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K
+
regulates Ca
2+
to drive inflammasome signaling:
dynamic visualization of ion flux in live cells
JR Yaron
1,2
, S Gangaraju
1
, MY Rao
1
, X Kong
1
, L Zhang
1
,FSu
1
, Y Tian
1
, HL Glenn
1
and DR Meldrum*
,1
P2X
7
purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in
assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome
regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real time. The use of
KS6, a novel sensor for selective and sensitive dynamic visualization of intracellular potassium flux in live cells, multiplexed with
the intracellular calcium sensor Fluo-4, revealed a coordinated relationship between potassium and calcium. Interestingly, the
mitochondrial potassium pool was mobilized in a P2X
7
signaling, and ATP dose-dependent manner, suggesting a role for
mitochondrial sensing of cytosolic ion perturbation. Through treatment with extracellular potassium we found that potassium
efflux was necessary to permit sustained calcium entry, but not transient calcium flux from intracellular stores. Further,
intracellular calcium chelation with BAPTA-AM indicated that P2X
7
-induced potassium depletion was independent of calcium
mobilization. This evidence suggests that both potassium efflux and calcium influx are necessary for mitochondrial reactive
oxygen generation upstream of NLRP3 inflammasome assembly and pyroptotic cell death. We propose a model wherein potassium
efflux is necessary for calcium influx, resulting in mitochondrial reactive oxygen generation to trigger the NLRP3 inflammasome.
Cell Death and Disease (2015) 6, e1954; doi:10.1038/cddis.2015.277; published online 29 October 2015
NLR family, pyrin domain-containing 3 (NLRP3) is the most
extensively studied among the inflammasome family of
caspase-1-activating complexes and is critical to the innate
immune response to infection, damage and pathophysiologi-
cal dysfunction.
1
A likely reason for the widespread interest
in NLRP3 is its responsiveness to extracellular ATP,
pore-forming toxins, biological particulate matter, synthetic
nanoparticles, vaccine adjuvants and pathogens including
bacteria, fungi and viruses.
2–7
There is ongoing debate about
how such a diverse array of stimuli are able to converge on a
single pathway, despite the apparent differences in their mode
of action.
Proposed mechanisms for regulating the activation of the
NLRP3 inflammasome pathway are varied and controversial.
1
Among the most popular proposed mechanisms is the flux of
cellular ions. The asymmetric distribution of ions in cellular
compartments establishes a gradient such that, under
conditions of membrane permeability, ions rapidly diffuse
across the gradient without energy input.
8
Cells benefit from
asymmetric ion distribution by using it to affect rapid processes
such as neuronal action potentials.
8
Recent work has
implicated potassium flux as the common trigger in regulating
NLRP3 inflammasome activity.
9
Indeed, it has been under-
stood for over two decades that potassium flux regulates the
processing of interleukin (IL)-1β, a downstream effect of
inflammasome activation.
10,11
While potassium is the most
commonly studied ion posited to regulate the NLRP3 pathway,
calcium flux has gained popularity in recent years because
intervention in calcium mobilization has inhibitory effects on
inflammasome activity.
12–14
Both ions are permeant to the
non-specific cation channel formed by plasma membrane
expressed purinergic receptor P2X, ligand-gated ion channel,
7 (P2X
7
) which is activated by external ATP. However, it is
currently not known how or whether the two ions relate to each
other in the context of inflammasome regulation.
1,12
In addition to ion flux, mitochondrial reactive oxygen species
(mROS) signaling has been proposed as a critical regulator of
NLRP3 activation.
15
Mitochondrial dysfunction and loss of
mitochondrial membrane potential lead to a rapid increase in
mROS production, which has been described to activate the
inflammasome through the activity of thioredoxin-interacting
protein (TXNIP).
16
In support of this mechanism, most known
NLRP3-activating stimuli induce ROS generation and specific
mitochondria-targeted ROS scavengers have been shown
to inhibit inflammasome assembly.
17
The existence of a
convergent pathway involving ion flux, particularly of potas-
sium, and ROS generation in triggering the assembly of the
inflammasome has been suggested, however such a link has
remained elusive.
18,19
In this study, we tested the hypothesis that P2X
7
purinergic
receptor activation with extracellular ATP induces mitochon-
drial ROS generation and this effect is mediated by cytosolic
and mitochondrial potassium depletion. We applied a novel
intracellular potassium sensor to characterize the real-time
dynamics of potassium mobilization in the mouse macrophage
cell line J774A.1 after stimulation with ATP. By co-localizing
1
Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, 85287 AZ, USA and
2
Biological Design Graduate Program,
School of Biological and Health Systems Engineering, Ira A. Fulton Schools of Engineering, Arizona State University, Tempe, 85287 AZ, USA
*Corresponding author: DR Meldrum, Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, 1001 S. McAllister Avenue, PO Box
877101, Tempe, AZ 85287-7101, USA. Tel: 480 727 9397; Fax: 480 965 2337; E-mail: Deirdre.Meldrum@asu.edu
Received 08.5.15; revised 27.8.15; accepted 31.8.15; Edited by G Amarante-Mendes
Abbreviations: NLRP3, NLR family, pyrin domain-containing 3; IL-1β, interleukin-1 beta; P2X
7
, purinergic receptor P2X, ligand-gated ion channel, 7; ROS, reactive
oxygen species; mROS, mitochondrial reactive oxygen species; TXNIP, thioredoxin-interacting protein; LPS, lipopolysaccharide
Citation: Cell Death and Disease (2015) 6, e1954; doi:10.1038/cddis.2015.277
&
2015 Macmillan Publishers Limited All rights reserved 2041-4889/15
www.nature.com/cddis
the sensor signal to mitochondria using a mitochondria-
specific dye, we observed a P2X
7
-dependent mitochondrial
potassium depletion that was sensitive to pharmacological
and ionic inhibition. Temporally, mitochondrial potassium
mobilization occurred before potassium efflux-dependent
mitochondrial ROS generation. Further study identified a
critical role for calcium influx upstream of mitochondrial ROS
generation, inflammasome assembly and pro-inflammatory
cytokine release. We report here the first-ever multiplexed
imaging of intracellular potassium and calcium in live cells and
our finding that potassium efflux is required for sustained
calcium influx, while calcium chelation had no effect on the
kinetics of potassium efflux. We propose that mitochondrial
ROS generation is a downstream effect of potassium
efflux-dependent calcium influx and defines a coordinated, ion
flux-driven regulation of the NLRP3 inflammasome via
oxidative signaling.
