ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination

Article20 February 2019free access Source DataTransparent process ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination Guangming Ren orcid.org/0000-0002-7841-185X State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Xuanyi Zhang orcid.org/0000-0003-2814-3313 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Yang Xiao orcid.org/0000-0003-3629-0946 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Wen Zhang State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Search for more papers by this author Yu Wang State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China An Hui Medical University, Hefei, China Search for more papers by this author Wenbing Ma State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Xiaohan Wang State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Pan Song Beijing Institute of Radiation Medicine, Beijing, China Search for more papers by this author Lili Lai Beijing Institute of Radiation Medicine, Beijing, China Search for more papers by this author Hui Chen State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Yiqun Zhan State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Jianhong Zhang State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Institute for Immunology and School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Miao Yu orcid.org/0000-0003-2963-8254 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Changhui Ge Beijing Institute of Radiation Medicine, Beijing, China Search for more papers by this author Changyan Li orcid.org/0000-0001-9959-1661 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Ronghua Yin Corresponding Author [email protected] orcid.org/0000-0001-8119-3657 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Xiaoming Yang Corresponding Author [email protected] State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Search for more papers by this author Guangming Ren orcid.org/0000-0002-7841-185X State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Xuanyi Zhang orcid.org/0000-0003-2814-3313 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Yang Xiao orcid.org/0000-0003-3629-0946 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Wen Zhang State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Search for more papers by this author Yu Wang State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China An Hui Medical University, Hefei, China Search for more papers by this author Wenbing Ma State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Xiaohan Wang State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Pan Song Beijing Institute of Radiation Medicine, Beijing, China Search for more papers by this author Lili Lai Beijing Institute of Radiation Medicine, Beijing, China Search for more papers by this author Hui Chen State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Yiqun Zhan State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Jianhong Zhang State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Institute for Immunology and School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Miao Yu orcid.org/0000-0003-2963-8254 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Changhui Ge Beijing Institute of Radiation Medicine, Beijing, China Search for more papers by this author Changyan Li orcid.org/0000-0001-9959-1661 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Ronghua Yin Corresponding Author [email protected] orcid.org/0000-0001-8119-3657 State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Search for more papers by this author Xiaoming Yang Corresponding Author [email protected] State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China Search for more papers by this author Author Information Guangming Ren1, Xuanyi Zhang1, Yang Xiao1, Wen Zhang1,2, Yu Wang1,3, Wenbing Ma1, Xiaohan Wang1, Pan Song4, Lili Lai4, Hui Chen1, Yiqun Zhan1, Jianhong Zhang1,5, Miao Yu1, Changhui Ge4, Changyan Li1, Ronghua Yin *,1 and Xiaoming Yang *,1,2 1State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China 2Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China 3An Hui Medical University, Hefei, China 4Beijing Institute of Radiation Medicine, Beijing, China 5Institute for Immunology and School of Medicine, Tsinghua University, Beijing, China *Corresponding author. Tel: +86 10 66930293; E-mail: [email protected] *Corresponding author. Tel: +86 10 61777000; E-mail: [email protected] EMBO J (2019)38:e100376https://doi.org/10.15252/embj.2018100376 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases. Synopsis ABRO1 binds to NLRP3 in an S194 phosphorylation-dependent manner upon