The IL-1-like IL-33 is inactivated after maturation by caspase-1

Corinne Cayrola,b and Jean-Philippe Girarda,b,1

aCentre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France; and bUniversite´de Toulouse, Universite´Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France

Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved April 10, 2009 (received for review December 13, 2008) IL-33 is a chromatin-associated cytokine of the IL-1 family that has that nuclear IL-33 possesses transcriptional regulatory proper- recently been linked to many diseases, including asthma, rheuma- ties and associates with chromatin in vivo (5). Recently, we found toid arthritis, atherosclerosis, and cardiovascular diseases. IL-33 that IL-33 mimics Kaposi sarcoma herpesvirus for attachment to signals through the IL-1 -related ST2 and drives chromatin, and docks, through a short chromatin-binding pep- production of pro-inflammatory and T helper type 2-associated tide, into the acidic pocket formed by the histone H2A-H2B in mast cells, T helper type 2 lymphocytes, , dimer at the surface of the nucleosome (22). Together, our , invariant natural killer T cells, and natural killer cells. findings suggested IL-33 is a dual-function protein that may play It is currently believed that IL-33, like IL-1␤ and IL-18, requires important roles as both a cytokine and an intracellular nuclear processing by caspase-1 to a mature form (IL-33112–270) for biolog- factor (5, 22). A similar duality of function has previously been ical activity. Contrary to the current belief, we report here that shown for IL-1␣ and chromatin-associated cytokine HMGB1. full-length IL-331–270 is active and that processing by caspase-1 IL-1␣, a cell-associated cytokine, exhibits potent pro- results in IL-33 inactivation, rather than activation. We show that activities mediated by the cell surface full-length IL-331–270 binds and activates ST2, similarly to IL-33112– IL-1 receptors (1) but also functions intracellularly by translo- 270, and that cleavage by caspase-1 does not occur at the site cating to the nucleus and regulating transcription (23). HMGB1 initially proposed (Ser111), but rather after residue Asp178 between regulates transcription and chromatin structure in the nucleus the fourth and fifth predicted ␤-strands of the IL-1-like domain. (24) but also functions extracellularly as a cytokine when se- Surprisingly, the caspase-1 cleavage site (DGVD178G) is similar to creted by activated during inflammation (25) or the consensus site of cleavage by caspase-3, and IL-33 is also a released by necrotic cells (26). IL-1␣ has also been shown to be substrate for this apoptotic caspase. Interestingly, we found that released by dying cells (1, 27–29), and HMGB1 and IL-1␣ were full-length IL-33, which is constitutively expressed to high levels by thus defined as endogenous ‘‘danger signals’’ or ‘‘alarmins’’ that endothelial cells in most normal human tissues, can be released in may alert the immune system after tissue damage during trauma the extracellular space after endothelial cell damage or mechanical or infection (30). injury. We speculate that IL-33 may function, similarly to the It is currently believed that IL-33, like classical IL-1 family prototypical alarmins HMGB1 and IL-1␣, as an endogenous danger members IL-1␤ and IL-18, requires maturation by caspase-1 for signal to alert cells of the innate immune system of tissue damage optimal biological activity. This is based on the initial report by during trauma or infection. Schmitz et al., who indicated caspase-1 cleaves IL-33 after residue Ser111, resulting in the production of mature IL-33112–270 inflammation ͉ ͉ endothelial cell (4). Most of the studies that have been published to date used a recombinant IL-33 protein corresponding to this mature form. Here, we show that full-length IL-33 is biologically active, ytokines of the IL-1 family play a major role in a wide range 1–270 and that cleavage by caspase-1 does not occur at the site initially of inflammatory, infectious, and autoimmune diseases (1, 2). C proposed but rather after residue Asp within the IL-1-like MEDICAL SCIENCES IL-33 [previously known as nuclear factor from high endothelial 