www.sciencemag.org/cgi/content/full/1156995/DC1
Supporting Online Material for
Innate Immune Activation Through Nalp3 Inflammasome Sensing of Asbestos and Silica
Catherine Dostert, Virginie Pétrilli, Robin Van Bruggen, Chad Steele, Brooke T Mossman, Jürg Tschopp*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 10 April 2008 on Science Express
DOI: 10.1126/science.1156995 This PDF file includes
Materials and Methods Figs. S1 to S7 Table S1 References
Supporting Online Material (SOM)
Experimental procedures
Reagents
For in vitro studies, crocidolite asbestos was purchased from SPI-CHEM, silica powder
(1.5 micron diameter) from Alfa Aesar and diesel particulate matter (DEP) from NIST
(Standard Reference Material). Cigarette smoke extract (CSE) was prepared as described
previously (1). Nigericin, uric acid, cytochalasin D, N-acetyl-L-cysteine, apocynin,
rotenone, thenoyltrifluoroacetone (TTFA), deferoxamine mesylate, uricase and z-VAD-
fmk were purchased from Sigma. Latrunculin A was obtained from Calbiochem and DPI
from Alexis. R837 and ultrapure LPS were obtained from Invivogen. Anti-human
cleaved IL-1β (2021L) was purchased from Cell Signaling, and anti-IL1β p35 (AL177)
from Alexis (Axxora plateform). The antibody against mouse IL-1β was a gift from
Roberto Solari, Glaxo. The antibody against human caspase-1 (p10) was purchased from
Imgenex. The antibody against mouse caspase-1 (p20) was a generous gift from Dr. Peter
Vandenabeele (Ghent University). The ELISA kits for human cytokine detection were
obtained from BD Biosciences. All tissue culture reagents were bought from Invitrogen.
Mice
Nalp3-/-, ASC-/- and Ipaf-/- mice were described previously (2, 3). Nalp3-/-, ASC-/- and
Ipaf-/- mice are in C57Bl6 background.
Generation of THP1 cells expressing shRNA
THP-1 stably expressing shNALP3, caspase-1, ASC, MyD88, p22phox and thioredoxin
were obtained as previously described (4).
Cell preparation
Peritoneal macrophages were obtained by injecting 8 to 12 week-old mice of indicated
genotypes i.p. with 10% thioglycollate solution, followed by peritoneal lavage 3 days
later. Bone-marrow macrophages were derived form tibia and femoral bone marrow cells
as described elsewhere (5).
In general, macrophages were plated at a density of 106 cells in 12-well dishes and non-
adherent cells were removed after 2 h. Cells were cultured in DMEM complemented with
10% FCS, 1 mM sodium pyruvate, 100 UI/ml penicillin/streptomycin and 2 mM L-
glutamine.
Mouse macrophages were primed overnight with 100 ng/ml ultra-pure LPS (Invivogen).
THP-1 cells were cultured in RPMI complemented with 10% FCS and 50 µM 2-
mercaptoethanol. For experiments, THP-1 were differentiated 3 hours with 0.5 µM PMA.
Human monocytes were purified as previously described (6) and differentiated for 7 days
with human recombinant M-CSF (ReliaTECH GMBH) before treatment with ultrapure
LPS 18 h before the experiment. Eight hundred thousand cells were treated for 6 h with
the indicated stimuli. KCl (130 mM) was added at the same time as the stimulation. Cell
extracts and precipitated supernatants were analysed by Western blotting (3).
Scanning Electron Microscopy (SEM). To determine whether THP-1 cell
phagocytised fibers and particles, cells were grown on Thermanox plastic coverslips
(Nalge Nunc International, Naperville, IL) in 12-well plates as described above, and
treated with particles for 6 h. Coverslips were fixed in 2% glutaraldehyde in PBS, rinsed
briefly with PBS followed by 0.05 M cacodylate buffer (pH 7.2), and post-fixed in 1%
osmium tetroxide in 0.05 M cacodylate buffer at 4°C for 30-45 min. After 3-5 rinses in
cacodylate buffer, they were incubated in 1% tannic acid in 0.05 M cacodylate buffer for
1 h, rinsed briefly in buffer, then distilled water and incubated in 0.5% uranyl acetate in
water for 1 h. Samples were then dehydrated in graded ethanols from 35% to 100%.
