home: cell press · web viewhuman flag-ips-1, myc-cathepsin d, myc-tbk1, myc-fadd, flag-caspase-8...
TRANSCRIPT
Immunity, Volume 38
Supplemental Information
Poly IC Triggers a Cathepsin D- and PS-1-DependentPathway to Enhance Cytokine Productionand Mediate Dendritic Cell Necroptosis
Jian Zou, Taro Kawai, Tetsuo Tsuchida, Tatsuya Kozaki, Hiroki Tanaka, Kyung-
Sue Shin, Himanshu Kumar, and Shizuo Akira
Figure S1, Related to Figure 1
(A) GM-DCs derived from wild-type (WT) or IPS-1-deficient (IPS-1 KO) mice were
stimulated with poly IC (pIC) for the indicated periods. Cell lysates were blotted with
an anti-phosphorylated IRF3 antibody.
(B) FL-DCs were stained with anti-CD11b-PE, anti-B220-APC and anti-CD24-FITC
antibodies. B220-CD11b+ cells were separated into CD11bhiCD24lo or
CD11bloCD24hi cDCs by cell sorting.
(C) Total RNA prepared from CD11bhiCD24lo and CD11bloCD24hi cells was
subjected to RT-PCR analyses for the expression levels of Ifih1 (MDA5), Tlr3
(TLR3) or Actb (-actin).
(D) CD11bloCD24hi cells derived from WT, TLR3-deficient (TLR3 KO) or TRIF-
deficient (TRIF KO) mice were stimulated with poly IC, and the concentrations of
IFN- and IL-12p40 in the culture supernatants were measured by ELISA. Data
represent means ± SD (n=3).
(E) GM-DCs (left) or CD11bloCD24hicDCs (right) derived from WT or IPS-1 KO mice were cocultured with CD8+ T cells derived from OT-I mice in the presence of
OVA and poly IC or CpG ODN for 4 d. The concentrations of IFN- in the culture
supernatants were measured by ELISA. Data represent means ± SD (n=3).
(F) GM-DCs were stimulated with high molecular weight poly IC (H) or low
molecular weight poly IC (L) with (right) or without (left) transfection. The
concentrations of IFN- in the culture supernatants were measured by ELISA. Data
represent means ± SD (n=3). C: control.
Figure S2, Related to Figure 4
(A) Lysosomes were isolated from WT or IPS-1-deficient (KO) GM-DCs with or
without poly IC stimulation. The lysosomes were lysed and blotted with anti-
Cathepsin D or anti-LAMP1 antibody.
(B) CD11bhiCD24lo and CD11bloCD24hi cells derived from WT or KO mice were
stimulated with poly IC or LPS, and immunostained with an anti-Cathepsin D
antibody.
(C) GM-DCs were stimulated with poly IC and immunostained with anti-Cathepsin
D and anti-IPS-1 antibodies.
Figure S3, Related to Figure 5
(A) GM-DCs were treated with the indicated siRNAs. Total RNA was prepared and
analyzed for the expressions of Ctsd (Cathepsin D [Cat D]), Ctsb (Cathepsin B [Cat
B]), Ctsl (Cathepsin L [Cat L]) and Actb (-actin) by RT-PCR.
(B) GM-DCs treated with the indicated siRNAs were stimulated with poly IC. The
concentrations of IFN- (left panel) and IL-6 (right panel) in the culture
supernatants were measured by ELISA. Data represent means ± SD (n=3).
(C) GM-DCs treated with the indicated siRNAs were stimulated with LPS. The
concentrations of IL-6 in the culture supernatants were measured by ELISA. Data
represent means ± SD (n=3).
(D) CD11bhiCD24lo or CD11bloCD24hi cells with or without Pep A pretreatment
were cocultured with CD4+ or CD8+ T cells derived from OT-II or OT-I mice in the
presence of OVA and poly IC, respectively. The concentrations of IFN- in the
culture supernatants were measured by ELISA. Data represent means ± SD (n=3).
(E) Schematic representation of Caspase-8. The arrows indicate the cleavage sites of
Caspase-8 by Cathepsin D (upper). The lower panels show sequence alignments of
mutants of Caspase-8.
(F) HEK293 cells were transiently transfected with Flag-Caspase-8, Flag-Caspase-8
L237A, or Flag-Caspase-8 L237A-M383A-L443A, together with Myc-empty or
Myc-Cat D. Cell lysates were immunoprecipitated with an anti-Flag antibody and
immunoblotted with an anti-Flag antibody.
Figure S4, Related to Figure 6
(A) GM-DCs pretreated with z-VAD-fmk were stimulated with poly IC. The cells
were stained with a FITC-anti-Annexin V antibody and PI. The population of dead
cells was analyzed by flow cytometry. Data represent means ± SD (n=3).
(B) GM-DCs were treated with the indicated siRNAs. Total RNA was prepared and
analyzed for the expressions of Ripk3 (Rip-3) and Actb (-actin) by RT-PCR.
(C) GM-DCs pretreated with the indicated inhibitors were stimulated with poly IC.
