Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation

Abstract

The NLRP3 inflammasome is activated by a wide range of stimuli and drives diverse inflammatory diseases. The decrease of intracellular K⁺ concentration is a minimal upstream signal to most of the NLRP3 activation models. Here, we found that cellular K⁺ efflux induces a stable structural change in the inactive NLRP3, promoting an open conformation as a step preceding activation. This conformational change is facilitated by the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker also facilitates the ensemble of NLRP3^PYD into a seed structure for ASC oligomerization. The introduction of the NLRP3 PYD-linker-FISNA sequence into NLRP6 resulted in a chimeric receptor able to be activated by K⁺ efflux–specific NLRP3 activators and promoted an in vivo inflammatory response to uric acid crystals. Our results establish that the amino-terminal sequence between PYD and NACHT domain of NLRP3 is key for inflammasome activation.

Fig. 1.. K⁺ efflux induces cells with multiple NLRP3 puncta.

(A) IL-1β release and intracellular K⁺ concentration from LPS-primed bone marrow–derived macrophages (BMDM) activated for 30 min with different doses of nigericin. n = 4 to 6 independent experiments for each concentration of nigericin. (B) IL-1β release from LPS-primed BMDMs activated for 30 min with nigericin (10 μM) in the presence of increasing concentrations of KCl. (C) IL-1β release from LPS-primed BMDMs activated for 30 min with nigericin (10 μM, top) or with C. difficile toxin B (TcdB; 1 μg/ml, bottom) in the absence/presence of 40 mM KCl, RbCl, CsCl, or LiCl. n = 4 independent experiments; Kruskal-Wallis test. (D) Representative fluorescent micrographs of human embryonic kidney (HEK) 293T cells expressing YFP-NLRP3 (green) and stained for nuclei [4′,6-diamidino-2-phenylindole (DAPI); blue] treated for 30 min with nigericin (10 μM) in the absence/presence of 140 mM KCl. Scale bar, 10 μm. (E) Representative fluorescent micrographs of HEK293T cells (left) and Nlrp3^−/− immortalized macrophages (iMos; right) expressing YFP-NLRP3 (green) and ASC (stained in red) and stained for nuclei (DAPI, blue) and treated for 30 and 60 min with nigericin (10 μM), respectively. Scale bar, 20 μm (left) and 10 μm (right). Arrows denote ASC specks; HEK293T cells were stably expressing YFP-NLRP3 and transfected for ASC.

Fig. 2.. K⁺ efflux modifies the NLRP3 structure.

(A) Average BRET signal for YFP-NLRP3-Luc expressed in HEK293T cells before and after stimulation with nigericin (10 μM, denoted by a gray box) in the absence (left, middle) or presence (right) of 140 mM KCl; note that kinetics of BRET have been plotted with the same scale, but relatively at the same level to ease comparison. n = 51, 4, and 6 for nigericin 15 min, nigericin 85 min, and nigericin + KCl, respectively; Mann-Whitney test. (B) NLRP3 BRET signal variation after treatment for 15 min with nigericin (10 μM) versus basal conditions without nigericin in the presence of increasing concentrations of KCl. n = 3 to 5 independent cell culture wells for each KCl concentration. (C) Average BRET signal for YFP-NLRP3-Luc expressed in HEK293T cells before and after stimulation with nigericin (10 μM) in the absence/presence of 40 mM RbCl or 40 mM LiCl; note that kinetics of BRET have been plotted with the same scale, but relatively at the same initial level to ease comparison. n = 12 independent cultures from four independent experiments; Mann-Whitney test. (D) Average BRET signal for YFP-NLRP3-Luc expressed in HEK293T cells (top and middle) or HEK293T-P2X7 receptor (bottom), before and after stimulation with valinomycin (50 μM), BB15C5 (50 μM), or ATP (3 mM), in the absence/presence of 140 mM KCl; note that kinetics of BRET have been plotted with the same scale, but relatively at the same initial level to ease comparison. n = 6 to 18 independent cell cultures; Mann-Whitney test.

Fig. 3.. The PYD domain is dispensable for NLRP3 activation in response to K⁺ efflux.

