Pharmacologic inhibition of the NLRP3 inflammasome preserves
cardiac function after ischemic and non-ischemic injury in the mouse
SUPPLEMENTAL MATERIAL
METHODS
Synthesis of the NLRP3 inflammasome inhibitor
5-chloro-2-methoxybenzoic acid was reacted with 2-phenylethanamine in
the presence of 1-Ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDCI) to
form the amide intermediate, 5-chloro-2-methoxy-N-(2-phenylethyl)benzamide, which was then treated with chlorosulfonic acid and aqueous
ammonium hydroxide to afford 5-chloro-2-methoxy-N-[2-(4sulfamoylphenyl)-ethyl]-benzamide (16673- 34-0). As vehicle, we used a
1:10 in poly(ethylene glycol)400 (PEG400) solution diluted in dimethyl
sulfoxide (DMSO). The inhibitor was administered intraperitoneally.
Additional details are included in a prior publication. 1
Infarct size assessment
Infarct size was measured as previously described. 1, 2 Mice were sacrificed
and the hearts were quickly removed and mounted on a Langendorff
apparatus. A solution of 0.9% NaCl containing 2.5 mM CaCl2 was used to
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perfuse the coronary artery. After the heart was perfused, the coronary artery
was closed again, and approximately 1 ml of 1% Evans blue dye (SigmaAldrich) was injected as a bolus through the aorta until the heart “not-atrisk” turned blue. The hearts were removed from the Langendorff apparatus,
frozen, and cut into 6 transverse slices of equal thickness, about 1 mm, from
apex to base. A 10% TTC isotonic phosphate buffer (pH 7.4) solution was
used to incubate the slides at room temperature for 30 minutes. The infarcted
tissue (appearing white), the viable tissue (red), and the non-risk area (blue)
were measured by computer morphometry using Image Pro Plus 6.0
software (Media Cybernetics, Silver Spring, MD).
Doppler echocardiography
Echocardiography was performed to measure cardiac dimensions and
function, as previously described. 2 Mice underwent transthoracic
echocardiography at baseline (before treatment), 7 days, or 10 days later
(prior to sacrifice). Echocardiography was performed with the Vevo770
imaging system (VisualSonics Inc, Toronto, Ontario, Canada) and left
ventricular (LV) end-diastolic diameter (LVEDD), LV end-systolic
diameters (LVESD) were measured at M-Mode. LV fractional shortening
(LVFS) was also calculated.
2
Use of glyburide as a comparison for ischemia-reperfusion studies
The NLRP3 inflammasome inhibitor is an intermediate compound in the
synthesis of glyburide. The inhibitor, however, lacks the cyclohexylurea
moiety and therefore, is not a sulfonylurea. Due to this change, the inhibitor
has no effects on insulin release. 1 The inhibition of the ATP-sensitive K+
(KATP) channel in pancreatic β-cells by glyburide mediates insulin release,
and it has also been reported to interfere with naturally occurring
cardioprotective mechanisms (i.e. preconditioning) and therefore behaves as
a potentially cardiotoxic agent in the setting of ischemia-reperfusion. In this
study we used glyburide (132.5 mg/kg)(a dose equimolar with the NLRP3
inhibitor, N=6) given at reperfusion as an additional group in order to
determine the effects of glyburide in this model. Mice underwent the
surgical coronary ligation for 30 minutes followed by reperfusion, and were
then sacrificed at 24 hours for infarct size assessment.
