Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury
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eLife assessment
In this important study, Di et al., examine the mechanism by which potassium channels are activated prior to NLRP3 inflammasome activation. The main strength of the study is that it uses a combination of cell culture work and a mouse model to address the cell biology of inflammasome activation. However, certain aspects of the study including the characterization of inflammasome activation and the evidence to support the role of Rab11a in the translocation of TWIK2 are incomplete.
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Abstract
Potassium efflux via the two-pore K + channel TWIK2 is a requisite step for the activation of NLRP3 inflammasome, however, it remains unclear how K + efflux is activated in response to select cues. Here, we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in the extrusion of K + . We showed that ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP-ligated purinergic receptor P2X7 each prevented endosomal fusion with the plasmalemma and K + efflux as well as NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-depleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. We conclude that Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 localization and activity at the cell surface and the downstream activation of the NLRP3 inflammasome. Results show that endosomal trafficking of TWIK2 to the plasmalemma is a potential therapeutic target in acute or chronic inflammatory states.
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eLife assessment
In this important study, Di et al., examine the mechanism by which potassium channels are activated prior to NLRP3 inflammasome activation. The main strength of the study is that it uses a combination of cell culture work and a mouse model to address the cell biology of inflammasome activation. However, certain aspects of the study including the characterization of inflammasome activation and the evidence to support the role of Rab11a in the translocation of TWIK2 are incomplete.
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Reviewer #1 (Public Review):
This important study by Di et al., focuses on the mechanism by which potassium channels are activated prior to NLRP3 inflammasome activation. Using confocal- and electron-microscopy studies the authors demonstrate that the potassium channel, TWIK2, located in the endosomal compartment during basal conditions, is translocated onto the plasmalemma upon ATP stimulation. The authors suggest that this translocation triggers potassium efflux and subsequent NLRP3 inflammasome activation. Using Rab11a-deficient cells, the authors also show an essential role for Rab11a in this process.
This is a well written mechanistic study that has novel findings that are of interest to the inflammasome field. It addresses a long-standing question in the field, the exact mechanism by which potassium channel is activated upon …
Reviewer #1 (Public Review):
This important study by Di et al., focuses on the mechanism by which potassium channels are activated prior to NLRP3 inflammasome activation. Using confocal- and electron-microscopy studies the authors demonstrate that the potassium channel, TWIK2, located in the endosomal compartment during basal conditions, is translocated onto the plasmalemma upon ATP stimulation. The authors suggest that this translocation triggers potassium efflux and subsequent NLRP3 inflammasome activation. Using Rab11a-deficient cells, the authors also show an essential role for Rab11a in this process.
This is a well written mechanistic study that has novel findings that are of interest to the inflammasome field. It addresses a long-standing question in the field, the exact mechanism by which potassium channel is activated upon treatment with NLRP3 stimuli. However, to make the conclusions more convincing, the authors should include additional stimuli such as pore-forming toxins, LPS transfection, and/or infections with bacterial pathogens to show that the Rab11a-dependent TWIK2 translocation is a universal requirement for initiation of potassium efflux by multiple stimuli and not specific to ATP. Similarly, the authors should include important controls in their inhibitor/siRNA experiments to show that the cells are still functional and the defects they observe are specific to NLRP3 inflammasome.
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Reviewer #2 (Public Review):
Previous work by the same group has shown that the potassium channel TWIK2 contributes to the activation of the NLRP3 inflammasome in macrophages. In this manuscript, the authors provide new insights into the biology of TWIK2 and show that TWIK2 translocated to the plasma membrane of macrophages following stimulation with ATP. They show that ATP stimulation induced exocytosis, via a process dependent on the purinergic receptor P2X7, the presence of calcium and vesicle fusion. Genetic deletion of P2X7, depletion of calcium, and pharmacological inhibition of vesicle fusion collectively contributed to the inhibition of current changes and NLRP3 inflammasome activation. The authors also show that the endosomal protein Rab11a translocated to the plasma membrane following ATP stimulation and that Rab11a …
Reviewer #2 (Public Review):
Previous work by the same group has shown that the potassium channel TWIK2 contributes to the activation of the NLRP3 inflammasome in macrophages. In this manuscript, the authors provide new insights into the biology of TWIK2 and show that TWIK2 translocated to the plasma membrane of macrophages following stimulation with ATP. They show that ATP stimulation induced exocytosis, via a process dependent on the purinergic receptor P2X7, the presence of calcium and vesicle fusion. Genetic deletion of P2X7, depletion of calcium, and pharmacological inhibition of vesicle fusion collectively contributed to the inhibition of current changes and NLRP3 inflammasome activation. The authors also show that the endosomal protein Rab11a translocated to the plasma membrane following ATP stimulation and that Rab11a contributed to NLRP3 inflammasome activation. Depletion of Rab11a in macrophages prevented lung injuries and NLRP3 inflammasome activation in mice treated with LPS.
The major strength of the work is the use of a combination of cell culture work and a mouse model to address the cell biology of inflammasome activation.
The weakness is that the current set of data is not able to fully support the conclusion that Rab11a, P2X7 and calcium influx mediate the translocation of TWIK2 to the plasma membrane. The characterisation of inflammasome activation is also partial. If these weaknesses can be addressed, the authors would have achieved their aims and increased the impact of their work in the field of inflammasome biology. -
Reviewer #3 (Public Review):
Here, the authors aim to uncover the mechanism by which the K+ efflux channel TWIK2 contributes to activation of the canonical NLRP3 inflammasome, as a follow on from their 2018 publication identifying TWIK2 as an essential factor in ATP-induced inflammasome activation. They firstly use immunofluorescence to identify TWIK2 trafficking to the membrane following ATP challenge, and is found to colocalise with early and recycling endosomes during homeostasis. The strengths of the paper are the finding that TWIK2 localisation in cells may be altered by ATP. Biophysical investigation of membrane potential identifies extracellular Ca2+ as essential for NLRP3 activation, and the calcium-dependent small GTPase Rab11a was found to colocalise with the plasma membrane upon ATP treatment. Finally, mice harbouring Rab11a …
Reviewer #3 (Public Review):
Here, the authors aim to uncover the mechanism by which the K+ efflux channel TWIK2 contributes to activation of the canonical NLRP3 inflammasome, as a follow on from their 2018 publication identifying TWIK2 as an essential factor in ATP-induced inflammasome activation. They firstly use immunofluorescence to identify TWIK2 trafficking to the membrane following ATP challenge, and is found to colocalise with early and recycling endosomes during homeostasis. The strengths of the paper are the finding that TWIK2 localisation in cells may be altered by ATP. Biophysical investigation of membrane potential identifies extracellular Ca2+ as essential for NLRP3 activation, and the calcium-dependent small GTPase Rab11a was found to colocalise with the plasma membrane upon ATP treatment. Finally, mice harbouring Rab11a siRNA-treated macrophages were found to exhibit reduced inflammation in response to induction of sepsis, further reinforcing the potential of Rab11a targeting for novel therapeutics. However, mechanistic exploration do not provide direct evidence on TWIK2 trafficking or the involvement of Rab11a specifically with NLRP3 inflammasomes, and results with non-specific inhibitors needs to be supported by further experiments.
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