(A) Representative fluorescent micrographs of HEK293T cells expressing YFP-ΔPYD-NLRP3 (green) and stained for nuclei (DAPI, blue) treated for 30 min with nigericin (10 μM) in the absence/presence of 140 mM KCl. Scale bar, 10 μm. (B) Average BRET signal for YFP-ΔPYD-NLRP3-Luc expressed in HEK293T cells before and after stimulation with nigericin (10 μM) in the absence/presence of 140 mM KCl. n = 5 to 17 independent cell cultures, from three to five independent experiments; note that kinetics of BRET have been plotted with the same scale, but relatively at the same initial level to ease comparison; Mann-Whitney test. (C) IL-1β release from Nlrp3^−/− immortalized macrophages (iMos) treated for 16 hours with doxycycline (1 μg/ml) and LPS (100 ng/ml) to induce the expression of YFP-ΔPYD-NLRP3 and then activated for 60 min with nigericin (10 μM). n = 3 independent experiments. (D) Representative fluorescent micrographs of HEK293T cells expressing YFP-ΔPYD-NLRP3 or full-length NLRP3 as indicated (green) and ASC (red), stained for nuclei (DAPI, blue) and treated for 30 min with nigericin (10 μM). Scale bar, 20 μm.

Fig. 4.. Model of ASC^PYD-NLRP3-NEK7 oligomer.

(A) The structure on the left corresponds to a ribbon plot showing an oligomer complex of 11 NLRP3 monomers, 11 NEK7, and 11 ASC^PYD chains. The representation in the right shows the superposition of 11 NLRP3 monomers of the oligomer complex of the modeled inflammasome structure. PYD domains are placed at different distances of the NACHT domain depending on the conformation of the linker (shown in dark red in the right representation). (B) Oligomer complex of 11 NLRP3 monomers, 11 NEK7, and 11 ASC^PYD chains. Linker fragment between PYD domains and FISNA domains of each NLRP3 is shown in dark red. FISNA domains of NLRP3 are shown in gray-blue, highlighting the continuous connectivity through FISNA-FISNA and FISNA-NACHT interfaces. This model presents a compatible structure to promote ASC^PYD filament formation.

Fig. 5.. The sequence between PYD and NATCH domains is important for NLRP3 activation in response to K⁺ efflux.

(A) IL-1β release from Nlrp3^−/− immortalized macrophages (iMos) treated for 16 hours with doxycycline (1 μg/ml) and LPS (100 ng/ml) to induce the expression of different truncations of NLRP3 as indicated and then activated for 60 min with nigericin (10 μM) in the absence/presence of 40 mM KCl. n = 2 to 5 independent experiments; Mann-Whitney test. (B) Western blot for IL-1β, caspase-1, GSDMD, NLRP3, and β-actin from cell lysates and supernatants of Nlrp3^−/− iMos expressing YFP-Δ92–120-NLRP3 treated as in (A) in the absence/presence of MCC950 (10 μM); representative of n = 2 independent experiments. (C) Percentage of ASC specking Nlrp3^−/− iMos expressing different truncations of NLRP3 treated as in (A). n = 3 to 27 independent experiments; Mann-Whitney test. (D) Representative fluorescent micrographs of Nlrp3^−/− iMos (top) and HEK293T cells (bottom) expressing different truncations of YFP-NLRP3 as indicated (green) and iMos stained for ASC [red, arrowheads denote ASC specks quantified in (C)] and nuclei (DAPI, blue), treated for 60 and 30 min with nigericin (10 μM), respectively. Scale bar, 10 μm. Quantification of HEK293T cells with NLRP3 punctuate staining is shown on the right of micrographs. n = 3 to 10 independent experiments. (E and F) Average BRET signal for YFP-Δ92–120-NLRP3-Luc, YFP-Δ92–132-NLRP3-Luc, YFP-Δ92–148-NLRP3-Luc (E), or YFP-Δ149-180-NLRP3-Luc, YFP-Δ181–217-NLRP3-Luc (F) expressed in HEK293T cells before and after stimulation with nigericin (10 μM, indicated by an arrow) in the absence/presence of 140 mM KCl or MCC950 (10 μM); note that kinetics of BRET have been plotted with the same scale, but relatively at the same initial level to ease comparison. n = 2 to 19 independent cell cultures; Mann-Whitney test.

Fig. 6.. NLRP3 PYD-linker-FISNA sequence renders NLRP6 sensitive to K⁺ efflux.