priming, subsequently acting as a scaffold to assemble the BRISC. NLRP3 activation signals then trigger the BRISC-dependent removal of K63-linked ubiquitin on NLRP3 LRRs, followed by assembly of the NLRP3 inflammasome. Abro1−/− and Brcc3−/− macrophages show specific defective activation of the NLRP3 inflammasome. ABRO1 binds to NLRP3 upon priming, which dependents on the phosphorylation of NLRP3 at S194 and the activity of JNK kinase. ABRO1 recruits BRISC to NLRP3, and mediates the BRISC-dependent removal of K63-linked ubiquitin on NLRP3 LRRs upon stimulation with activators. Mice lacking ABRO1 or BRCC3 exhibit severely reduced inflammation in response to in vivo models of NLRP3 activation. Introduction The NOD-like receptor family protein 3 (NLRP3) inflammasome is a critical component of innate immunity (Schroder & Tschopp, 2010). It is capable of sensing cellular stress derived from a wide variety of stimuli, including invading pathogens, pore-forming toxins, endogenous danger signals, metabolic dysfunction, and pollutant particles (Abderrazak et al, 2015). Consequently, dysregulated NLRP3 inflammasome activation is associated with both heritable and acquired inflammatory diseases (Menu & Vince, 2011; de Torre-Minguela et al, 2017). Successful NLRP3 inflammasome activation requires two sequential steps: priming and activation (He et al, 2016). The key priming event is the NF-κB-mediated transcription of pro-IL-1β and NLRP3 (Bauernfeind et al, 2009). The following activation step is characterized by the assembly of the NLRP3 inflammasome complex and the subsequent proteolytic processing of pro-IL-1β and pro-IL-18 by autocatalytically activated pro-caspase-1. The regulation of NLRP3 inflammasome activation has been extensively investigated. A number of studies have reported that regulation of NLRP3 inflammasome activity occurs by modification of NLRP3 at the transcription level (Fernandes-Alnemri et al, 2013; Huai et al, 2014). Nonetheless, accumulating evidence also indicates that post-translational modifications (PTMs; Baker et al, 2017), especially ubiquitination and phosphorylation, modulate NLRP3 and ASC (apoptosis-associated speck-like protein containing a CARD) to facilitate the assembly of inflammasome complexes, which are controlled by protein turnover (Yan et al, 2015b), protein distribution and location (Subramanian et al, 2013), and the intricate protein–protein interactions (Stutz et al, 2017). It has been reported that NLRP3 is poly-ubiquitinated in resting macrophages with mixed Lys-48 and Lys-63 ubiquitin chains (Juliana et al, 2012; Py et al, 2013). Lys48-linked polyubiquitin plays a major role in NLRP3 stability. Several E3 ubiquitin ligases, such as MARCH7 (Yan et al, 2015b), TRIM31 (Song et al, 2016), and FBXL2 (Han et al, 2015), have been shown to promote Lys48-linked ubiquitination of NLRP3, leading to its degradation (Baker et al, 2017). In addition, previous studies have shown that an additional decrease in ubiquitinated NLRP3 can be induced by inflammasome activation signals (Juliana et al, 2012). Inhibition of NLRP3 deubiquitination by pharmacologic administration of nonspecific DUB inhibitors almost completely blocks NLRP3 activation in both mouse and human cells (Juliana et al, 2012; Lopez-Castejon et al, 2013), indicating that deubiquitination is required not only for NLRP3 stability but also for NLRP3 inflammasome activation. Subsequently, the deubiquitination enzyme BRCA1/BRCA2-containing complex subunit 3 (BRCC3, also known as BRCC36 in human) is identified as a critical regulator of NLRP3 ubiquitination and inflammasome activity by knockdown experiments in vitro (Py et al, 2013). However, the in vivo role of the BRCC3 in NLRP3 deubiquitination and the underlying molecular mechanisms are still unknown. Moreover, a previous study has reported that mice lacking Abraxas brother 1 (ABRO1, also known as Abraxas 2 and KIAA0157), a scaffold to assemble the BRCC3 complex (also known as BRCC36 isopeptidase complex [BRISC]), exhibit a markedly decreased severity of LPS-induced injury and display significantly reduced mortality, which indicates that BRCC3 complex is an important regulator of cellular responses to LPS (Zheng et al, 2013). LPS-induced inflammatory cytokines including interleukin 1-beta (IL-1β) and IL-18 play a key role in LPS-induced septic shock. Neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality (Vanden Berghe et al, 2014). However, Zheng et al (2013) have shown that ABRO1 knockout results in a decreased induction of Il1b mRNA in blood leukocytes by LPS treatment, and LPS plus ATP-induced caspase-1 activation is not impaired in Abro1−/− macrophages. As