Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression

Abstract

The IL-1 family cytokines are regulated on transcriptional and posttranscriptional levels. Pattern recognition and cytokine receptors control pro-IL-1beta transcription whereas inflammasomes regulate the proteolytic processing of pro-IL-1beta. The NLRP3 inflammasome, however, assembles in response to extracellular ATP, pore-forming toxins, or crystals only in the presence of proinflammatory stimuli. How the activation of gene transcription by signaling receptors enables NLRP3 activation remains elusive and controversial. In this study, we show that cell priming through multiple signaling receptors induces NLRP3 expression, which we identified to be a critical checkpoint for NLRP3 activation. Signals provided by NF-kappaB activators are necessary but not sufficient for NLRP3 activation, and a second stimulus such as ATP or crystal-induced damage is required for NLRP3 activation.

Figure 1. Caspase-1 activation and the formation of ASC pyroptosomes require priming

(A) C57BL/6 expressing ASC-YFP left untreated or stimulated as indicated and counterstained for membranes (choleratoxin subunit B (red)) and nuclei (Hoechst dye (blue)) were analyzed by confocal microscopy. (B) ASC-CFP pyroptosome formation after LPS priming and subsequent ATP stimulation was analyzed by epifluorescence microscopy. (C) Immunoblot for cleaved caspase-1 in supernatants from cells stimulated as in (B). Data are representative of three independent experiments (error bars, s.d. in B).

Figure 2. PRR activation is required for NLRP3 inflammasome activation

(A) Immunoblot of caspase-1 in wild type or knock-out macrophages primed for 4h with LPS (200 ng/ml), Pam2CysK4 (50 ng/ml), TLR4 activating (UT18), or TLR4 blocking (MTS510) antibodies and subsequently stimulated with ATP (1h). Cells from wild-type or knock-out macrophages were left untreated or stimulated for 4h with (B) poly(I:C) (1 µg/ml), Pam2CysK4 (50 ng/ml), or R848 (0.5 µg/ml) or with (C) Pam2CysK4 (50 ng/ml), MDP (10 µg/ml), LPS (200 ng/ml), or transfected with poly(dA-dT). ATP was added for 1h as indicated. (D) Wild type or TNFR1/2 dKO macrophages were stimulated with TNFα as indicated or with LPS as in (A) and analyzed for caspase-1 activation. Immunoblot analyses of cleaved caspase-1 from supernatants are show. Data are from one representative experiment out of three (A, B) or two (C–D).

Figure 3. NLRP3 induction involves NF-κB activity

Caspase-1 immunoblot from supernatants of wild type macrophages pretreated with (A) cycloheximide or (B) Bay11-7082 as indicated for 1h followed by LPS (200 ng/ml, 4h) and stimulated with ATP (1h). (C) Messenger RNA expression of ASC or NLRP3 in LPS primed or untreated macrophages. Cells were pretreated with Bay11-7082 for 1h where indicated. (D) HEK293T were transfected with pcDNA3-MyD88 or control (pcDNA3) together with a NLRP3 promoter reporter and assessed for luciferase activity after 20h. (E) Immunoblots for NLRP3, pro-IL-1β and β-actin in lysates from C57BL/6 macrophages treated with LPS for 6h as indicated, or, (F) treated with LPS (200 ng/ml) for the indicated periods of time. Controls are lysates from NLRP3-KO macrophages with and without heterologous NLRP3 expression. Data are from one representative experiment of three (A-D) or of two (E, F) experiments (error bars, s.d.).

Figure 4. Stable NLRP3 expression is sufficient for priming-independent NLRP3 activation

Macrophages were stimulated for 4h and ATP or nigericin were added for an additional 1h as indicated. (A) Confocal microscopy of caspase-1-KO macrophages expressing ASC-CFP and NLRP3-YFP. (B) Expression of ASC-CFP and NLRP3-YFP assessed by GFP immunoblot after GFP immunoprecipitation. (C) ASC pyroptosome quantification in caspase-1-KO macrophages expressing ASC-CFP and NLRP3-YFP or ASC-CFP and ASC-YFP (D) Immortalized C57BL/6 (left) or NLRP3-KO (right) macrophages with or without stable expression of NLRP3-mCitrine were left untreated or primed with LPS (200 ng/ml) for (4h), and stimulated with nigericin for 1h as indicated. Caspase-1 immunoblots of combined supernatant and cell lysates are shown. NLRP3 expression was verified by immunoblotting for GFP and NLRP3. One representative experiment out of three (A, C-D) or two (B) is shown.