il-37 final supplement v2 - nature research...il-37 cells. the raw-il-37 (open bars) and the...
TRANSCRIPT
![Page 1: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/1.jpg)
Supplementary Material
accompanying the manuscript
Interleukin 37 is a fundamental inhibitor of innate immunity
Marcel F Nold, Claudia A Nold-Petry, Jarod A Zepp, Brent E Palmer,
Philip Bufler & Charles A. Dinarello
Nature Immunology: doi:10.1038/ni.1944
![Page 2: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/2.jpg)
2
Supplementary Figure 1 Effect of siRNA to IL-37 on TNF and IL-1Ra in PBMCs.
PBMCs were transfected with concentration-matched pairs of siIL-37 (open bars) or
scrambled (solid bars), then stimulated with 1 µg/ml LPS or 10 ng/ml Pam3CSK4 or left
untreated for 24 h. Thereafter, protein concentrations of TNF (a) and IL-1Ra (b) were
determined in culture supernatants by electrochemiluminescence assay. The graphs
display mean ± SEM cytokine concentrations, n = 7 independent experiments; *, P <
0.05 and **, P < 0.01 for siIL-37 compared to scrambled. The numbers above the bars
indicate fold changes comparing siIL-37 to scrambled; the numbers next to the error
bars are individual P-values.
Nature Immunology: doi:10.1038/ni.1944
![Page 3: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/3.jpg)
3
Supplementary Figure 2 Cytokine production in THP-1 and A549 cells transfected
with IL-37b. THP-1 (a-c) or A549 (d) cells were transfected with either the pIRES IL-37b
plasmid (open bars) or mock-transfected with pIRES lacking IL-37b (solid bars). (a-c)
THP-1 cells were then either differentiated with PMA (b,c) or not (a); n = 6; *, P < 0.05;
**, P < 0.01; ***, P < 0.001 for mock-transfected vs. IL-37b expression. (a) IL-8
concentrations after treatment with LPS (1 µg/ml), IL-1β (25 ng/ml), or vehicle. (b,c) IL-
1α and IL-1β in PMA-differentiated THP-1 macrophages after incubation with LPS, IL-1β,
or vehicle. (d) Immunofluorescence of IL-37b in transiently transfected A549 cells;
similar images were obtained during each of the transfection experiments.
Nature Immunology: doi:10.1038/ni.1944
![Page 4: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/4.jpg)
4
Supplementary Figure 3 Confocal microscopy of mock-transfected A549 cells
(control for Fig. 4a). A549 cells were transfected, then stimulated as indicated. 24 h
later, images of the expression and localization of IL-37/FLAG (Cy3, yellow), phospho-
Smad3 (FITC, green), cell membranes (Alexa Fluor 633, red), and nuclei (DAPI, blue,
shown only in overlays) were obtained by confocal microscopy. One representative of
three independently performed experiments is shown.
Nature Immunology: doi:10.1038/ni.1944
![Page 5: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/5.jpg)
5
Supplementary Figure 4 Inhibition of Smad3 increases IL-1α production in RAW-
IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell
clones were treated with the indicated concentration of SIS3 for 30 min. Thereafter, 100
ng/ml LPS were added to the cultures. IL-1α was measured in cell lysates 24 h later.
Data are depicted as means of percent changes conferred by treatment with SIS3 (i.e.
difference between LPS alone and LPS + SIS3) ± SEM, n = 6 independent experiments;
*, P < 0.05 and ***, P < 0.001 for RAW-IL-37 vs mock-transfected cells.
Nature Immunology: doi:10.1038/ni.1944
![Page 6: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/6.jpg)
6
Supplementary Figure 5 Silencing of Smad3 protein by lentivirally delivered
shRNA. THP-1 cells were incubated with lentivirus containing either scrambled shRNA
or shRNA to Smad3. After selection of positive cells with puromycin, single cell clones
were derived, expanded, and then tested for production of Smad3 by immunoblotting.
The depicted clones were selected out of a total of 10 scrambled- and 12 shSmad3-
transfected clones and used in the experiments.
Nature Immunology: doi:10.1038/ni.1944
![Page 7: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/7.jpg)
7
Supplementary Figure 6 Changes in kinase phosphorylation in THP-1 cells
conferred by IL-37b. After transfection with either the IL-37b construct or the mock
plasmid, THP-1 macrophages were stimulated with 1 µg/ml LPS plus 50 ng/ml IFN-γ.
Lysates were harvested after 10, 20, or 120 min and the phosphorylation status of the
indicated kinases was determined using the Phospho-Kinase Array followed by
densitometry. The numbers indicate phosphorylation sites as follows: 1, p70 S6 KT389;
2, p53S392; 3, p53S46; 4, p70 S6 KT229; 5, p70 S6 KT421; and 6, p53S15. Mean changes
in od/mm2 ± SEM are shown, n = 5 independent experiments, comparing LPS plus IFN-
γ-stimulated IL-37b-expressing cells with mock-transfected cells. Red and orange bars
and labels depict decreases in cytokine concentrations by 33 to 67% and more than
67%, respectively; green bars and labels represent increases by more than 33%.
