α-Hemolysin of uropathogenic E. coli regulates NLRP3 inflammasome activation and mitochondrial dysfunction in THP-1 macrophages

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
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α‑Hemolysin of uropathogenic
E. coli regulates NLRP3
inammasome activation
and mitochondrial dysfunction
in THP‑1 macrophages
Vivek Verma1, Parveen Kumar2, Surbhi Gupta1, Sonal Yadav1, Rakesh Singh Dhanda3,
Henrik Thorlacius4 & Manisha Yadav1,4*
Hemolysin expressing UPEC strains have been associated with severe advanced kidney pathologies,
such as cystitis and pyelonephritis, which are associated with an inammatory response. Macrophages
play an important role in regulating an inammatory response during a urinary tract infection. We
have studied the role of puried recombinant α‑hemolysin in inducing inammatory responses and
cell death in macrophages. Acylation at lysine residues through HlyC is known to activate proHlyA
into a fully functional pore‑forming toxin, HlyA. It was observed that active α‑hemolysin (HlyA)
induced cleavage of caspase‑1 leading to the maturation of IL‑1β, while inactive α‑hemolysin
(proHlyA) failed to do so in THP‑1 derived macrophages. HlyA also promotes deubiquitination,
oligomerization, and activation of the NLRP3 inammasome, which was found to be dependent on
potassium eux. We have also observed the co‑localization of NLRP3 within mitochondria during
HlyA stimulations. Moreover, blocking of potassium eux improved the mitochondrial health in
addition to a decreased inammatory response. Our study demonstrates that HlyA stimulation
caused perturbance in potassium homeostasis, which led to the mitochondrial dysfunction followed
by an acute inammatory response, resulting in cell death. However, the repletion of intracellular
potassium stores could avoid HlyA induced macrophage cell death. The ndings of this study will help
to understand the mechanism of α‑hemolysin induced inammatory response and cell death.
A urinary tract infection (UTI) is one of the most common bacterial infections and is the second most common
nosocomial infection1. Advanced stages of UTI involve uropathogenic Escherichia coli (UPEC) infection in the
bladder (cystitis) and kidney (pyelonephritis) leading to sepsis and kidney damage. Such infections are one of
the leading causes of death worldwide1–3. E. coli, generally a commensal bacteria residing inside the distal colon4,
accounts for about 80% of the community-acquired infections of the urinary tract5. A UPEC mediated UTI is
further complicated due to the emergence of the drug-resistant UPEC strain6. erefore, it is very important
to understand the pathogenesis of a UPEC mediated UTI to nd an eective cure for such dreadful infections.
UPEC, which is thought to have arisen from distal gut microora, has to pass through the challenges of the
distinctly dierent habitats of the bladder, kidney and blood to cause an ascending UTI7. With time, UPEC
has found ways to evade the immune system. CFT073, a virulent UPEC strain isolated from a pyelonephritis
patient, was shown to induce cell death in macrophages8. It has been shown that macrophages are important
for the recruitment of neutrophils during an experimental UTI9. UPEC, in comparison to its non-pathogenic
commensal partners, contains extra genes coding for virulence factors which help it during pathogenesis10. e
open
              
India. Department of Urology, University of Alabama At Birmingham, Hugh Kaul Genetics Building,
Birmingham, AL, USA.       
Sweden. Department of Clinical Sciences, Section of Surgery, Malmö, Skåne University Hospital, Lund University,
Malmö, Sweden. *email: manisha.dhanda@gmail.com

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resulting virulence factors can be secreted toxins, such as α-hemolysin (HlyA), cytotoxic necrotizing factor 1
(CNF1), secreted auto-transporter toxin (SAT) and membrane-bound proteins (mbriae: type 1 m, P m, S
m, agellin, lipopolysaccharides and capsule)10.
α-hemolysin (HlyA) is an important toxin in the pathogenesis of UPEC, which mediates more severe forms of
UTI such as cystitis and pyelonephritis11. An inactive α-hemolysin (non-acylated) matures within the cytoplasm
to form an active toxin (acylated) through HlyC (acyltransferase) mediated fatty acylation, before being released
into the extracellular environment12,13. HlyA has a concentration dependent eect on the epithelial cells14 of the
kidney. At higher concentrations, it causes hemolysis of erythrocytes and other cells, thereby helping UPEC to
cross mucosal barriers to cause harm to immune cells and causing the bacteria to access the nutrients of the
hosts15,16. On the other hand, at lower concentrations it leads to apoptosis of host cells, including the immune
cells (neutrophils and T lymphocytes) and kidney cells (renal cells and bladder epithelial) to ultimately cause
exfoliation17–19. Furthermore, HlyA causes a Ca2+ imbalance in renal epithelial cells, followed by the synthesis
of inammatory cytokines IL-6 and IL-820.
An inammatory response is mediated by various pattern recognition receptors (PRRs). Inammasomes are
multi-protein complexes acting as cytosolic PRRs, which are activated during various infections21,22. CFT073, the
UPEC strain, which kills macrophages as mentioned earlier, has been shown to activate the nod-like-receptor
pyrin domain-containing 3 (NLRP3) inammasome8. Contrasting reports have also shown that CFT073 blocks
the activation of NLRP3 inammasome through the virulence factor TcpC23. Aer activation of NLRP3 by
various stimuli, it oligomerizes with apoptosis-associated speck-like protein containing CARD (ASC) and pro-
caspase-1 (48kDa) to mediate an autocatalytic cleavage of procaspase-1 into its functional form, i.e. caspase-1
(10kDa and 20kDa)24,25. A cleaved form of caspase-1, in turn, cleaves proInterleukin (proIL)-1β into its mature
and secretable form that is IL-1β (17kDa)24,25. Interestingly, CFT073 hlyA mutant could not trigger cell death
and IL-1β release in mouse macrophages. However in humans, this mutant strain partially reduced the level of
UPEC-triggered macrophage cell death8. In addition, a random transposon mutant library screen showed that
HlyA had a prominent role in CFT073-triggered cell death in human macrophages26. Bhakdi etal. have shown
that E. coli HlyA induces IL-1β release and cell death into monocytes27. Very recently, it has been shown that the
UPEC α-hemolysin changes the mitochondrial dynamics in a calcium inux dependent manner in rat Sertoli
cells causing mitochondrial dysfunction28. Also, HlyA disrupted cell membrane lead to the release of DAMP
(danger associated molecular pattern), which induces a pro-inammatory response in testicular macrophages28.
Similarly, Schaale etal. mentioned that UPEC exhibits completely dierent behavior in humans and rodents8.
erefore, the current study was designed to study the role of uropathogenic E. coli α-hemolysin in the activation
of NLRP3 inammasome and mitochondrial health, with respect to cell death in human macrophages. For this,
recombinant puried E. coli α-hemolysin was used to understand the functional eect of HlyA in macrophages
derived from a human leukemic cell line, THP-1.
Results
HlyA promotes caspase‑1 cleavage and IL‑1β maturation along‑with oligomerization and deu‑
biquitination of NLRP3 in THP‑1 derived macrophages. Prior to the use of recombinant hemolysin,
the levels of endotoxin in our preparations of HlyA and proHlyA were checked to avoid the synergistic eect of
lipopolysaccharide contamination during various stimulations. e amount of endotoxin contamination in the
preparation of HlyA and proHlyA was found to be 0.0121 ± 0.0002 and 0.0119 ± 0.0001 ng/ml, respectively29.
Recently, Schwarz etal. have reported a minimum amount of endotoxin required to elicit an immune response
in human immune cells to be 0.02ng/ml30. erefore, an insignicant amount of endotoxin contamination was
found in our preparations of HlyA and proHlyA, unable to produce a synergistic eect during stimulations.
Reports suggest that E. coli strains carrying α-hemolysin increase the production of IL-1β from various cell
types8,26,31,32. Similarly, we found that the levels of proIL-1β and procaspase-1 were not signicantly dierent
aer stimulation and an eect was seen on the cleavage of both proteins (Fig.1A) (Supplementary Information
1). HlyA induce signicant cleavage of IL-1β (P ≤ 0.01) and caspase-1 (P ≤ 0.01) in THP-1 derived macrophages
(THP-1m), whereas proHlyA failed to do so (Fig.1A–C). Nigericin (an ionophore previously reported to promote
the cleavage of IL-1β and caspase-1 through activation of NLRP3 in a potassium (K+) dependent manner) was
used as a positive control in our experiment, because α-hemolysin of UPEC also promotes K+ perturbations in
an intracellular mileu33,34.
Deubiquitination of NLRP3 is required for the activation and interaction of ASC, which is rather inhibited
due to the ubiquitination of NLRP3 in resting cells (i.e. primarily macrophages)35,36. erefore, we looked at
the oligomerization of NLRP3 during stimulation with HlyA and proHlyA. We have observed that proHlyA
failed to initiate the oligomerization of NLRP3, whereas both HlyA and nigericin induced oligomerization of
NLRP3 with ASC (Fig.1D) (Supplementary Information 1). is indicates that the pore forming property of an
active α-hemolysin is important in initiating the NLRP3 inammasome formation. Additionally, we checked
the ubiquitination status of NLRP3 during HlyA and proHlyA stimulations. It was found that both HlyA and
nigericin promote the deubiquitination of NLRP3, whereas proHlyA failed to do so (Fig.1E) (Supplementary
Information 1).
HlyA promotes oligomerization and deubiquitination of NLRP3 in a potassium dependent
manner. Klo etal. reported that HlyA promotes intracellular potassium perturbances34 and that potassium
oscillations in cytosol play an important role in the regulation of inammasomes37–39. us, we hypothesized
that HlyA induced inammasomes activation might involve K+ perturbations. erefore, as suggested earlier, we
used a 140mM extracellular potassium (KCl) concentration and 100M of glibenclamide to inhibit the eect of
potassium eux generated during action of pore-forming toxins (PFTs) on NLRP3 inammasome activation39.