178 domain. Consequently, caspase-1 processing results in inactiva- venule, or NF-HEV (3)] is the most recent addition to the IL-1 tion of IL-33, rather than activation. We also demonstrate that family (4, 5). Based on animal model studies and analyses of full-length IL-33 can be released in the extracellular space diseased tissues from patients, IL-33 has been proposed to 1–270 after endothelial cell damage or injury. We discuss the possibility represent a promising therapeutic target for several important that IL-33 may function as an endogenous danger signal, like the diseases, including asthma and other allergic diseases (4), rheu- prototypical alarmins IL-1␣ and HMGB1. matoid arthritis (5, 6), atherosclerosis (7), and cardiovascular diseases (8, 9). IL-33 has been shown to signal through the IL-1 Results receptor-related protein ST2 (4) and to drive production of The 20–22 kDa Caspase-1 Cleavage Product of IL-33 Does Not Corre- cytokines [both pro-inflammatory and T helper type 2 (Th2)- spond to the IL-1-Like Domain. As previously reported by Schmitz associated cytokines] and in mast cells, Th2 lym- et al. (4), we confirmed that in vitro translated human IL-33 can phocytes, basophils, eosinophils, invariant natural killer (NK) T be cleaved by caspase-1 to a 20–22 kDa form, as determined by cells, and NK cells (4, 10–19). IL-33 signaling has also been SDS/PAGE (Fig. 1A). As expected, pro-IL-1␤ was also cleaved shown to require the IL-1 receptor (IL-1R) accessory protein, by caspase-1 to the IL-1␤ mature form. Cleavage was abrogated IL-1RAcP, indicating that IL-33 shares with IL-1 not only in both cases by the caspase-1 inhibitor Ac-YVAD-CHO (Fig. structural homology but also signaling pathways (11, 14). We initially discovered IL-33 as a nuclear factor abundantly expressed in endothelial cells of high endothelial venules in Author contributions: C.C. and J.-P.G. designed research; C.C. performed research; C.C. and lymphoid organs (3, 5), but we (20) and others (21) have recently J.-P.G. analyzed data; and J.-P.G. wrote the paper. found that IL-33 is also constitutively expressed to high levels in The authors declare no conflict of interest. the nucleus of endothelial cells in other human tissues. These This article is a PNAS Direct Submission. observations indicated that IL-33 is widely expressed along the 1To whom correspondence should be addressed. E-mail: [email protected]. vascular tree and that endothelial cells constitute a major cellular This article contains supporting information online at www.pnas.org/cgi/content/full/ source of IL-33 in most human tissues (20, 21). We also showed 0812690106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812690106 PNAS ͉ June 2, 2009 ͉ vol. 106 ͉ no. 22 ͉ 9021–9026 Downloaded by guest on September 26, 2021 A like domain, as initially proposed. Western blot analysis with antibodies specific for the N- and C-terminal domains of IL-33 Caspase 1 : - Caspase 1 : - Ac-YVAD- : -----+ MW Ac-YVAD- : -----+ MW [see supporting information (SI) Fig. S1] clearly revealed that CHO (kDa) CHO (kDa) antibodies directed against the first 15 amino-terminal residues 150 150 76 76 of IL-33 (IL-33-Nter) recognize the 20–22 kDa caspase-1 cleav- 52 52 age product, whereas the Nessy-1 mAb, which is directed to the 38 β 38 IL-33 31 pro-IL-1 31 C-terminal IL-1-like domain (IL-33179–270), recognizes a smaller cleaved 24 mature 24 fragment of 12–13 kDa, migrating close to the gel front and β IL-33 17 IL-1 17 found at very low levels compared with the full-length IL-33 protein (Fig. 1C). We used an IL-33 protein tagged with myc-epitope at its carboxy-terminus in these experiments, and B Caspase 3 : - Western blot analysis with an anti-myc mAb detected the small MW Ac-DEVD-CHO : -----+(kDa) 12–13 kDa fragment but not the 20–22 kDa form (Fig. 1C), 150 allowing us to independently confirm that the 20–22 kDa 76 52 caspase-1 cleavage product corresponds to the N-terminal, 38 rather than C-terminal, part of IL-33. IL-33 31 24 cleaved IL-33 Caspase-1 Cleaves IL-33 After Residue Asp178 Within the IL-1-Like 17 Cytokine Domain. The size of the N-terminal fragment generated after caspase-1 processing indicated to us that cleavage must

