SUPPLEMENTARY FIGURE 1 0 20 50 80 100 IL-17RD (ng) S1a S1b S1c IL-17RD β-actin kda S1d - si sc Il17rd Il17ra rig/s15-574 - 458-361 bp S1f S1g S1h
S1i S1j Supplementary Figure 1. Knockdown of IL-17RD enhances TLR-induced pro-inflammatory signalling. (a-c) Assay of luciferase reporter activity in HEK293 cells transfected with (a) MyD88, Mal, TRIF, TRAM and Myc-tagged IL-17RD (0 ng) with a NF-κB luciferase reporter plasmid (60 ng) or (b,c) IKKi, TBK1 and IL-17RD (0 ng) and PFR-luciferase (60 ng) with (b) IRF3- Gal4 (30 ng) or (c) IRF7-Gal4 (25 ng) constructs. TK Renilla was measured to determine transfection efficiency. (a, top panel) Immunoblotting of cell lysates with indicated antibodies to show IL-17RD expression. (d) Assay of NF-κB reporter activity in HEK293 TLR4 or TLR3 cells transfected with scrambled or IL-17RD-specific sirna for 48 h followed by stimulation with LPS (100 ng/ml) or poly(i:c) (25 μg/ml). (d, inset) RT-PCR indicating knockdown with IL-17RD sirna specifically targets Il17rd mrna. (f) Knockdown of Il17rd mrna in (left panel) U373 and (right panel) THP-1 cells stably transduced with lentiviral constructs encoding control or IL-17RD-specific shrna. (g,h) Levels of (g) Il8 mrna in THP-1 knockdown cells treated with LPS (100 ng/ml) and (h) Ifnb1 mrna in U373 knockdown cells treated with poly(i:c) (25 μg/ml) for 6 h were evaluated by qpcr. (i) qpcr indicating knockdown of Il17rd mrna in PBMCs. (j) Supernatants from PBMCs transfected with scrambled or IL-17RD-specific sirna for 48 h followed by stimulation with LPS (100 ng/ml) and poly(i:c) (5 μg/ml) measured for IL-8 and IFN-β production by sandwich ELISA. Data are presented as the mean +/- S.E.M. of three independent experiments and were subjected to two-tailed paired Student's t test, *, p < 0.05; **, p < 0.01; ***, p < 0.001. Images have been cropped for presentation. Full size images are presented in Supplementary Fig. S12.
SUPPLEMENTARY FIGURE 2 S2a S2b S2c S2d Il17rd +/+ Il17rd / Probe 0 30 60 120 0 30 60 120 comp LPS (min) NF-κB/Oligo binding Free Probe Supplementary Figure 2. IL-17RD-deficient cells show increased production of pro-inflammatory cytokines upon TLR stimulation. (a) WT and Il17rd / MEFs were infected with EMCV for 24 h and IL-6, KC and RANTES levels in supernatants were measured by ELISA. (b) Cxcl1 and Il6 mrna levels in WT and Il17rd / BMDMs treated with LPS (100 ng/ml) for 6 h. (c) Ifnb1 mrna levels in WT and Il17rd / MEFs infected with EMCV for 24 h were assayed by qpcr. (d) WT and Il17rd / MEFs were stimulated with LPS (100 ng/ml) for 0, 30, 60 and 120 mins and nuclear extracts assayed by EMSA for NF-κB/DNA binding. Nuclear extracts (from Il17rd / cells stimulated with LPS for 30 min) were also pre-incubated for 15 min with the unlabelled competitor (comp) oligonucleotide. (a-c) Data are presented as the mean +/- S.E.M. of three independent experiments and were subjected to two-tailed paired Student's t test, *, p < 0.05; **, p < 0.01; ***, p < 0.001 or is a representative (d) of 3 independent experiments. Full size images are presented in Supplementary Fig. S13.
SUPPLEMENTARY FIGURE 3 S3a Il17rd +/+ Il17rd / LPS (min) 0 5 15 30 60 240 0 5 15 30 60 240 S3b p-erk ERK β-actin Il17rd +/+ Il17rd / LPS (min) 0 5 15 30 60 240 0 5 15 30 60 240 p-erk ERK β-actin kda kda Supplementary Figure 3. IL-17RD inhibits TLR4-induced phosphorylation of ERK. (a,b) WT and Il17rd / (a) MEFs and (b) BMDCs were treated with LPS (100 and 10 ng/ml, respectively) for 0, 5, 15, 30, 60 and 240 mins. Cell lysates were subject to SDS-PAGE followed by immunoblotting with indicated antibodies. Data are representative of 3 independent experiments. Images have been cropped for presentation. Full size images are presented in Supplementary Fig. S14.
