) for 6 h. (F)ImmunoblotanalysisofIL-1b p17 and caspase-1 p10 in supernatants and pro–IL-1b, procaspase-1, b-arrestin1, and b-actin in lysates of peritoneal macrophages treated as in (E). Casp1 p10, active form of caspase-1; MOI, multiplicity of infection; Pro- Casp1, procaspase-1. The data represent at least three experiments [means 6 SD in (A), (C), and (E)]. *p , 0.05 (Student t test).

cilitate internalization of GPCRs upon activation (22), and P2X7R, the receptor for extracellular ATP, belongs to GPCR su- perfamily (23), expression of P2X7R on the cell surface was analyzed. However, no significant difference was observed between WT and Arrb12/2 macrophages (Supplemental Fig. 2C), suggesting that b-arrestin1 deficiency has no effect on the internalization of P2X7R. Because pyroptosis is induced during inflammasome activation, LDH release from the stimulated macrophages was examined to determine whether cell death is affected by b-arrestin1 deficiency. As expected, similar to the pattern of IL-1b production and caspase-1 activation, LDH release was also reduced in Arrb12/2 macrophages upon various stimuli (Fig. 1D). FIGURE 1. NLRP3 inflammasome–mediated IL-1b production and These data suggest that b-arrestin1 deficiency results in decreased caspase-1 activation is reduced in b-arrestin1–deficient macrophages. (A NLRP3 inflammasome-mediated caspase-1 activation and active and B) Production of IL-1b (A) and IL-6 (B) by peritoneal macrophages IL-1b production. from WT mice or Arrb12/2 mice primed with LPS for 4 h and then either left unstimulated (Mock) or stimulated with 5 mM ATP (30 min), 20 mM b-arrestin1 is also required for NLRC4, but not AIM2, nigericin (Nig, 30 min), or 250 mg/ml MSU crystals (MSU, 3 h). (C) inflammasome activation b Immunoblot analysis of IL-1 p17 and caspase-1 p10 in supernatants and b pro–IL-1b, procaspase-1, b-arrestin1, and b-actin in lysates of peritoneal To define whether the effect of -arrestin1 was confined to NLRP3 macrophages treated as in (A). (D) Cell viability was detected by LDH re- or extended to other inflammasome types, we assessed the func- lease assay. Casp1 p10, active form of caspase-1; Pro-Casp1, procaspase-1. tions of b-arrestin1 in NLRC4 and AIM2 inflammasome activa- The data represent at least three experiments [means 6 SD in (A), (B), and tion. To activate the NLRC4 inflammasome, LPS-primed WT and 2 2 (D)]. *p , 0.05 (Student t test). Arrb1 / macrophages were infected with different multiplicities 1870 REGULATION OF INFLAMMASOME ACTIVATION BY b-arrestin1 of infection of S. typhimurium (22). Similar to the NLRP3 assembled by ASC, which is thought to mediate caspase-1 acti- inflammasome, the NLRC4 inflammasome–mediated IL-1b pro- vation (5). We next examined ASC pyroptosome formation in WT duction and caspase-1 activation were impaired in Arrb12/2 and Arrb12/2 peritoneal macrophages. On the one hand, ASC macrophages (Fig. 2A, 2B). S. typhimurium was reported to ac- pyroptosome was isolated and then cross-linked with noncleavable tivate the NLRP3 inflammasome as well, but the stimulation protein cross-linking agent disuccinimidyl suberate in the ATP-, condition was different from NLRC4 (13). Therefore, to further S. typhimurium–, or poly(dA:dT)-stimulated macrophages. Incon- characterize the role of b-arrestin1 in NLRC4 activation, another sistent with IL-1b production and caspase-1 activation, deficiency NLRC4 inflammasome stimulator, purified flagellin from S. typhi- of b-arrestin1 resulted in reduced NLRP3- and NLRC4-mediated murium, was also tested, and the results were similar to those ASC pyroptosome formation (Fig. 3A, 3B). In contrast, AIM2- with S. typhimurium infection (Fig. 2C, 2D). However, upon the mediated ASC pyroptosome was unaffected in Arrb12/2 macro- intracellular delivery of poly(dA:dT) (24, 25), which activates phages (Fig. 3B). On the other hand, confocal microscopy was AIM2 inflammasome, b-arrestin1deficiencyhadnoeffectonIL-1b applied for the detection of the endogenous ASC, which showed secretion and caspase-1 activation (Fig. 2E, 2F). LDH release showed that .78% of the WT macrophages contained the ASC pyropto- the similar trend as the IL-1b secretion during activation of these some after stimulation with LPS and ATP, compared with only inflammasomes (Supplemental Fig. 3). Thus, b-arrestin1 is also re- 33.1% in Arrb12/2 macrophages. Flagellin-induced ASC pyrop- quired for NLRC4 inflammasome activation. tosome formation was also