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It was observed that HlyA induced the cleavage of IL-1β and caspase-1 was inhibited in the presence of gliben-
clamide and high extracellular KCl (140mM) (Fig.2A–C) (Supplementary Information 1). However, no signi-
cant eect was seen on the pro- forms of IL-1β and caspase-1 levels. is indicates that NLRP3 inammasome
formation is inhibited due to the blockage of potassium eux as proposed by Arlehamn etal.39.
e interaction between NLRP3 and ASC was also investigated by immunoprecipitation of NLRP3 inam-
masome complex by anti-ASC antibody. It was found that NLRP3 oligomerization, due to HlyA stimulation,
was inhibited in the presence of glibenclamide (100M) and higher extracellular K+ (140mM KCl) (Fig.2D)
(Supplementary Information 1). e interaction of NLRP3 with ASC is downstream to the deubiquitination
process, therefore the ubiquitination of NLRP3 during these potassium interventions was also investigated.
Interestingly, we observed that ubiquitination is also a K+ dependent process during HlyA stimulations, because
in the presence of glibenclamide and higher extracellular K+, deubiquitination of NLRP3 was inhibited (Fig.2E)
Figure1. HlyA induces cleavage of IL-1β and caspase-1 with simultaneously aecting oligomerization and
deubiquitination of NLRP3. THP-1m were stimulated with nigericin (30min), HlyA and proHlyA (2h) as
indicated. Mock shows resting macrophages without any stimulation in all blots. (A) Immunoblots showing
pro- and cleaved forms of IL-1β and caspase-1, GAPDH was used as an endogenous control. (B) Bar graphs
showing integrated densitometric value (IDV) of cleaved IL-1β (p17) and (C) cleaved caspase-1 (p20). Results
were expressed as Mean IDV ± SEM and analyzed by using one-way ANOVA with Bonferroni’s post test. (D)
Co-immunoprecipitation of NLRP3 with ASC shows oligomerization of NLRP3 with ASC during various
stimulations. ASC was precipitated with ASC antibody and further NLRP3 was detected by immunoblotting.
ASC was also checked in input lysates through immunoblotting. (E) Shows endogenous ubiquitination of
NLRP3 during stimulation of THP-1m with HlyA and proHlyA for 2h. ASC was also detected in input lysates
through immunoblotting. Blots are representative of three independent experiments. P value is shown as
**p ≤ 0.01, n. s = non signicant.

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(Supplementary Information 1). erefore, these results demonstrated that oligomerization and deubiquitination
of NLRP3 inammasome is dependent on intracellular K+ during HlyA stimulations.
NLRP3 co‑localizes in mitochondria during HlyA stimulation. NLRP3 inammasome plays an
important role in the processing of IL-1β in cytosol to initiate a cascade of pro-inammatory responses. Zhou
etal. reported the presence of NLRP3 in mitochondria and its activation during mitochondrial dysfunction,
leading to cell death in response to various stimulators, such as nigericin, monosodium urate crystals and
alum40. We have not come across any report which shows an interaction of NLRP3 with mitochondria upon
Figure2. Oligomerization and deubiquitination of NLRP3 is dependent on intracellular K+ concentration
during HlyA stimulation. THP-1m cells were treated with 140mM of potassium chloride (KCl) and 100M
of glibenclamide (Gli) for 30min followed by stimulation with HlyA for 2h. (A) Immunoblots showing pro
and cleaved forms of IL-1β and caspase-1; GAPDH was used as endogenous control. (B) Bar graphs showing
integrated densitometric value (IDV) of cleaved IL-1β and (C) cleaved caspase-1, under dierent HlyA
stimulation, as mentioned. Results were expressed as Mean IDV ± SEM and analyzed by using one-way ANOVA
with Bonferroni’s post test. (D) Co-immunoprecipitation with anti-ASC antibody and immunoblotting with
anti-NLRP3 antibody shows oligomerization of NLRP3 with ASC during various stimulations and treatments
as mentioned. ASC was also checked in input lysates by immunoblotting. (E) Co-immunoprecipitation with
anti-ASC antibody and immunoblotting with anti-Ubiquitin antibody shows ubiquitination status of NLRP3
during various stimulations and treatments as mentioned earlier. ASC was also checked in input lysates by
immunoblotting. Blots are representative of three independent experiments. P value is shown as ***p ≤ 0.001, n.
s = non signicant.