Results
P2X
7
receptor-dependent potassium efflux induces
inflammasome activation in J774A.1 macrophages. Our
first objective was to determine the response of the J774A.1
mouse monocyte/macrophage cell line to extracellular ATP.
As expected, immunoblotting indicated that untreated
J774A.1 lacks proIL-1βwhile maintaining constitutive levels
of procaspase-1 (Figure 1a). Upon priming with E. coli
lipopolysaccharide (LPS), proIL-1βprotein becomes highly
expressed. Release of active caspase-1 p10 and mature
IL-1βp17 was detected in concentrated supernatants of LPS-
primed J774A.1 after treatment with 3 mM extracellular ATP.
The release of both active components was abolished in the
presence of high extracellular potassium (to suppress the
intracellular–extracellular concentration gradient) as well
as the selective, competitive, P2X
7
receptor antagonist
A438079.
20
The requirement for potassium efflux in
inflammasome-mediated pyroptotic cell death was confirmed
by propidium iodide staining and live-cell imaging (Figure 1b).
Combined LPS and ATP treatment resulted in a time-
dependent accumulation of cells positive for propidium iodide
that was inhibited in the presence of 130 mM extracellular
potassium. Further, tagging of activated caspase-1 with the
fluorescent marker FLICA revealed the assembly of the
inflammasome as indicated by the presence of classical
perinuclear caspase-1-positive specks that were significantly
suppressed by high extracellular potassium and treatment
with A438079 (Figures 1c and d). We performed co-
localization experiments to confirm that the triggered
structure was the NLRP3 inflammasome and contained
ASC as expected (Supplementary Figure S1). Thus,
J774A.1 exhibits the 1st/2nd signal (LPS priming and ATP
stimulation, respectively) behavior representative of the
potassium efflux-dependent NLRP3 inflammasome pathway
in macrophages.
ATP-induced calcium influx regulates the NLRP3 inflam-
masome. We next sought to determine the role of
ATP-induced calcium influx on inflammasome activation.
A previous study has shown that intracellular calcium
chelation with BAPTA-AM suppresses IL-1βprocessing and
release upon ATP-induced inflammasome activation.
14
In agreement with this observation, we found that BAPTA-
AM significantly suppressed ATP-induced IL-1βprocessing
and release as indicated by ELISA in J774A.1 cell super-
natants (Figure 2a). It has not yet been reported whether
calcium chelation suppresses IL-1βprocessing and release
upstream or downstream of inflammasome assembly, though
some reports propose a possible calcium influx-dependent
lysosomal exocytosis pathway for IL-1βrelease.
21
FLICA was
used to observe ATP-induced inflammasome assembly as
indicated by perinuclear caspase-1 specks. While stimulation
with 3 mM ATP resulted in perinuclear caspase-1 speck
appearance indicative of inflammasome assembly, chelation
with BAPTA-AM significantly inhibited inflammasome forma-
tion (Figures 2b and c). These results suggest that calcium
influx regulates ATP-induced NLRP3 activation upstream of
inflammasome formation.
Direct visualization of potassium mobilization in macro-
phages with a novel intracellular sensor. To better under-
stand the intracellular potassium dynamics triggered by ATP-
induced NLRP3 inflammasome activation, we utilized KS6
(Figure 3a), a novel intracellular potassium sensor that
predominantly localizes to mitochondria, although back-
ground staining can be variably observed in other cellular
compartments in a concentration- and cell type-specific
manner.
22
KS6 is sensitive to potassium in the physiological
range (Figure 3b). Further, KS6 is highly selective for
potassium against other biological monovalent and divalent
ions such as iron(II), iron(III), copper, manganese, calcium,
zinc, magnesium and sodium.
22
We first confirmed the
sensor localization in J774A.1 cells. Live-cell imaging
revealed a strongly enriched signal from KS6 in the
mitochondrial matrix as verified by co-staining with Mito-
Tracker Green FM. Co-localization analysis with the CoLoc2
FIJI plugin determined a Spearman’s rank correlation value of
0.799, indicating a predominantly mitochondrial localization
(Figures 3c–f). Initial studies characterizing the performance
of the sensor in HeLa and U87 cells at concentrations of 2 μM
yielded predominant co-localization with mitochondria with
only minor background staining in other cellular
compartments.
22
Based on consultation with the sensor
development team, 5 μM was chosen as the appropriate
concentration for J774A.1 cells. We found that loading with
5μM KS6 enabled dynamic potassium imaging of both
mitochondrial and cytosolic potassium content with no
phenotypic effect on cell viability in J774A.1 cells. Thus,
KS6 is suitable for detection of subcellular potassium content.
We next confirmed that whole-cell KS6 signal responds to
ATP-induced P2X
7
activation. P2X
7
engagement results in the
opening of a non-specific cation pore and potassium efflux
down the intracellular–extracellular potassium concentration
gradient.