impaired in Arrb12/2 macrophages, with 31.4–60.8% in WT cells, whereas AIM2 inflammasome– b -arrestin1 is critical for NLRP3- and NLRC4-mediated ASC dependent ASC pyroptosome formation was intact with b-arrestin1 pyroptosome formation deficiency (Fig. 3C, 3D). Collectively, these data indicate that For NLRP3, NLRC4, and AIM2 inflammasomes, once activated, b-arrestin1 is involved in the NLRP3 and NLRC4 inflammasome Downloaded from they recruit ASC to form ASC pyroptosome, a single large structure assembly to activate caspase-1. http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. NLRP3- and NLRC4- but not AIM2-mediated ASC pyroptosome formations are impaired in b-arrestin1–deficient macrophages. (A) ASC oligomerization and redistribution assay in LPS-primed peritoneal macrophages from WT mice or Arrb12/2 mice left untreated (Mock) or stimulated with ATP. Immunoblot (IB) analysis of ASC in cross-linked pellets (upper panels) and ASC, b-arrestin1, and b-actin in cell lysates (lower panels). (B) ASC oligomerization assay in LPS-primed peritoneal macrophages from WT mice or Arrb12/2 mice left untreated (Mock) or infected with S. typhimurium (S. typhi, multiplicity of infection of 50) or transfected with 1 mg poly(dA:dT). Immunoblot (IB) analysis of ASC in cross-linked pellets (upper panels) and ASC, b-arrestin1, and b-actin in cell lysates (lower panels) are shown. (C) Immunofluorescence microscopy of LPS-primed peritoneal macrophages treated with ATP, flagellin, poly(dA:dT), or left unstimulated (Mock) and then stained for ASC, NLRP3, and DNA (with DAPI). Scale bars, 10 mm. (D) Percentage of macrophages containing ASC foci from WT mice or Arrb12/2 mice treated as in (C). The quantification represents the mean of three independent experiments, with at least 200 cells counted in each experiment. The data represent at least three experiments [means 6 SD in (D)]. *p , 0.05 (Student t test). The Journal of Immunology 1871 b-arrestin1 specifically interacts with NLRP3 and NLRC4 and b-arrestin1 is required for NLRP3 and NLRC4 inflammasome promotes their self-oligomerization activation but not for AIM2 inflammasome activation. b Because b-arrestins have been reported to function as adaptors in To determine the association of -arrestin1 with NLRP3 in a more various signaling pathways (26), the requirement of b-arrestin1 physiological setting, we immunoprecipitated endogenous NLRP3 for full inflammasome activation led us to examine the potential from BMDCs and then evaluated the immunoprecipitates for the b interaction between b-arrestin1 and the NLRP3 inflammasome presence of -arrestin1 upon different stimulation. In accordance b components. We found that b-arrestin1 specifically interacted with with the interaction of -arrestin1 with NLRP3 in HEK293T cells, b NLRP3, but not ASC or procaspase-1, when they were coex- endogenous -arrestin1 was associated with NLRP3 under the pressed in HEK293T cells (Fig. 4A). Given that NLRP3 has three physiological condition (Fig. 4D). Because the active inflam- different functional domains (PYD, NACHT, and LRR domains), masome complexes are also found in the insoluble fraction (11), the b we further examined the domains of NLRP3 accounting for the NLRP3/ -arrestin1 association was also detected in this part. Upon b association with b-arrestin1. As shown in Fig. 4B, b-arrestin1 had LPS plus ATP stimulation, a fraction of -arrestin1 sedimented in strong association with the PYD domain and weak binding to the the insoluble part and showed an interaction with NLRP3, which NACHT and LRR domains of NLRP3. Because we determined precipitated more upon ATP stimulationcomparedwithLPSpriming b that b-arrestin1 is indispensible for not only NLRP3 inflamma- alone (Fig. 4D). Taken together, our findings suggest that -arrestin1 some activation but also NLRC4 inflammasome activation, we specifically interacts with NLRP3 and NLRC4. sought to determine whether other inflammasome sensors would It has been reported that NLRs, including NLRP3 and NLRC4, interact with b-arrestin1. Of the proteins tested, we found that once activated, undergo self-oligomerization to form a molecular platform, which recruits the adaptor protein ASC (27). To further