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HlyA stimulation. erefore, the localization of NLRP3 in response to HlyA stimulation was investigated. Our
data shows the presence of NLRP3 in mitochondria during HlyA stimulations (arrow in Fig.3A).
To conrm the presence of NLRP3 in mitochondria, we isolated the mitochondrial fractions from THP-1m
aer stimulation with HlyA. NLRP3 was found in the mitochondrial fractions isolated from HlyA- and nigericin-
stimulated THP-1m (Lane 3 and 4, Fig.3B) (Supplementary Information 1). However, NLRP3 was not detected in
mitochondrial fractions of unstimulated THP-1m (Lane 2, Fig.3B). e presence of NLRP3 was also conrmed
as a positive control for NLRP3 detection in the cytoplasmic extracts of unstimulated THP-1m (Lane 1, Fig.3B).
An anti-Tubulin antibody was used to check the purity of isolated mitochondrial fractions and to eliminate the
possibility of the contamination with cytoplasmic NLRP3. An anti-VDAC-1 antibody was used as a loading
control for mitochondrial fractions (Fig.3B) (Supplementary Information 1). Voltage-dependent anion channel
(VDAC-1) is an ion channel in the outer membrane of the mitochondria. e densitometry analysis showed that
NLRP3 in mitochondrial fractions of HlyA- and nigericin-treated cells was signicantly higher (P < 0.01) than
the control group (unstimulated) (Fig.3C).
Figure3. NLRP3 colocalizes in mitochondria during HlyA stimulation. (A) THP-1m cells were stimulated
with HlyA for 2h and then stained with Mitotracker (red) for mitochondria and DAPI (blue) for nucleus.
NLRP3 was probed by anti-NLRP3 antibody and detected by secondary Alexa uor 488 (green) and observed
under ×40 objective through confocal microscopy (Scale = 5m). White arrows indicate the colocalization
(yellow) of NLRP3 (green) with mitochondria (red). Figures are representative of 3 independent experiments.
(B) THP-1m cells were treated with HlyA (2h) and nigericin (30min) as indicated and then followed for
preparation of cytoplasmic extract and mitochondria isolation. Cytoplasmic extracts and mitochondrial
fractions were immunoblotted for the presence of NLRP3, tubulin and VDAC1 proteins. Immunoblot shows
the presence of NLRP3 in mitochondrial and cytoplasmic fractions whereas NLRP3 is present in mitochondrial
fractions of HlyA stimulated cells, while absent in mitochondria from unstimulated THP-1m cells. Tubulin was
immunoblotted to check the purity of mitochondrial fractions for contamination of cytoplasmic content and
VDAC1 was used as a loading control for mitochondrial fractions. Blots are representative of 3 independent
experiments. (C) Bar graph showing integrated densitometric values (IDV) of NLRP3 normalized to VDAC1
in Mitochondrial fractions. Comparisons between multiple groups were made using one-way ANOVA with
Bonferroni’s post test. P value is shown as **p ≤ 0.01.

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Mitochondrial dysfunction is dependent on intracellular potassium ion concentration during
HlyA stimulation of THP‑1m. e eect of HlyA stimulation on the mitochondrial membrane potential
(Ψm) was analyzed using the lipophilic cationic probe JC1 (Fig.4). JC1m onomers give a green uorescence at
529nm. It enters inside mitochondria based on the membrane potential. Once inside the mitochondria, the JC1
form aggregates, this gives a red uorescence at 590nm. So Ψm was measured as a red/green uorescence ratio.
It was found that the HlyA stimulation signicantly reduced Ψm as compared to the resting THP-1m (control)
(p < 0.05) (Fig. 4), whereas pre-treatment of THP-1m cells with glibenclamide and high potassium containing
medium signicantly increased Ψm during HlyA stimulation. Carbonyl cyanide 3-chlorophenylhydrazone
(CCCP), an oxidative phosphorylation inhibitor, was used as a control to induce depolarization of the mito-
chondria (Fig.4). erefore, it was concluded that the K+ concentration perturbations produced during HlyA
stimulation induce mitochondrial dysfunction and repletion of potassium ion could restore the mitochondrial
homeostasis.
α‑hemolysin induces oxidative stress in THP‑1m, whereas repletion of potassium brought
glutathione‑redox status back to normal. GSH (Glutathione) is the major antioxidant defense against
reactive oxygen species (ROS). e antioxidant function of GSH is determined by the redox-active thiol (-SH)
of cysteine that becomes oxidized, when GSH reduces the target molecule41. e ratio of reduced to oxidized
glutathione within cells is oen used as a marker of oxidative stress42. NLRP3 inammasome activated caspase-1
promotes multiple pathways causing mitochondrial disassembly, resulting in dissipation of mitochondrial mem-
brane potential, mitochondrial permeabilization and mitochondrial ROS production43. erefore, we sought to
determine the GSH:GSSG (Glutathione disulde) ratio to check the oxidative stress induced by α-hemolysin in
THP-1m. It was found that during HlyA stimulation GSH level was reduced (Fig.5A) in order to tackle the oxi-
dative stress and is oxidized to GSSG (Fig.5B), thus leading to a low GSH:GSSG ratio (Fig.5C). Upon inhibition
of the K+ eux by using glibenclamide and a high K+ containing medium, the GSH:GSSG ratio was signicantly
improved (Fig.5C).
HlyA promotes mitochondrial biogenesis in THP‑1m upon stimulation. In various pathologies,
it is evident that the increase in mitochondrial dysfunction is combated through mitochondrial biogenesis, in
which dysfunctional mitochondria are replaced with healthy mitochondria to promote cell survival44. erefore,
we investigated the mitochondrial DNA copy number through quantitative real-time PCR. It was found that
mtDNA copies were signicantly (p = 0.0125) increased during HlyA stimulation of THP-1m, which was not
seen on the inhibition of K+ eux (Fig.6), indicating a possible role of potassium eux in the regulation of
mitochondrial biogenesis.
Figure4. HlyA induces mitochondrial dysfunction in a potassium dependent manner. e graph shows
red/green uorescence ratio as a measurement of mitochondrial membrane potential (Ψm) during various
stimulations of THP-1m. Cells were seeded and dierentiated in 96-well clear well black plate and then
stimulated with HlyA (2h) alone or in combination with 30min pretreatment of cells by 100M glibenclamide
(Gli) and 140mM KCl as indicated, followed by staining with JC1 dye for 30min. JC1 remains as a monomer
in the cytoplasm, where it gives a green uorescence, while on its directional uptake inside the mitochondria,
promoted by membrane potential, leads to the formation of JC1 aggregates, which uoresce at red uorescence.
Fluorescence was taken at Ex 485 and Em 530 for green aggregates and Ex 488 and Em 590 for red aggregates.
e graph shows decreased mitochondrial membrane potential (Ψm) during HlyA stimulation, whereas in the
case of pretreatment with Gli and KCl along with HlyA stimulation, Ψm was increased. Comparisons between
multiple groups were made using one-way ANOVA with Bonferroni’s post test. P value is shown as **p ≤ 0.01,
***p ≤ 0.001.

