Supplementary Material

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

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 Pam 3 CSK 4 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.

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.

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.

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.

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.

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 K T389 ; 2, p53 S392 ; 3, p53 S46 ; 4, p70 S6 K T229 ; 5, p70 S6 K T421 ; and 6, p53 S15. Mean changes in od/mm 2 ± SEM are shown, n = 5 independent experiments, comparing LPS plus IFNγ-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%.

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.

9 Cytokine concentrations 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 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.

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-β 1 <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.

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.