ABRO1 is an important subunit of the BRCC3 complex, and removal of ABRO1 leads to the loss of BRCC3 deubiquitinase (DUB) activity (Feng et al, 2010), these results imply that the BRCC3 complex probably does not contribute to NLRP3 inflammasome activation upon LPS plus ATP challenge (Zheng et al, 2013). Thus, the role of the BRCC3 complex in the NLRP3 inflammasome is controversial, and further studies are required to clarify the contribution of BRCC3 complex to NLRP3 inflammasome activation. BRCC3 is a JAB1/MPN/Mov34 (JAMM) domain-containing Zn2+ metalloprotease DUB (Cooper et al, 2009). In the cytoplasm, BRCC3 forms a multiprotein complex (BRISC) with ABRO1, NBA1, and BRE that specifically cleaves “Lys(K)-63”-linked ubiquitin (Feng et al, 2010). ABRO1 is a paralog of a BRCA1-interacting protein, Abraxas, and they share 39% sequence homology within their amino-terminal regions (Wang et al, 2007). ABRO1 contains a structural domain that interacts with subunits NBA1, BRE, and BRCC36 (Hu et al, 2011) but lacks the PSXXF domain that serves as a specific recognition motif for the BRCT domain of BRCA1 (Wang et al, 2007). Therefore, ABRO1 does not interact with BRCA1 but serves as a scaffold protein and recruits the rest of the components, including NBA1, BRE, and BRCC3, to form the BRISC (Feng et al, 2010). So far, only a few substrates of BRISC have been identified, including the type 1 interferon (IFN) receptor chain 1 (IFNAR1) (Zheng et al, 2013), the essential spindle assembly factor nuclear mitotic apparatus (NuMA) (Yan et al, 2015a), the poly (ADP-ribose) polymerase tankyrase 1 (Tripathi & Smith, 2017), and the HIV-1 Tat protein (Xu et al, 2018). Here, we describe a specific function of ABRO1 as a regulator of the NLRP3 inflammasome complex in macrophages. Our results generate a model involving that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, leading to ABRO1-dependent recruitment of BRISC which mediates deubiquitination of NLRP3 upon inflammasome activation. Importantly, we provide clear genetic evidence from knockout mice that BRISC plays a key role in the regulation of NLRP3 inflammasome. Results Abro1−/− macrophages show specific defective activation of the NLRP3 inflammasome To further investigate the potential functions of ABRO1, we generated Abro1-deficient (Abro1−/−) mice by the partial deletion of exon 1 of the murine Abro1 locus (Fig EV1A and B). In line with a previous report, Abro1−/− mice appeared normal and showed no anatomical abnormalities (Zheng et al, 2013). Based on the observation that Abro1−/− mice and cells display abnormalities in response to LPS stimulation (Zheng et al, 2013), we investigated the role of ABRO1 in macrophages. The development and proliferation abilities were comparable between Abro1+/+ and Abro1−/− bone marrow-derived macrophages (BMDMs; Fig EV1C and D). LPS-induced production of IL-6 and TNF-α in condition medium was comparable between Abro1+/+ and Abro1−/− BMDMs (Fig EV1E). However, Abro1−/− BMDMs generated significantly less IL-1β and IL-18 compared with Abro1+/+ BMDMs in response to LPS priming and ATP stimulation (Fig 1A and B). ABRO1 overexpression greatly enhanced IL-1β secretion in Abro1+/+ BMDMs, and re-expression of exogenous ABRO1 protein in Abro1−/− macrophages restored IL-1β secretion to a similar level observed in Abro1+/+ cells in response to inflammasome activation (Fig 1C). Moreover, knockdown of ABRO1 significantly reduced IL-1β release in human monocyte-derived macrophages (HMDMs) treated with LPS plus ATP (Fig 1D). Similarly, significant decreases of IL-1β production were also observed in response to other NLRP3 inflammasome stimuli, including nigericin, silica, monosodium urate crystals (MSU), muramyl dipeptide (MDP), and aluminum (Alum) in Abro1−/− macrophages (Fig 1E). These results indicate that activation of the NLRP3 inflammasome is defective in Abro1−/− macrophages. Importantly, IL-1β defects in Abro1−/− BMDMs were not due to altered inflammasome-related core protein levels (Fig EV1F), LPS unresponsiveness (depicted by LPS/TLR4 signaling pathway activation, Il1b, Tnfa, and Il6 transcript expression levels, TNF-α, and IL-6 release; Fig EV1G, H, and E), nor reduced macrophage viability (Fig EV1I). Of note, Nlrp3−/− BMDMs showed almost a complete loss of IL-1β release, while the IL-1β release is generally only reduced by about 70% in Abro1−/− BMDMs relative to wild-type cells after LPS plus ATP or LPS plus nigericin treatment (Fig EV2A). Therefore, our results suggest that ABRO1 is required for effective activation of the NLRP3 inflammasome and IL-1β secretion upon stimulation with the NLRP3 ligands. Click here