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Quenching of mitochondrial ROS reduced IL‑1β release in HlyA stimulated THP‑1m. Zhou
etal. demonstrated mitochondrial ROS as an essential mediator of NLRP3 inammasome activation during
stimulations, with known NLRP3 activators40. We investigated the role of mitochondrial ROS in NLRP3 activa-
tion by assessing the levels of IL-1β in cell culture medium (Supplementary Information 1), aer stimulations.
HlyA stimulation induced robust IL-1β release from THP-1m as compared to the mock (p ≤ 0.0001) (Fig.7).
Whereas, cells treated with MitoTEMPO along with HlyA stimulation showed a signicant decrease in IL-1β
release (p ≤ 0.0001), in comparison to the cells treated with HlyA alone (Fig.7). ese observations are similar to
the outcomes of the study by Zhou etal.40. Additionally, Heid etal. observed the abrogation of IL-1β release by
using MitoTEMPO in nigericin and ATP treated cells45. Our data also conrms the role of mitochondrial ROS
in NLRP3 mediated IL-1β release during UPEC HlyA stimulation.
UPEC α‑hemolysin induced cell death is reversed by maintaining potassium homeosta‑
sis. Inammasome activation leads to cytokine release and is accompanied by pyroptosis, leading to tis-
sue damage. At lower concentrations, α-hemolysin was observed to induce apoptosis in host cells, including
immune cells (neutrophils and T lymphocytes) and kidney cells (renal cells and bladder epithelial), ultimately
causing exfoliation17–19. Here we assessed LDH release, in terms of percentage cytotoxicity in THP-1m cells
upon HlyA stimulation for 2h, with K+ concentration interventions. An α-hemolysin induced cell death in
THP-1m (% cytotoxicity = 24.01 ± 0.08) was detected (Fig.8). HlyA was reported to trigger K+ perturbances in
the cells34. On the other hand, K+ concentration was reported to play an important role in assembly and activ-
ity of inammasome37–39 and that the inammasome activation can be prevented by blocking K+ eux37–39.
We have found that a higher concentration of K+ (140mM) in cell culture medium reduces cell death (% cyto-
toxicity = 10.51 ± 0.30) (Fig. 8). Additionally, blockage of potassium eux through glibenclamide also caused
a decrease in cell death (% cytotoxicity = 16.38 ± 1.15) during HlyA stimulation (Fig.8). erefore, LDH assay
results showed that upon α-hemolysin stimulation, cells undergo pyroptosis, which can be reversed through
inhibition of potassium eux.
Figure5. α-Hemolysin induces oxidative stress in mitochondria of THP-1m and inhibition of potassium
eux brought glutathione-redox status to normal. GSH:GSSG estimation was performed to evaluate the eect
of α-hemolysin (HlyA) on THP-1m mitochondrial redox state. Additionally, the eect of glibenclamide and
potassium chloride were also assessed on hemolysin induced oxidative stress in THP-1m. THP-1m pre-treated
with glibenclamide (100M, 30min prior to stimulation) and KCl (140mM, 30min prior to stimulation)
were stimulated with HlyA for 2h. Data shown is the average of three independent experiments. Comparisons
between multiple groups were made using one-way ANOVA with Bonferroni’s post test. P value is shown as
*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

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Discussion
α-hemolysin is an important virulence factor of UPEC, especially associated with severe upper tract patho-
genesis of the urinary tract such as cystitis and pyelonephritis. Since it is associated with more than 50% of
cases of severe UTI, therefore, it is thought to play an important role in the pathogenesis of UPEC10,46. Fur-
thermore, hlyA-positive UPEC strains cause more tissue damage than hlyA-negative strains leading to severe
Figure6. Mitochondrial Biogenesis was increased in THP-1m cells upon stimulation with HlyA. mtDNA copy
number was quantied through quantitativereal-time PCR in THP-1m cells. THP-1m cells were stimulated
with HlyA for 2h and prior to stimulation of THP-1m, cells were treated with 140mM of potassium chloride
(KCl) and 100M of glibenclamide (Gli) for 30min as indicated. mtDNA copy numbers were normalized to
the nuclear DNA copy number of 18S and represented as delta-Ct values. Lower delta-Ct indicates an increase
in gene expression and vice versa. e graph shows a signicant (*p = 0.01) increase in mtDNA only during
HlyA stimulation, as compared to the mock and other stimulations of HlyA, where cells were pretreated with
Gli and KCl. Results are representative of three biological and three technical replicates. n.s = non signicant.
Comparisons between multiple groups were made using one-way ANOVA with Bonferroni’s post test.
Figure7. Cytokine IL-1β release during α-hemolysin stimulation along with mitoROS inhibition and blockage
of potassium eux. (A) ELISA was performed to assess IL-1β release in cell culture supernatants during HlyA
stimulation, along with mitochondrial ROS inhibitor (MitoTEMPO 20M) and potassium eux inhibition
(140mM KCl and Glibenclamide 100M). Data is represented as IL-1β concentration in pg/ml (Mean ± SEM).
For statistical analysis, one-way ANOVA with Bonferroni’s test for multiple comparisons was used. P value
is shown as ***p ≤ 0.001. (B) Immunoblot showing the cleaved form of IL-1β in acetone precipitated culture
supernatants.

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clinical complications11,46. Earlier studies pointed towards the role of HlyA in inammatory cell death8,31,47. We
have characterized the role of HlyA in NLRP3 inammasome activation by employing a recombinant puried
active HlyA along with an inactive pro-hlyA, which was used to challenge human THP-1m. It was observed that
HlyA promoted cytolytic activity and induced cleavage of IL-1β and caspase-1 in THP1m cells (Fig.1A–C). We
also demonstrated HlyA induced oligomerization of NLRP3 inammasome (Fig.1D). It is evident that HlyA
induces deubiquitination of NLPR3, which is required for NLRP3 to be activated and initiate a pro-inammatory
response, in the form of IL-1β release (Fig.1E). Craven etal. observed that NLRP3 dependent IL-1β release
was inhibited in cells treated with 140mM KCl during S. aureus α-hemolysin stimulation in THP-1 cells48.
We observed that UPEC α-hemolysin induced IL-1β release and NLRP3 inammasome oligomerization was
dependent on K+ eux (Fig.2A,B,D). Interestingly, deubiquitination of NLRP3 was also aected in a similar way
(Fig.2E). Furthermore, it was shown that NLRP3 co-localizes within mitochondria followed by HlyA stimulation
and causes mitochondrial dysfunction.
e NLRP3 inammasome can be activated by diverse stimuli, which shows the involvement of many
upstream receptors sensing dierent ligands causing similar cellular events, leading to the activation of NLRP3
inammasome49. ough the exact mechanism remains unknown, literature suggests that NLRP3 inammasome
assembly activation involves potassium eux from the cells, production of mitochondrial ROS, deubiquitination
of NLRP3 followed by translocation to mitochondria, which triggers the release of mitochondrial DNA or car-
diolipin from mitochondria; nally cathepsin-B release from lysosome into cytoplasm, followed by pyroptosis50.
NLRP3 inammasome is activated in response to UPEC infection and IL-1β is released in a caspase-1 dependent
manner8,51. Mutant studies showed that the HlyA-positive UPEC strain induces NLRP3 expression and caspase-1
activation in bladder epithelial cells8. Further overexpression of HlyA in UPEC led to increased inammasome
activation and IL-1β secretion in the mouse, inducing inammatory cell death in urothelial cells31. We observed
that recombinant puried active HlyA induces caspase-1 activation and IL-1β maturation in THP-1m. In addi-
tion, we have also seen that HlyA induces deubiquitination of NLRP3 and its further assembly with ASC and
pro-caspase-1. ese results indicate that HlyA is one of the virulence factors of UPEC, which leads to the activa-
tion of NLRP3 inammasome. To understand the mechanism of NLRP3 inammasome activation during HlyA
stimulation, we further looked into the eect of high extracellular K+ and K+ channel inhibitors (glibenclamide).
Potassium ion eux is a characteristic of apoptotic cells and leads to caspase activation52,53; inhibiting K+ eux
delayed apoptosis through interference in cytochrome c release52,54–56, which implicated the association of K+
eux with mitochondrial membrane integrity56. Potassium concentration in cells was shown to impact the health
and functionality of cells; changes in intracellular K+ concentration have an eect on cell survival, mitochondrial
health and immune response of neutrophils against bacteria56–58. Besides, HlyA was shown to alter signalling of
host cells and aect viability and function of eector phagocytes19,59–61. Moreover, HlyA was reported to trigger
K+ perturbances in the cells34. K+ concentration plays an important role in assembly and activity of inamma-
some; inammasome activation can be prevented by blocking K+ eux37–39. We found that the maturation of
both caspase-1 and IL-1β was inhibited by preventing K+ eux using K+ ion channel inhibitor (glibenclamide)
and high K+ concentration in the media (Fig.2A–C). Similarly, Planillo etal. found that intracellular concentra-
tion of K+ regulates NLRP3 mediated caspase-1 activation during various stimulations with PFTs38. Moreover,
inhibition of K+ eux during HlyA stimulation could have led to the ubiquitination of NLRP3, which further
impeded its oligomerization and activation (Fig.2D,E). erefore, we may speculate that deubiquitination,
Figure8. α-hemolysin induced cell death in THP-1 macrophages, reversed by inhibition of potassium
eux. LDH release assay was performed to evaluate the eect of α-hemolysin (HlyA) on THP-1m cell death.
Additionally, the eect of glibenclamide and potassium chloride were assessed on hemolysin induced cell death
of THP-1m. THP-1m pre-treated with glibenclamide (100M, 30min prior to stimulation) and KCl (140mM,
prior 30min to stimulation) were stimulated with α-hemolysin for 2h. Data is represented as percentage
LDH release (Mean ± SEM). Comparisons between multiple groups were made using one-way ANOVA with
Bonferroni’s post test. P value is shown as ** ≤ 0.01.