23
We probed this response by demonstrating a live-
cell titration between normal (130 mM) and intermediate
(50 mM) concentrations of extracellular potassium added to
the cell culture medium (Figure 4a). In control experiments, we
found that addition of equimolar (130 mM) concentrations of
extracellular sodium had no effect on KS6 sensor response,
indicating insensitivity to osmolarity effects (Supplementary
K
+
regulates Ca
2+
flux in inflammasome signaling
JR Yaron et al
2
Cell Death and Disease
Figure 1 P2X
7
-induced potassium efflux regulates NLRP3 inflammasome assembly and pyroptotic cell death. (a) Immunoblot analysis of procaspase-1 p45 and activated
p10 fragments, and proIL-1β(34 kD) and mature (17 kD) fragments in the lysates and concentrated supernatants of J774A.1 cells primed for 4 h with 1 μg/ml LPS and stimulated
with 3 mM ATP for 30 min with or without addition of 130 mM extracellular KCl or 25 μM of the P2X
7
antagonist A438079. (b) Propidium iodide in J774A.1 cells primed with LPS for
4 h and stimulated with ATP in the presence or absence of 130mM extracellular KCl. (c) Caspase-1 FLICA staining (green) in J774A.1 cells untreated or primed for 4 h with 1μg/ml
LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 130 mM extracellular KCl or 25 μM A438079. Arrows: caspase-1 specks indicative of inflammasome
assembly. Scale bar represents 50 μM. Nuclei are stained with NucBlue Fixed DAPI solution (blue). (d) Image cytometry analysis of inflammasomes detected by Caspase-1
FLICA. J774A.1 cells untreated or primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 130 mM extracellular KCl or 25 μM
A438079. Bar graph represents mean counts and standard error from at least 4000 cells for each condition from at a minimum of three fields in two independent experiments.
Statistics were calculated by one-way ANOVA with Tukey’spost hoc analysis
Figure 2 Calcium influx is an upstream regulator of IL-1βrelease and NLRP3 inflammasome assembly. (a) ELISA analysis of released IL-1βfrom J774A.1 cells primed with
1μg/ml LPS for 4 h and stimulated with 3 mM ATP for 30 min. Where indicated, cells were pretreated with 100 μM BAPTA-AM before addition of ATP. Statistics were calculated by
one-way ANOVA with Tukey’spost hoc and represent the mean and standard error of two independent experiments. (b) Cells were prepared as in (a), except for the addition of
caspase-1 FLICA 1 h before the addition of ATP (green). Arrows indicate perinuclear caspase-1 specks. Blue fluorescence indicates nuclei stained with NucBlue Fixed DAPI
solution. Scale bar represents 50 μM. (c) Image cytometry analysis of inflammasomes detected by caspase-1 FLICA. Bar graph represents mean counts and standard error from
at least 4000 cells for each condition from at a minimum of three fields in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey’spost hoc
analysis
K
+
regulates Ca
2+
flux in inflammasome signaling
JR Yaron et al
3
Cell Death and Disease
Figure S2). Next, we tested whether differing concentrations of
extracellular ATP would result in a dose-dependent opening of
the P2X
7
pore. It was recently reported that membrane
permeability is dose dependently related to P2X
7
receptor
activation, but it is unknown whether potassium efflux is
similarly dose dependent.
24
We observed a dose dependence
of both potassium efflux and membrane permeability as
indicated by the response in the KS6 potassium sensor and
uptake of the membrane impermeable DNA dye TO-PRO-3,
respectively (Figures 4b and c). Both events were dependent
on P2X
7
activity as inhibition with A438079 suppressed both
events (Figures 4d and e). Importantly, our single cell
microscopic data confirm previous reports that the threshold
for potassium concentration required for ATP-induced inflam-
masome activity is approximately 50-60% of basal levels,
corresponding to a total cellular potassium concentration of
about 60–80 mM. Taken together, these experiments demon-
strate the ability to directly visualize P2X
7
-dependent intracel-
lular potassium dynamics in live macrophages.
Extracellular ATP mobilizes mitochondrial potassium
downstream of P2X
7
engagement. Mitochondrial potassium
represents a significant portion of total cellular potassium, as
its concentration is nearly twice (200–300 mM) that of the
cytosol (100–150 mM).
25
Because extracellular ATP is
detected at plasma membrane-localized P2X
7
receptors
resulting in cytosolic potassium efflux, we sought to determine
whether the mitochondrial potassium pool was mobilized by
macrophage purinergic signaling. We used KS6 and
co-localized the signal with MitoTracker Green FM to observe
the kinetics of mitochondrial potassium in live J774A.1 cells
(Figure 5). Real-time monitoring of KS6 signal during P2X
7
engagement with 3 mM extracellular ATP revealed both
cytosolic and mitochondrial potassium depletion. The deple-
tion of cytosolic and mitochondrial potassium was suppressed
by 130 mM extracellular potassium or by inhibition of P2X
7
with
A438079. Notably, the loss of potassium occurred prior to the
shrinking and disintegration morphology indicative of mito-
chondrial damage.
Mitochondrial ROS generation is essential for pyroptosis
in J774A.1 macrophages. We next determined that mROS
was necessary for the assembly and function of the
inflammasome. LPS-primed J774A.1 cells were treated with
ATP with and without pre-treatment with the mitochondria-
localized reactive oxygen scavenger MitoTEMPO. Previous
studies have shown that MitoTEMPO is effective in inhibiting
pyroptosis and release of IL-1β.
17
Here, we show that the
assembly of the inflammasome speck, as indicated by FLICA
labeling for caspase-1, is strongly inhibited in the presence of
MitoTEMPO (Figures 6a and b). We further validate the need
for mROS in inflammasome function by demonstrating an
inhibition of caspase-1 p10 and IL-1βp17 processing and
release when cells are treated with ATP in the presence of
MitoTEMPO as detected by immunoblotting (Figure 6c).
Finally, we demonstrate that pyroptotic cell death requires
mROS by measurement of lactate dehydrogenase activity in
cell supernatants (Figure 6d). These results demonstrate the
need for mROS in the assembly and activity of the
inflammasome.