0 0 0 0 7 7 7 7 occur within the IL-1-like domain. Inspection of this region 2 2 2 - -2 - - 1 1 1 1 L 3 L 3 L 3 L 3 3 3 3 3 revealed the existence of a potential cleavage site (DGVD178G), C R - R - R - R - L L L R I R IL R I R I similar to the consensus site of cleavage by caspase-3, and located between the fourth and fifth predicted ␤-strands of the IL-1-like Caspase 1 : - - ++ - - + + - - + + - - + + domain (Fig. 2A). The possibility that caspase-1 may cleave IL-33 Ac-YVAD-CHO : - --+ - --+ - - - + - --+ MW (kDa) at this position was further supported by the observation that an 76 in vitro translated fragment corresponding to IL-33 residues 52 38 1–178 co-migrated on SDS/PAGE with the 20–22 kDa caspase-1 31 cleavage product (Fig. 2B). To provide definitive proof for 24 caspase-1 cleavage of IL-33 after residue Asp178, a single muta-

17 tion was introduced that replaced Asp178 by an alanine. As shown in Fig. 2C, this mutation totally prevented the processing of Fluo.gel WB IL-33 WB IL-33 WB full-length IL-33 by caspase-1. We concluded that caspase-1 Nter Nessy-1 myc cleaves IL-331–270 at the DGVD178G site. Mutation of Asp178 to alanine also abrogated cleavage of IL-33 by caspase-3 (Fig. 2D), Fig. 1. Processing of full-length IL-33 by caspase-1 generates a 20–22 kDa indicating that caspase-1 and caspase-3 cleave IL-331–270 at the cleavage product that does not correspond to the IL-1-like domain. (A) Re- same site within the IL-1-like domain. We then asked whether ␤ combinant caspase-1 cleaves full-length IL-33 and pro-IL1 in vitro. Fluores- cleavage of IL-33 at this site occurs in cells undergoing apoptosis. cently labeled were incubated for2hat37°Cwith increasing amounts of recombinant caspase-1 (0.05, 0.15, 0.5, or 1 unit) and then ana- WT IL-33 and the IL-33D178A mutant were expressed in U2OS lyzed by SDS/PAGE and fluorography. Cleavage was abrogated by the epithelial cells (Fig. S2), and apoptosis was induced by treatment caspase-1 inhibitor Ac-YVAD-CHO. (B) Recombinant caspase-3 cleaves full- with the DNA-damaging agent doxorubicin. Western blot anal- length IL-33 in vitro. Fluorescently labeled IL-33 was incubated with increasing ysis revealed that WT IL-33, but not the IL-33D178A mutant, is amounts of recombinant caspase-3 (0.05, 0.15, 0.5, or 1 unit) as described in A. cleaved during doxorubicin-induced apoptosis and that cleavage Cleavage was abrogated by the caspase-3 inhibitor Ac-DEVD-CHO. (C) The can be prevented by pretreatment with the pan-caspase inhibitor 20–22 kDa caspase-1 cleavage product of IL-33 is recognized by IL-33 Nter Z-VAD-fmk (Fig. 2E). We next looked at endogenous native antibodies but not by antibodies to the C terminus (Nessy-1, anti-myc). Fluo- IL-33. As endothelial cells constitute a major cellular source of rescently labeled or unlabeled IL-33 proteins, containing a C-terminal myc- IL-33 in human tissues (20, 21), we selected human primary epitope tag, were cleaved with 0.5 unit of caspase-1 as described in A and analyzed by fluorography (fluorescent IL-33) or Western blot (unlabeled IL-33) endothelial cells as a cellular system for these experiments. with IL-33 Nter, Nessy-1, or anti-myc antibodies. Western blot analysis revealed that endogenous IL-33 migrates as a 30–31 kDa band, the identity of which was validated after knockdown of IL-33 expression with specific siRNAs (Fig. 2F). 1A). We then tested the possibility that IL-33 may also be a Treatment of the endothelial cells with the apoptosis-inducing substrate for other caspases. We found that incubation of in vitro agent staurosporine resulted in a complete maturation of en- translated full-length IL-33 protein with caspase-3, the proto- dogenous IL-33 that was prevented by pretreatment with the typic apoptotic caspase, led to the production of a 20–22 kDa pan-caspase inhibitor Z-VAD-fmk (Fig. 2G). We concluded that native IL-33 is processed by endogenous caspases during apo- fragment very similar in size to the fragment observed after ptosis in primary human endothelial cells. caspase-1 cleavage (Fig. 1B). Like cleavage by caspase-1, cleav- age of IL-33 by caspase-3 was specific, as it was not observed in Full-Length IL-331–270 Binds and Activates the IL-33 Receptor ST2. As the presence of the caspase-3 inhibitor Ac-DEVD-CHO. We our data indicated that, unlike IL-1␤ and IL-18, cleavage of concluded that full-length IL-331–270 protein can be cleaved by IL-33 by caspase-1 does not lead to the production of the mature both caspase-1 and caspase-3 in vitro and that processing results IL-1-like domain, we then asked whether, similarly to the IL-1␣ in both cases in the generation of a 20–22 kDa cleavage product. precursor (1, 31), full-length IL-331–270 may possess biological Schmitz et al. (4) suggested caspase-1 cleaves IL-331–270 after activity. We first tested the capacity of full-length IL-331–270 to residue Ser111, resulting in the production of mature IL-33112–270, bind to the ST2 receptor in pull-down experiments. We found corresponding to the C-terminal IL-1-like cytokine domain. that a human ST2-Fc fusion protein precipitates in vitro syn- Unexpectedly, we discovered that the 20–22 kDa caspase-1 thesized full-length IL-331–270, similarly to the isolated IL-1-like cleavage product does not correspond to the C-terminal IL-1- domain IL-33112–270 (Fig. 3A). In contrast, the amino-terminal