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oligomerization and activation of NLRP3 depend on K+ eux during HlyA stimulation. It would be interesting
to elucidate further, that how K+ eux regulates NLRP3 inammasome activation or which biomolecules are
modied during this process in order to regulate its activation. Almost all PFTs have been reported to work via
K+ eux mechanism for NLRP3 activation62. E. coli α-hemolysin induced cell death was decreased in THP-1
monocytes on inhibition of P2X7R receptor, known to be responsible for facilitation of K+ eux during pore
formation63. Whereas S. aureus α-hemolysin induced IL-1β release was found to be independent of P2X7R, but
again dependent on intracellular K+ concentration64.
Despite energy production, mitochondria play an important role in cell death, innate immune response,
cell metabolism and signaling by regulating the production of ROS. erefore, mitochondria dysfunction has
been observed during many diseases such as cancer, diabetes and infections, where pathogens specically target
mitochondria to gain hold of the cells and avoid cell death65,66. Bacterial virulence factors, mainly toxins, induce
dysfunction in mitochondria to avoid apoptosis and phagocytosis to multiply inside the cells65,67. Earlier reports
showed bacterial PFTs impede mitochondrial function by disturbing mitochondrial membrane potential and
leakage of cytochrome c, inducing either apoptosis or necrosis68,69. Similarly, UPEC mediates programmed necro-
sis by impairing mitochondrial function. e UPEC HlyA, a PFT, causes mitochondrial fragmentation and loss
of mitochondrial membrane potential, which nally results in mitochondrial dysfunction in sertoli cells of mice
in in-vitro28. Besides mitochondrial dysfunction, UPEC causes permanent loss of plasma membrane integrity
of sertoli cells, allowing the release of DAMPs, which cause activation of testicular macrophages by secretion
of pro-inammatory cytokines in-vitro28. We have also observed that stimulation with HlyA disturbed mito-
chondrial membrane potential and led to the depolarization of mitochondria in THP-1m (Fig.4). In addition,
NLRP3 was found to be co-localized within mitochondria, during stimulation of THP-1m with HlyA (Fig.3).
Activation of inammasome was seen in cases where mitochondrial activity was disrupted. When the VDAC1
of mitochondria was inhibited, it resulted in NLRP3 inammasome activation, clearly indicating the association
with mitochondrial dysfunction40. We observed that blockage of K+ eux (via glibenclamide or 140mM KCl)
prevented HlyA induced mitochondrial depolarization in THP-1m (Fig.4), and this blockage is also reported
to be associated with inhibition of NLRP3 inammasome activation37,39. erefore, we may interpret that UPEC
HlyA induced disruption of mitochondrial membrane potential is dependent on K+ eux and is also associated
with NLRP3 inammasome activation.
Studies have shown the importance of ROS, especially mitochondrial ROS, for activation of NLRP3
inammasome40, but the role of ROS in NLRP3 inammasome activation has not been well elucidated. Earlier,
it was thought that ROS produced by NOX is important for NLRP3 activation70. Other studies have shown that
inhibiting ROS has no eect on activation of NLRP3 and it just blocks priming of macrophages, i.e. mRNA
expression of pro-IL-1β and NLRP371. Further, ROS has a very short life span and acts only in the vicinity as
a messenger72. erefore, it was concluded that NLRP3 should be co-localized with mitochondria for ecient
activation by ROS produced. We observed that NLRP3 is co-localized with mitochondria in THP-1m, when
they were challenged with HlyA (Fig.3). Zhou etal. also observed that in the presence of NLRP3 inammasome
activators, NLRP3 get colocalized with mitochondria and mitochondria-associated markers, where it can sense
and regulate mitochondrial activity40. Mitochondrial ROS is an essential mediator of NLRP3 inammasome
activation during stimulations with known NLRP3 activators40. Similarly, Heid etal. saw ablation of IL-1β release
in macrophages by quenching mitochondrial ROS via MitoTEMPO during stimulation with nigericin and ATP45.
In contrast, Planillo etal. found role of intracellular K+ in NLRP3 activation, but they failed to observe any
signicant eect of mitochondrial dysfunction on IL-1β release38. Whereas we have found signicant reduction
in IL-1β release upon inhibition of Mitochondrial ROS (via MitoTEMPO) and potassium eux during HlyA
stimulation (Fig.7). is shows that intracellular K+ and mitochondrial health regulate IL-1β secretion during a
UPEC HlyA stimulation. Besides, heme is known to be associated with pathogenesis of hemolytic diseases, mainly
sepsis, as it induces inammation during infection via an unknown mechanism73,74. However, it was shown that
blocking the oxidative eect of heme can save the tissue from death in case of sepsis73,75. Recently, heme was
shown to be associated with NLRP3 inammasome activation in a mitochondrial ROS (mitoROS) dependent
manner, as blocking mitoROS avoided inammasome activation by heme76. erefore, we may interpret that
mitoROS may be an important factor which helps in NLRP3 co-localization with mitochondria. It would be
interesting to know how NLRP3 interacts with mitochondria and what could be the impacts of an association
between NLRP3 and mitochondria.
To further assess the mitochondrial health upon HlyA stimulation, we measured the reduced/oxidized glu-
tathione levels. e modulation of the redox microenvironment is an important regulator of immune cell activa-
tion and proliferation. Reduced glutathione (GSH) is considered to be one of the most important scavengers of
ROS and its ratio with oxidized glutathione (GSSG) may be used as a marker of oxidative stress77. Our results
showed that HlyA increased the oxidative stress in THP-1m, whereas inhibition of potassium eux during HlyA
stimulation improved GSH:GSSG ratio signicantly (Fig.5). It is well established that mitochondrial dysfunction
is associated with many chronic inammatory diseases and plays an important role in the pathogenesis of many
disorders78. Mutation of genes involved in mitophagy has been found in diseases, such as Parkinson’s and Crohn’s
disease79. Furthermore, a defect in mitophagy has been associated with increased production of IL-1β, a marker
of inammasome activation80. Mitophagy is also a cell defense mechanism to remove damaged mitochondria,
which might block inammasomes by inhibiting the activation of NLRP3 inammasome81. Autophagy works
through the removal of damaged mitochondria, a source of ROS, thus inhibiting the activation of the NLRP3
inammasome. Blocked mitophagy caused increased activation of the NLRP3 inammasome40. In addition, the
removal of the damaged mitochondria induces the formation of new mitochondria. Recent studies have shown
that mitochondrial biogenesis and mitophagy are coupled; for example, parkin regulates both mitophagy and
mitochondrial biogenesis82,83. HlyA has been shown to induce the production of nitric oxide (NO) through induc-
ible nitric-oxide-synthase (iNOS) pathway84 and NO induces PGC-1, a co-activator of mitochondrial replication,

