1. Volume 153, Issue 2, Pages 348-361 (April 2013)
The Adaptor MAVS Promotes NLRP3 Mitochondrial Localization and Inflammasome Activation 
Naeha Subramanian, Kannan Natarajan, Menna R. Clatworthy, Ze Wang, Ronald N. Germain 
Cell 
Volume 153, Issue 2, Pages (April 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
NLRP3 Is Cytosolic in the Resting State and Localizes to Mitochondria upon Activation
(A and B) Representative confocal immunofluorescence images (A) and quantification (B) of NLRP3 colocalized with Mitotracker in HEK293T cells stably expressing NLRP3. Expression of NLRP3 was induced with doxycycline (DOX; 1 μg/ml), and cells were either untreated or treated with 15 μM nigericin for 45 min prior to imaging.
(C and D) Representative immunofluorescence images (C) and quantification (D) of NLR colocalized with Mitotracker in HEK293T cells expressing NLRP3 or NLRP4 under transient low-expression conditions. Cells were transfected with 100 ng plasmid DNA for 8 hr, and were either untreated or treated with 15 μM nigericin for 45 min prior to imaging.
(E) Subcellular fractionation of WT and ASC KO BMDMs. BMDMs were either untreated (Mock) or primed with LPS (1 μg/ml for 4 hr) prior to stimulation with 7.5 μM nigericin for 30 min (Nig). Mitochondrial (M) and cytosolic (C) fractions were fractionated and analyzed for expression of NLRP3 by immunoblot. Purity of the fractions was assessed by blotting for Complex 1 (mitochondrial protein) and ERK-2 (cytosolic protein).
Imaging data are representative of several images from three independent experiments. Data points on graphs represent independent fields (with three to five cells per field). Error bars are mean ± SEM, and p values from an unpaired t test (two-tailed) are shown. Mito, Mitotracker; coloc, colocalization channel. See also Figure S1 and Table S1.
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4. Figure 2
NLRP3 Possesses an N-Terminal Sequence that Controls Mitochondrial Association and ASC Speckle Formation
(A) Panel of N-terminal deletions of NLRP3. “----” indicates the deleted sequence. Structured region of the NLRP3 PYD encompasses residues 6–90.
(B) Representative immunofluorescence images of HEK293-ASC-YFP cells expressing WT and N-terminal deletions of NLRP3 under transient low-expression conditions (100 ng plasmid DNA for 8 hr), and activated with 15 μM nigericin for 45 min prior to imaging. Representative images of unstimulated cells are shown in Figure S2.
Imaging data are representative of several images from four independent experiments. Mito, Mitotracker. See also Figure S2.
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5. Figure 3
Effect of N-Terminal Deletions of NLRP3 on Mitochondrial Recruitment, ASC Speckle Formation, and IL-1β Secretion
(A and B) Quantification of ASC speckle formation in HEK293-ASC-YFP cells transiently transfected with low levels of WT or N-terminal deletions of NLRP3 (as in Figures S2 and 2B), and either untreated (A) or treated with 15 μM nigericin (B) for 45 min prior to imaging.
(C) Quantification of colocalization of WT and N-terminal deletion mutants of NLRP3 with Mitotracker in HEK293-ASC-YFP cells transfected as above, and either untreated or treated with 15 μM nigericin for 45 min prior to imaging.
(D) Minimum N-terminal sequence (2–7) necessary for NLRP3 mitochondrial association and ASC aggregation is indicated in red.
(E) ELISA for IL-1β in supernatants of PMA-differentiated (non-LPS-primed) THP-1 cells stably expressing NLRP3 WT or NLRP3 Δ 2-21, and stimulated with the indicated NLRP3 activators. Expression of NLRP3 was induced for 12 hr with DOX (1 μg/ml), prior to differentiation with 100 nM PMA for 3 hr.
(F) Subcellular fractionation of HEK293T cells stably expressing either NLRP3 WT or NLRP3 Δ NLRP3 expression was induced with 1 μg/ml DOX for 6 hr prior to stimulation with nigericin for 45 min.
Data points in (A) and (B) represent the percentage of all transfected cells (pooled from multiple fields) containing a fully developed, incompletely developed, or no ASC speckle. At least 20 cells were evaluated for each construct. Data points in (C) represent independent fields (three to five cells per field). Data are representative of three independent experiments. Error bars are mean ± SEM, and p values from an unpaired t test (two-tailed) are shown. ND; not detectable. See also Figure S3.