Samples were critical point dried using liquid CO2 as the transition fluid in a Samdri PVT-
3B critical point dryer (Tousimis Research Corporation, Rockville, MD). Specimens were
mounted on aluminum specimen stubs using conductive graphite paint and after drying
were sputter-coated for 4-5 min. with gold and palladium in a Polaron sputter coater
(Model 5100). Specimens were then examined with a JSM 6060 scanning electron
microscope (JEOL USA, Inc., Peabody, MA) and a JEOL 1210 STEM with energy
dispersive x-ray capability.
ROS detection
We assessed intracellular ROS using the ROS-specific fluorescent probe 2'7'-
dichlorofluorescin diacetate (H2DCFDA, BioChemika, Fluka). Cells were loaded for 10
min with 10 µM H2DCFDA, washed twice with PBS and exposed to asbestos, MSU,
ATP and H2O2. Fluorescence was recorded in 96-well plates over time with a Titertek
FluoroskanII using a FITC filter (excitation 485nm, emission 538nm).
In vivo inhalation exposures to asbestos
Nalp3+/+ and Nalp3-/- littermates in the C57Bl6 background (8-12 weeks old) were
exposed to chrysotile asbestos (NIEHS reference sample at approximately 7 mg/m3 air) or
clean air (sham groups) in separate inhalation chambers for 6 h per day for a total of 8
days as described previously (7, 8). Mice were killed on day 9 (n=4-5 mice per group),
the time point of peak inflammation, mucin production and chemokine/cytokine
production in response to asbestos by an intraperitoneal injection of sodium
pentobarbital, and lungs were lavaged and differential cell counts on each BALF sample
after cytospins performed as described previously (8) (9). To quantify cytokine and
chemokine levels in BALF supernatants, a multiplex suspension protein array was
performed using the Bio-Plex Protein Array System and a Mouse Cytokine 22-plex Panel
(Bio-Rad) (8). Other lung lobes were perfused and inflated under pressure with
phosphate-buffered saline, fixed in 4% paraformaldehyde, and embedded in paraffin for
histology (H&E stained sections) and mucin production as confirmed by Alcian
blue/periodic acid-Schiff (PAS) staining in distal bronchioles using a blind code system
by a board-certified pathologist (K.J. Butnor) (7). The percentage of bronchioles affected
and severity of mucous metaplasia after evaluation of >10 distal bronchioles per mouse
were determined. The numbers of Alcian-blue/PAS positive cells were scored in
individual bronchioles using a 1-4 scale for severity: 1= no positive cells; 2= 1-25 %
positive cells; 3= 26-50% positive cells; and 4= 51-100% positive cells. Data from all
studies were analysed by ANOVA using the Student-Newman-Keul's procedure for
adjustment of multiple pair-wise comparisons or the nonparametric Kruskal-Wallis test.
Values of p<0.05 were considered statistically significant.
SN
Cell
Asbestos
Ø Silica MSUMSU +zVAD
-Casp1
-Casp1p10
-Casp1
-Casp1p10 (longer exposure)
DEP
Suppl. Fig.1:
THP1 cells were stimulated with 0.1 mg/ml asbestos, 0.5 mg/ml silica, 0.5mg/ml DEP
and 0.1 mg/ml MSU. Media supernatants (SN) were analysed for the presence of
caspase-1 and its cleaved p10 subunit, and cell extracts (Cell) for the presence of caspase-
1 by Western blotting.
-Nalp3
-Casp1
-ASC
moc
ksh
Nal
p3sh
ASC
sh c
aspa
se1
-tubulin
sh M
yD88
-MyD88
-ASC short
-non specific
Suppl. Fig.2:
THP1 cells were i nfected with lentivirus carrying various shRNA. The efficacy of
knockdown was determined by Western blot analyses.
-IL-1β p17
-proIL-1β
WT Nalp3 -/-
-Casp1
-Casp1
-Casp1 p20
.2 .1
Asbestos
.15
MSU
MyD88 -/-
SN
Cell
.2 .1
Asbestos
- .15
MSU
.2 .1
Asbestos
- .15 (mg/ml)
MSU
Ø
Longexposure
Suppl. Fig.3:
Peritoneal macrophages from Nalp3-/- mice (Nalp3-/-
) or littermate controls (Nalp3+/+
mice), as we ll as M yD88-/-
mice were s timulated as indicated with asbestos or MSU
(mg/ml). Media supernatants (SN) a nd cell extracts (Cell) were analysed by Western
blotting as indicated for IL-1 and caspase-1 processing.