(A) IL-1β release from Nlrp3^−/− immortalized macrophages (iMos) treated for 16 hours with doxycycline (1 μg/ml) and LPS (100 ng/ml) to induce the expression of YFP-NLRP6, YFP-NLRP3, or YFP-chimeric NLRP3/6 as indicated and then activated for 60 min with nigericin (10 μM) in the absence/presence of 40 mM KCl; n = 4 independent experiments; Mann-Whitney test. (B) Western blot for IL-1β, caspase-1, NLRP3/6, and β-actin, from cell lysates and supernatants of Nlrp3^−/− iMos expressing YFP-NLRP3, YFP-NLRP3(1–217)-NLRP6(196–892), or YFP-NLRP6 treated as in (A); representative of n = 3 independent experiments. (C) IL-1β release [enzyme-linked immunosorbent assay (ELISA), left; Western blot, right] from Nlrp3^−/− iMos expressing the chimera YFP-NLRP3(1–217)-NLRP6(196–892) treated as in (A) in the absence/presence of MCC950 (10 μM) or Ac-YVAD-AOM (YVAD; 100 μM). For the ELISA, n = 4 to 6 independent experiments; Mann-Whitney test. (D) Percentage of ASC specking in Nlrp3^−/− iMos expressing the chimera YFP-NLRP3(1–217)-NLRP6(196–892) treated as in (A). n = 7 to 8 independent experiments; Mann-Whitney test. (E) Percentage of ASC specking in HEK293T cells expressing ASC and the chimera YFP-NLRP3(1–217)-NLRP6(196–892) treated for 30 min with nigericin (10 μM) in the absence/presence of 140 mM KCl. n = 5 to 15 independent experiments; Kruskal-Wallis test. (F) Representative fluorescent micrographs of HEK293T cells expressing the chimera YFP-NLRP3(1–217)-NLRP6(196–892) (green) and stained for nuclei (DAPI, blue), treated as in (E). Scale bar, 10 μm. Right: Quantification of YFP-NLRP3(1–217)-NLRP6(196–892) cells with a punctuate staining. n = 4 independent experiments; Mann-Whitney test.

Fig. 7.. NLRP6 with the PYD-linker-FISNA NLRP3 sequence is activated by MSU crystals and imiquimod.

(A) IL-1β release from Nlrp3^−/− immortalized macrophages (iMos) expressing different truncations of NLRP3 as indicated treated for 16 hours with doxycycline (1 μg/ml), LPS (100 ng/ml), and MSU crystals (300 μg/ml) in the absence/presence of 40 mM KCl. n = 3 to 4 independent experiments; Mann-Whitney test. (B) IL-1β release from Nlrp3^−/− iMos treated as in (A) but expressing different chimeric NLRP6/3 receptors as indicated. n = 3 to 4 independent experiments; Mann-Whitney test. (C) IL-1β release from Nlrp3^−/− iMos expressing different truncations of NLRP3 as indicated, treated for 16 hours with doxycycline (1 μg/ml) and LPS (100 ng/ml) to induce the expression of different NLRP3 truncations as indicated and then activated for 1 hour with imiquimod (100 μM) in the absence/presence of 40 mM KCl. n = 3 to 4 independent experiments; Mann-Whitney test. (D) IL-1β release from Nlrp3^−/− iMos treated as in (C) but expressing different chimeric NLRP6/3 receptors as indicated. n = 3 to 4 independent experiments; Mann-Whitney test. (E) Quantification of HEK293T cells expressing YFP-NLRP3 treated for different times with imiquimod (100 μM, for 0.5, 2, 4, or 6 hours) or nigericin (10 μM, 30 min) in the absence/presence of 40 mM KCl. n = 3 to 4 independent experiments; Mann-Whitney test. (F) Average BRET signal for YFP-NLRP3-Luc expressed in HEK293T cells before and after stimulation with imiquimod (100 μM, 6 hours), in the absence or presence of 40 mM KCl. n = 6 to 9 independent measurements; Kruskal-Wallis test.

Fig. 8.. NLRP6 with the NLRP3 PYD-linker-FISNA sequence induces an inflammatory response to MSU crystals.

(A) Protocol followed in the experimental in vivo assay (top). IL-1β and IL-18 in the peritoneal lavage of Nlrp3^−/− after an intraperitoneal (i.p.) injection with Nlrp3^−/− iMos expressing YFP-NLRP6, the chimera YFP-NLRP3(1–217)-NLRP6(196–892), or YFP-NLRP3 and then challenged with an intraperitoneal injection of MSU crystals for 16 hours (bottom). n = 7 to 11 mice, each one represented by a dot; Mann-Whitney test. (B) CXCL1, CXCL10, and CCL2 chemokines in the peritoneal lavage of Nlrp3^−/− mice treated as in (A). n = 8 to 11 mice, each one represented by a dot; Mann-Whitney test. (C) Quantification of CD11b⁺, Ly6G⁺, and F4/80⁻ cells in the peritoneal lavage of Nlrp3^−/− mice treated as in (A). n = 5 to 11 mice, each one represented by a dot; Mann-Whitney test.