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6. Figure 4
The Mitochondrial Adaptor MAVS Mediates NLRP3 Mitochondrial Localization
(A and C) Representative confocal immunofluorescence images (A) and quantification (C) of NLRP3 colocalized with Mitotracker in HEK293T cells treated with control siRNA or MAVS siRNA prior to overexpression of NLRP3. siRNA transfected into cells appears as punctate spots shown in blue.
(B and D) Representative immunofluorescence images (B) and quantification (D, left) of HEK293T cells treated with control siRNA or MAVS siRNA prior to transient low expression of NLRP3 and stimulation with 15 μM nigericin for 45 min. Immunoblot and corresponding densitometric quantification for efficiency of MAVS knockdown are shown (D, right).
(E) Immunoblot showing NLRP3 association with MAVS in BMDMs upon treatment with activators of NLRP3, but not activators of NLRC4 or NOD2/NLRP1. In some experiments, MAVS expression in lysates decreases upon stimulation with ATP for unknown reasons.
(F) Immunoblot showing progressive loss of MAVS association by N-terminal deletion mutants of NLRP3 in HEK293T cells. “|” indicates removal of an irrelevant lane(s) here and throughout. Cells were transiently transfected with low levels of FLAG-tagged WT or N-terminal deletions of NLRP3, and treated with 15 μM nigericin for 45 min prior to lysate preparation.
(G) Immunoblot showing MAVS association of NLRP4 with the added N terminus of NLRP3 (NLRP4 + 2–11) in HEK293T cells. Cells were transiently transfected with the indicated FLAG-tagged constructs (1 μg DNA/106 cells for 20 hr) prior to lysate preparation. Error bars are mean ± SEM.
(H) Immunoblot showing association of the indicated in vitro translated FLAG-tagged NLR proteins with purified MAVS. Immunoblots are representative of two independent experiments. Nig, nigericin; flg, flagellin; MDP, muramyldipeptide. Imaging data are representative of several images from two independent experiments. Data points on graphs in (C) and (D) represent individual fields with three to five cells per field (C) or 30–40 cells per field (D). Error bars are mean ± SEM, and p values from an unpaired t test (two-tailed) are shown. See also Figure S4 and Movies S1 and S2.
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7. Figure 5
Silencing MAVS in BMDMs Reduces IL-1β Secretion in Response to NLRP3 Activators
(A–E) ELISA for IL-1β in supernatants of control or MAVS-silenced, LPS-primed BMDMs treated with the indicated concentrations of ATP and nigericin for 20 min (A and B), transfected poly I:C and poly dA:dT for 6 hr (C, E), or transfected flagellin for 2 hr (D).
(F) Immunoblot for efficiency of MAVS knockdown in BMDMs. MAVS-2 siRNA specifically silenced MAVS and was used for assessing IL-1β secretion shown in (A)–(E).
(G) Quantitative RT-PCR for IFN-β (G, left) and pro-IL-1β (G, right) expression in human monocytes treated with NLRP3 activator LPS-ATP or RIG-I activator influenza A strain HKX31 (multiplicity of infection = 1). mRNA expression was normalized to the housekeeping gene GAPDH. ELISA and RT-PCR data are representative of two independent experiments. Error bars are mean ± SEM, and p values from an unpaired t test (two-tailed) are shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant; ND, not detectable.
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8. Figure 6
MAVS Is Required for Optimal Inflammasome Activation by Noncrystalline NLRP3 Activators In Vitro
(A–F) ELISA showing IL-1β secretion from WT and MAVS −/− macrophages. LPS-primed BMDMs were treated with the indicated concentrations of ATP (A) or nigericin (B) for 20 min, alum crystals for 4 hr (C), transfected poly I:C for 6 hr (D), flagellin for 2 hr (E), or poly dA:dT for 6 hr (F). Error bars are mean ± SEM.
(G) Immunoblots showing processing of IL-1β and caspase-1 and ASC oligomerization in WT and MAVS −/− macrophages. LPS-primed BMDMs were treated with transfected poly I:C (2 μg/106 cells) for 6 hr, ATP (2.5 mM), and nigericin (7.5 μM) for 20 min, alum crystals (200 μg/ml) for 6 hr or transfected flagellin (1 μg/106 cells) for 2 hr. Cell culture supernatants (Sup), cell lysates, and crosslinked pellets from whole-cell lysates were analyzed by immunoblotting as indicated. Different doses of each NLRP3 activator were examined and representative blots at subsaturating doses are shown. “∗” indicates nonspecific bands observed at higher exposures.
