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).