Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury
- Department of Pharmacology and Regenerative Medicine, The University of Illinois College of Medicine, United States
- Shanghai Frontiers Science Center of Drug Target Identification and Delivery, School of Pharmacy, Shanghai Jiao Tong University, China
- Fluorescence Imaging Core, The University of Illinois College of Medicine, United States
- Department of Chemistry, University of Chicago, United States
Figures
Figure 1 with 3 supplements
Intracellular TWIK2 plasmalemma translocation on ATP challenge.
(A) Visualization of intracellular TWIK2 plasmalemma translocation post-ATP challenge. TWIK2-GFP plasmids were transfected into RAW 264.7 cells for 48 hr and cells were imaged with confocal …
Figure 1—figure supplement 1
Electron microscopy assessment of TWIK2 plasmalemma translocation.
TWIK2 plasmalemma translocation from immunogold labeling electron microscopy before (A) and after ATP (B) (5 mM, 30 min) challenge in RAW 264.7 macrophages. TWIK2 (10 nm gold particles) was …
Figure 1—video 1
Plasma membrane translocation of intracellular TWIK2 (control).
TWIK2-GFP plasmids were transfected into RAW 264.7 cells for 48 hr and cells were imaged with confocal microscope in the presence (Figure 1—video 2) or absence (Figure 1—video 1) of extracellular …
Figure 1—video 2
Plasma membrane translocation of intracellular TWIK2 upon ATP challenge.
Figure 2
Endosomal localization of intracellular TWIK2 determined with fluorescent immunostaining of macrophages.
TWIK2 intracellular localization was determined with fluorescent immunostaining with TWIK2 antibody along with other various antibodies against some specific vesicular proteins and imaged with …
Figure 3
Association of P2X7-dependent ATP-induced exocytosis with plasmalemma potassium efflux and NLRP3 inflammasome activation.
(A, B) Exocytic event is linked to plasmalemma potassium efflux. (A) Exocytosis was evaluated with measurement of whole-cell plasma membrane capacitance (Cm) reflecting the membrane surface changes. …
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Figure 3—source data 1
P2X7-dependent ATP-induced exocytic event is linked to plasmalemma potassium efflux and NLRP3 inflammasome activation.
Related to Figure 3C. Inhibition of vesicle–plasmalemma fusion prevents NLRP3 inflammasome activation in macrophages. (C) Representative results of western blot from three independent experiments showing reduced caspase 1 activation (reduced Casp-1 p20, C). Monocyte-derived macrophages (MDMs) pretreated with vesicle–plasmalemma fusion inhibitor Vacuolin (10 µM, 2 hr) were primed with lipopolysaccharide (LPS; 3 hr) and subsequently challenged with ATP (5 mM) for 30 min. Cell lysates or pellets were immunoblotted with indicated antibodies (anti-TWIK2 or anti-IL1β).
- https://cdn.elifesciences.org/articles/83842/elife-83842-fig3-data1-v2.zip
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Figure 3—source data 2
P2X7-dependent ATP-induced exocytic event is linked to plasmalemma potassium efflux and NLRP3 inflammasome activation.
Related to Figure 3E. Representative results of western blot from three independent experiments showing reduced IL-1β maturation (reduced IL-1β p17). Monocyte-derived macrophages (MDMs) pretreated with vesicle–plasmalemma fusion inhibitor Vacuolin (10 µM, 2 hr) were primed with lipopolysaccharide (LPS; 3 hr) and subsequently challenged with ATP (5 mM) for 30 min. Cell lysates or pellets were immunoblotted with indicated antibodies (anti-TWIK2 or anti-IL1β).
- https://cdn.elifesciences.org/articles/83842/elife-83842-fig3-data2-v2.zip
Figure 4
ATP-induced potassium current and NLRP3 inflammasome activation in Ca2+-dependent manner.
(A, B) Ca2+ dependent of ATP-induced potassium efflux current. Whole-cell current was recorded with patch clamp in monocyte-derived macrophages (MDMs) from three mice with or without pretreatment of …
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Figure 4—source data 1
Ca2+-dependent plasmalemma potassium efflux and NLRP3 inflammasome activation.
Related to Figure 4C. Extracellular Ca2+-dependent NLRP3 inflammasome activation in macrophages. Monocyte-derived macrophages (MDMs) were primed with lipopolysaccharide (LPS) and subsequently challenged with ATP and cell lysates or pellets were immunoblotted with indicated antibodies (anti-Caspase 1 or anti-IL1β). Representative western blotting results from three independent experiments showing reduced caspase 1 activation (reduced Casp-1 p20) in the absence of extracellular Ca2+.
- https://cdn.elifesciences.org/articles/83842/elife-83842-fig4-data1-v2.zip
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Figure 4—source data 2
ATP-induced potassium current and NLRP3 inflammasome activation in Ca2+-dependent manner.