The cell viability was measured by cell titer-Glo luminescent cell viability kit. Data
represent means ± SD (n=3).
Figure S5, Related to Figure 7
(A) GM-DCs treated with the indicated siRNAs were stimulated with poly IC. The
concentrations of HMGB1 in the culture supernatants were measured by ELISA.
Data represent means ± SD (n=3).
(B) GM-DCs were stimulated with poly IC together with HSP70, oxLDL, acLDL or
acBSA at the indicated concentrations. After 24 h, the concentrations of IFN- in the
culture supernatants were measured by ELISA. Data represent means ± SD (n=3).
(C) GM-DCs derived from WT or MyD88 and TRIF doubly deficient (DKO) mice
were stimulated with poly IC together with recombinant HMGB1 at the indicated
concentrations. After 24 h, the concentrations of IFN- in the culture supernatants
were measured by ELISA. Data represent means ± SD (n=3).
(D) RAW 264.7 cells were stimulated with 50 μg/ml of Etoposide for 24 h. The
supernatants were collected and then incubated with streptavidin beads with or
without biotinylated poly IC for 4 h. The bound proteins were eluted by heat
incubation and analyzed by immunoblotting with an anti-HMGB1 antibody.
(E) Recombinant HMGB1 was preincubated with an anti-HMGB1 neutralizing
antibody, and then IP with streptavidin beads with or without biotinylated poly IC for
4 h. The bound proteins were eluted by heat incubation and analyzed by
immunoblotting with an anti-HMGB1 antibody.
Supplemental Experimental Procedures
Reagents and Plasmids
High and low molecular weight poly IC were purchased from InvivoGen.
LysoTracker Red DND-99 and Alexa Fluor 488-conjugated rabbit anti-goat IgG
antibody were purchased from Invitrogen. LPS from Salmonella minnesota Re-595,
anti-Flag M2 affinity agarose gel, anti-Flag M2-peroxidase (HRP) antibody,
Cytochalasin D, Bafilomycin A-1, acridine orange, Piceatannol, Pep A, CA-074Me,
Z-FF-fmk, protein A-Sepharose, CHX, recombinant murine TNF- and recombinant
human HMGB1 were purchased from Sigma-Aldrich. Anti-c-myc (9E10), anti-
Cathepsin D (G19) and anti-TOM20 (FL-145) antibodies were purchased from Santa
Cruz Biotechnology. Anti-phosphorylated-Syk, anti-Syk, anti-Caspase-8 and anti-
IPS-1 antibodies were purchased from Cell Signaling Technology. Anti-RIP-1, Alexa
Fluor 647-conjugated anti-mouse CD107a (LAMP1), anti-K63-linked ubiquitin and
anti-HMGB1 antibodies were purchased from BD Biosciences, BioLegend,
Millipore and MBL, respectively. Nec-1 was purchased from Enzo Life Sciences.
EthD-III was purchased from PromoKine. The ELISA kit for IFN- and HMGB1
were purchased from PBL Biomedical Laboratories and Shino-Test Corporation,
respectively. Mouse IL-6, IL-12p40 and IFN- ELISA kits were purchased from
R&D Systems.
Plasmids
Human Flag-IPS-1, Myc-Cathepsin D, Myc-TBK1, Myc-FADD, Flag-
Caspase-8 full-length (FL) and Flag-Caspase-8 a.a. 1-237 (C8 1-237) were amplified
by PCR and ligated into pFlag-CMV6 or pEF-BOS to generate Flag- and Myc-
tagged expression constructs, respectively. A series of mutant Caspase-8 expression
plasmids were generated via a KOD-Plus-mutagenesis kit (Toyobo). The expression
plasmids and reporter plasmids were described previously (Kawai et al., 2005).
Reporter Assay, Immunoprecipitation, and Immunoblot Analysis
HEK293 cells plated on 24-well plates (1×105 cells/well) were transiently
transfected with 50 ng of luciferase reporter plasmid together with a total of 1.0 mg
of plasmid using Lipofectamine 2000 (Invitrogen). The luciferase activities in total
cell lysates were measured with a Dual-Luciferase Reporter Assay System
(Promega). A Renilla luciferase reporter plasmid (50 ng) was used as an internal
control. HEK293 cells (1×106) were transiently transfected with a total of 10 mg of
plasmids. Cell lysates were precipitated with anti-Flag or anti-Myc beads or protein
A-Sepharose preconjugated with the first antibodies, and immunoblotted with the
respective antibodies (Kawai et al., 2005). Poly IC was conjugated with biotin (Label
IT Biotin Nucleic Acid Labeling Kit; Mirus). Biotinylated poly IC was attached to
streptavidin-agarose beads (Pierce streptavidin agarose resin; Thermo Scientific).
Recombinant HMGB1 was incubated with biotinylated poly IC-streptavidin beads
for 4 h at 4°C. The precipitates were eluted by boiling with Laemmli sample buffer,
and analyzed by immunoblot with respective antibodies.
