Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Dec 5;9(1):5182.
doi: 10.1038/s41467-018-07573-4.

NLRP3 lacking the leucine-rich repeat domain can be fully activated via the canonical inflammasome pathway

Affiliations

NLRP3 lacking the leucine-rich repeat domain can be fully activated via the canonical inflammasome pathway

Iva Hafner-Bratkovič et al. Nat Commun. .

Abstract

NLRP3 is a cytosolic sensor triggered by different pathogen- and self-derived signals that plays a central role in a variety of pathological conditions, including sterile inflammation. The leucine-rich repeat domain is present in several innate immune receptors, where it is frequently responsible for sensing danger signals and regulation of activation. Here we show by reconstitution of truncated and chimeric variants into Nlrp3-/- macrophages that the leucine-rich repeat domain is dispensable for activation and self-regulation of NLRP3 by several different triggers. The pyrin domain on the other hand is required to maintain NLRP3 in the inactive conformation. A fully responsive minimal NLRP3 truncation variant reconstitutes peritonitis in Nlrp3-/- mice. We demonstrate that in contrast to pathogen-activated NLRC4, the constitutively active NLRP3 molecule cannot engage wild-type NLRP3 molecules in a self-catalytic oligomerization. This lack of signal amplification is likely a protective mechanism to decrease sensitivity to endogenous triggers to impede autoinflammation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The LRR domain is not important for activation of NLRP3. a Schematic representation of truncation variants based on the predicted domain organization of the NLRP3. Variants are labeled either as 1-last amino acid residue or by just the last amino acid residue number. b The response of truncated variants with variable numbers of LRRs to nigericin (IL-1β, left; IL-18, right). Immortalized mouse bone marrow–derived macrophages from Nlrp3−/− mice with stable integration of the designated constructs were primed with LPS (100 ng/ml) and doxycycline (0.05 μg/ml) for 11 h. After priming, the medium was exchanged, and the cells were stimulated with nigericin (5 μM) for 1 h. Representative of 3 independent experiments is shown. The mean and the s.e.m. are shown of 2 biological replicates which are combined to show expression in d. c Cells were primed as in b. After priming, SiO2 (0.2 mg/ml) and alum (0.5 mg/ml) were added for 6 h. Representative of 3 independent experiments is shown. The mean and the s.e.m. of 3 biological replicates are shown. d Western blot analysis of primed and doxycycline-treated iBMDMs. NLRP3 was detected with the Cryo-2 antibody, which binds to the pyrin domain. Representative of 3 independent western blots is shown
Fig. 2
Fig. 2
Determination of the minimal fully responsive NLRP3 variant. a Schematic representation of truncation variants based on the predicted domain organization of the NLRP3. b Immortalized mouse bone marrow–derived macrophages from NLRP3-deficient mice with stable integration of the designated constructs were primed with LPS (100 ng/ml) and doxycycline (1 μg/ml) for 11 h. After priming, the medium was exchanged, and the cells were stimulated with nigericin (5 μM) for 1 h. c Western blot of NLRP3 variants. d Selected cell lines were stimulated as in b, and caspase-1 autoproteolytic activation was followed by western blotting. e Selected cell lines were primed as in b. After priming, SiO2 (0.2 mg/ml) and alum (0.5 mg/ml) were added for 6 h. f Cell lines were primed as in b. After priming, the medium was replaced, and imiquimod (20 μg/ml) was added for 24 h. g Stable Nlrp3−/− iBMDMs harboring different NLRP3 variants and wild-type iBMDMs were primed with LPS and doxycycline for 11 h, after which the medium was replaced, and the inhibitors KCl (130 mM), glyburide (100 µM), and DPI (50 µM) were added for 0.5 h before nigericin was added (5 μM). h Nlrp3−/− iBMDMs were primed as before, inhibitors G5, MCC950 (950), Cy-09 and shikonin (sh) were added prior nigericin stimulation. Representative of 3 (b, e, f) or 2 (c, d, g, h) independent experiments is shown. The mean and the s.e.m. of 3 (b, eh) or 8 (ctrl in h) biological replicates are shown. 1–686 variant corresponds to MiniNLRP3
Fig. 3
Fig. 3