-IL-1β p17
-pro-IL-1β
SN
Cell
Ipaf +/+
.2 .1 .05
Asbestos (µ
g/ml)
Ø
Silica (µ
g/ml)
.15
MSU (µg/ml)
.2 .1 .05
Ipaf -/-
.2 .1 .05
Asbestos (µ
g/ml)
Ø
Silica (µ
g/ml)
.15 (mg/ml)
MSU (µg/ml)
.2 .1 .05
Suppl. Fig.4:
Peritoneal macrophages from Ipaf-/- mice (Ipaf-/-
) or littermate control mice (Ipaf+/+
)
were stimulated as indicated with asbestos, silica or MSU (mg/ml). Media supernatants
(SN) and cell extracts (Cell) were analysed by Western blotting as indicated in the text.
-IL-1β p17
-pro-IL-1β
SN
Cell
Ø
Asbestos MSU
- 0.5 - - 0.5 - 0.5 - µM cytochalasin D
- - 2 - - 2 - 2 µM latrunculin A
-IL-1β p17
Suppl. Fig.5:
THP1 cells were stimulated for 6 h with asbestos or MSU in the presence or absence of
cytochalasin D or latrunculin A. Media supernatants (SN) and cell extracts (Cell) were
analysed by Western blotting as indicated in the text.
0
10
20
30
40
50
60
70
0 30 60 90 120 150 180Time (min)
H2DC
FDA
(AU) /
ATPAsbestosMSUH2O2
A
5
15
25
H2DC
FDA
(AU)
AsbestosAsbestos +KCl
MSU +KClMSU
+KCl
B C
2
4
6
8
10
12
AsbestosAsbestos +NAc
MSU +NAcMSU
+NAc
/
/
H2DC
FDA
(AU)
10
20
Suppl. Fig.6:
(A) THP1 cells were stimulated with ATP (5 mM), asbestos (0.2 mg/ml), MSU (0.2
mg/ml) or H2O2 (1 mM) and ROS production was monitored with the fluorescent probe
H2DCFDA over time. (B,C) Cells were treated as indicated with N-acetyl-L-cysteine (25
mM) or KCl (130 mM).
-IL-1β p17SN
Cell
Ø
-pro-IL-1β
- + - + - + - + DPI
MSU AsbestosWT sh
Nalp3
WT shNalp
3
Ø
Ø
Asbestos
A
B
DPIApocyn
in
Rotenone
TTFA
-IL-1β p17SN
Cell -pro-IL-1β
-
Suppl. Fig.7:
(A) THP1 cells were stimulated for 6 h with MSU (0.1 mg/ml) or asbestos (0.1 mg/ml) in
the presence or absence of DPI (25 M). (B) THP1 cells were stimulated for 6 h with
asbestos (0.1 mg/ml) after 20 min pretreatment with DPI (20 M), apocynin (100 M
overnight), rotenone (100 M) or TTFA (100 M). Media supernatants (SN) and cell
extracts (Cell) were analysed by Western blotting as indicated in the text.
STable I
Mucin Production in Bronchiolar Epithelium (Alcian blue/PAS staining)
*Significantly different from sham of same genotype. †Significantly different from +/+
Asb
% Bronchioles Affected
Severity
Sham Nalp3+/+
0 ± 0
1 ± 0
Sham Nalp3-/-
0 ± 0
1 ± 0
Asb Nalp3+/+
11.25 ± 5.154
1.75 ± 0.25*
Asb Nalp3-/-
45 ± 9.354*†
2.2 ± 0.2*
References
1. S. Carnevali et al., Am J Physiol Lung Cell Mol Physiol 284, L955 (2003). 2. S. Mariathasan et al., Nature 430, 213 (2004). 3. F. Martinon, V. Petrilli, A. Mayor, A. Tardivel, J. Tschopp, Nature 440, 237
(2006). 4. S. Papin et al., Cell Death Differ 14, 1457 (2007). 5. A. Didierlaurent et al., Mol Cell Biol 26, 735 (2006). 6. L. Agostini et al., Immunity 20, 319 (2004). 7. T. Sabo-Attwood et al., Am J Pathol 167, 1243 (2005). 8. A. Haegens et al., J Immunol 178, 1800 (2007). 9. R. F. Robledo et al., Am J Pathol 156, 1307 (2000).