Nature Immunology: doi:10.1038/ni.1944
![Page 8: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/8.jpg)
8
Supplementary Figure 7 IL-37b expression affects RAW cell differentiation. RAW-
IL-37 and mock-transfected (MT) cells were detached from the flasks, plated into 6-well
polystyrene plates and allowed to grow overnight. (a) Live cells 20 h after plating. Cells
were then either left untreated for control (b) or stimulated with 10 ng/ml LPS (c) and
photographed another 21 h later. The images are representative of 9 independently
performed experiments.
Nature Immunology: doi:10.1038/ni.1944
![Page 9: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/9.jpg)
9
Supplementary Table 1 Cytokine concentrations in RAW cells after stimulation
with TLR agonists. RAW-IL-37 (= C17) and mock-transfected (MT) cells were stimulated
with the listed TLR ligands for 24 h. Concentrations of stimuli are given in µg/ml.
Supernatants (IL-6, IFN-γ, TNF, MIP-1α, MIP-2) and lysates (IL-1α) were then assayed
by electrochemiluminescence or ELISA. The Table displays absolute cytokine
concentrations in pg/mg total protein (pg/mg t.p.) from one representative of 3
independent experiments.
Cytokine concentra-tions in pg/mg t.p.
IL-1α MIP-2 TNF IL-6 MIP-1α IFN-γ
Stimulus µg/ml MT C17 MT C17 MT C17 MT C17 MT C17 MT C17
Ctrl 6 2 0.1 0.01 66 60 0.3 1 555 682 3 3
LPS O55 0.1 4438 52 399 22.2 19103 2046 852 22 663 615 9 12
Pam 0.01 1543 8 77 9.5 4531 1786 82 2 456 626 11 11
Pam 0.1 1944 23 431 21.4 4312 1813 206 11 557 524 12 9
poly(I:C) 0.03 758 4 26 8.7 5060 1985 214 10 577 634 8 14
poly(I:C) 0.1 1082 7 38 12 21932 2796 239 15 619 602 16 16
LPS R5 0.01 2489 26 209 16 4411 1522 527 15 518 779 6 8
LPS R5 0.1 3222 32 297 17 6314 1852 562 16 488 649 5 9
Flagellin 0.01 12 3 0.2 0.01 91 118 0.4 1 666 714 2 6
Flagellin 0.1 18 5 0.8 0.01 901 198 0.4 1 452 708 3 7
MALP-2 0.01 1679 12 351 14.4 5304 1076 116 8 704 671 6 6
MALP-2 0.1 2005 15 455 16.1 11843 1009 145 8 768 643 8 7
poly(U) 1 10 2 0.1 0.01 76 69 0.4 1 794 800 7 9
poly(U) 5 12 3 0.3 0.01 113 77 0.4 1 614 961 8 7
CpG 1 1899 10 279 15.2 5464 1585 34 7 512 526 6 9
CpG 5 2241 34 219 24.4 5005 2372 57 15 501 511 10 12
Nature Immunology: doi:10.1038/ni.1944
![Page 10: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/10.jpg)
10
Panel 6a test SDF-1 0.002 BCA-1 0.044 Panel 6b & c het hom test IL-27 0.014 all cytokines <0.05 <0.05 ANOVA on ranks IL-4 0.491 I-309 0.26 Panel 6d IL-2 0.631 IL-1α 0.007 unp. t-test IL-7 0.289 MIP-1α 0.002 Rank-sum test M-CSF 0.116 MIP-2 0.005 Rank-sum test TIMP-1 0.011 IL-1α 0.392 Panel 6e MIP-1α 0.303 IL-1β <0.001 <0.001 RANTES 0.013 all IL-6 0.012 0.008 all One Way MIG 0.038 unpaired TNF 0.287 0.287 ANOVA IL-1Ra 0.024 t-test MCP-1 0.001 <0.001 sTREM-1 0.392 KC 0.077 0.021 MCP-5 0.251 IP-10 0.019 Panel 7a MCP-1 0.026 MIP-1α 0.002 Rank-sum test IL-23 0.034 MIP-2 0.01 unp. t-test KC 0.024 IL-1α 0.279 Rank-sum test IL-1β 0.008 IL-1β 0.021 Rank-sum test MIP-2 0.014 IL-6 <0.001 unp. t-test IL-17 0.026 TGF-β1 <0.001 unp. t-test eotaxin 0.017 IL-6 <0.001
Supplementary Table 2 Individual P-values and statistical tests of Figs. 6 and 7a.
Nature Immunology: doi:10.1038/ni.1944
![Page 11: IL-37 final Supplement V2 - Nature Research...IL-37 cells. The RAW-IL-37 (open bars) and the mock-transfected (solid bars) RAW cell clones were treated with the indicated concentration](https://reader033.vdocuments.mx/reader033/viewer/2022053007/5f0ad5747e708231d42d911e/html5/thumbnails/11.jpg)
11
comparing to symbol IL-1β IL-6 MCP-1 KC WT + scr WT + siS3 # 0.16 0.13 0.55 0.197 WT + scr IL-37tg + scr * 0.003 <0.001 0.01 <0.001
IL-37tg + scr IL-37tg + siS3 0.038 0.049 0.005 0.037
comparing to symbol IL-17 TNF IFN-γ
WT + scr WT + siS3 # 0.495 0.017 0.345 WT + scr IL-37tg + scr * 0.002 0.015 0.021
IL-37tg + scr IL-37tg + siS3 0.039 0.047 0.195
Supplementary Table 3 Individual P-values of Fig. 8 b and c.
Nature Immunology: doi:10.1038/ni.1944