Human variants phenocopy corresponding mouse variants. a Nlrp3−/− iBMDMs with stably integrated human NLRP3 variants were primed with LPS (100 ng/ml) and doxycycline (1 μg/ml) for 11 h and stimulated with nigericin (10 μM) or ATP (5 mM) for 45 min. Supernatants were analyzed for IL-1β maturation (left) and LDH activity (right). b Cells were unprimed (doxycycline-treated) or primed (LPS, doxycycline) for 12 h and stimulated for 1 h, and cell supernatants were analyzed for mature IL-1β and caspase-1 p20 subunit. Cell lysates were analyzed for expression of pro-caspase-1, pro-IL-1β, ASC, and NLRP3 variant. c Supernatants from b were analyzed for TNF-α and IL-6 concentrations. d, e LPS-primed and nigericin-treated Nlrp3−/− iBMDMs with human NLRP3 were analyzed for the formation of ASC specks. Nuclei are depicted in blue (DAPI), ASC in green, and actin in red; the bar represents 10 μm. To provide an estimate of ASC speck formation (in %) (e), four random 138 × 110 μm2 frames were recorded for each condition, the number of ASC specks was divided by the number of nuclei within each frame. Representative of 3 (ae) independent experiments is shown. The mean and the s.e.m. of 3 (a) or 2 (c) biological replicates and 4 random frames (e) are shown. 1–688 variant corresponds to MiniNLRP3
Fig. 4
Fig. 4
MiniNLRP3 clusters after stimulation and facilitates peritonitis in Nlrp3−/− mice. a Tagged and untagged versions of NLRP3 variants were primed for 11 h with LPS and doxycycline (0.5 μg/ml) and stimulated for 1 h with nigericin (5 μM). Supernatants were analyzed for IL-1β maturation and LDH activity. b Expression of murine NLRP3-YFP-tagged variants in dormant and activated conditions. Cells were untreated (ctrl), LPS and doxycycline-treated, and LPS, doxycycline and nigericin (5 μM)-treated (1 h). Nuclei are depicted in blue (Hoechst) and NLRP3-YFP variants in yellow; the scale bar represents 10 μm. c Cells were primed as in a. After priming, the pan-caspase inhibitor (Z-VAD-FMK) was added for 30 min before stimulation with nigericin for 30 min. Cells were lysed, and proteins were immunoprecipitated with antibodies against fluorescent protein, * non-specific band (likely light chain). Representative of 3 (a, b) or 2 (c) independent experiments is shown. The mean and s.e.m. of 3 biological replicates are shown a. d Primed and doxycycline-stimulated Nlrp3−/− iBMDMs carrying either the empty vector, 1–665, 1–686, or full-length mouse NLRP3 were injected into the peritoneal cavity of the Nlrp3−/− mice, and neutrophil infiltration was analyzed 24 h later. Four animals were used per condition. The mean and the s.e.m. are depicted. Statistical values: n.s. p> 0.05, *p < 0.05. The two-tailed nonparametric t-test without/with Welch correction was used for pairwise comparison. 1–686 variant corresponds to MiniNLRP3
Fig. 5
Fig. 5
NLRP3 (1–667) senses nigericin-induced perturbation. a Alignment of NLRP3 protein sequences in the region between 1–665 and 1–686 from different species. b Single and double point mutants were transduced into Nlrp3−/− iBMDMs, and after selection, the cells were primed for 12 h and stimulated with nigericin (5 μM) for 1 h. c NLRP3 variants were expressed in HEK293. Cell lysates were incubated with control beads (CB) or ATP beads (AB). NLRP3 binding to ATP beads was also followed in the presence of CY-09 ( + CY-09) and detected by western blot. d Intramolecular BRET of the human NLRP3 variants was followed in the HEK293 cells after nigericin was added (left). The arrow indicates the time of injection of nigericin or buffer. The bar graph (right) represents the slope of the curve after nigericin addition. To calculate the slope, the signals were normalized to the resting BRET signal (the signal before nigericin addition) to annihilate differences in BRET responses. n.s.: p > 0.05. (c) Representative of 3 (d) or 2 (b, c) independent experiments is shown. The mean and the s.e.m. (b) or s.d. (d) of 3 biological replicates are shown. Individual points of d are depicted in Supplementary Fig. 24. MiniNLRP3 is represented by mouse 1–686 or human 1–688 variants
Fig. 6
Fig. 6
MiniNLRP3 (1–686) supports CAPS-associated constitutive activation. Truncated variants were introduced on a pathological substitution (a-R258W; b-T346M) background, and constitutive activation was followed by IL-1β release upon LPS priming. Representative of 3 (a) or 2 (b) independent experiments is shown. The mean and s.e.m. of 3 (a, b) biological replicates are shown
Fig. 7
Fig. 7