P2X
7
-dependent potassium and calcium ion flux is
essential for mitochondrial mROS production. Direct
visualization revealed that the mitochondrial potassium pool
Figure 3 KS6 localizes to predominantly mitochondria in live cells. (a) Chemical structure of the intracellular potassium sensor KS6. (b) Spectrofluorophotometric
characterization of KS6 sensor signal response to potassium titration in solution. (c) J774A.1 cells were stained with KS6 intracellular potassium sensor and MitoTracker Green
FM before imaging by confocal microscopy. (d) Inset of boxed region from (c) displaying the overlap of MitoTracker Green FM and KS6. (e) Signal from MitoTracker Green FM.
(f) Signal from KS6. Arrows indicate discrete mitochondria clearly stained for both probes. Scale bar represents 20 μMin(c) and 10 μMin(d)
K
+
regulates Ca
2+
flux in inflammasome signaling
JR Yaron et al
4
Cell Death and Disease
responds to receptor-mediated changes in intracellular
potassium, hence, we sought to determine if ion flux had an
effect on pro-inflammatory mitochondrial signaling. Using the
mitochondria-targeted ROS probe MitoSOX, we investigated
whether potassium efflux and calcium influx had an effect on
ROS production (Figures 6e and f). Results indicated a
substantial increase in MitoSOX signal when LPS-primed
cells were stimulated with ATP. In support of a role for P2X
7
signaling in this response, inhibition of the channel with
A438079 reduced levels of mROS production to that of basal
levels seen in LPS-primed J774A.1 cells. Importantly, both
potassium efflux and calcium influx were necessary for the
generation of mROS as treatment with 130 mM extracellular
KCl (Figure 6e) or BAPTA-AM (Figure 6f) resulted in strong
suppression of MitoSOX oxidation.
Comparing the kinetics of MitoSOX oxidation and potassium
efflux in the mitochondria, we find that potassium mobilization
is a rapid event and likely occurs upstream of ROS generation.
While this is difficult to directly correlate due to KS6 exhibiting
rapid response dynamics while MitoSOX converts by a
comparatively slow process, the seconds-scale response of
potassium efflux is notably quicker than the apparent minutes-
scale generation of ROS. These results taken together
suggest that calcium and potassium flux triggered by P2X
7
activation results in mitochondrial ion imbalance and mROS
generation upstream of NLRP3 inflammasome activation.
ATP-induced potassium efflux is required for calcium
influx. While both potassium and calcium are implicated in
ATP-induced inflammasome activation, it is unclear whether
there is a relationship between them.
1
To investigate the
dynamics of ATP-induced ion flux, we performed multiplexed
imaging of potassium and calcium by combining the KS6
intracellular potassium sensor with Fluo-4, a commercially
available calcium indicator (Figure 7). Results showed that
LPS priming alone had no dramatic effect on ion content;
calcium transients were apparent but overall signal was
stable for both calcium and potassium. Upon ATP stimulation,
a rapid calcium signal spike occurred, followed by a second,
more sustained increase in calcium signal. This bi-phasic
Figure 4 Real-time P2X
7
-dependent intracellular potassium dynamics observed with KS6. (a) Kinetic trace of potassium efflux from J774A.1 cells stimulated with 5 mM ATP
at the indicated time point in the presence of 0 mM additional KCl (normal DMEM medium), 50 or 130 mM additional extracellular KCl. Traces represent the mean and standard
error of 10–20 cells in each field. (b) Response at 40 min of potassium efflux (top panel) or TO-PRO-3 uptake (bottom panel) of J774A.1 cells primed for 4 h with1 μg/ml LPS and
treated with 1, 3 or 5 mM extracellular ATP. Bars represent mean and standard deviation of 20 cells in each condition. Statistics were performed by one-way ANOVA with Fischer’s
LSD comparison test. *Indicates Po0.05 and **** indicates Po0.0001. (c) Representative fields at the indicated time points of LPS-primed J774A.1 loaded with KS6 (red) and
treated with 1, 3 or 5 mM extracellular ATP in the presence of TO-PRO-3 (cyan). Scale bar represents 50 μM. Pre-treatment of LPS-primed, ATP-stimulated J774A.1
macrophages with the P2X
7
inhibitor A438079 suppresses (d) potassium efflux (e) and membrane permeability. Traces represent mean and standard error for five representative
cells. Results are representative of at least two experiments
K
+
regulates Ca
2+
flux in inflammasome signaling
JR Yaron et al
5
Cell Death and Disease
calcium response is indicative of the kinetics associated with
rapid, endoplasmic reticulum-stored calcium ahead of plasma
membrane-localized calcium entry from the extracellular
environment.
26
Importantly, potassium flux occurred concur-
rently with the second phase of calcium influx, but was stable
through the initial calcium spike. Calcium chelation with
BAPTA-AM resulted in a suppression of calcium dynamics,
but had no effect on the ability for ATP to induce potassium
depletion. This suggests that potassium flux is upstream of
calcium flux. Addition of extracellular potassium had no effect
on the initial calcium spike after ATP addition, but suppressed
the second, sustained calcium rise. Together, these results
suggest that ATP-induced potassium efflux is upstream and
necessary for plasma membrane-associated calcium influx,
but not transient store-associated calcium spikes.
Discussion
In this study, we investigated the question of how ion flux and
mitochondrial reactive oxygen interact upstream of inflamma-
some assembly. Both of these phenomena are recognized as
key mediators of inflammasome regulation and have been
separately suggested as the common induction mechanism
for inflammasome assembly. Despite the proposed link
between potassium efflux and mitochondrial signaling result-
ing in inflammasome assembly, this association had yet to be
observed.
1,18,19,27
Further, there has been limited evidence
regarding the relationship between calcium and potassium.
Thus, it has been unclear how ion flux and oxidative signaling
contribute to inflammasome regulation.