9022 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812690106 Cayrol and Girard Downloaded by guest on September 26, 2021 A DGVDG Hs …170SNESG KMLM183… DGVDG Mm …167ASQSG KKVM180… 1 65 112 270 CBM N-terminal domain IL-1-like cytokine domain

A A B 0 8 C 0 8 D 0 8 7 7 7 7 7 7 2 1 -2 1 -2 1 L - - 3 1 3 1 3 1 3 D 3 1 3 D R 3 3 3 3 3 3 R ------IL IL IL IL IL IL

Caspase 1: - - + + - Caspase 1: - ++ - + + Caspase 3 : - ++ - + + Ac-YVAD-: - --+ - Ac-YVAD-: - - + - - + Ac-DEVD- : - - + - - + CHO CHO : CHO MW (kDa) 52 38 31 IL-33 IL-33

24 cleaved cleaved * IL-33 17 IL-33

pcDNA3 IL-33 E 1-270 IL-33D178A FG 1-178 SiRNA 1-270 Doxorubicin : - + + - ++ - + + 3 Staurosporine : - rl -3 + + Z-VAD-fmk : - - t L MW + I Z-VAD-fmk : IL-33 - - - C - IL-33 - + + (kDa) + + 47.8 35.9 IL-33 IL-33 cleaved 24.5 cleaved IL-33 18.7 IL-33 PARP cleaved PARP