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thus regulating mitochondrial biogenesis85,86. We observed that HlyA induces mitochondrial biogenesis, which
might be a stress response by the THP-1m cells to remove dysfunctional mitochondria to control inammation
(Fig.6). However, HlyA did not induce mitochondrial biogenesis during blockage of K+ eux, which suggests
that HlyA induced mitochondrial damage is protected by the inhibition of K+ eux. Additionally, our ndings
suggest that the cell death induced by HlyA can be signicantly reduced by inhibiting the activation of NLRP3
inammasome, achieved by blocking K+ eux through glibenclamide or by repleting intracellular K+ concen-
tration (Fig.8).
We may conclude that UPEC HlyA can induce the formation of the NLRP3 inammasome by initiating
deubiquitination of NLRP3, whereas proHlyA failed to do so. erefore, we could suggest that the pore-forming
property of UPEC α-hemolysin is necessary to initiate a pro-inammatory response. In addition, results showed
that HlyA induced activation of the NLRP3 inammasome depends on the concentration of intracellular K+ and
mitochondrial ROS during the stimulations of THP-1m. Similarly, K+ eux due to HlyA stimulation resulted
in the prevalence of dysfunctional mitochondria and localization of NLRP3 to the mitochondria for MitoROS
sensing. It would be interesting to further investigate the contribution of mitochondrial dysfunction in detail
during HlyA-induced NLRP3 inammasome activation. During HlyA stimulation of THP-1m, an increase in
mitochondrial biomass was observed, which could be a strategic stress response, but inhibition of K+ eux
neutralized this stress.
Materials and methods
Materials. Glibenclamide (Cat# G0639, Sigma-Aldrich, India) and Nigericin (Cat# 481990, Merck, India)
were used in stimulation protocol. For RNA isolation and cDNA synthesis, TRI Reagent (Sigma cat # T9424;
St Louis, Missouri, U.S.A) and RevertAid First Strand cDNA Synthesis Kit (ermo Scientic Cat# K1622;
Waltham, Massachusetts, U.S.A) were used, respectively. Protein estimation was done using a bicinchoninic
acid (BCA) kit (Cat# 71285, Merck, India). Antibodies used were anti-NLRP3 antibody (Cat# NBP1-77080,
Novus biological, USA), anti-Ubiquitin antibody (Cat# P497, Biolegend, California, USA), anti-ASC (Cat#
NBP1-78977, Novus biological, USA), anti-VDAC antibody (Cat# 820701, Biolegend, California, USA), anti-
Tubulin antibody (Cat# ab6046, Abcam, Cambridge, USA), anti-IL-1β antibody (ab2105, Abcam, Cambridge,
USA), anti-Caspase-1 antibody (Cat# NBP1-45433, Novus biological, USA) and anti-GAPDH antibody (Cat#
SC47724, Santa Cruz, California, USA). HRP-labelled anti-mouse (Cat# SC2005, Santa Cruz, California, USA)
and anti-rabbit secondary antibody (Cat# ab6721, Abcam, Cambridge, USA) were used for immunoblotting.
Clarity Western ECL substrate (Cat# 1705060, Bio-Rad, California, USA) was used for chemiluminescence. For
confocal microscopy, Mitotracker Red CMXRos (Cat# M7512, ermo Fisher Scientic, USA), Alexa uor 488
(Cat# A-11012, ermo Fisher Scientic, USA), VECTASHIELD Antifade Mounting Medium (Cat# H-1000,
Vector laboratories, USA) and JC-1 Mitochondrial Membrane Potential Probe (Cat# T3168, ermo Fisher Sci-
entic, USA) were used. For GSH and GSSG estimation O-Phthaladehyde (OPT) (Cat # 27329, SRL) and N-Eth-
ylmaleimide (NEM) (Cat # 78503, SRL) were used. For enzyme-linked immunosorbent assay (ELISA) of IL-1β
cytokine, Human IL-1β GENLISA ELISA kit (Cat# KB1063, Krishgen Biosystems) was used and MitoTEMPO
(Cat# SML0737, Sigma-Aldrich, India) was used for mitoROS inhibition. For LDH release Assay Cayman LDH
cytotoxicity assay kit (Cat # 10009172) was used.
Toxin preparation. Acylated Active (HlyA) and inactive α-hemolysin (proHlyA) were recombinantly pro-
duced according to the previous method29. Recently, we have reported a simple method for the recombinant pro-
duction of hexa-histidine tagged active and inactive α-hemolysin by cloning only hlyA and hlyC genes of operon
hlyCABD29. Fatty acid acylation of HlyA at lysine 564 and 590 residue is an important step, which is performed
by HlyC12. erefore, we have cloned hlyA and hlyC simultaneously for the production of active α-hemolysin,
while hlyA was cloned alone to produce inactive α-hemolysin (non-acylated). Over-expression was achieved
with 1mM isopropyl-1-thio-β--galactopyranoside (IPTG) at 18°C for 6h. Both active and inactive forms of
HlyA were puried by a batch purication method as described earlier29. Protein was subjected to desalting
and concentration through Amicon Ultra-0.5ml centrifugal lters (Merck) for 50kDa molecular weight cut o
(MWCO). Protein was eluted in 1X PBS supplemented with 20mM CaCl2. Puried active and inactive HlyA
were quantied through SDS-PAGE and then further subjected to hemolysis assay. An insignicant amount of
endotoxin contamination was found in the preparations when subjected to LAL Assay to produce any synergistic
eect29. erefore, in further experiments, wherever HlyA was required, a batch of HlyA showing more than
90% of activity was used.
Cell culture. THP-1 cells (cat # TIB-202, ATCC, Manassas, VA) were cultured in RPMI-1640 medium sup-
plemented with 10% heat inactivated fetal bovine serum, 2mM -glutamine, 1mM sodium pyruvate and 10mM
HEPES (cat#15630080, Life Technologies, Carlsbad, CA). THP-1 cells were dierentiated into macrophage-like
cells (THP-1m) by culturing for 48h in a medium containing 25nM Phorbol 12-myristate 13-acetate (PMA)
(Sigma-Aldrich, St Louis, MO) and followed by 24h of rest before any stimulation87.
Stimulation protocol. THP-1m were incubated in RPMI 1,640 medium with HlyA and proHlyA along
with control (mock/without any stimulation) for 2 h27; 200ng of HlyA and proHlyA was used for stimula-
tions per million of THP-1m in 1ml of medium27. Nigericin was used at 15M concentration for 30min for
stimulations40. For experiments involving K+ concentration interventions, 140mM potassium chloride (KCl)
was added to the medium 30min prior to the stimulation with HlyA39,88. Additionally, THP-1m cells were
treated with 100M of glibenclamide (Gli) for 30min prior to incubation with HlyA wherever indicated39,89.