The effect of potassium blockade is often used as a test for
basic characterization of both new activators and inhibitors of
the inflammasome because of its long-standing and appar-
ently ubiquitous participation in linking stimulus detection and
inflammasome assembly. Details of how potassium affects the
inflammasome have not been adequately described, however,
because of technical limitations in potassium measurement.
The most common methods for determining the role of
potassium in various aspects of the inflammasome pathway
are either blockade with high extracellular potassium or
quantitation of potassium content by spectroscopy or photo-
metry of bulk populations lysed in nitric acid.
9,18,28
While high
extracellular potassium is effective for determining how
blockade of potassium efflux affects downstream phenotypes,
which we have also used in this study, it obscures intermediate
responses and is unable to reveal cellular kinetics. Likewise,
bulk cell potassium determination can provide only low-
resolution kinetic details and completely obscures the
contribution of individual cells or subpopulations in the
response to stimuli. The latter point was recently identified to
be an essential characteristic of inflammasome-associated
response by macrophages. Specifically, it was observed by
single cell analysis that IL-1βwas released in a bursting
manner only from cells dying by pyroptosis.
29
This opposes
the long-standing paradigm of secretion by various contro-
versial pathways.
30
This observation highlights the importance
of investigating inflammasome-associated cellular processes
at the single cell level and avoiding reliance on bulk cell
determination methods.
Figure 5 P2X
7
activation results in mitochondrial potassium mobilization. J774A.1 cells were primed for 4 h with 1 μg/ml LPS and loaded with 5 μM KS6 (red) and 10 nM
MitoTracker Green FM (green). Real-time confocal microscopy was performed to track the potassium dynamics after stimulation with 3 mM extracellular ATP with or without the
P2X
7
inhibitor A438079. Results revealed a rapid, receptor-dependent mobilization of potassium as indicated by a reduction in KS6 signal in the co-localized space with
MitoTracker Green FM that was sensitiveto inhibition with A438079. Subsequent to the mobilization, mitochondria appeared to fragment. Fields are representative of at least 3–5
experiments. Scale bar represents 20 μM
K
+
regulates Ca
2+
flux in inflammasome signaling
JR Yaron et al
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Cell Death and Disease
While calcium indicators are well established and exten-
sively used, existing methods for probing potassium are
lacking. To date, Arlehamn et al.
31
have reported the only
live-cell imaging experiments on potassium in macrophages
stimulated to undergo inflammasome assembly by infection
with Pseudomonas. A major limitation to this study, however, is
their application of PBFI for live cell potassium readout. PBFI is
a potassium sensor that exhibits nearly equivalent sensitivity
to sodium ions as it does to potassium ions, which makes its
readout difficult to interpret.
32
Further, PBFI has a Kd of
o10 mM and its response saturates at approximately 50 mM,
a concentration lower than the threshold for inflammasome
activation as reported by others and reaffirmed in this study.
Accordingly, upstream dynamics are obscured by a saturated
sensor response and a depletion of cellular potassium can
only be detected upon cell death with PBFI. Finally, PBFI is
necessarily excited with UV light and therefore induces
phototoxic damage to cells under observation, potentially
obscuring the effects of stimulus-induced cell death. For the
current studied we applied KS6, an improved intracellular
Figure 6 Mitochondrial ROS is essential for ATP-evoked inflammasome activity and is driven by P2X
7
-dependent cation flux in J774A.1 cells. (a) Caspase-1 FLICA staining
(green) in J774A.1 cells primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 500 μM MitoTEMPO treatment. Arrows point to
caspase-1 specks indicative of inflammasome assembly. Scale bar represents 50 μM. Nuclei are stained with NucBlue Fixed DAPI solution (blue). (b) Image cytometr y analysis of
inflammasomes detected by caspase-1 FLICA. Bar graph represents mean counts and standard error from atleast 4000 cells for each condition from at a minimum of three fields
in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey’spost hoc analysis. (c) Immunoblot analysis of pro-caspase-1 p45 and activated p10
fragments, and proIL-1β(34 kD) and mature (17 kD) fragments in the lysates and concentrated supernatants of J774A.1 cells primed for 4 h with 1 μg/ml LPS and stimulated with
3 mM ATP for 30 min with or without pretreatment with 500 μM MitoTEMPO. (d) Assessment of lactate dehydrogenase (LDH) activity in the supernatants of J774A.1 cells primed
with 1 μg/ml LPS and stimulated with ATP for 30min with or without pretreatment with 500 μM MitoTEMPO. Results are fold-change versus LPS primed cells and error bars
represent standard error of two independent experiments. ** Indicates Po0.01 by one-way ANOVAwith Tukey’spost hoc comparison. (eand f) J774A.1 cells were left untreated,
primed with LPS for 4 h, or primed with LPS and treated with 3 mM ATP as indicated. MitoSOX (red) was added to all samples 15 min after ATP addition and incubated for an
additional 15 min before live-cell imaging. (e) Evaluation of the role of P2X
7
and potassium efflux in mROS generation. 130 mM KCl and 25 μM A438079 were added 15–20 min
before ATP addition. (f) Evaluation of the role of calcium influx in mROS generation. 100 μM BAPTA-AM was added 15–20 min before ATP addition. Nuclei are stained with
Hoechst 33342 (blue). Scale bar is 50 μm. Results are representative of at least two independent experiments
K
+
regulates Ca
2+
flux in inflammasome signaling
JR Yaron et al
7
Cell Death and Disease
potassium sensor, which addresses the drawbacks of PBFI.
22
KS6 is excited by visible light, is strongly selective for
potassium relative to sodium and other monovalent and
divalent ions, readily taken up by live cells, and is sensitive to
potassium across a supraphysiological range. Here we show
that KS6 can be effectively used for observing dynamic
responses of potassium in live cells and, further, that it can be
multiplexed with other intracellular indicators for analytes such
as calcium.