NLRP3, NLRC4, and NLRP12, but not AIM2, interacted with Downloaded from b b-arrestin1 (Fig. 4C). This is consistent with the findings that investigate whether -arrestin1 plays a role in NLRP3 and NLRC4 self-oligomerization, we expressed Flag-tagged and hemagglutinin- tagged NLRP3 or NLRC4 in the presence or absence of b-arrestin1 in HEK293T cells. When immunoprecipitated with anti-Flag Ab, b-arrestin1 enhanced the interaction between different tagged NLRP3 proteins (Fig. 4E). The same effect was found on NLRC4 self-oligomerization (Fig. 4F). Thus, this assay indicates that http://www.jimmunol.org/ b-arrestin1 may promote self-oligomerization of NLRP3 and NLRC4 proteins. b-arrestin1 regulates NLRP3 and NLRC4 inflammasomes in vivo To determine whether the effect of b-arrestin1 on NLRP3 and NLRC4 inflammasome activation was also critical in vivo, different mouse models were adopted. Under steady-state, the physiological levels of peritoneal macrophages and neutrophils were by guest on October 1, 2021 similar between WT and Arrb12/2 mice (Supplemental Fig. 4). In the MSU-induced peritoneal inflammatory mouse model, which is an NLRP3 inflammasome–induced peritonitis model (9), the production of IL-1b and IL-6 and the influx of neutrophils into the peritoneal cavity were determined. Compared with WT littermates, b-arrestin1 deficiency resulted in attenuated IL-1b production (Fig. 5A) and neutrophil infiltration (Fig. 5C), whereas IL-6 production was intact (Fig. 5B). In contrast, b-arrestin1 had no effect on zymosan-induced neutrophil influx, which is known to induce peritonitis in an IL-1b–independent manner. Additionally, log-phase S. typhimurium–induced inflammation in vivo depends on NLRC4 inflammasome. During S. typhimurium infection, peritoneal IL-1b production was significantly reduced in Arrb12/2 mice (Fig. 5D), but not IL-6 production (Fig. 5E). b FIGURE 4. -arrestin1 specifically interacts with NLRP3 and NLRC4 b-arrestin1 deficiency also rescued the weight loss caused by and promotes their self-oligomerization. (A–C) Immunoprecipitation (IP) S. typhimurium (Fig. 5F), with similar bacteria loads in the spleens and immunoblot (IB) analysis of transfected proteins in lysates of 2/2 HEK293T cells. (A) Interaction of hemagglutinin (HA)-tagged b-arrestin1 and livers of WT and Arrb1 mice (Fig. 5G), suggesting that the and Flag-tagged NLRP3 inflammasome components. (B) Interaction of Flag- weight loss was enhanced by excessive inflammation instead of tagged NLRP3 proteins and HA-tagged b-arrestin1. (C) Interaction of HA- bacteria proliferation. These results indicate the pivotal function tagged b-arrestin1 and 3xFlag-tagged NLRP3, NLRP12, NLRC4, and of b-arrestin1 in NLRP3 and NLRC4 inflammasome activation AIM2. (D) Interaction of endogenous b-arrestin1 with NLRP3 in both Triton in vivo. X-100–soluble (S) and Triton X-100–insoluble (I) fractions from BMDCs left unstimulated or stimulated with LPS or LPS plus ATP. (E)Immuno- Discussion precipitation (IP) and immunoblot (IB) analysis of transfected HA-tagged Although several regulators have been found involved in either NLRP3 and Flag-tagged NLRP3 in lysates of HEK293T cells with or without b-arrestin1. (F) IP and IB analysis of transfected HA-tagged NLRC4 NLRP3 or NLRC4 inflammasome, our present study suggests a b and Flag-tagged NLRC4 in lysates of HEK293T cells with or without critical role of -arrestin1 in activation of both inflammasomes. b-arrestin1. Data are representative of three independent experiments. anti- These regulators function in diverse ways, including modification N3, anti-NLRP3; I, Triton X-100–insoluble fraction; S, Triton X-100–solu- of the inflammasome components, scaffolding inflammasome as- ble fraction. sembly, or changing the localization of inflammasomes (28–30). 1872 REGULATION OF INFLAMMASOME ACTIVATION BY b-arrestin1

FIGURE 5. b-arrestin1 regulates NLRP3 and NLRC4 inﬂammasomes in vivo. (A and B) Production of IL-1b and IL-6 in peritoneal lavage ﬂuid from 2/2 C 2/2

Arrb1 mice and WT littermates 6 h after i.p. injection of MSU crystals (n = 6–8). ( ) Quantification of neutrophil influx in WT and Arrb1 mice 6 h Downloaded from after treatment with sterile PBS alone or sterile PBS supplemented with 1 mg MSU crystals or 0.5 mg zymosan (n = 5–8). (D–G) WT and Arrb12/2 mice were peritoneally infected with 1 3 102 CFU log-phase S. typhimurium in 100 ml sterile PBS, and weight loss was monitored until day 5 (F). At day 5 postinfection, peritoneal fluid was collected for detection of IL-1b (D) and IL-6 (E), and the bacteria loads in the spleen and liver were determined (G). Data are representative of at least two experiments (means 6 SD). *p , 0.05, **p , 0.01 (Student t test).