S4a SUPPLEMENTARY FIGURE 4 IL-17RD Full Length IL-17RD ΔC S4b N N C SEFIR IL-17RD SEFIR N SEFIR C - + - + - + TLR3-FLAG - - + + - - IL-17RD WT-Myc - - - - + + IL-17RD ΔC -Myc C S4c - + - - - + + + Mal -FLAG - - + - - + - - IL-17RD -Myc - - - - + - - + IL-17RD ΔC -Myc - - - + - - + - IL-17RD SEFIR -Myc - 100-55 - 15-36 - 100-60 - 15-130 - 36-45 kda S4d - + - - - + + + TRIF -FLAG - - + - - + - - IL-17RD -Myc - - - - + - - + IL-17RD ΔC -Myc - - - + - - + - IL-17RD SEFIR -Myc S4e kda - + - + TLR3 -FLAG - - + + IL-17RD SEFIR -Myc -130-130 kda -55-55 kda
S4f S4g S4h - + - - + + MyD88 -FLAG - - + - + - IL-17RD WT-Myc - - - + - + IL-17RD E350A-Myc kda S4i S4j - + - - + + MyD88 -FLAG - - + - + - IL-17RD WT-Myc - - - + - + IL-17RD Y330F-Myc Supplementary Figure 4. The SEFIR domain is critical for the inhibitory function of IL-17RD. (a) Schematic depicting structure of wild-type IL-17RD and generated mutants. (b) HEK293 cells were transfected for 24 h with TLR3-FLAG and IL-17RD-Myc or IL-17RD ΔC-Myc (1 μg). Cell lysates were immunoprecipitated with an anti-flag antibody, followed by immunoblotting with indicated antibodies. (c,d) HEK293 cells were transfected for 24 h with (c) Mal-FLAG or (d) TRIF- FLAG with IL-17RD-Myc, IL-17RD ΔC-Myc or IL-17RD SEFIR-Myc (1 μg). Cell lysates were immunoprecipitated with an anti-flag antibody, followed by immunoblotting with indicated antibodies. (e) HEK293 cells were transfected for 24 h with IL-17RD SEFIR-Myc and TLR3-FLAG. Cell lysates were immunoprecipitated with an anti-flag antibody, followed by immunoblotting with indicated antibodies. (f,g,i) Assay of luciferase reporter activity in HEK293 cells transfected with MyD88 (50 ng) with a NF-κB luciferase reporter plasmid (60 ng) and (f) IL-17RA, IL-17RA SEFIR, Act1, Act1 SEFIR, IL-17RD or IL-17RD SEFIR (50 ng) or (g) IL-17RD and IL-17RD E350A or (i) IL-17RD and IL-17RD Y330F. (h,j) HEK293 cells were transfected for 24 h with MyD88-FLAG with IL-17RD- Myc and (h) IL-17RD E350A-Myc or (j) IL-17RD Y330F-Myc. Cell lysates were immunoprecipitated with an anti-flag antibody, followed by immunoblotting with indicated antibodies. Data are (b-e,h,j) representative of 3 independent experiments or (f,g,i) presented as the mean +/- S.E.M. of three independent experiments and were subjected to two-tailed paired Student's t test, *, p < 0.05; **, p < 0.01; ***, p < 0.001. Full size images are presented in Supplementary Fig. S15. kda
S5a - + - - + - MyD88-FLAG - - + - - + MyD88 TIR-FLAG - - - + + + IL17RD SEFIR-Myc SUPPLEMENTARY FIGURE 5 S5b kda S5c MyD88+ TRAF6+ - + - - + + + MyD88 TIR-FLAG EV - - RA RASEF Act ActSEF RD RDSEF IB: HA IB: HA - - + - + - + TRAF6-HA - - - + - + + IL-17RD SEFIR-Myc IP: MyD88 IB: TRAF6 IP: MyD88 IB: MyD88 IP: MyD88-130 -72-130 -72-35 IL-17RA Act1 Act1 SEFIR IL-17RA SEFIR MyD88 kda IB: HA IL-17RD IL-17RD SEFIR TRAF6-45 kda
