Supporting Information

Lefrançais et al. 10.1073/pnas.1410700111 SI Materials and Methods Western Blot Analysis. Proteins were fractionated by SDS/PAGE, − − Animals. C57BL/6 wild-type and Rag2 / mice were purchased electroblotted and detected with mAb to human IL-33-Cter − − from Charles River Laboratories. IL-33 / mice have been pre- (305B, 1/1,000, Alexis Biochemicals) or goat antiserum to mouse − − viously described (1). Rag2γc / mice from the National Institute IL-33-Cter (AF326, 1/500; R&D), followed by HRP-conjugated of Allergy and Infectious Diseases (2) were obtained via Taconic goat anti-mouse or donkey anti-goat polyclonal antibodies Biosciences. All mice were handled according to institutional (1/10000; Promega), and finally an enhanced chemiluminescence guidelines under protocols approved by the IPBS and FRBT kit (Amersham ECL Prime; GE Healthcare). Quantitative Western (C2EA-01) animal care committees (project no. 00663.01). blots were performed to quantify the six IL-33 forms used in cellular bioassays, using a Typhoon TRIO instrument (Amersham, GE Plasmid Construction and Protein Production. IL-33 deletion Healthcare). A standard curve was performed using purified mutants were amplified by PCR using the human (NM_033439) recombinant IL-3395–270 protein. IL-33/NF-HEV cDNA as template. The PCR fragments, thus obtained, were cloned into plasmid pcDNA3.1 (Invitrogen). The IL-33 Activity Assays and ELISA. In vitro translated full-length IL-33 or mature forms (up to 6 μL lysate per well, 18-h treatment) were human IL-33 , IL-33 , IL-33 , IL-33 , and K71G K77K78GG F94G R106G used to stimulate IL-33-responsive MC/9 mast cells (105 cells per IL-33 mutants were generated by PCR and cloned into the L108G well in 96-well plates), KU812 basophil-like chronic myelogenous same expression vector. All primer sequences are available upon leukemia cells (ATCC; 5 × 105 cells per well in 96-well plates) and request. Wild-type and mutant IL-33 proteins were synthesized cultured ILC2s (5 × 104 cells per well in 96-well plates). Cytokine in vitro in rabbit reticulocyte lysate (RRL) using the TNT- levels in culture supernatants were determined using DuoSet IL-6, Coupled Transcription/Translation system (Promega). For ex- IL-5, and IL-13 ELISA (R&D Systems). The concentration of IL-5 pression in Escherichia coli, cDNAs encoding mature forms of and IL-13 in BAL fluids and lung homogenate supernatants were IL-3395–270 and IL-33109–270 were subcloned into expression analyzed by ELISA (ELISA MAX Deluxe Sets, Biolegend for IL-5 vector pET-15b (Novagen). Recombinant proteins were pro- and Quantikine, R&D Systems for IL-13) according to the man- duced in E. coli BL21pLysS (Novagen) and purified using Ni- ufacturer’s instructions. NTA agarose column (Qiagen). His-Tag was removed by cleavage with thrombin and proteins were further purified by gel In Vivo Treatment of Mice. C57BL/6 mice were treated daily i.p. filtration (FPLC; GE Healthcare). Endotoxin levels were <0.01 with 4 μg recombinant human IL-3395–270, IL-33109–270, or PBS endotoxin units/μg of protein as determined by the Limulus for 7 d. 24 h after the last injection, BAL fluids (three lavages of amebocyte lysate QCL-1000 method (Lonza). lungs with three aliquots of 0.6 mL of PBS) were collected and stored at −80 °C for cytokine assays. Upper right lungs were Culture and Activation of Mast Cells. Human mast cells (hMCs) collected and homogenized in 1 mL of PBS with Ultra-Turrax + were obtained from CD133 peripheral blood precursors cul- (IKA Laboratory). Lung homogenates were centrifuged at 16000 tured in the presence of stem cell factor (SCF) and IL-6 as rpm for 15 min at 4 °C and the supernatants were stored at −80 °C previously described (3). After 2 mo of culture, cells were tested for cytokine analysis. Protein concentrations in lung supernatants + + + phenotypically (tryptase ,CD117,FceRI ) and functionally were quantified using NanoDrop (Nanodrop 1000 Spectropho- (β-hexosaminidase release in response to FceRI cross-linking) tometer; Thermo Fischer). To obtain lung single-cell suspensions, before use for experiments. Supernatants from activated mast lower right lungs were digested with 2 mg/mL collagenase D and cells, treated with either anti-IgE (2.5 μg/mL) or substance P 0.1 mg/mL DNase I for 60 min at 37 °C and meshed through μ (10 μmol/L) for 30 min, were collected and used for IL-33 a70- m cell strainer. Leukocyte population was enriched by using cleavage assays. Mouse bone marrow mast cells (BMMCs) (4) a Lympholyte M gradient (Cedarlane Laboratories) at 1,300 rpm were obtained from femur and tibia bone marrow cell suspensions for 20 min at room temperature. The total number of cells in lungs cultured for 10 wk in DMEM (ATCC) in the presence of IL-3 and BAL fluids was counted with a hemocytometer. Left lung was (10 ng/mL) and SCF (50 ng/mL). BMMCs or MC/9 mast cells fixed in PFA 4% overnight at 4 °C for histological analysis. For induction of allergic airway inflammation, mice were anesthetized (ATCC) were activated by treatment with PMA (20 nM to 10 μM) by isoflurane inhalation and 12.5 μg Alternaria alternata extract and ionomycin (2 μM) for 3 h, and supernatants were collected. (Greer Laboratories) was administered intranasally (i.n.) in 50 μL Protein Cleavage Assays with Mast Cell Proteases. In vitro translated PBS on days 0, 1, and 2. Tryptase inhibitor NM was used in some proteins produced in rabbit reticulocyte lysate (RRL) were in- experiments (2 mg/kg, administered i.n. on days 0, 1, and 2). BAL cubated with mast cell chymase (0.14 mU to 4.5 mU, Sigma) or fluids were collected 24 h after the final Alternaria challenge and tryptase (0.24 mU to 12 mU, Calbiochem) in 15 μL assay buffer analyzed by flow cytometry. μ + μ ’ (2 l RRL lysate 5 L PBS) for 30 at 37 °C. Cleavage assays Analysis of Cell Surface Markers by Flow Cytometry. ILC2s were 3– 4 with activated mast cells (6.10 3.10 mast cells) were performed identified in lung single-cell suspensions and BAL fluids using a for 1h-2h at 37 °C using hMCs, BMMCs, or MC/9 mast cells combination of FITC-conjugated antibodies allowing the exclu- supernatants. In some experiments, mast cell proteases super- sion of T, B, natural killer (NK), NKT, mast cells, basophils, natants were preincubated (20 min, 37 °C) with cysteine protease dendritic cells and macrophages. Lineage antibodies included inhibitor E64 (50 μM; Sigma), serine protease inhibitor AEBSF CD4 (clone GK1.5), CD19 (clone 1D3), CD45R (clone RA3- (8 mM; Calbiochem), tryptase inhibitor Nafamostat mesylate 6B2), NK1.1 (clone PK136), CD3 (clone 17A2), CD11b (clone (NM, 1μM; Enzo Life Sciences) or chymase inhibitors chymos- M1/70) (BD Bioscience), Ter119 (clone Ter119), Ly-6G (clone tatin (CS, 100 μM; Sigma) and CGI (50 μM; Calbiochem), before RB6-8C5), FceRIa (clone MAR-1) and CD11c (clone N418) adding in vitro translated proteins. Cleavage products were an- (eBioscience). Antibodies CD45-PerCp (clone 30F11), CD90.2- alyzed by SDS/PAGE and Western blot. APCCy7 (clone 53–2.1) (BD Bioscience), Sca-1-APC (clone D7),