Related to Figure 4D. Extracellular Ca2+-dependent NLRP3 inflammasome activation in macrophages. Monocyte-derived macrophages (MDMs) were primed with lipopolysaccharide (LPS) and subsequently challenged with ATP and cell lysates or pellets were immunoblotted with indicated antibodies (anti-IL1β). Representative western blotting results from three independent experiments showing reduced caspase 1 activation (reduced Casp-1 p20) and IL-1β maturation (reduced IL-1β p17) in the absence of extracellular Ca2+.
- https://cdn.elifesciences.org/articles/83842/elife-83842-fig4-data2-v2.zip
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Figure 4—source data 3
ATP-induced potassium current and NLRP3 inflammasome activation in Ca2+-dependent manner.
Related to Figure 4G. Reduced caspase 1 activation in the presence of Ca2+ chelator BAPTA-AM in monocyte-derived macrophages (MDMs). MDMs were primed with lipopolysaccharide (LPS; 3 hr) and then were pretreated with or without BAPTA-AM (10 µM) for 30 min and subsequently challenged with ATP (5 mM) for 30 min and cell lysates were immunoblotted with anti-Caspase 1. Representative western blotting results from three independent experiments showing reduced caspase 1 activation (reduced Casp-1 p20) when cells were treated with BAPTA-AM.
- https://cdn.elifesciences.org/articles/83842/elife-83842-fig4-data3-v2.zip
Figure 5 with 1 supplement
Rab11a mediates endosomal TWIK2 plasmalemma translocation and NLRP3 inflammasome activation on ATP challenge of macrophages.
(A) Confocal images of Rab11a immunostaining in mouse monocyte-derived macrophages (MDMs) from three mice before and after ATP challenge. Rab11a (green) distribution was identified with fluorescent …
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Figure 5—source data 1
Rab11a mediates endosomal TWIK2 plasmalemma translocation and NLRP3 inflammasome activation on ATP challenge in macrophages.
Related to Figure 5I. Rab11a-dependent NLRP3 inflammasome activation induced by ATP in macrophages. Inhibited NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) treated with siRNA targeting mouse Rab11a (siRab11a). Representative results of western blot from three independent experiments showing reduced caspase 1 activation (reduced Casp-1 p20) and IL-1β maturation (reduced IL-1β p17) and Rab11a knocking down after cells were treated with siRab11a in MDMs, but the NLRP3 expression was not affected by siRab11a treatment. MDMs pretreated with siRab11a for 48 hr were primed with lipopolysaccharide (LPS; 3 hr) and subsequently challenged with ATP (5 mM) for 30 min. Cell lysates were immunoblotted with indicated antibodies (anti-TWIK2 or anti-IL1β or anti-Rab11a or anti NLRP3).
- https://cdn.elifesciences.org/articles/83842/elife-83842-fig5-data1-v2.zip
Figure 5—figure supplement 1
Relative mRNA expression of vesicle fusion proteins in monocyte-derived macrophages (MDMs).
mRNA of various vesical fusion proteins were assessed by Qrt-PCR for the following: Rab27a and b; Rab11a and b; Rab6a and b; Synaptotagmin7-1 and 2 (Syt7-1 and 2); Vamp2 and 3. The highest …
Figure 6 with 1 supplement
Rab11a deficiency in macrophages prevents sepsis-induced NLRP3 inflammasome activation and inflammatory lung Injury in mice.
(A) Schematic illustration of the experiments. Lung macrophages (Mac) were depleted with clodronate liposomes (for 48 hr) and then reconstituted intratracheally with monocyte-derived macrophages …
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Figure 6—source data 1
Rab11a deficiency in macrophages prevents sepsis-induced NLRP3 inflammasome activation and inflammatory lung injury in mice.
Related to Figure 6B. Lung macrophages (Mac) were depleted with clodronate liposomes and then reconstituted via intratracheal route with monocyte-derived macrophages (MDMs) treated with either siRNA of Rab11a or siRNA control as illustrated. The mice were injected with lipopolysaccharide (LPS; intra-peritoneal injection, i.p.) after 24 hr of macrophage reconstitution. Lungs were harvested for evaluation of NLRP3 inflammasome activation and lung inflammation. NLRP3 inflammasome activation (indicated by caspase 1 activation and IL-1β maturation) in the murine lung was assessed by immunoblotting.
- https://cdn.elifesciences.org/articles/83842/elife-83842-fig6-data1-v2.zip
Figure 6—figure supplement 1
Endosomal TWIK2 plasmalemma translocation and resultant NLRP3 inflammasome activation.
TWIK2 is basally active potassium channel in both early endosome (EE) and recycling endosome (RE). Extracellular ATP (e[ATP]) activates P2X7 and induces Ca2+ influx via P2X7 which activates Ca2+-sens…