Effects of the NLRP3 inflammasome inhibitor on primary adult
cardiomyocytes in vitro
A primary adult rat cardiomyocyte culture was established in vitro as
previously described. 2 Briefly, cardiomyocytes from adult male Wistar rats
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(~300 g)(Harlan Sprague- Dawley Inc.,Indianapolis, IN) were isolated using
collagenase type II (Worthington Collagenase type II-285 U/mg CLS-242B13250). Following tissue digestion, the freshly isolated cardiomyocytes
were plated with Medium 199 (Life-Technologies, Gran Island, NY)
containing 2mM L-carnitine, 5mM creatine, 5mM taurine, 5mM glucose,
0.1µM insulin, and 1% penicillin-streptomycin. After 1 hour, the myocytes
were treated with Escherichia coli 0111:B4 LPS (25 ng/mL; Sigma-Aldrich)
for 2 hours followed by ATP (5 mM) for 30 minutes, to induce NLRP3
inflammasome formation. The NLRP3 inhibitor (400µM), was added 30
minutes prior ATP. Formation of the NLRP3 inflammasome in the
cardiomyocytes was determined and quantified by caspase-1 activity
(enzymatic activity assay) as previously described. 3
Effect of the NLRP3 inflammasome inhibitor in response to
monosodium urate and cholesterol crystals in murine macrophages in
vitro
Murine macrophages, J774A.1 cells, were primed with LPS (1µg/mL) for 4
hours and then the NLRP3 inflammasome formation was triggered with
monosodium urate crystals (MSU) (200µg/ml; InvivoGen, San Diego-CA)
or cholesterol crystals (CC) (500µg/ml; Sigma-Aldrich) for 6 hours. The
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NLRP3 inflammasome inhibitor (400µM) was added at the time of the
crystal administration. The supernatants were collected, and levels of IL-1β
were measured with a mouse IL-1β ELISA kit (R&D system DuoSet,
Pittsburgh, PA) as the read out.
Effects of the NLRP3 inflammasome inhibitor in a mouse model of
cryopyrinopathy
Bone marrow-derived macrophages (BMDM) from wild type (WT) and
Nlrp3 A350V/CreT transgenic mice 4 which express an inducible mutated
form of NLRP3, were isolated as previously described. 5 This mouse strain
was donated by Dr. Hal Hoffman (UCSD) and has increased expression of
IL-1 following tamoxifen treatment, activating the expression of the
recombinase Cre, which mediates DNA recombination to permit the
expression of the mutant/active NLRP3 (for more details refer to Brydges
S.D. et al). 4 The mutant NLRP3 gene, once recombination is induced by the
tamoxifen-dependent Cre recombinase, spontaneously oligomerizes forming
an active inflammasome. After isolation of the femur and the tibia from wild
type and Nlrp3 A350V/CreT mice, bone marrow cells were extracted, and
red blood cells were lysated using the RBC lysis buffer (eBioscience, San
Diego CA). Cells were cultured for 5 days in DMEM 10% FBS
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supplemented with 30% L929 to induce macrophage differentiation, then
plated for the experiment. 5 To induce the mutated NLRP3, the BMDM were
exposed to 1µM 4-hydroxytamoxifen (TAM) (Sigma-Aldrich) for 48 hours,
then LPS (1µM) was administrated for 24 hours. The NLRP3 inflammasome
inhibitor (400 µM) was added at the time of TAM. The supernatants were
collected, and IL-1β levels were measured with a mouse IL-1β ELISA kit
(R&D system DuoSet).
RESULTS
Effects of glyburide on infarct size
When tested in the I/R infarct model, glyburide given at reperfusion (at a
dose equimolar to that of the NLRP3 inflammasome inhibitor) had no
protective effects on infarct size and cardiac troponin I levels (Supplemental
Figure 1), thus suggesting that the presence of the cyclohexylurea moiety
negates the cardioprotective effects seen with the NLRP3 inflammasome
inhibitor.
Effects of the NLRP3 inflammasome inhibitor on primary cultured
adult cardiomyocytes in vitro
Treatment of primary isolated cardiomyocytes with LPS/ATP induced
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formation of the NLRP3 inflammasome as shown with increased caspase-1
activity when compared to the treatment in control (Supplemental Figure 2).
Administration of the NLRP3 inflammasome inhibitor prevented caspase-1
activation (p=0.01)(Supplemental Figure 2).
Effect of the NLRP3 inflammasome inhibitor on IL-1 production
following stimulation with monosodium urate or cholesterol crystals
MSU crystals and CC induced NLRP3 inflammasome activation in J774A.1
cells, proven by increased levels of released IL-1β when compared to the
control treated cells (Supplemental Figure 3). Phagocytosis of crystalline
material, here being the MSU crystals and CC, leads to active swelling of
phagosomes, followed by phagosomal destabilization and rupture, thus
resulting in the release of phagosomal contents in the cytosolic compartment
and activation the NLRP3 inflammasome. 6 The NLRP3 inflammasome
inhibitor prevented the inflammasome activation shown by reduced IL-1β
levels in the supernatant when compared to the positive controls
(Supplemental Figure 3).