Mice
TRIF-, TLR3- and MyD88/TRIF-deficient mice were described previously
(Yamamoto et al., Science 301:640-3, 2003). OT-I transgenic mice (C57BL/6) were
provided by W. R. Heath.
Reagents
An anti-phosphorylated-IRF3 antibody was purchased from Cell Signaling
Technology. An anti-LAMP1 antibody was purchased from BD Bioscience.
Recombinant HSP70 was purchased from Enzo Life Sciences. Oxidized low-density
lipoprotein (oxLDL) and acetylated low-density lipoprotein (acLDL) were purchased
from Biomedical Technologies. Maleimide-activated BSA (acBSA) was purchased
from Adar Biotech. Etoposide was purchased from Sigma-Aldrich.
Cell Sorting and Isolation
FL-DC subsets were sorted based on CD24 and CD11b expression using a
FACSAria (BD Biosciences). Naive CD8+ T cells were separated from OT-I
transgenic mouse spleens using MACS beads (Miltenyi Biotec), in accordance with
the manufacturer’s instructions.
Immunoprecipitation and Immunoblot Analysis
For stimulation, RAW 264.7 cells were plated in 6-well culture plates
(3×106/well) for 2 h, washed twice with Opti-MEM (Gibco Life Technologies) and
then stimulated with 50 g/ml Etoposide for 24 h. The supernatants were
immunoprecipitated with biotinylated poly IC-streptavidin beads for 4 h at 4°C.
Recombinant HMGB1 was precultured with an anti-HMGB1 neutralizing antibody
(Shino-Test Corporation) at 4°C for 4 h, and then immunoprecipitated with
biotinylated poly IC-streptavidin beads. The immunoprecipitates were eluted by
boiling with Laemmli sample buffer, and analyzed by immunoblotting with an anti-
HMGB1 antibody (MBL).
RT-PCR
Total RNA was isolated using the TRIzol reagent and reverse-transcribed with
RTase (Toyobo) according to the manufacturer’s instructions. PCR was performed
with the following primers: Ifih1, 5’-ATGTTCGTGGAGGCCCTAGAG-3’ and 5’-
CGAAGCAGCTGACACTTCCTT-3’; Tlr3, 5’-ATGAAAGGGTGTTCCTCTTATC-
3’ and 5’-GAGGGCGAATAACTTGCCAATT-3’; Actb, 5’-
GACATGGAGAAGATCTGGCACCACA-3’ and 5’-
ATCTCCTGCTCGAAGTCTAGAGCAA-3’.
CTL Responses
OT-I transgenic CD8+ T cells (1×105) were cocultured with DCs (1×105) in
the presence of OVA protein (100 g/ml), with or without poly IC or CpG DNA
supplementation. After 4 d, IFN- production in the culture supernatants was
analyzed by ELISA.
Lysosome Isolation
Lysosomes were isolated from GM-DCs using a Lysosome Enrichment Kit
(Thermo Scientific) according to the manufacturer’s instructions.
RNA Interference
Double-stranded RNA duplexes corresponding to mouse Cathepsin D,
Cathepsin L, Cathepsin B, and Rip-3 were purchased from Invitrogen. The targeting
sequences were as follows: Cathepsin D sense, 5’-
CCAGACAAGUAUAUACUCAAGGUAU-3’, Cathepsin L sense, 5’-
GCUAAUGACACAGGGUUCGUGGAUA-3’, Cathepsin B sense, 5’-
GAGGUGUCUGCUGAAGACCUGCUUA-3’, Rip-3 sense, 5’-
UGGAAGACACGGCACUCCUUGGUAU-3’. Negative control oligo was
purchased from Invitrogen. GM-DCs were electroporated with 100nM siRNA via
Neon transfection system (Invitogen) according to the manufacturer’s instructions.
At 48 h after transfection, the cells were used for further experiments. Knockdown of
Ctsd was verified by RT-PCR with the primers 5’-
TCCATGTAAGTCTGACCAGTC-3’ and 5’-GAAGCCACTCAGGCAGATTGT-3’;
Knockdown of Ctsl was verified by RT-PCR with the primers 5’-
ATGAATCTTTTACTCCTTTTGGC-3’ and 5’-CGTTACAGCCCTGATTGCCTT-
3’; Knockdown of Ctsb was verified by RT-PCR with the primers 5’-
ACCTGCATTCACACCAATGGC-3’ and 5’-TTAGAATCTTCCCCAGTACTG-3’;
Knockdown of Ripk3 was verified by RT-PCR with the primers 5’-
ATGTCTTCTGTCAAGTTATGG-3’ and 5’-TGCCCACACGAGGATCCCAAAG-
3’.
Cell Viability Assays
GM-DCs (1×105 cells/well) were seeded into 96-well plates and treated with
poly IC, or recombinant murine TNF-a plus Etoposide for 24 h. Nec-1 or Pep A was
added 1h prior to stimulation. The viable cells per well was determined by measuring
intracellular levels of ATP using the cell titer-Glo luminescent cell viability assay kit
(Promega) according to the manufacturer’s instructions. Luminescence was
measured on a Centro LB960 microplate reader (Berthold Technologies).