Volume 68, Issue 1, Pages 185-197.e6 (October 2017) NLRP3 Phosphorylation Is an Essential Priming Event for Inflammasome Activation  Nan Song, Zhao-Shan Liu, Wen Xue, Zhao-Fang Bai, Qian-Yi Wang, Jiang Dai, Xin Liu, Yi-Jiao Huang, Hong Cai, Xiao-Yan Zhan, Qiu- Ying Han, Hongxia Wang, Yuan Chen, Hui-Yan Li, Ai-Ling Li, Xue-Min Zhang, Tao Zhou, Tao Li  Molecular Cell  Volume 68, Issue 1, Pages 185-197.e6 (October 2017) DOI: 10.1016/j.molcel.2017.08.017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 185-197.e6DOI: (10.1016/j.molcel.2017.08.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 NLRP3 Phosphorylation at S194 Is Required for Inflammasome Activation (A) NLRP3 inflammasome was reconstituted in HEK293T cells by co-expressing of human wild-type (WT) or NLRP3-S198A (left) or corresponding mouse NLRP3 proteins (right) plus Flag-tagged pro-IL-1β, ASC and pro-caspase-1. The activity of NLRP3 was indicated by cleaved IL-1β in whole-cell lysates (WCL). (B) Scheme of NLRP3 domain structure and the alignment of NLRP3 orthologs. The phosphorylated serine residue is underlined in red. (C–E) NLRP3 knockdown iBMDMs were transduced with lentivirus expressing mouse WT or NLRP3-S194A, respectively. These iBMDMs were treated with LPS (200 ng/mL, 4 hr), nigericin (7.5 μM, 1 hr), or LPS plus nigericin, respectively. The supernatants and WCL were immunoblotted (C). Coomassie blue staining was provided as the loading control for the supernatants. IL-1β secretion (D) and LDH release (E) were measured in the supernatant of iBMDMs in (C) as indicated. (F–J) iBMDMs expressing mouse WT or NLRP3-S194A were primed and treated with indicated stimuli. The supernatants and WCL were immunoblotted (F and G). The release of LDH (H) and the secretion of IL-1β (I), TNF-α (J) were measured in the supernatant of indicated iBMDMs in (F and G). Experiments were repeated at least three times, and representative data are shown. Values, mean ± SD. ∗∗∗p < 0.005 (two tailed t test). See also Figure S1. Molecular Cell 2017 68, 185-197.e6DOI: (10.1016/j.molcel.2017.08.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 NLRP3 Inflammasome Activation Is Disrupted in Nlrp3S194A/S194A Mice (A–D) LPS-primed Nlrp3+/+ (WT) and Nlrp3S194A/S194A (KI) BMDMs were left untreated or treated with indicated stimuli for NLRP3 (A), AIM2, pyrin, and NLRC4 (B). The supernatants and WCL were immunoblotted. The IL-1β secretion (C) and LDH release (D) were measured. For each experimental group, three supernatant samples were analyzed. (E and F) ELISA of IL-1β (E) and quantification of peritoneal exudate cells (F) in the peritoneal cavity fluid of Nlrp3+/+ (+/+), Nlrp3S194A/S194A (S194A/S194A), and Nlrp3−/− (−/−) mice that were intraperitoneally (i.p.) injected with 1 mg monosodium urate for 6 hr. (G–I) ELISA of serum IL-1β (G), TNF-α (H), and IL-1α (I) in indicated mice after intraperitoneal injection of 20 mg/kg LPS for 3 hr. (J) Survival of Nlrp3+/+, Nlrp3S194A/S194A, and Nlrp3−/− mice subjected to 10 mg/kg LPS was monitored. Experiments were repeated at least three times, and representative data are shown. Values, mean ± SD (C and D). Horizontal bar indicates mean ± SD, n = 8 (E–I). ∗∗p < 0.01; ∗∗∗p < 0.005 (two tailed t test [C–I], or log-rank test [J]). See also Figure S2. Molecular Cell 2017 68, 185-197.e6DOI: (10.1016/j.molcel.2017.08.