For instance, deubiquitinase BRCC3, which can deubiquitinate flammation via NLRC4 inflammasome. Thus, in our experiment, http://www.jimmunol.org/ the LRR domain of NLRP3, is essential for the full activation mice were infected with S. typhimurium by i.p. injection and of NLRP3 inflammasome (30). Similarly, protein kinase Cd,an similar results were observed as with MSU-induced peritonitis. NLRC4 kinase, is required for NLRC4 inflammasome activation These results may suggest that excessive inflammation, such as via phosphorylation on NLRC4 (31). Besides, it has been reported undue IL-1b secretion, but not the pathogen alone, is responsible that GBP5 is a unique activator of NLRP3-ASC assembly, limited for the pathogen-induced pathogenicity. to bacterial cell wall components but not crystalline agents or In previous studies, it was suggested that b-arrestins may dsDNA (31). With yeast two-hybrid, Tschopp and colleagues (32) function as negative regulators to suppress NF-kB activation found that heat shock protein 90 was required for the activity through direct interaction with IkBa, then preventing its phos- by guest on October 1, 2021 of several NLRs, including NLRP3, NLRC4, and Nod2. In this study, phorylation and degradation (33), or interaction with TRAF6 and our results suggest that b-arrestin1 is required for the full activation of preventing its autoubiquitination and activation of NF-kB (34). bothNLRP3andNLRC4inflammasomes through interacting with Afterward, other findings indicated that the functions of b-arrestins NLR proteins and promoting their self-oligomerization. We found that are pleiotropic (35, 36). Nevertheless, we found that b-arrestin1 had b-arrestin1 could interact with three domains of NLRP3 and full- no effect on IL-6 and pro–IL-1b production, but it regulated IL-1b length NLRC4 in HEK293T cells. In the endogenous immunopre- secretion by regulating inflammasome activation. The contradicted cipitation assay, the b-arrestin1/NLRP3 interaction was detected in effects of b-arrestins on NF-kB activation and inflammatory cyto- both the Triton X-100–soluble and Triton X-100–insoluble fractions kine secretion may be explained by the different cell types and upon different stimulations. activation states adopted. During inflammasome activation, in addition to IL-1b secretion Another b-arrestin family member b-arrestin2, 78% identical and caspase-1 activation, there is another important marker, ASC to b-arrestin1 at the amino acid level, was involved in NLRP3 pyroptosome formation. Interestingly, only one ASC pyroptosome inflammasome inhibition induced by omega-3 fatty acids (v-3 is formed per cell, which is crucial for the subsequent procaspase- FAs) (37). Although endogenous b-arrestin2 could interact with 1 recruitment and activation (5). In our study, two different NLRP3, this interaction only occurred in the presence of v-3 strategies were adopted, and both showed a remarkable impair- FAs. When macrophages were primed with LPS together with v-3 ment of ASC pyroptosome formation in Arrb12/2 macrophages FAs, v-3 FAs induced b-arrestin2/NLRP3 interaction and inhib- upon NLRP3 and NLRC4 inflammasome activation, whereas ited nigericin-induced NLRP3 inflammasome activation. How- AIM2-induced ASC pyroptosome formation was intact. The de- ever, when macrophages were stimulated with LPS and nigericin fect of ASC pyroptosome formation in b-arrestin1 deficiency in the absence of v-3 FAs, b-arrestin2 deficiency had no effect on macrophages was supposed to result in subsequent attenuated IL- NLRP3 inflammasome activation. Therefore, the suppressive ef- 1b secretion. fect of b-arrestin2 on NLRP3 inflammasome relied on the presence In vivo, b-arrestin1 also played a critical role for NLRP3 and of