Fig. 2. Caspase-1 processing of IL-33 occurs after residue Asp178 within the IL-1-like domain. (A) Primary structure of human IL-33. The N-terminal domain involved in IL-33 nuclear activities and the IL-1-like domain, with its 12 predicted ␤-strands (black boxes), are indicated. The sequence surrounding the caspase-1 and caspase-3 cleavage site (Asp178) is shown for both human (Hs) and mouse (Mm) IL-33. CBM, chromatin-binding motif (aa 40–58). (B) An IL-331–178 deletion protein generated by in vitro translation (not tagged with myc-epitope) co-migrates on SDS/PAGE with the 20–22 kDa caspase-1 cleavage product of IL-33. Fluorescently labeled IL-33 protein was incubated with 0.5 units of caspase-1 for2hat37°C(with or without prior incubation with Ac-YVAD-CHO inhibitor) and analyzed by SDS/PAGE and fluorography. (C and D) Mutation of Asp178 to alanine abrogates cleavage of IL-33 by both caspase-1 (C) and caspase-3 (D). Fluorescently labeled IL-331–270 and IL-33D178A proteins were incubated with recombinant caspase-1 (C) or caspase-3 (D) as described in B. The Ac-YVAD-CHO and Ac-DEVD-CHO inhibitors were used at 100 ␮M. Asterisk indicates non-specific band. (E) Mutation of Asp178 to alanine abrogates cleavage of IL-33 by endogenous caspases during doxorubicin-induced apoptosis. U20S cells were transfected with IL-331–270 or IL-33D178A expression vectors and treated 24 h later with doxorubicin in the presence or absence of the pan-caspase inhibitor Z-VAD-fmk. Proteins were analyzed 24 h later by Western blot analysis with IL-33 mAb 305B. (F) Endogenous IL-33 in primary human endothelial cells (treated with control or IL-33 siRNA) was detected by Western blot analysis with IL-33 mAb 305B. (G)

Endogenous IL-33 is cleaved by endogenous caspases in endothelial cells treated with the apoptosis-inducing agent staurosporine. Endothelial cells were treated MEDICAL SCIENCES with staurosporine in the presence or absence of the pan-caspase inhibitor Z-VAD-fmk. Proteins were analyzed by Western blot analysis with IL-33 mAb 305B or PARP mAb (used as a control). IL-331–270 and IL-331–178 proteins (not tagged with myc-epitope), generated by in vitro translation, are shown (Right).

part of IL-33, IL-331–111, did not bind to the ST2-Fc chimera NF␬B-GFP reporter construct (Fig. 3D). We concluded protein. These results indicated that full-length IL-331–270 spe- that, similarly to IL-33112–270, full-length IL-331–270 is able to bind cifically binds to the ST2 receptor. Binding of full-length IL-33 and activate the ST2 receptor. to ST2 was also observed using endogenous native IL-33 ob- tained from endothelial cells extracts (Fig. 3B), indicating the The 2 Caspase-1 Cleavage Products, IL-331–178 and IL-33179–270,DoNot natively folded forms behave similarly to the in vitro generated Activate ST2. As we found caspase-1 processing of IL-33 occurs forms regarding ST2 binding. within the IL-1-like domain, we predicted the 2 caspase-1 We next investigated the capacity of full-length IL-331–270 to cleavage products, IL-331–178 and IL-33179–270, would not exhibit signal through ST2 by using a previously described NF-␬B- biological activity. To confirm this prediction, we tested the dependent reporter assay (4, 14). HEK293T cells, transiently capacity of IL-331–178 and IL-33179–270 to activate ST2 using the transfected with an expression vector for human ST2 together NF␬B-dependent GFP reporter assay. Immunofluorescence with an NF␬B-GFP reporter gene construct, were stimulated (Fig. 4A) and FACS (Fig. 4B) analyses revealed that, whereas with full-length IL-331–270, IL-331–111, or IL-33112–270. As previ- treatment of the cells with full-length IL-331–270 resulted in ously reported (4, 14), a small population of cells expressed GFP significant activation of ST2-dependent signaling, treatment with in the absence of IL-33 stimulation. However, stimulation of cells the 2 caspase-1 cleavage products did not activate the ST2- with full-length IL-331–270 or the IL-1-like domain IL-33112–270, dependent NF␬B-GFP reporter gene. These results were con- but not IL-331–111, led to a significant increase in the number of firmed using another bioassay, IL-33-dependent secretion of GFPϩ cells (Fig. 3C). Quantification of the results by FACS IL-6 by the line MC/9 (32). Full-length IL-33 signifi- analysis revealed that full-length IL-331–270 and the IL-1-like cantly induced IL-6 secretion by MC/9 cells whereas the 2 domain IL-33112–270 possess a similar capacity to activate the caspase-1 cleavage products had no effect (Fig. 4C). We con-