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Lysate preparation of THP‑1 cells. Cytoplasmic extract was prepared by lysing cells using cold Radio-
immunoprecipitation assay (RIPA) lysis buer (150mM NaCl, 1% Nonidet P-40, 0.5% Sodium deoxycholate,
0.1% Sodium dodecyl sulphate and 25mM Tris)90 (pH 7.4) supplemented with protease inhibitor cocktail for
15min on ice. Homogenous lysis was achieved by passing cell suspension through a 28 gauge needle syringe
(Dispovan, India) and then lysates were cleared by centrifugation at 13,000rpm for 20min. e supernatant was
collected as cytoplasmic extract and stored at − 80°C until further use.
Western blotting. Protein was estimated using a BCA kit (Merck) according to the manufacturer’s instruc-
tions. 50µg of protein was resolved on 15% or 8% SDS-PAGE gel (as required to detect desired molecular weight
of protein) and subsequently transferred onto PVDF membrane (GE Healthcare Life Sciences), kept overnight
at 25V and at 4°C. e various protein molecules were probed with specic primary antibodies (IL-1β, Cas-
pase-1, NLRP3, Ubiquitin, ASC, VDAC-1, Tubulin and GAPDH) wherever required, followed by HRP-labelled
anti-mouse and anti-rabbit secondary antibodies. e Clarity Western ECL substrate (Bio-Rad) was used to
develop the blot by chemiluminescence. GAPDH and VDAC-1 were used as loading controls in immunoblot-
ting for cytoplasmic extracts and mitochondrial fractions, respectively. Tubulin was immunoblotted to check for
contamination of cytoplasmic content in mitochondrial fractions. Quantication was carried out using ImageJ
soware (NIH).
Cytokine measurement. Dierentiated THP-1m cells were stimulated with HlyA for 2h. Before stimula-
tion, cells were incubated in high K+ containing media and glibenclamide, as indicated earlier in the stimulation
protocol. One group was treated with MitoTEMPO (20µM for 1h), to see the eect of mitochondrial ROS dur-
ing HlyA stimulation on IL-1β release. e expression of cytokine IL-1β was estimated by ELISA using IL-1β
ELISA kit (Cat# KB1063, Krishgen Biosystems). Aer incubations, the culture medium was harvested, ltered
(0.2m lters) and ELISA was performed following the manufacturer’s instructions. Each experiment was per-
formed three times and statistical analysis was done.
Acetone precipitation of cell culture supernatant. For detecting IL-1β release through Western blot-
ting, cell culture supernatants from various stimulations were subjected to acetone precipitation of proteins.
Protein was precipitated by adding four volumes of chilled acetone to the sample (culture medium) followed by
incubation at − 20 °C for 1 h. e mixture was then centrifuged at 13,000rpm for 15min and the pellet obtained
was dissolved in 1% SDS supplemented with 1× protease inhibitor.
Confocal microscopy. PMA treated THP-1 cells were seeded and dierentiated into THP-1m in 8-well
chamber slides at a concentration of 105 cells/well, as mentioned earlier. THP-1m were stimulated with 200ng of
HlyA for 2h. Aer 2h, THP-1m were washed thrice with 1× PBS and incubated with 200nM Mitotracker Red
CMXRos for 45min at 37°C, followed by washing thrice with PBS for 5min each. e cells were washed twice
with 1× PBS and subsequently xed with 4% paraformaldehyde (pH 7.4) for 30min at 37°C. Further, the cells
were permeabilized using 0.15% Triton X-100 in 1× PBS for 10min at room temperature in darkness. ereaer,
the cells were washed thrice with PBS followed by blocking with 1% BSA in PBST for 30min at room tempera-
ture in darkness. e cells were subsequently incubated with anti-NLRP3 antibody at a concentration of 20g/
ml in 1% BSA in PBS overnight at 4°C in darkness. e cells were again washed thrice with 1× PBS for 5min
and incubated with a secondary antibody Alexa uor 488 at a concentration of 2g/ml in 1× PBS with 1% BSA
for 2h at room temperature in darkness. Subsequently, the cells were washed thrice with 1× PBS for 5min and
incubated with DAPI at 300nM concentration in 1× PBS for 5min, followed by washing thrice in 1× PBS. e
cells were mounted with VECTASHIELD Antifade Mounting Medium. Confocal imaging was performed with a
Nikon A1 laser scan confocal microscope with Plan Apooptics, equipped with an argon laser. Data was analyzed
using the NIS Elements Advanced Research soware.
Mitochondria isolation. Mitochondria isolation from THP-1m cells was performed using a method of
Clayton etal.91 with minor modications. THP-1m cells were seeded in 75 cm2 asks and dierentiated into
THP-1m, as mentioned previously in the cell culture section of methods. Two asks per treatment were used
further for mitochondria isolation (~ 2 × 107 cells). Aer harvesting cells, 9ml of ice-cold RSB hypotonic buer
(10mM NaCl, 1.5mM MgCl2 and 10mM Tris–HCl [pH 7.5]) was added to resuspend the pellet. e suspended
cells were thereaer transferred to a 15ml dounce homogenizer and kept on ice for 10–15min. e cells were
then broken mechanically by using a dounce homogenizer with a tight tting Teon pestle. Five cycles of 10–15
strokes, followed by a rest of 1min on ice, were used to break open the cells. Immediately thereaer, 6ml of
2.5× MS homogenization buer (525mM mannitol, 175mM sucrose, 12.5mM Tris–HCl [pH 7.5] and 2.5mM
EDTA [pH 7.5]) was added. e mixture was immediately invert-mixed 6–8 times to maintain tonicity and to
prevent agglutination of organelles by sealing the mouth of the homogenizer with paralm; the homogenate was
then transferred to a 50ml centrifuge tube. e volume of homogenate was raised up to 20ml by adding 1× MS
homogenization buer (210mM mannitol, 70mM sucrose, 5mM Tris–HCl [pH 7.5] and 1mM EDTA [pH
7.5]). e mixture was centrifuged at 1,500g for 5min at 4°C to separate nuclei, unbroken cells and larger mem-
brane fragments. e supernatant was transferred and 15ml of 1× MS homogenization buer was added and
centrifuged at 1,500g for 5min at 4°C to remove cytoplasmic and nuclear content. is step was repeated one
more time. Supernatant was transferred to a fresh and sterile centrifuge tube and mitochondria were pelleted
down by centrifuging the tube at 13,000g for 15min at 4°C. Aerward, the mitochondrial pellet was washed
twice using 10ml of 1× MS homogenization buer. Ultimately, the mitochondrial pellet was used for immuno-