to expand this figure. Figure EV1. Abro1−/− macrophages show normal proliferation, differentiation and LPS responses A, B. Generation of Abro1−/− mice. Targeted disruption of the Abro1 locus by replacing partial exons 1 with neo cassette (A). PCR analysis of tail biopsy genomic DNA and immunoblot analysis of ABRO1 expression (B). C, D. Abro1+/+ and Abro1−/− BMDMs were isolated as described. The total cell number (C) and percentage of CD11b+F4/80+ macrophages (D) were analyzed at each time point as indicated. E–I. Abro1+/+ and Abro1−/− BMDMs were stimulated with LPS as indicated. CBA analysis of TNF-α and IL-6 in supernatants (E). Immunoblot analysis of indicated proteins in cell lysates with specific primary antibodies (F, G). Real-time PCR analysis of Il1b, Tnfa, and Il6 mRNA levels (normalized to Gapdh) (H). The cellular ATP level was measured by CellTiter-Glo Luminescent Cell Viability Assay (The luminescence values of Abro1+/+ BMDMs at 0 h were set to 100%) (I). Data information: Data are presented as mean ± SEM from three independent experiments performed in triplicate wells (C–E, H, I) or are representative of three independent experiments (F, G). Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Abro1−/− macrophages show defective activation of the NLRP3 inflammasome A, B. LPS-primed Abro1+/+ and Abro1−/− BMDMs were treated with various doses of ATP. CBA analysis of IL-1β (A) and ELISA of IL-18 (B) in the culture supernatants. C. CBA analysis of IL-1β secretion from Abro1+/+ and Abro1−/− BMDMs transduced with GFP- or ABRO1-GFP-expressing lentiviruses prior to stimulation with LPS and ATP. Cell lysates were immunoblotted with anti-ABRO1 antibody. D. ELISA analysis of IL-1β secretion from HMDMs transduced with lentiviruses expressing shCon or shABRO1 prior to stimulation with control medium (Con), LPS or LPS plus ATP. Cell lysates were immunoblotted with anti-ABRO1 and anti-GAPDH antibodies. E. Abro1+/+ and Abro1−/− BMDMs were left untreated (Con), treated with LPS alone (LPS) or pretreated with LPS, and then stimulated with nigericin, silica, MSU, MDP, Alum, poly(dA:dT), flagellin, or LTx (Anthrax Lethal Factor). CBA analysis of IL-1β secretion in the culture supernatants. F. Representative immunoblot analysis of cleaved caspase-1 and IL-1β in culture supernatants (SN) of LPS-primed Abro1+/+ and Abro1−/− BMDMs treated with ATP, nigericin, MSU, flagellin, or poly(dA:dT). G. Flow cytometric analysis of caspase-1 activity in LPS-primed Abro1+/+ and Abro1−/− BMDMs stimulated with nigericin for indicated times. H. Immunostaining of endogenous ASC specks in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with nigericin or poly(dA:dT). Scale bars, 10 μm. I. Quantification of ASC specks from (H) was performed by counting cells in five random areas of each image in triplicate experiments and described as a percentage of ASC specks for total cell nuclei. At least 100 cells from each treatment condition were quantified. UD, undetectable. J. LPS-primed Abro1+/+ and Abro1−/− BMDMs were left untreated or treated with ATP. Immunoblot analysis of NLRP3 and ASC protein in cell lysates immunoprecipitated with anti-ASC antibody. Data information: Data are presented as mean ± SEM from three independent experiments performed in triplicate wells (A–E, G, I) or are representative of three independent experiments (F, H, J). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant; two-tailed unpaired t-test (C–E, I) or two-way ANOVA with Bonferroni post-test (A, B, G). Source data are available online for this figure. Source Data for Figure 1 [embj2018100376-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Abro1−/− macrophages show defective activation of the NLRP3 inflammasome CBA analysis of IL-1β secretion from LPS-primed WT, Abro1−/−, and Nlrp3−/− BMDMs treated with ATP or nigericin. Immunoblot analysis of the ASC oligomerization in uncross- or cross-linked pellets from Abro1+/+ and Abro1−/− BMDMs stimulated with LPS and nigericin. Data information: Data are presented as mean ± SEM from three independent experiments performed in triplicate wells (A) or are representative of three independent experiments (B). **P < 0.01, ***P < 0.001; two-tailed unpaired t-test (A). Source data are available online for this figure. Download figure Download PowerPoint We next investigated whether ABRO1 was important for the activation of other inflammasomes, namely the NLRP1 (activated with anthrax lethal toxin), NLRC4 (activated with Salmonella flagellin), and AIM2 (activated with poly(dA:dT)) inflammasomes (Baroja-Mazo et al, 2014). As shown in Fig 1E, Abro1−/− and Abro1+/+ BMDMs generated comparable levels of IL-1β