Exchange of the PYD with the CARD domain leads to constitutive activation. a Chimeric CARD-NLRP3 variants and b corresponding interacting domains (PYD, CARD) were expressed in Nlrp3−/− iBMDMs by doxycycline (a: 0, 0.005, 0.01, 0.02, and 0.05 μg/ml; b: 0, 0.01, 0.05, 0.1, 0.5, 1, and 5 μg/ml) and primed with LPS (100 ng/ml) and then tested for their constitutive and nigericin-induced activation (5 μM, 1 h, note that 0 μg/ml dox is omitted in the latter case), as followed by IL-1β release. c For ASC speck labeling, cells were primed with doxycycline (0.5 μg/ml) and LPS (100 ng/ml) for 10 h after which the cells were fixed and labeled with an antibody against ASC. The formation of ASC specks in primed and nigericin-treated (1 h, 10 μM) cells harboring NLRP3 is shown as the control. The bar represents 10 μm. d CARD-NLRP3 chimeras were introduced into Asc/ iBMDMs, and expression of the chimeras was induced with doxycycline. Constitutive activation was followed by measuring IL-1β release. Representative of 3 (a, b) or 2 (c, d) independent experiments is shown. The mean and the s.e.m. of 2 or 3 (a, b, d) biological replicates are shown. n.d. – below detection limit
Fig. 8
Fig. 8
Constitutively active variants fail to engage wild-type NLRP3 in seeded oligomerization. Two possible mechanisms of NLRP3 activation: Triggers induce simultaneous conformational change in NLRP3 molecules that assemble into oligomer (I), or triggers induce conformational change in a few NLRP3 molecules, which recruit nascent NLRP3 molecules to form an oligomer (II). ad To estimate the protein levels of NLRP3, wild-type iBMDMs transduced with empty vector (a), wild-type (b), or R258W (c) or T346M (d) AU1-tagged NLRP3 variants were analyzed for NLRP3 content by Cryo-2 labeling in various testing conditions. NLRP3 levels were first normalized to actin and then to the basal endogenous NLRP3 level in the non-LPS and non-doxycycline-treated condition. Analysis of 3 biological replicates is provided in the charts. One blot per NLRP3 variant is shown. All blots used for analysis can be found in the Supplementary Fig. 18, arrow indicates the position of NLRP3 variants and * an unspecific band. e Activation of wild-type endogenous NLRP3 in wild-type iBMDMs was followed in the presence of constitutively active pathological variants. Cells were first treated with doxycycline (0, 0.5, 1, and 2 μg/ml) for 12 h, then with LPS (100 ng/ml) for 6 h, and afterward with nigericin or buffer (1 h). Representative of 3 (e) independent experiments is shown. The mean and the s.e.m. of 2 (0 μg/ml dox) or 3 biological replicates are shown
Fig. 9
Fig. 9
The role of NLRP3 domains in inflammasome autoregulation and activation. a The LRR domain is redundant for sensing and inflammasome formation and does not restrict the NLRP3 molecule in the inactive conformation. Instead, the sensing and inflammasome-forming regions are located N-terminally to the LRR domain, consisting of the PYD, the NBD, and associated domains and neighboring segments. b Before activation, NLRP3 is locked in the inactive form by the interactions of PYD with NLRP3 (92–665). Upon activation, inhibitory interactions are released, and several molecules of NLRP3 in the active conformation form an oligomer (green arrows). Unlike NLRC4, the activated NLRP3 molecule is unable to engage NLRP3 molecules in an inactive conformation (red arrows) in catalytic autoactivation, decreasing sensitivity and potential autoimmune activation by endogenous triggers

Similar articles

Cited by

References

    1. Kayagaki N, et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature. 2015;526:666–671. doi: 10.1038/nature15541. - DOI - PubMed
    1. Shi J, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature. 2015;526:660–665. doi: 10.1038/nature15514. - DOI - PubMed
    1. Coll RC, O’Neill L, Schroder K. Questions and controversies in innate immune research: what is the physiological role of NLRP3? Cell Death Discov. 2016;2:16019. doi: 10.1038/cddiscovery.2016.19. - DOI - PMC - PubMed
    1. Mariathasan S, et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature. 2006;440:228–232. doi: 10.1038/nature04515. - DOI - PubMed
    1. Gurcel L, Abrami L, Girardin S, Tschopp J, van der Goot FG. Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell. 2006;126:1135–1145. doi: 10.1016/j.cell.2006.07.033. - DOI - PubMed

Publication types

MeSH terms