S5d hil-17rd MyD88 IL-17RD SEFIR ΔBox1 Box1 Box2 355 RPKVFLCYSSKDGQNHMNVVQCFAYFLQDFCGCEVALDLWEDFSLCREGQREWVIQKIHE 160 RFDAFICYCPSD----IQFVQEMIRQLEQTN-YRLKLCVSDRDVLP--GTCVWSIA---- S5e hil-17rd MyD88 hil-17rd MyD88 hil-17ra MyD88 415 SQFIIVVCSKGMKYFVDKKNYKHKGGGRGSGKGELFLVAVSAIAEKLRQAKQSSSAALSK 209 SELIEKRCRR-MVVVVSDDYLQSKE----CDFQTKFALSLSPGAHQKRLIPIKYKAMKKE IL-17RD SEFIR ΔBox3 Box3 * * * 475 FIAVY-FDYSCEGDVPGILDLSTKYRLMDNLPQLC 264 FPSILRFITVCDYTNP-----CTKSWFWTRLAKAL Box1 Box2 377 PRKVWIIYSADHPLYVDVVLKFAQFLLTACGTEVALDLLEEQAISEAGVMTWVGRQKQEM 160 RFDAFICYCPSDIQFVQEMIR----QLEQTNYRLKLCVSDRDVLPGTCVWSIAS----EL hil-17ra MyD88 437 VESNSKIIVLCSRGTRAKWQALLGRGAPVRLRCDHGKPVGDLFTAAMNMILPDFKRPACF 212 IEKRCRRMVVVVSDD-----YLQSKECDFQTKFALSLSPG----AHQKRLIP-IKYKAMK Box3 hil-17ra MyD88 497 GTYVVCYFSEVSCDGDVPDLFGAAPRYPLMDRFEEVY 262 KEFPSILRFITVCDYTNP-----CTKSWFWTRLAKAL Supplementary Figure 5. Selective Targeting of MyD88 by the SEFIT domain of IL-17RD (a-c) HEK293 cells were transfected for 24 h with (a) MyD88-FLAG or MyD88-TIR-FLAG with IL-17RD SEFIR-Myc or (b) MyD88- TIR-FLAG with TRAF6-HA and IL-17RD SEFIR-Myc or (c) MyD88, TRAF6-HA with IL-17RA-FLAG (RA), IL-17RA SEFIR-FLAG (RASEF), Act1-FLAG (Act), Act1 SEFIR-FLAG (ActSEF), IL-17RD-Myc (RD) and IL-17RD SEFIR-Myc (RDSEF). Cell lysates were subject to SDS-PAGE followed by immunoblotting with indicated antibodies. (d,e) Sequence allignment of (d) the IL-17RD SEFIR and MyD88 TIR domains or (e) the IL-17RA SEFIR and MyD88 TIR domains using ClustalW2 (http://www.ebi.ac.uk/tools/msa/clustalw2/) and BoxShade software (http://www.ch.embnet.org/software/box_form.html). Boxes 1, 2 and 3 of TIR and SEFIR domains are labelled and outlined. The black and grey shading represent amino acid identity and similarity respectively. Forward arrow indicates starting amino acid (after Start site) of IL-17RD SEFIR Box1 truncation mutant and reverse arrow indicates last amino acid of IL-17RD SEFIR Box3 truncation mutant (before STOP codon). Asterisks indicate mutated residues found in IL-17RD SEFIR T496P, IL-17RD SEFIR K497R and IL-17RD SEFIR L504Fpoint mutants. Data (a-c) are representative of 3 independent experiments. Full size images are presented in Supplementary Fig. S16.
S6 Wash #1 Wash #2 Wash #3 - + - + - + - + rmyd88 -GST + + + + + + + + ril-17rd -Myc SUPPLEMENTARY FIGURE 6 IP: GST IP: GST IB: GST - 35 kda Supplementary Figure 6. Recombinant IL-17RD fails to interact with recombinant MyD88 in vitro. Myc-tagged recombinant IL-17RD (ril-17rd-myc) was incubated with recombinant MyD88 (rmyd88-gst) for 1 h at 4 o C, followed by 1 h incubation with a specific anti-gst antibody prior to incubation for 1 h with agarose A/G beads. Immunoprecipitates were washed 3 times and washes were saved and protein concentrated using Amicon Ultra filters. Sample buffer was added to immunoprecipitates (beads) and flow-through wash samples, the samples were boiled and subject to SDS- PAGE followed by immunoblotting with indicated antibodies. Data are representative of 2 independent experiments. Images have been cropped for presentation. Full size images are presented in Supplementary Fig. S17.