in response to anthrax lethal toxin (LTx), flagellin and poly(dA:dT), indicating that ABRO1 is specific for NLRP3 inflammasome activation. The maturation and secretion of pro-caspase-1 and pro-IL-1β are critical steps for inflammasome activation (Franchi et al, 2009). We therefore measured the cleavage of pro-IL-1β and pro-caspase-1. As expected, pro-caspase-1 and pro-IL-1β were processed to their respective mature forms in LPS-primed Abro1+/+ BMDMs stimulated with ATP, nigericin, and MSU. However, these effects were significantly inhibited in Abro1−/− BMDMs (Fig 1F). In line with these results, significantly reduced active caspase-1 levels were consistently observed in Abro1−/− BMDMs compared with Abro1+/+ BMDMs in response to LPS priming followed by ATP stimulation (Fig 1G). However, flagellin and poly(dA:dT) treatment promoted normal IL-1β and caspase-1 maturation in Abro1−/− BMDMs (Fig 1F), which reconfirmed that ABRO1 was not required for NLRC4 and AIM2 inflammasome activation. The formation of the adaptor protein ASC speck is another hallmark of inflammasome activation. Once the inflammasome is active, ASC will oligomerize as part of the inflammasome complex to form a single large (up to 2 μm) perinuclear focus per cell (Fernandes-Alnemri et al, 2007). Therefore, we investigated whether ABRO1 deficiency affected ASC speck formation. Compared with Abro1+/+ BMDMs, the amounts of ASC specks induced by LPS plus nigericin were significantly reduced in Abro1−/− BMDMs, whereas ASC speck formation after poly(dA:dT) transfection was unaffected (Fig 1H and I). We also examined the impact of ABRO1 deficiency on ASC oligomerization. The results showed that ASC oligomerization was significantly reduced in Abro1−/− BMDMs (Fig EV2B). Moreover, ABRO1 deficiency weakened the interaction between NLRP3 and ASC (Fig 1J). These results provided robust evidence that ABRO1 was necessary for optimal formation of the NLRP3-ASC complex during NLRP3 inflammasome activation. We also examined whether ABRO1 affected the upstream events of NLRP3 inflammasome activation, including K+ efflux, Ca2+ mobilization, mitochondrial dysfunction, and ROS generation (He et al, 2016). Our results showed that these upstream pathways for NLRP3 activation were unaffected in Abro1−/− BMDMs after the respective activator treatment (Fig EV3A–D). It is postulated that ABRO1 acts downstream of K+ efflux, Ca2+ flux, and mitochondrial damage to control NLRP3 activation. Click here to expand this figure. Figure EV3. Normal K+ efflux, Ca2+ flux, mitochondrial ROS, and membrane potential in Abro1−/− BMDMs Flow cytometric analysis of intracellular K+ level detected by K+-sensitive fluorescence indicator Asante Potassium Green-2 AM (APG-2) in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with ATP for 15 min. Flow cytometric analysis of intracellular Ca2+ level detected by Ca2+-sensitive fluorescence indicator Fluo-4 AM in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with ATP for 15 min. Flow cytometric analysis of mitochondrial ROS (MitoSOX Red) in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with nigericin for 15 min. Flow cytometric analysis of membrane potential (MitoTracker DeepRed) in LPS-primed Abro1+/+ and Abro1−/− BMDMs left untreated or treated with ATP for 15 min. Data information: Data are presented as mean ± SEM from three independent experiments performed in triplicate wells (A–D). Download figure Download PowerPoint ABRO1 deficiency attenuated NLRP3-dependent responses in vivo To assess the role of ABRO1 in the regulation of the NLRP3 inflammasome in vivo, different NLRP3-dependent inflammatory models were employed. Injection MSU (i.p) into mice can induce NLRP3-dependent IL-1β production and recruitment of inflammatory cells in the peritoneal cavity (Shenoy et al, 2012). As Fig 2A shows, IL-1β secretion detected in the lavage fluid was significantly decreased in Abro1−/− mice (about 60%) and Nlrp3−/− mice (about 80%) relative to wild-type mice after MSU injection, but inflammasome-independent TNF-α secretion was unaffected in both knockout mice. Both Abro1−/− and Nlrp3−/− mice also exhibited impaired recruitment of neutrophils and inflammatory monocytes to the peritoneal cavity compared with wild-type mice (Fig 2B). In addition, caspase-1 activity in recruited Abro1−/− immune cells was markedly suppressed (Fig 2C). We also detected significantly lower production of IL-1β and recruitment of inflammatory cells in the lavage fluid of Abro1−/− mice in another NLRP3-dependent peritonitis mouse model induced by Alum (Jin et al, 2013; Fig 2D and E). Finally, ABRO1 deficiency markedly increased mice survival in an L