Lefrançais et al. www.pnas.org/cgi/content/short/1410700111 1of5 CD25-eFluor450 (clone PC61.5), CD127-Pe (clone A7R34), Hematopoietic progenitor cell enrichment kit; Stem Cell Tech- − + KLRG1-APC (clone 2F1), ICOS-PE (clone 7E-17G9) (eBio- nologies). Subsequently, Lin CD45 cells were selected by using science) and T1/ST2 biotinylated (clone DJ8, MD Biosciences) magnetic beads conjugated to anti-mouse CD45 monoclonal − + followed by streptavidin-PeCy7 staining (BD Bioscience) were antibody (Miltenyi Biotech). Purity of Lin CD45 cells was ana- also included for further characterization of ILC2s. Eosinophils lyzed by staining isolated cells with lineage antibodies and anti- + were identified by using CD45-PerCp (clone 30F11), SiglecF-Pe bodies against ST2, CD25, CD127, Sca-1 and CD90.2. Lung CD45 − (clone E20-2440) (BD Bioscience) and CD11c-APC (clone N418) Lin ILC2s were cultured at a density of 3.105 cells/mL in RPMI (eBioscience). Antibodies were diluted in a FcR blocker (CD16/ medium complemented with 10% FCS, 1% Penicillin/Streptomy- CD32 clone 2.4G2; BD bioscience) for 30 min in the dark at 4 °C. cin, 50 μM β-mercaptoethanol, 20 ng/mL recombinant mouse IL-2 Isotype and single-stain controls were included. Dead cells were (rmIL-2) and 10 ng/mL rmIL-7 (R&D Systems). After 72 h, surface excluded by using viability dye eFluor506. Samples were acquired markers of lung ILC2s were analyzed by flow cytometry. on a LSRII flow cytometer using DiVa software (BD Bioscience) and analyzed using FlowJo software (Tree Star). Histology. Histological evaluation was performed on formalin- fixed mouse tissues. Five μm paraffin-embedded sections of the Isolation and Culture of ILC2 Cells. ILC2s were isolated from pooled lung were prepared and stained with hematoxylin and eosin for − − lungs of Rag2 / mice treated with daily i.p. injections of 4 μgof morphological evaluation. Periodic-acid-Schiff (PAS) staining rhIL-3395–270 for 7 d. Lineage-negative cells were enriched by was used to detect the presence of mucus in lung tissue sections. magnetically depleting lineage-positive cells using biotin-conju- gated antibodies to CD5, CD11b, CD19, CD45R, Ly-6G/C, Statistical Analyses. The Student t test was used for comparison Ter119, and 7–4 and Easysep D magnetic particles (Mouse between two groups. All data are represented as mean ± SEM.

