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: jurg.tschopp@unil.ch

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).