(H) Immunoblot for NLRP3 following stimulation with LPS (1 μg/ml for 4 h). “∗” indicates a nonspecific band. Data are representative of two independent experiments. Error bars are mean ± SEM, and p values from an unpaired t test (two-tailed) are shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant; ND, not detectable. Casp-1, caspase-1. See also Figures S5 and S7.
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9. Figure 7
MAVS Is Essential for NLRP3 Inflammasome Function In Vivo During Acute Tubular Necrosis
(A) Percentage weight loss of WT, MAVS −/−, and ASC −/− mice injected intraperitoneally with 250 mg folic acid/kg body weight (n = 5 mice per group).
(B) Measurement of BUN (blood urea nitrogen) in sera of mice with ATN. (C) Representative hematoxylin-eosin stained images from kidneys of WT, MAVS −/−, and ASC −/− mice at 36 hr after folic acid treatment.
(D and E) Representative confocal immunofluorescence images (D) and quantification (E) of neutrophil influx at the corticomedullary junction in kidneys of mice with ATN.
(F and G) Representative confocal immunofluorescence images (F) and quantification (G) of IL-1β-positive cells at the corticomedullary junction. The sum of neutrophils (E) or IL-1β-positive cells (G) counted in five independent high power fields (200×) at the corticomedullary junction of each mouse kidney is shown. Data points in (B, E, and G) represent individual mice. Error bars are mean ± SEM, and p values from a two-tailed t test are shown. Results are representative of three independent experiments. FA, folic acid. See also Figure S6.
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10. Figure S1
NLRP3 Localizes to Mitochondria upon Overexpression in HEK293T Cells Independent of ASC, Related to Figure 1
(A) Confocal immunofluorescence images of HEK293T cells transiently overexpressing NLRP3, NLRP2, or NLRP4 showing colocalization of NLRP3, but not NLRP2 or NLRP4, with mitochondria. Mito, Mitotracker; coloc, colocalization channel.
(B) Quantification of NLR colocalized with Mitotracker Red in HEK293T cells transfected as in (A).
(C) Immunoblot (left) and quantitative RT-PCR (right) for ASC in HEK293T and THP-1 cells showing absence of ASC expression at the protein and mRNA level in HEK293T cells. Equal numbers of THP-1 and HEK293T cells (1 × 105 per lane) were used. mRNA expression levels were normalized to the housekeeping gene GAPDH.
(D) Confocal immunofluorescence images of HEK293-ASC-YFP cells transiently overexpressing NLRP3, NLRP4, or NOD1, showing colocalization of NLRP3, but not NLRP4 or NOD1, with mitochondria and the ASC speckle. Mito, Mitotracker.
(E–I) Quantification of NLR colocalized with Mitotracker Red (E), NLR colocalized with the ASC speckle (F), ASC colocalized with Mitotracker Red (G), percentage of transfected cells containing an ASC speckle (H) and ASC colocalized with NLR (I) in HEK293-ASC-YFP cells transfected as in (D). Images are representative of several independent experiments. Data points on graphs represent independent fields (with 3-5 cells per field). Data in (H) represent the percentages of all transfected cells (pooled from multiple fields) containing an ASC speckle. At least 20 cells were evaluated for each NLR. Error bars are mean ± SEM, and p values from an unpaired t test (two-tailed) are shown.
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11. Figure S2
Effect of N-Terminal Deletions of NLRP3 on ASC Speckle Formation under Unstimulated Conditions, Related to Figure 2
Representative immunofluorescence images of HEK293-ASC-YFP cells expressing WT and N-terminal deletions of NLRP3 under transient low-expression conditions, showing progressive loss of mitochondrial localization and ASC aggregation of NLRP3 mutants under unstimulated conditions. HEK293-ASC-YFP cells were transiently transfected with low levels of NLRP3 WT, Δ 2-3, Δ 2-5, Δ 2-7, Δ 2-9, Δ 2-11 or Δ Related quantification of ASC speckle formation is shown in Figure 3A. Data are representative of several images from four independent experiments. Mito, Mitotracker.