Detection of extracellular ATP by the P2X
7
purinergic
receptor is a prototypical stimulus for NLRP3 inflammasome
activation.
10,28
Potassium efflux and ROS generation have
independently been proposed as downstream effects of P2X
7
activation.
33
By live-cell imaging, we identified a rapid
and robust mitochondrial potassium mobilization associated
with P2X
7
engagement. P2X
7
is expressed on the plasma
membrane and its sensing activity is therefore localized
distally to mitochondrial responses.
34
We propose that
mitochondria mobilize their potassium pool as a response to
changes in cytosolic potassium levels, which is directly
responsive to P2X
7
activity. This provides additional support
for the observation that mitochondria respond to cytosolic
potassium levels, as it was previously shown that mitochondria
are capable of sequestering and buffering cytosolic
potassium.
35
A mitochondrial potassium buffering mechanism
is further supported by our finding that efflux is ATP dose
dependent. P2X
7
has multiple ATP-sensing sites that have
been shown to dose dependently affect the level of pore
dilation permissive to ion flux.
36
It is still not well understood
how mitochondria respond to cytosolic potassium, but we
speculate that this may occur either through mitochondrial
potassium channels or transient leakage associated with
membrane potential changes.
37
A pharmacologic screen with
readout from KS6 will help to identify channels and signaling
pathways that result in mitochondrial sensitivity to cytosolic
potassium. Our data suggest that the magnitude of P2X
7
activation dictates the degree of potassium efflux, and
therefore inflammasome assembly responses.
Intracellular ion homeostasis is essential for maintenance of
mitochondrial integrity as mitochondrial membrane potential is
heavily dependent on charge distribution.
8,37
We hypothe-
sized that the mitochondrial potassium loss we observed
would be correlated with mitochondrial reactive oxygen
production. In support of this, we found that blockade with
Figure 7 Real-time, multiplexed visualization of ATP-induced potassium and calcium dynamics. J774A.1 cells were primed for 4 h with 1 μg/ml LPS, stained with KS6 and
Fluo-4 DIRECT and both fluorophores were imaged simultaneously by confocal microscopy. (a) Representative fields for each condition showing Fluo-4 and KS6 signal
responses. 16-color pseudocolor look-up tables were used for visualization. Blue indicates low signal intensity and red indicates high signal intensity. Scale bar represents 25 μM.
(b) Mean and standard error for 30 representative cells in each condition. Where indicated cellswere stimulated with 3 mM ATP. Inhibitors were added 15–20 min before imaging.
Results are representative of at least two experiments
K
+
regulates Ca
2+
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JR Yaron et al
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Cell Death and Disease
high extracellular potassium or the P2X
7
inhibitor A438079
suppresses the elevated levels of mitochondrial ROS
observed with ATP treatment. Likewise, we found that
potassium blockade and calcium chelation suppress mROS
generation. We report here the first-ever multiplexed imaging
of potassium and calcium in live cells. This approach allowed
us to demonstrate that potassium efflux is upstream of, and
necessary for, sustained calcium influx in response to ATP
treatment. These results suggest a mechanism whereby
potassium efflux triggered by P2X
7
activation regulates
calcium influx, ultimately resulting in mROS generation
leading to NLRP3 inflammasome activation, possibly through
mitochondrial calcium overload (Figure 8). Additional studies
may help to better define the potassium-sensitive link between
mitochondrial signaling and NLRP3 inflammasome assembly.
We propose that rapidly activating intermediates such as
TXNIP or Syk may be appropriate targets for providing this link
based on the association between both of these targets and
mitochondrial ROS in the NLRP3 inflammasome signaling
pathway.
6,12,16,38,39
Recently, a study was published by Katsnelson et al.
40
reporting that potassium efflux induced NLRP3 inflammasome
assembly independently of calcium influx. Among the conclu-
sions of this paper are that (1) potassium efflux is sufficient for
inflammasome assembly independent of calcium flux and
(2) calcium flux antagonists, such as BAPTA, inhibit the
inflammasome by an off-target mechanism dissociated from
their calcium-related effects. Here, we report that potassium
efflux leads to calcium influx and that both ion fluxes
are necessary for ATP-induced inflammasome assembly.
To demonstrate this, we showed that inhibition of potassium
efflux also inhibited calcium influx and that calcium chelation
had no effect on potassium efflux. Our results do not directly
contradict the results of Katsnelson et al. as they did not
interrogate the effects of potassium efflux inhibition on calcium
content and their calcium and potassium experiments were
performed on independent populations of cells with disparate
temporal resolution. Also, Katsnelson et al.
40
fail to reproduce
earlier data showing that inflammasome activity is inhibited in
calcium-free medium as reported by Murakami et al.
13
Regarding off-target inhibitor effects, Katsnelson et al.
propose that chelation of zinc ions by BAPTA may cause
calcium-independent inflammasome inhibition. This mechan-
ism of inhibition would be contrary to the previously described
promotion, not inhibition, of inflammasome assembly due to
zinc ion chelation.
41
This study establishes a previously unknown relationship
between potassium and calcium during purinergic receptor-
dependent activation of the NLRP3 inflammasome. Namely,
potassium efflux is the dominant regulatory ion upstream of
calcium influx, both of which are required for mitochondrial
oxidative signaling leading to NLRP3 inflammasome activa-
tion. This finding reconciles the observation that intervention in
calcium signaling can modulate inflammasome signaling, but
treatment with calcium ionophores is insufficient for stimulat-
ing IL-1βprocessing and release.
10,13
This study also provides
the first highly selective, real-time observation of ATP-induced
potassium dynamics as well as the first multiplexed imaging of
calcium and potassium in live cells. We propose that future
work toward elucidating the NLRP3 inflammasome pathway
will benefit from application of real-time visualization of
potassium and calcium ion dynamics.