Effects of the NLRP3 inflammasome inhibitor in a mouse model of
cryopyrinopathy
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BMDM from wild type and Nlrp3 A350V/CreT mice treated with LPS alone
released a low amount of IL-, showing the lack of NLRP3 inflammasome
activation (Supplemental Figure 4). Increased levels of IL-1 were found
only in the supernatant of the Nlrp3 A350V/CreT BMDM treated with
tamoxifen, which induced the mutated NLRP3, and the LPS treated cells
compared to the WT cells, reflecting the activation of the NLRP3
inflammasome. Treatment with the NLRP3 inflammasome inhibitor
prevented IL-1β release after tamoxifen and LPS stimulation in the
transgenic-derived cells (Supplemental Figure 4). These data together show
the inhibitory properties of the molecule on the NLRP3 inflammasome and
also suggest a direct interaction with the sensor.
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References
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Federici M, Van Tassell BW, Zhang S, Abbate A. A novel pharmacologic
inhibitor of the NLRP3 inflammasome limits myocardial injury after
ischemia-reperfusion in the mouse. J Cardiovasc Pharmacol. 2014; 63: 316322.
2. Abbate A, Salloum FN, Vecile E, Das A, Hoke NN, Straino S, BiondiZoccai GG, Houser JE, Qureshi IZ, Ownby ED, Gustini E, Biasucci LM,
Severino A, Capogrossi MC, Vetrovec GW, Crea F, Baldi A, Kukreja RC,
Dobrina A. Anakinra, a recombinant human interleukin-1 receptor
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Salloum FN, Kannan HR, Menna AC, Voelkel NF, Abbate A. The
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C, Putnam CD, Boyle DL, Firestein GS, Horner AA, Soroosh P, Watford
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WT, O'Shea JJ, Kastner DL, Hoffman HM. Inflammasome-mediated disease
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5. Weischenfeldt J, Porse B. Bone Marrow-Derived Macrophages (BMM):
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SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure 1. Effects of glyburide in the ischemia/reperfusion
(I/R) model. (A) Schematic representation of the experimental design; (B)
Mean±SEM percent of left ventricle (LV) at risk of infarction (Area at risk)
following I/R event; (C) Mean± SEM percent of LV infarct size 24 hours
following I/R event evaluated by triphenyl tetrazolium chloride (TTC) stain;
(D) Mean±SEM of serum cardiac troponin I levels 24 hours after
ischemia/reperfusion, #p<0.01 vs sham. N= 4-6 per group.
Supplemental Figure 2. The NLRP3 inhibitor prevents caspase-1
activity in primary cultured rat cardiomyocytes (CM). (A) Schematic
representation of the experimental design. CM were treated with LPS
(25ng/ml) for 2 hours and then ATP (5mM) for 30 minutes to induce
NLRP3 inflammasome formation. The NLRP3 inflammasome inhibitor
(NLRP3i) (400µM) was administrated 30 minutes prior ATP. (B) Caspase-1
activity was measured as read out of inflammasome activation, #p=0.02
LPS/ATP vs control, *p=0.01 LPS/ATP/NLRP3i vs LPS/ATP-treated cells.
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Supplemental Figure 3. Effect of the NLRP3 inflammasome inhibitor on
IL-1β production following stimulation with uric acid or cholesterol
crystals. (A) Schematic representation of the experimental design. Murine
macrophage cells (J774A.1) were treated with LPS (1µg/ml) for 4 hours and
MSU (200µg/ml) or CC (500µg/ml) for 6 hours to induce NLRP3
inflammasome formation. NLRP3 inflammasome inhibitor (NLRP3i)
(400µM) was administrated at the moment of the crystal administration. (B)
IL-1β levels were measured in the supernatant, *p=0.05 LPS/MSU/NLRP3i
vs LPS/MSU; #p=0.003 LPS/CC/NLRP3i vs LPS/CC.
Supplemental Figure 4. Effect of the NLRP3 inflammasome inhibitor on
IL-1β production in bone marrow derived macrophages (BMDM) from
genetically modified mice with constitutively active NLRP3. (A)
Schematic representation of the experimental design. BMDM were extracted
from WT and NLRP3 transgenic and treated with tamoxifen (TAM) (1µM)
for 48 hours and LPS (1µM) for 24 hours to induce NLRP3 inflammasome
formation. NLRP3 inflammasome inhibitor (NLRP3i) (400µM) was
administrated with TAM. (B) IL-1β levels were measured in the supernatant,
*p<0.0001 LPS/TAM/NLRP3i vs LPS/TAM.
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