Fig. 1h Control shrna IL-17RD shrna LPS (min) 0 5 15 30 60 240 0 5 15 30 60 240 p-iκbα - 36 shrna Ctrl oe (Hek) - 100 IκBα IB: IL-17RD - 36-100 p-ikk - 85 IB: IL-17RD (lighter exposure) IKK - 85-45 β-actin - 45 Supplementary Figure 7: Full blots relating to Figure 1
Fig. 1i Control shrna IL-17RD shrna Poly(I:C) (min) 0 45 90 120 240 360 0 45 90 120 240 360 p-irf3-55 IRF3-55 shrna Ctrl oe - 100 IB: IL-17RD - 100 p-tbk1-84 - 45-45 TBK1-84 β-actin Supplementary Figure 7 (continued) : Full blots relating to Figure 1
2d Il17rd +/+ Il17rd / LPS (min) 0 5 15 30 60 240 0 5 15 30 60 240 -polyubiquitinated IκBα Il17rd +/+ Il17rd / p-iκbα -36 IB: IL-17RD -80 p-ikk -85 IKK -85 p-irf3-50 IRF3-50 β-actin Supplementary Figure 8: Full blots relating to Figure 2
2e Il17rd Il17rd 2f +/+ / Poly(IC) Probe 0 60 120 0 60 120 IgG SS LPS (min) (min) 0 15 30 60 90 240 0 15 30 60 90 240 pirf3 Il17rd +/+ Il17rd / -50 NF-κB/Oligo binding p-iκbα -36 Free Probe IκBα -36 2g Il17rd +/+ Il17rd / Probe Comp 0 30 120 0 30 120 Poly(I:C) (min) p-ikk -85 PRD I/III binding IKK -85 Free Probe β-actin IκBα Supplementary Figure 8 (continued): Full blots relating to Figure 2
2h 2i Il17rd +/+ EV 0 15 30 60 Il17rd / EV mil-17rd Poly(I:C) (min) 0 15 30 60 0 15 30 60 p-tbk1-84 TBK1-84 p-irf3-55 IRF3-55 Myc β-actin Supplementary Figure 8 (continued): Full blots relating to Figure 2
4a 4c - + - + - + MyD88-FLAG - - + + - - IL-17RD Full length -Myc - - - - + + IL-17RD ΔC -Myc 4e - + - + - + TLR4-FLAG - - + + - - IL-17RD WT-Myc - - - - + + IL-17RD ΔC -Myc - 100-110 - 40-100 - 60-40 Supplementary Figure 9: Full blots relating to Figure 4
4f - + - - - + + + MyD88 -FLAG - - + - - + - - IL-17RD -Myc - - - - + - - + IL-17RD ΔC -Myc 4g - + - + TLR4 -FLAG - - + + IL-17RD SEFIR -Myc - - - + - - + - IL-17RD SEFIR -Myc -130-130 Supplementary Figure 9 (continued): Full blots relating to Figure 4
5a Il17rd 5b 5c +/+ Il17rd / LPS (min) 0 5 20 60 0 5 20 60 IP: MyD88 IB: TLR4-110 Il17rd +/+ Il17rd / LPS(min) 0 5 30 60 0 5 30 60 Il17rd +/+ Il17rd / Poly(I:C) (min) 0 30 90 180 0 30 90 180 IP: MyD88 IB: TRAF6-60 IP: TRAF6 IB: Ub - 50 IP: TRAF6 IB: Ub -50-40 IP: MyD88 IB: MyD88 IP: TRAF6 IB: TRAF6-50 IP: TRAF6 IB: TRAF6 IB: TLR4-110 IB: TRAF6-50 IB: TRAF6 IB: MyD88-40 IB: TRAF6-60 - 45-42 - 45 Supplementary Figure 10: Full blots relating to Figure 5