1. Pichery M, et al. (2012) Endogenous IL-33 is highly expressed in mouse epithelial barrier 3. Gaudenzio N, Laurent C, Valitutti S, Espinosa E (2013) Human mast cells drive memory tissues, lymphoid organs, brain, embryos, and inflamed tissues: In situ analysis using CD4+ T cells toward an inflammatory IL-22+ phenotype. J Allergy Clin Immunol 131(5): a novel Il-33-LacZ gene trap reporter strain. J Immunol 188(7):3488–3495. 1400–1407. 2. Cao X, et al. (1995) Defective lymphoid development in mice lacking expression of the 4. Gurish MF, et al. (1992) Differential expression of secretory granule proteases in mouse common cytokine receptor gamma chain. Immunity 2(3):223–238. mast cells exposed to interleukin 3 and c-kit ligand. J Exp Med 175(4):1003–1012.

B 1-266

MC/9 sup

A CS NM CS + vehicle AEBSF NM Full length IL-33 Mm

MW (kDa) MW 55 (kDa) 40 55 40 35 Full length MmIL-331-266 35 25 Full length MmIL-33 1-266 25 Cleaved MmIL-33 forms Cleaved MmIL-33 forms 15 15

10 10

Fig. S1. Murine full-length IL-33 protein is a substrate for mast cell proteases. In vitro translated full-length murine IL-331–266 (2.5 μL lysate) was incubated with supernatants of activated mouse bone-marrow–derived mast cells (BMMCs) or MC/9 mast cells for 1 h at 37 °C. (A) Cleavage of full-length IL-33 by supernatants from activated bone-marrow–derived murine mast cells. Murine mast cells were differentiated from bone marrow multipotential stem cells by culture in the presence of SCF and IL-3, and release of serine proteases from secretory granules was induced by stimulation with ionomycin and PMA. (B) Murine full-length IL-33 protein is processed by mast cell tryptase. Activated MC/9 mast cells were pretreated with vehicle, chymase inhibitor chymostatin (CS), tryptase inhibitor nafamosat mesylate (NM), or serine protease inhibitor AEBSF for 1 h at 37 °C. Proteins were migrated on SDS/PAGE and revealed by Western blot with goat anti-mouse IL-33–Cter antibodies.

Lefrançais et al. www.pnas.org/cgi/content/short/1410700111 2of5 A PBS 109-270 0.47% 29.3% 68.9% 73.4% 93.8% ST2 Sca-1 CD25 CD45 Lin CD90.2 CD127 CD127 Isotype Isotype Isotype Isotype Isotype Isotype

B C BAL ) 5 Number of 10 ILC2 (x CD117 ICOS KLRG1 PBS 109-270

D E PBS 109-270 Lung Lung 4.24% 19.3% IL-5 pg/mg total protein IL-13 pg/mg total protein SiglecF CD11c PBS 109-270 PBS 109-270

F BAL BAL G Pre Post 24.4%45.8% 94.6%3.46% IL-5 pg/ml IL-13 ng/ml 29.7% 1.82%

PBS 95-270 PBS 95-270 CD45 Lin

Fig. S2. IL-33 mature forms IL-33109–270 and IL-3395–270 are potent inducers of ILC2s, eosinophils and type-2 cytokines in vivo. (A) Representative FACS plots of ILC2 population from lungs of C57BL/6 mice treated with PBS or rhIL-33109–270. C57BL/6 mice received 4 μg rhIL-33109–270 or PBS for 7 consecutive days via i.p. + injection. At day 8, lungs were collected and processed to single cell suspensions for immunostaining and flow cytometry analysis. ILC2s were gated on CD45 − + + + + + Lin ST2 CD90.2 Sca-1 CD127 CD25 .(B) Representative histograms of CD117, ICOS, and KLRG1 expression at the surface of lung ILC2 populations (black lines).