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 NLRP3 Is Phosphorylated during Priming (A) Unprimed or LPS-primed iBMDMs were treated with 7.5 μM nigericin for 15 min or 200 μg/mL monosodium urate for 2 hr. NLRP3 was immunoprecipitated. Immunoblot analysis of S194 phosphorylated NLRP3 (pNLRP3S194) and total NLRP3 was performed. (B–D) iBMDMs were treated with indicated TLR agonists for 3 hr or left untreated (B). iBMDMs were treated with 100 ng/mL LPS, 500 ng/mL Lfn-FliC, or transfected with 2 μg/mL poly(dA:dT), respectively (C). iBMDMs were treated with 100 ng/mL LPS for indicated time courses (D). Immunoprecipitation (IP) and immunoblot analysis were performed as in (A). (E) Nlrp3−/− BMDMs were electroporated with indicated plasmids, followed by treatment with 100 ng/mL LPS for 8 hr. Caspase-1 cleavage was analyzed by immunoblot analysis. (F) The secretion of IL-1β was measured in the supernatant of indicated BMDMs in (E). For each experimental group, three supernatant samples were analyzed. Experiments were repeated at least three times, and representative data are shown. Values, mean ± SD. ∗∗∗p < 0.005 (two tailed t test). See also Figures S3 and S4. Molecular Cell 2017 68, 185-197.e6DOI: (10.1016/j.molcel.2017.08.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 NLRP3 Is Directly Phosphorylated by JNK1 during Priming (A) In vitro kinase assays using GST-mouse NLRP31–532 and recombinant JNK1 or JNK2. Samples were analyzed by immunoblotting and Coomassie blue staining. Mut, S194A. KD, kinase dead. (B) Jnk1+/+ or Jnk1−/− BMDMs were primed with 200 ng/mL LPS for 4 hr or left untreated. NLRP3 was immunoprecipitated. The IP samples and input (5%) were immunoblotted. (C–F) LPS-primed Jnk1+/+ (WT) or Jnk1−/− (KO) BMDMs were left untreated or treated with indicated stimuli for NLRP3 (C), AIM2, pyrin, and NLRC4 (D). The supernatants and WCL were immunoblotted. The IL-1β secretion (E) and LDH release (F) were measured in the supernatants of indicated BMDMs in (C and D). For each experimental group, three supernatant samples were analyzed. (G) Nlrp3−/− BMDMs electroporated with indicated plasmids were pretreated with or without SP600125 (40 nM) for 1 hr, followed by 100 ng/mL LPS stimulation for 8 hr. Caspase-1 cleavage was analyzed by immunoblotting. Experiments were repeated at least three times, and representative data are shown. Values, mean ± SD. ∗∗∗p < 0.005 (two tailed t test). See also Figures S5 and S6. Molecular Cell 2017 68, 185-197.e6DOI: (10.1016/j.molcel.2017.08.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 NLRP3 S194 Phosphorylation Is Required for Inflammasome Assembly (A) Representative images of ASC specks in LPS-primed Nlrp3+/+ and Nlrp3S194A/S194A BMDMs treated with indicated stimuli. ASC, green; nuclei, blue. White arrows indicate ASC specks. Bars, 10 μm (left). The percentage of cells containing an ASC speck was quantified (right). At least 100 BMDMs from each genotype were analyzed. (B) LPS-primed BMDMs were treated with nigericin. Triton X-insoluble extracts were cross-linked and immunoblotted for the detection of ASC oligomerization. Both Triton X-soluble and -insoluble fractions were immunoblotted. (C) LPS-primed Nlrp3+/+ and Nlrp3S194A/S194A BMDMs were pretreated with Z-YVAD-FMK and then stimulated with nigericin. Cell lysates were fractionated by gel filtration chromatography and immunoblotted. (D) Representative images of ASC specks in LPS-primed Jnk1+/+ or