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12. Figure S3
NLRP3 N-Terminal Deletions Show Self-Association and Expression Similar to the WT Protein, Related to Figure 3
(A) Immunoblot showing similar self-association ability of WT and N-terminal deletions of NLRP3. HEK293T cells were transiently transfected with 0.5 μg of the indicated HA and FLAG-tagged NLRP3 constructs. Proteins in lysates were immunoprecipitated with anti-HA antibody, and immunoprecipitates were probed with antibodies to the FLAG and HA tags.
(B) Immunoblot showing similar expression of WT and N-terminal deletions of NLRP3. HEK293T cells were transiently transfected with low levels (transient low expression) of the indicated FLAG-tagged constructs. Immunoblots are representative of two independent experiments.
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13. Figure S4
The N Terminus of NLRP3 Mediates Its Association with MAVS, Related to Figure 4
(A and B) Representative confocal images (A) and quantification (B) showing colocalization of NLRP3 WT but not the Δ 2-21 mutant with MAVS upon activation with nigericin. Expression of NLRP3 in HEK293T cells stably expressing the WT or Δ 2-21 deletion under a tetracycline-inducible (TRE) promoter, was induced with 1 μg/ml doxycycline (DOX), and cells were treated with 15 μM nigericin for 45 min prior to imaging. coloc, colocalization channel.
(C) Immunoblot showing association of WT NLRP3, but not the Δ 2-21 mutant of NLRP3 with MAVS upon activation with nigericin. Expression of NLRP3 in HEK293T cells was induced with DOX, and cells were treated with 15 μM nigericin. NLRP3 was immunoprecipitated from lysates; immunoprecipitates were separated by SDS-PAGE and probed with antibody to MAVS. ∗ represents a nonspecific band.
(D) Immunoblot showing association of in vitro translated WT NLRP3, but not Δ 2-5 or Δ 2-7 N-terminal deletions of NLRP3, with purified MAVS. FLAG-tagged NLRP3 WT, Δ 2-5, and Δ 2-7 proteins were prepared by in vitro translation using a reticulocyte lysate system and immunoprecipitated with anti-FLAG antibody followed by anti-mouse Dynabeads. Bead-bound NLRP3 proteins were incubated with purified MAVS protein and precipitates were examined for the presence of NLRP3 and MAVS.
(E and F) Immunoblots showing NLRP3 association with MAVS in BMDMs upon treatment with activators of NLRP3, but not activators of NLRC4, NOD2/NLRP1, or AIM2. LPS-primed BMDMs were treated with the indicated concentrations of ATP, nigericin (NLRP3 activators), transfected flagellin (NLRC4 activator), transfected MDP (NOD2/NLRP1 activator), or transfected plasmid DNA/poly dA:dT (AIM2 activators). Proteins in lysates were immunoprecipitated with antibody to MAVS; immunoprecipitates and lysates were separated by SDS-PAGE and probed with antibody to NLRP3.
(G) Corresponding assay for lactate dehydrogenase (LDH) released in supernatants of BMDMs treated as in (F) showing that NLRP3-MAVS association in immunoprecipitates does not correlate with cell death due to inflammasome activation. Data are represented either as fold increase in LDH release over untreated cells (G, top) or as % LDH release relative to maximum release (G, bottom). Error bars are mean ± SEM. Nig, nigericin; pdA:dT, poly dA:dT; flg, flagellin; MDP, muramyldipeptide.
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14. Figure S5
MAVS Is Required for Optimal Inflammasome Activation in Response to Noncrystalline NLRP3 Activators In Vitro, Related to Figure 6
(A and B) Immunoblots (left) and corresponding densitometric quantification (right) for IL-1β showing reduced processing of pro-IL-1β to its cleaved, active form in MAVS −/− macrophages compared to WT macrophages. LPS-primed bone marrow-derived macrophages (A) or LPS-primed peritoneal macrophages (B) were treated with NLRP3 activators ATP and nigericin for 20 min. Pro-IL-1β in lysates and cleaved IL-1β in supernatants was detected by immunoblot. Nig, nigericin; flg, flagellin.