Materials and Methods
Cell culture. The mouse macrophage cell line J774A.1 (TIB-67) was obtained
from ATCC (Manassas, VA, USA) and cultured in DMEM containing 10% FBS,
100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Grand Island, NY, USA) at
37 °C with 5% CO
2
in a humidified atmosphere. Cells were passaged by scraping
and viability and density were assessed by Trypan Blue dye exclusion on a
Countess automated cell counter (Life Technologies, Grand Island, NY, USA).
KS6 potassium sensor loading. KS6 (ex/em 561/630 nm) was kept in a
1-mM DMSO stock solution stored at 4 °C. To facilitate consistent dye distribution,
Figure 8 Proposed mechanism for ion flux-dependent regulation of the NLRP3 inflammasome. Activation of the P2X
7
receptor pathway with extracellular ATP results in the
exchange of potassium and calcium across the plasma membrane, dominantly regulated by efflux of potassium from the cytosol to the extracellular space. Influx of calcium
causes a mitochondrial calcium overload resulting in mitochondrial destabilization and mROS generation, which activates the NLRP3 inflammasome through an unknown
mechanism, but possibly by involvement of TXNIP or Syk.
6,12,16,38,39
P2X
7
receptor activation also results in a mitochondrial potassium efflux that may be involvedin mitochondrial
destabilization and mROS generation. Inhibition of potassium efflux prevents calcium influx and downstream mROS generation. Likewise, chelation of intracellular calcium
prevents mROS generation
K
+
regulates Ca
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flux in inflammasome signaling
JR Yaron et al
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Cell Death and Disease
stock KS6 was combined 1:1 with 10% Pluronic F127 and mixed thoroughly by
pipetting before loading.
42
The mixture was added 1:100 to each well of a chamber
slide for a final KS6 concentration of 5 μM and incubated for 30–60 min at 37 °C.
Where indicated, cells were subsequently stained with 10 nM MitoTracker Green FM
(Molecular Probes, Eugene, OR, USA). Co-localization analysis was performed
using ImageJ/FIJI version 1.49 f and the CoLoc 2 plugin.
43
KS6 was developed in-
house
22
(Center for Biosignatures Discovery Automation, Arizona State University,
Tempe, AZ, USA).
Live-cell imaging. Cells seeded in an 8-chamber μ-slide (Ibidi, Verona, WI,
USA) were primed with 1 μg/ml E. coli O111:B4 LPS (Sigma-Aldrich, St. Louis, MO,
USA) for 2–4 h followed by the addition of inhibitors or other treatment as indicated,
then stimulation with 1–5 mM ATP (Sigma-Aldrich). Alternately, cells were stimulated
by addition of 20 μM nigericin (#11437, Cayman Chemical, Ann Arbor, MI, USA).
Samples were imaged on a Nikon Ti microscope equipped with a C2si confocal
scanner (Nikon Instruments, Melville, NY, USA) and Tokai Hit stage-top incubator
(Tokai Hit Co., Shizuoka, Japan). Excitation laser lines were 408, 488, 561 and
639 nm and emission was collected by photomultipliers filtered for the standard
DAPI, FITC, TRITC and Cy5 bandwidths. Objectives used were × 20 air 0.75 NA,
× 60 oil immersion 1.4 NA or × 60 water immersion 1.2 NA, all from Nikon. Where
indicated, cells were imaged in the presence of 5 μM TO-PRO-3 (Molecular
Probes). For calcium imaging, cells were loaded with 1 × Fluo-4 DIRECT solution
(Molecular Probes) and incubated for 30–60 min before imaging. For analysis of
mitochondrial ROS production, MitoSOX red (Molecular Probes) was added 15 min
after stimulation with ATP.
Caspase-1 FLICA assay. J774A.1 cells were seeded at a density of
1–2×10
5
per well in 200 μl of complete DMEM and grown overnight. The following
day cells were primed for 4 h with 1 μg/ml E. coli O111:B4 LPS. During the last hour
of priming, cells were loaded with 1 × FAM-YVAD-FMK (Caspase-1 FLICA;
Immunochemistry Technologies, Bloomington, MN, USA). Inhibitors, as indicated,
were added during the last 15–20 min of priming. Cells were stimulated with 3 mM
ATP for 30 min, subsequently washed 2 × with warm DMEM and fixed in 2%
formaldehyde solution, prepared in PBS from powdered paraformaldehyde, for
10 min at room temperature. Cells were permeabilized in 0.25% Triton X-100 for
10 min, then counterstained with NucBlue DAPI fixed cell stain (Life Technologies),
according to the manufacturer’s instructions in PBS, then rinsed 2x with PBS and
submerged in 200 μl mounting medium (90% glycerol in PBS and 0.1% NaN
3
).
Samples were imaged by laser-scanning confocal microscopy as a series of 0.5 μM
z-stacks on a Nikon Ti microscope equipped with a Nikon C2si confocal scanner
controlled by the Nikon Elements AR software. Caspase-1 FLICA was excited at
488 nm and emission was collected in the FITC channel while NucBlue was excited
at 408 nm and emission was collected in the DAPI channel. Stacks were prepared
as maximum intensity projections using ImageJ/FIJI.