5d IB: HA/Myc - + - + + + + MyD88 -FLAG - - + + + + + TRAF6 -HA - - - - + - - IL-17RD -Myc - - - - - + - IL-17RD ΔC -Myc - - - - - - + IL-17RD SEFIR -Myc - 100-60 5e - + - - - + + + MyD88 -FLAG - - + - - + - - IL-17RD SEFIR-Myc - - - + - - + - IL-17RD SEFIR ΔBox1-Myc - - - - + - - + IL-17RD SEFIR ΔBox3-Myc 5g - + - - - - - + + + + + MyD88 -FLAG - - + - - - - + - - - - IL-17RD SEFIR -Myc - - - + - - - - + - - - IL-17RD SEFIR T496P -Myc - - - - + - - - - + - - IL-17RD SEFIR K497R -Myc - - - - - + - - - - + - IL-17RD SEFIR L504F -Myc - - - - - - + - - - - + IL-17RD SEFIR T496P/K497R -Myc - 15-36 -36 IB: HA - 60-40 -36-36 - 40-50 - 15-45 Supplementary Figure 10 (continued): Full blots relating to Figure 5
6a LPS (min) 0 1 5 20 60 6b IP: IL-17RD IB: MyD88-25 IL-17RD sirna - - - + Scrambled sirna - - + - LPS (min) 0 20 20 20 IP: IL-17RD IB: IL-17RD - 100 IB: IL-17RD IP: IL-17RD IB: TLR4-110 IB: TLR4-110 IB: MyD88-55 - 45 Supplementary Figure 11: Full blots relating to Figure 6
Fig. S1a NT 0 20 50 80 100 IL-17RD (ng) IL-17RD β-actin Supplementary Figure 12: Full blots relating to Figure S1
Fig. S2d Il17rd +/+ Il17rd / Probe 0 30 60 120 0 30 60 120 comp LPS (min) NF-κB/Oligo binding Free Probe Supplementary Figure 13: Full blots relating to Figure S2
Fig. S3a Fig. S3b Il17rd +/+ Il17rd / Il17rd +/+ Il17rd / LPS (min) 0 5 15 30 60 240 0 5 15 30 60 240 LPS (min) 0 5 15 30 60 240 0 5 15 30 60 240 p-erk p-erk ERK ERK β-actin β-actin Supplementary Figure 14: Full blots relating to Figure S3
Fig. S4b - + - + - + TLR3-FLAG - - + + - - IL-17RD WT-Myc - - - - + + IL-17RD ΔC -Myc Fig. S4C - + - - - + + + Mal -FLAG - - + - - + - - IL-17RD -Myc - - - - + - - + IL-17RD ΔC -Myc - - - + - - + - IL-17RD SEFIR -Myc - 100-15 - 55-130 - 15-36 - 100-60 - 15-36 - 45 Supplementary Figure 15: Full blots relating to Figure S4
Fig. S4d - + - - - + + + TRIF -FLAG - - + - - + - - IL-17RD -Myc - - - - + - - + IL-17RD ΔC -Myc - - - + - - + - IL-17RD SEFIR -Myc Fig. S4e - + - + TLR3 -FLAG - - + + IL-17RD SEFIR -Myc -55-130 -55-130 Supplementary Figure 15 (continued): Full blots relating to Figure S4
Fig. S4h - + - - + + MyD88 -FLAG - - + - + - IL-17RD WT-Myc Fig. S4j - + - - + + MyD88 -FLAG - - + - + - IL-17RD WT-Myc - - - + - + IL-17RD E350A-Myc - - - + - + IL-17RD Y330F-Myc -35-35 -35-35 Supplementary Figure 15 (continued): Full blots relating to Figure S4
Fig. S5a - + - - + - MyD88-FLAG - - + - - + MyD88 TIR-FLAG - - - + + + IL17RD SEFIR-Myc Fig. S5b - + - - + + + MyD88 TIR-FLAG - - + - + - + TRAF6-HA - - - + - + + IL-17RD SEFIR-Myc IB: HA -35 IB: HA -35 Supplementary Figure 16: Full blots relating to Figure S5
Fig. S5c MyD88+ TRAF6+ EV - - RA RASEF Act ActSEF RD RDSEF IP: MyD88 IB: TRAF6 IP: MyD88-130 -72 IP: MyD88 IB: MyD88-35 -130-72 -35 IB: HA Supplementary Figure 16 (continued): Full blots relating to Figure S5
Fig. S6 Wash #1 Wash #2 Wash #3 - + - + - + - + rmyd88 -GST + + + + + + + + ril-17rd -Myc IP: GST IP: GST IB: GST - 35 Supplementary Figure 17: Full blots relating to Figure S6