Isotype controls are shown (shaded histograms). (C) Number of ILC2s in BAL fluids in response to rhIL-33109–270. n = 8–10 mice, two experiments. (D) Repre- + + − sentative FACS plots of eosinophil population gated as CD45 SiglecF CD11c cells in whole-lung tissues extracted from PBS or rhIL-33109–270-treated C57BL/6 mice. Data are representative of two independent experiments. (E) IL-5 and IL-13 levels in lungs in response to rhIL-33109–270. n = 8–10 mice, two experiments. (F) Type-2 cytokine levels in BAL fluids in response to rhIL-3395–270. n = 8–10 mice, two experiments. (G) Representative FACS plots of the isolation of ILC2s from pooled lungs of rhIL-3395–270-treated Rag2-deficient mice. (Left) FACS plot before magnetic separation; (Right) FACS plot following magnetic separation. Data are representative of five independent experiments.

Lefrançais et al. www.pnas.org/cgi/content/short/1410700111 3of5 A hGranzyme B dose time MW (kDa) 35

25

15

B hGranzyme B

MW (kDa) 55 35

25 ?

111-270

15 C Nuclear Activation IL-1-like Cytokine 1 Domain 66 Domain 111 Domain 270

111-270 70 80 90 100 LKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSS

Fig. S3. Human full-length IL-33 protein is a substrate for granzyme B. (A) In vitro translated full-length human IL-331–270 was incubated with purified human granzyme B (Left) increasing amounts up to 0.3 units; (Right) increased time from 5 min to 1 h. Proteins were separated by SDS/PAGE and revealed by Western blot with anti–IL-33–Cter mAb 305B. Blots are representative of two independent experiments. (B) Mutation of D110 to glycine in full-length IL-33 inhibits formation of the 18-kDa granzyme B cleavage product. In vitro translated full-length human IL-331–270 or single point mutants IL-33E92G (E92G) and IL-33D110G (D110G) were incubated with purified human granzyme B (0.3 units, 1 h at 37 °C). (C) Mapping of the granzyme B cleavage site in the central domain of IL-33 (amino acids 66–111). The sequence surrounding the granzyme B cleavage site is shown.

Lefrançais et al. www.pnas.org/cgi/content/short/1410700111 4of5 yoiedmi IL-33 domain cytokine rpae( tryptase i.S4. Fig. ernase al. et Lefrançais AEadrvae yWsenbo ihanti with blot Western by revealed and PAGE h IL-1 The B IL-33 , www.pnas.org/cgi/content/short/1410700111 79 – iectkn oano L3 sntcevdb atcl rtae.( proteases. cell mast by cleaved not is IL-33 of domain cytokine like – 270 112 n IL-33 and – 270 eeicbtdwt nraigaonso uiidhmnms elcyae( chymase cell mast human purified of amounts increasing with incubated were 107 – 270 eecmgae ntegl.Bosaerpeettv ftoidpnetexperiments. independent two of representative are Blots gels. the on comigrated were ) B – IL-33 IL-33 A IL-33 IL-33 IL-33 – trmb35.TeI-3mtr om eeae yms elcyae( chymase cell mast by generated forms mature IL-33 The 305B. mAb Cter 112-270 1-270 112-270 1-270 (kDa) (kDa) MW MW 70 15 25 35 40 55 70 15 25 35 40 55 10 : : : : ------++ + + ------+- + - - + ------++ + IL-33 + - 1-270 - - + - 112- - - - 270 - +- + - - + - IL-331-270 + 112-270

IL-3379-270 IL-3395-270

IL-33107-270 IL-33109-270 Tyts hTryptase hTryptase CyaehChymase hChymase A and B nvtotasae ullnt ua IL-33 human full-length translated vitro In ) A rtyts ( tryptase or ) B .Poen eesprtdo SDS/ on separated were Proteins ). A IL-33 , 95 – 270 n IL-33 and 1 – 270 n IL-1 and 109 – 270 5of5 )and – like