(C–F) ELISA showing reduced IL-1β secretion from MAVS −/− macrophages compared to WT macrophages in response to noncrystalline but not crystalline NLRP3 activators. LPS-primed BMDMs (C, D, F) or LPS-primed peritoneal macrophages (E) were treated with the indicated concentrations of transfected poly I:C for 6 hr (C); CPPD crystals for 4 hr (D, left) or 2 hr (D, right); MSU crystals for 4 hr (E), or transfected flagellin for 2 hr (F). IL-1β in supernatants was detected by ELISA. Error bars are mean ± SEM, and p values from an unpaired t test (two-tailed) are shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ND, not detectable.
(G) Representative confocal immunofluorescence images of WT and MAVS −/− BMDMs showing that MAVS −/− macrophages have normal MAM architecture. WT and MAVS −/− BMDMs were stained with Mitotracker Red, ER marker calnexin and MAM marker FACL4. Note that in addition to being cytosolic, FACL4 staining is also partly nuclear (Elholm et al., 2000; Horner et al., 2011).
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15. Figure S6
MAVS −/− Mice Are Normal in Priming of NLRP3 and Pro-IL-1β Expression in the Kidney and Are Protected from Acute Tubular Necrosis but Not Crystal-Induced Peritonitis, Related to Figure 7
(A) Representative flowcytometry plots for pro-IL-1β expression in kidney resident CD11b+ Gr1- monocytes showing that MAVS −/− mice are normal in priming of pro-IL-1β expression. WT, MAVS −/−, NLRP3 −/−, and ASC −/− mice were injected intraperitoneally either with saline (vehicle) for 24 hr, 250 mg/kg folic acid (FA) for 24 hr, or 3 mg/kg LPS for 12 hr, and kidneys were harvested. Total leukocytes were isolated and expression of pro-IL-1β in the CD11b+ Gr1- resident monocyte population was analyzed by FACS. Grey histograms, vehicle; Red histograms, LPS or FA.
(B) Representative immunoblots for NLRP3 showing that MAVS −/− mice are normal in priming of NLRP3 expression in the kidney. Mice were injected intraperitoneally either with saline (UT) or with 250 mg/kg folic acid (FA) for 24 hr, kidneys were harvested, homogenized, protein concentrations across samples were normalized by Bradford Assay, and 400 μg protein from each sample was analyzed for expression of NLRP3 by immunoblot.
(C) ATN histology score of WT, MAVS −/−, and ASC −/− mice, 36 hr after intraperitoneal injection of folic acid as assessed by scoring of tubulointerstitial damage on H&E stained kidney sections.
(D) Plots for peritoneal neutrophil recruitment from three independent experiments showing that MAVS −/− mice do not reproducibly show protection from crystal-induced peritonitis. WT and MAVS −/− mice were injected intraperitoneally with vehicle (control) or 1 mg (left and middle) or 0.5 mg (right) MSU crystals for 6 hr. Peritoneal cavities were washed with 5 ml PBS and the lavage was analyzed for neutrophil recruitment by FACS using the neutrophil marker Ly6G. Absolute numbers of neutrophils recruited to the peritoneum are shown.
Data points on graphs represent individual mice. Error bars are mean ± SEM, and p values from a two-tailed t test are shown. FA, folic acid.
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16. Figure S7
Proposed Model of NLRP3 Inflammasome Activation Mediated by MAVS, Related to Figure 6
In untreated macrophages, NLRP3 and ASC are cytosolic in the resting state (1). NLRP3 is activated by danger signals resulting in self-association and/or oligomerization via its central NACHT domain (2–3). The oligomerized NLRP3 is recruited to mitochondria via association of a short sequence at its N terminus with the adaptor protein MAVS (4). Alternatively, self-associated/partially oligomerized NLRP3 might first be recruited via association with MAVS to the mitochondria, that then serve as a platform for solid-phase oligomerization of NLRP3 on the mitochondrial surface (4). Mitochondria-associated and fully oligomerized NLRP3 then promotes recruitment of the cytosolic adaptor protein ASC by homotypic PYD-PYD domain interactions (5), subsequently resulting in recruitment and activation of caspase-1 followed by cleavage of pro-IL-1β to its active, secreted form. We propose a “two adaptor” model for NLRP3 inflammasome activation through a putatively sequential amplification process first involving mitochondrial recruitment of NLRP3 mediated by MAVS (and possibly other mitochondrial proteins), followed by ASC oligomerization and pyroptosome formation resulting in optimal inflammasome function.
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