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blotting. e pellet was dissolved in 50l of RIPA buer supplemented with 1× protease inhibitor cocktail and
10l of mitochondrial fraction was used per lane to detect various proteins as indicated.
Mitochondrial membrane potential assay. Dierentiated THP-1m cells were stimulated with HlyA for
2h. Prior to stimulation, the cells were incubated in high K+ containing media and glibenclamide as indicated
earlier in the stimulation protocol. Aer all incubations and stimulations of THP-1m, the cells were washed with
1× PBS and stained for 30min with 2M JC1 dye in RPMI-1640 medium. JC1, as a monomer, gives a green uo-
rescence at 529nm. JC1 monomers move inside energized mitochondria depending on the membrane potential,
resulting in the subsequent JC1 aggregates inside mitochondria, which gives a red uorescence at 590nm. Tecan
Innite 200 pro, a multi-well plate reader was used for red/green uorescence analysis of JC1 dye assay. e
experiment was performed three times and with two technical replicates.
GSH:GSSG estimation. Glutathione estimation was performed using the method of Singh etal.92. For the
measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a uorescent reagent. OPT has an
ability to react specically with GSH at pH 8 and GSSG at pH 12. N-ethylmaleimide (NEM) has been used to
prevent auto-oxidation of GSH during measurement of GSSG in the present protocol.
For the experiment, PMA treated THP-1 cells were seeded in the 6-well plate at a density of 1.5 × 106 cells/
well and the cells were stimulated with HlyA for 2h, as mentioned in the stimulation protocol. For K+ concentra-
tion interventions, 140mM potassium chloride (KCl) was added to the medium 30min prior to the stimulation
with HlyA39,88. Additionally, THP-1m cells were treated with 100M of glibenclamide (Gli) for 30min prior to
incubation with HlyA, wherever indicated39,89. Culture media was removed and washed with 1× PBS and then
1ml of 0.1M potassium phosphate buer with EDTA (ice cold KPE buer). e cells were scraped and collected
in 1.5ml centrifuge tubes. en, centrifugation was done at 500g for 10min at 4°C and the supernatant was
discarded; thereaer, 200µl of KPE buer with 1× protease inhibitor was added to dissolve the cell pellet. Aer
sonication, lysed samples were centrifuged at 18,000g for 10min at 4°C and the resulting supernatants were
collected in separate pre-cooled 1.5ml centrifuge tubes (10µl samples from each tube were collected in 0.5ml
tubes for protein estimation by BCA kit). Aer protein estimation, 10µg of protein sample was precipitated.
Initially, 80µl of protein sample was mixed with 20µl trichloroacetic acid (TCA) (50% stock concentration),
vortexed and kept in ice for 10min. e protein sample with TCA was centrifuged at 10,000g for 10min at 4°C;
the supernatant was transferred into a fresh 1.5ml centrifuge tube. For GSH estimation: 10µl supernatant with
equal volume of OPT (1mg/ml) and 180µl KPE buer (pH-8) in a black 96-well plate was added. For GSSG
estimation: 50µl of the supernatant was transferred into a new centrifuge tube, 0.5µlN-ethylmaleimide (stock
concentration: 4M) was added and mixed thoroughly, and incubated for 30min at room temperature to inhibit
GSH. 10µl of this sample, 10µl OPT and 180µl 0.1N NaOH (pH-12) were added in a black 96-well plate. e
plate was incubated in the dark for 10min. Fluorescence at λex: 355nm and λem: 420nm in a microplate reader
was taken and the results were analyzed.
Co‑immunoprecipitation. To check endogenous levels of oligomerization and ubiquitination of NLRP3
during various stimulations, co-immunoprecipitation was performed as follows. Cytoplasmic extract of THP-
1m aer all stimulation protocols, which were subsequently stored at −80°C, were thawed on ice. Cytoplasmic
extracts of THP-1m cells (500g) were incubated for each reaction with 1g of anti-ASC (Novus Biologicals)
antibody and 20l of Recombinant Protein A-Sepharose 4B beads (Invitrogen) overnight at 4°C on a rotary
invert mixer. e next day, sepharose beads were washed thrice with RIPA lysis buer followed by mixing sepha-
rose beads with 2× SDS PAGE protein sample buer (80mM Tris HCl (pH6.8), 10% (v/v) Glycerol, 2% SDS,
238mM β-Mercaptoethanol, 0.0006% (v/v) Bromophenol blue and 0.1M dithiothreitol [freshly added])93. e
samples were then boiled at 95°C for 5min and run on 8% resolving SDS-PAGE. To check endogenous levels of
ASC in input lysates (cytoplasmic extract), 30g of input lysates were run on 15% SDS-PAGE. For immunoblot-
ting, the protocol was followed as described earlier. Each experiment included three biological and two technical
replicates.
Mitochondrial copy number. Mitochondrial copy number was determined by quantitative real-time PCR
(RT-qPCR). Total RNA was isolated from stimulated THP-1m by TRI reagent (Sigma) as per the manufacturer’s
instructions and quantied by using Nanodrop (ND-1000). A 500ng of total RNA aer quantication was
subjected to cDNA synthesis using cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit (ermo
Scientic)) as per the manufacturer’s protocol. Each experiment included three biological and three technical
replicates. ABI 7,300 Real-Time PCR machine was used for quantication of mitochondrial DNA (mtDNA) by
using Mesa green PCR Master mix (SYBR) (Eurogentec). e Real-time qPCR reaction contained 7.5µl of 2×
Mesa green PCR Master mix, 1µl cDNA, 1µM of each primer and water to make a nal volume of 15µl. qPCR
Table 1. List of primers used for quantitative real time PCR (RT-qPCR).
S. no Gene Primer sequence Reference
1mtDNA Forward: 5′-CCC CAG CCA TAA CAC AGT ATC AAA C-3’
Reverse: 5′-GCC CAA AGA ATC AGA ACA GATGC-3’ 94
218S rRNA Forward: 5′-GTG GTG TTG AGG AAA GCA GACA-3′
Reverse: 5′-TGA TCA CAC GTT CCA CCT CATC-3′95

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conditions were: 50°C for 5min, 95°C for 10min, 40× (95°C for 15s and 60°C for 1min). e primers used for
mtDNA94 and human 18S rRNA95 are given in Table1. Human18S rRNAnuclear amplicon was used as a house-
keeping gene for internal control (or reference gene). mtDNA copy number was normalized to amplication of
an 18S nuclear amplicon and calculated as mentioned previously96. Values of mtDNA are expressed as delta-
Ct ± SD, lower delta-Ct indicates higher copy number. Normalization was done according to the given formula;
delta-Ct (mtDNA) = (Ct of mtDNA in treatments) – (Ct of 18S in treatments). ree independent experiments
were performed and statistical analysis was done.
LDH release assay. For LDH release assay, 50,000 cells/well were seeded in 96-well clear plate and were
allowed to dierentiate as mentioned in the cell culture method. Dierentiated THP-1m were stimulated with
HlyA for 2h; prior to stimulation, cells were incubated in high K+ containing media and glibenclamide, as
indicated earlier in the stimulation protocol. e experiment was performed according to the manufacturer’s
instruction. Aer 2h, the plate was centrifuged at 400g for 10min at room temperature. 100l of culture
medium was transferred into another well and 100l of LDH reaction solution was added. e resulting solu-
tion was incubated at 37˚C with gentle shaking for 30min and then the absorbance was taken at 490nm. e
experiment was performed thrice with two technical replicates.
Statistical analysis. All data sets were analyzed via one-way ANOVA followed by Bonferroni’s post-hoc
analysis using GraphPad soware (GraphPad). P values ≤ 0.05 were considered as statistically signicant.
Received: 4 December 2019; Accepted: 7 July 2020
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Acknowledgements
is work was supported by the Research & Development grant, University of Delhi (No: RC/2015/9677). MY
is a recipient of Fast-track and RGYI grant from the Department of Science and Technology/SERB (No: SR/
FT/LS-117/2011) and the Department of Biotechnology (No: BT/PR6301/GBD/27/396/2012) respectively. VV
and SY are recipients of CSIR fellowship and SG is recipient of ICMR fellowship. DST Fast-track fellowship to
PK is gratefully acknowledged. MY is recipient of ICMR-DHR international fellowship.Open access funding
provided by Lund University.
Author contributions
V.V., P.K., R.S.D. and M.Y. designed experiments and analyzed the data. V.V., S.G., P.K. and S.Y. performed
experiments. V.V., P.K., R.S.D. and M.Y. conceptualized and directed the study and wrote the manuscript. H.T.
contributed to the experimental design and helped to write the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-69501 -1.
Correspondence and requests for materials should be addressed to M.Y.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.

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