Immunofluorescence. Cells seeded in an 8-chamber μ-slide were primed for
4 h with 1 μg/ml E. coli O111:B4 LPS. Cells were additionally treated with the
caspase-1 inhibitor ac-YVAD-CHO (50 μM) for the last 30 min of priming to inhibit
cell detachment downstream of inflammasome assembly. For inflammasome
stimulation, cells were treated with 3 mM ATP for 1 h. Cells were fixed with 2%
formaldehyde solution, permeabilized in 0.25% Triton X-100 in PBS and blocked in
0.25% Triton X-100 in PBS containing 5% BSA at room temperature. Polyclonal
rabbit Caspase-1 p10 antibody (#SC-514, Santa Cruz Biotechnology, Dallas, TX,
USA) was added 1 : 100 overnight at 4 °C. Secondary antibody, AlexaFluor
488-conjugated anti-rabbit secondary antibody (#A-11034, Life Technologies), was
added 1 : 1000 at room temperature for 1 h. DAPI solution was added using
NucBlue and samples were covered with 150 μl mounting medium (90% glycerol,
10% (10 × ) PBS with 0.01% NaN
3
) and kept at 4 °C until imaging. Inflammasome
images were obtained as 0.5–1μm z-stacks and presented as maximum intensity
projections. For co-localization studies, after caspase-1 FLICA labeling (described
above), cells were immunolabeled similarly except fixed with 4% formaldehyde and
probed with NLRP3 antibody (#SC-66846, Santa Cruz Biotechnology) diluted
1 : 100 or ASC antibody (#AL-177, Adipogen, San Diego, CA, USA) diluted 1:200
overnight, followed by AlexaFluor 568-conjugated anti-rabbit secondary antibody
(#A-11036, Life Technologies) diluted 1 : 1000.
Image cytometry. Confocal z-stacks for nuclear and FLICA channels were
opened in FIJI (ImageJ v1.49 f, http://www.fiji.sc) and converted to maximum
intensity projections. Automatically detected Otsu thresholding was applied to each
channel resulting in a positive signal mask. To each mask, a binary watershed
algorithm was applied to separate closely neighboring objects. To get the final count
for nuclei and FLICA, the Analyze Particles function was applied to each channel
with a size threshold of 20 to infinity square pixels for nuclei and 5 to infinity square
pixels for FLICA foci. The results were analyzed in GraphPad Prism by one-way
ANOVA with Tukey’spost hoc analysis.
Lysate and supernatant protein collection. Cells were seeded in 6-well
plates (10
6
cells/well) and primed for 4 h with 1 μg/ml E. coli O111:B4 LPS in complete
DMEM containing 10% FBS. After priming, cells were washed 1 × with serum-free
DMEM and 1.1 ml of warm serum-free DMEM was added to each well. Where noted,
cells were treated with inhibitors for 15–30min. Inflammasome activation was triggered
by application of freshly prepared 3 mM ATP solution in serum-free DMEM for 30 min.
After stimulation, supernatants were collected and spun at 14 000 × gfor 15 min at 4 °
C to remove cellular debris and approximately 1 ml was transferred to fresh 1.5 ml
tubes. Ten microliters of StrataClean resin (Agilent, Santa Clara, CA, USA) was added
to each supernatant, mixed well and placed on a rotator in a 4 °C refrigerator for 1 h.
Concentrated supernatant protein was collected by pelleting the StrataClean resin,
removing the supernatant and heating the resin resuspended in 50 μl1× Laemmli
buffer at 95 °C for 5 min. Cell lysates were prepared by addition of 100 μlhot1×
Laemmli buffer to each well for 5–10 min, scraping and transferring samples to 1.5 ml
tubes and heating at 95 °C for 5 min.
Immunoblotting. Twelve microliters of concentrated supernatant or lysate was
separated on 4–12% Mini-Protean TGX gels (Bio-Rad, Hercules, CA, USA) at
100 V for 1 h. Proteins were transferred onto 0.2 μm nitrocellulose membranes
(LiCor, Lincoln, NE, USA) at 100 V for 1 h, and subsequently blocked in 5% non-fat
dry milk in PBS containing 0.2% Tween-20 for 1 h. Blocked membranes were
incubated in 5% BSA in PBS containing 0.2% Tween-20 and either 1 : 500 rabbit
polyclonal against Caspase-1 p10 (#SC-514, Santa Cruz) or 1 : 1000 goat
polyclonal against IL-1β(#AF-401-NA, R&D Systems, Minneapolis, MN, USA) and
rotated overnight at 4 °C. The following day, donkey anti-goat IRDye 800CW and
goat anti-rabbit IRDye 680RD secondary antibodies (Li-Cor) were applied at a
dilution of 1 : 15 000 with rocking for 1 h at room temperature. Membranes were
imaged on a Li-Cor Odyssey CLx on auto exposure with high quality setting.
Lactate dehydrogenase release assay. Cells were seeded in 96-well
plates and primed for 4 h with 1 μg/ml E. coli O111:B4 LPS. Cells were treated for
the last 15–30 min with 500 μM MitoTEMPO and stimulated for 30 min with 3 mM
ATP. Fifty microliters of supernatant was used for LDH activity assay with the
CytoTox96 Non-Radioactive Cytotoxicity Kit (Promega, Madison, WI, USA)
according to the manufacturer’s instructions.
ELISA. J774A.1 cells were seeded in 96-well plates at a concentration of 10
5
cells/well and incubated overnight. Cells were primed for 4 h with 1 μg/ml E. coli
O111:B4 LPS and subsequently stimulated for 30 min with 3 mM ATP in 100 μl
medium. Where indicated, cells were treated with 100 μM BAPTA-AM (Tocris,
Minneapolis, MN, USA) for 15 min before ATP treatment. Supernatants were
collected and released IL-1βwas evaluated with ELISA using the R&D Systems
DuoSet kit according to the manufacturer’s protocol. Developed plates were read on
a Biotek Synergy H4 mutli-mode plate reader with Gen5 software.
Statistical analysis. Statistics were performed where indicated with GraphPad
Prism version 6 (GraphPad, La Jolla, CA, USA) and procedures for each analysis
are described in the figure captions.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. Support for this work was provided by the Microscale Life
Sciences Center, an NIH NHGRI Center of Excellence in Genomic Science (5P50
HG002360 to DRM), and the NIH Common Fund LINCS (Library of Integrated
Network-Based Cellular Signatures) program (U01CA164250 to DRM).
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K
+
regulates Ca
2+
flux in inflammasome signaling
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11
Cell Death and Disease