v-3 FAs. b-arrestin2 was also engaged in the downregulation of NLRC4 inflammasome–dependent disease models. MSU crystals, inflammasome by the N-methyl-D-aspartate (NMDA) receptor (38). which cause peritonitis in a mouse model (9), were also used in In this work, aspartate, the stimulator of NMDA was found to our experiment, and we found that MSU-induced peritonitis was downregulate transcripts of pro–IL-1b, NLRP3, and procaspase-1, significantly reduced in Arrb12/2 mice. These findings indicate which required NMDA and b-arrestin2. b-arrestin2 was also critical that b-arrestin1 is required for the inflammation induced by NLRP3 for dampening LPS and D-galactosamine- induced fulminant hepa- inflammasome. titis in mice. As to the regulation of NF-kB, unlike b-arrestin2, NLRC4 is the major receptor for recognition of Gram-negative whose effect is relatively clear, the exact role of b-arrestin1 in bacteria, and log-phase S. typhimurium could cause severe in- regulating NF-kB remains to be further investigation. At least our The Journal of Immunology 1873 results suggest b-arrestin1 has no impact on NF-kB signaling in 16. Sutton, C., C. Brereton, B. Keogh, K. H. Mills, and E. C. Lavelle. 2006. b A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells peritoneal macrophages. Therefore, the role of -arrestin1 compared that mediate autoimmune encephalomyelitis. J. Exp. Med. 203: 1685–1691. with b-arrestin2 in the context of inflammasome activation could be 17. Zhou, L., I. I. Ivanov, R. Spolski, R. Min, K. Shenderov, T. Egawa, D. E. Levy, divergent. W. J. Leonard, and D. R. Littman. 2007. 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Corrections

Mao, K., S. Chen, Y. Wang, Y. Zeng, Y. Ma, Y. Hu, H. Zhang, S. Sun, X. Wu, G. Meng, G. Pei, and B. Sun. 2015. b-arrestin1 is critical for the full activation of NLRP3 and NLRC4 inﬂammasomes. J. Immunol. 194:1867–1873.

During the assembly of Fig. 2, incorrect Western blot data were unintentionally used for Fig. 2F. The corrected Fig. 2 is shown below. The ﬁgure legend was correct as published and is shown below for reference. The authors regret this inadvertent error, and they believe that this correction has no impact on the content, interpretations, or conclusions for Fig. 2 or the work as a whole. The ﬁgure has been corrected in the online version of the article, which now differs from the print version as originally published.

FIGURE 2. b-arrestin1 is required for NLRC4 but not AIM2 inﬂammasome–mediated IL-1b production and caspase-1 activation. (A) Production of IL-1b by LPS-primed peritoneal macrophages from WT mice or Arrb12/2 mice left untreated (Mock) or infected with various dilutions of S. typhimurium for 2 h. (B) Immunoblot analysis of IL-1b p17 and caspase-1 p10 in supernatants and pro–IL-1b, procaspase-1, b-arrestin1, and b-actin in lysates of peritoneal macrophages treated as in (A). (C) Production of IL-1b by LPS-primed peritoneal macrophages from WT mice or Arrb12/2 mice left untreated (Mock) or incubated with various dosages ﬂagellin for 6 h. (D) Immunoblot analysis of IL-1b p17 and caspase-1 p10 in supernatants and pro–IL-1b, procaspase-1, b-arrestin1, and b-actin in lysates of peritoneal macrophages treated as in (C). (E) Production of IL-1b by LPS-primed peritoneal macrophages from WT mice or Arrb12/2 mice transfected with various dosages of poly(dA:dT) for 6 h. (F)Immunoblot analysis of IL-1b p17 and caspase-1 p10 in supernatants and pro–IL-1b, procaspase-1, b-arrestin1, and b-actin in lysates of peritoneal macrophages treated as in (E). Casp1 p10, active form of caspase-1; MOI, multiplicity of infection; Pro-Casp1, procaspase-1. The data represent at least three experiments [means 6 SD in (A), (C), and (E)]. *p , 0.05 (Student t test).