Jnk1−/− BMDMs treated with indicated stimuli. Bars, 10 μm (left). The percentage of cells containing an ASC speck was quantified (right). At least 100 BMDMs from each genotype were analyzed. (E) LPS-primed BMDMs were pretreated with Z-YVAD-FMK and then stimulated with nigericin. Cell lysates were fractionated and immunoblotted. (F) LPS-primed Nlrp3+/+ (WT) and Nlrp3S194A/S194A (KI) BMDMs were stimulated as indicated. ASC was immunoprecipitated. The IP samples and input (5%) were immunoblotted. Experiments were repeated at least three times, and representative data are shown. Values, mean ± SD. ∗∗∗p < 0.005 (two tailed t test). See also Figure S7. Molecular Cell 2017 68, 185-197.e6DOI: (10.1016/j.molcel.2017.08.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Phosphorylation Facilitates the Self-Association of NLRP3 (A) Representative images of HEK293T cells expressing Flag-GFP-tagged human WT or S198A NLRP3 treated with or without nigericin. White arrows indicate oligomerized NLRP3. Bars, 20 μm. (B) HEK293T cells expressing Flag-GFP-tagged human WT or S198A NLRP3 were treated as indicated. Cell lysates were fractionated by gel filtration chromatography and immunoblotted. ∗Non-specific bands. (C) Schematic drawing of the GST pull-down assay for the detection of NLRP3 homotypic interaction. (D) HEK293T cells were transfected with WT or S194A NLRP3-Flag. Pre-cleared cell lysates were incubated with purified GST-NLRP3ΔLRR for 2 hr. NLRP3-Flag bound with GST-NLRP3ΔLRR were pulled down by glutathione beads and subjected to immunoblot analysis. (E) Nlrp3+/+, Nlrp3S194A/S194A, and Nlrp3−/− BMDMs were left untreated or treated with 100 ng/mL LPS for 1 hr. GST pull-down assay was performed as in (D). Experiments were repeated at least three times, and representative data are shown. See also Figure S7. Molecular Cell 2017 68, 185-197.e6DOI: (10.1016/j.molcel.2017.08.017) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 S194 Phosphorylation Promotes the Deubiquitination of NLRP3 (A) Immunoblot analysis of Flag-IP samples from HEK293T cells transfected with ubiquitin (Ub) and indicated vectors. (B) HEK293T cells were transfected with indicated vectors and treated with 2 μM Anisomycin or 40 nM SP600125 for 4 hr after 16 hr transfection. NLRP3 ubiquitination was analyzed. (C) HEK293T cells were transfected with indicated vectors and treated with or without 2 μM Anisomycin for 4 hr after 16 hr transfection. NLRP3 ubiquitination was analyzed. (D) iBMDMs expressing Flag-GFP-tagged mouse WT or NLRP3 S194A were treated as indicated. NLRP3 ubiquitination was analyzed. Ratio quantification was carried out by dividing the smear intensity of ubiquitination blots by that of corresponding NLRP3 bands. The ratio values were relative to the non-treated condition of each group. (E) WT, Nlrp3S194A/S194A, and Jnk1−/− BMDMs were primed with 200 ng/mL LPS for 4 hr. NLRP3 immunoprecipitates were analyzed. (F) Both WT and Nlrp3S194A/S194A BMDMs were pretreated with 2 μM Anisomycin or 40 nM SP600125 for 0.5 hr and then treated with 200 ng/mL LPS for 4 hr. NLRP3 ubiquitination and ratio quantification were performed as described in (E) and (D), respectively. Experiments were repeated at least three times, and representative data are shown. See also Figure S7. Molecular Cell 2017 68, 185-197.e6DOI: (10.1016/j.molcel.2017.08.017) Copyright © 2017 Elsevier Inc. Terms and Conditions