Cayrol and Girard PNAS ͉ June 2, 2009 ͉ vol. 106 ͉ no. 22 ͉ 9023 Downloaded by guest on September 26, 2021 IP ST2-Fc A A LRR IL-331-270 IL-33179-270 33-LI 1-178 INPUT 112-270 1-270 1-111 B IP ST2-Fc 1-111 112-270 1-270 IL-33 IL-33 IL-33 RRL

MW IL-33 IL-33 IL-33 RRL -+ -+ -+ -+ST2- (KDa) 1-270 47.8 Fc + + - - ST2-Fc 35.9 Input IL-33 - + + - extracts 24.5 18.7 IL-33 13.9 * WB anti myc WB anti IL-33 C B C RRL IL-331-270 IL-33112-270 IL-331-111 140 800 120 100 600 80 60 400 IL-6 (pg)

positive cells positive 40 200 % increase GFP 20 0 0 0 8 L 0 0 8 L 7 0 7 7 7 7 2 7 1 R -2 -2 -1 R - -2 - 1 9 1 1 9 1 R 3 7 3 R 3 7 3 -3 1 3 -3 1 3 L 3 - L 3 - I -3 IL I -3 IL IL IL RRL IL-331-270 D 140 Fig. 4. The 2 caspase-1 cleavage products, IL-331–178 and IL-33179–270, are not 120 able to activate ST2. (A and B) The capacity of IL-331–178 and IL-33179–270 to activate ST2-dependent signaling was analyzed using an ST2-dependent FSC FSC 100 NF␬B-GFP reporter gene. Assays were performed in HEK293T cells transfected

0 1 2 3 4 80 10 10 10 10 10 100 101 102 103 104 with plasmids pNF-␬B-hrGFP and pEF-BOS-hST2 using in vitro translated IL- GFP GFP 60 33 , IL-33 , and IL-33 proteins as described in Materials and IL-33 1–270 179–270 1–178 112-270 IL-331-111 40 Methods. Cells were analyzed for GFP expression by fluorescence microscopy positive cells positive ϩ

% increase GFP 20 (A) and flow cytometry (B). The percentage increase in GFP cells is shown 0 (Below). Results are shown as means and SDs of 3 independent transfection

FSC 0 0 1

FSC L 7 7 1 R -2 -2 -1 1 2 1 experiments. (C) The capacity of IL-33 and deletion mutants to activate the R 3 1 3 3 1 3 100 101 102 103 104 0 1 2 3 4 - 3 - 10 10 10 10 10 IL 3 L IL-33-responsive mast cell line MC/9 was analyzed by determining IL-6 levels in GFP L- I GFP I supernatants using an ELISA. Results are shown as means and SDs of 3 separate data points. Fig. 3. Full-length IL-331–270 is able to bind and activate the ST2 receptor. (A) Pull-down of full-length IL-331–270 with ST2-Fc fusion protein. Full-length (IL-33 ), C-terminal IL-1-like domain (IL-33 ), and N-terminal domain 1–270 112–270 membrane disruptions that are observed in cells subjected to (IL-331–111) proteins tagged with myc-epitope at their C terminus were incu- bated with ST2-Fc for 16 h at 4 °C and precipitated with protein-G agarose mechanical forces in vivo, has previously been shown to result in beads. The precipitates were separated by SDS/PAGE and analyzed by Western the release of growth factors such as basic FGF (33). Interest- blot with anti-myc antibody. Rabbit reticulocyte lysate (RRL)isanun- ingly, we found that full-length IL-33 was released from endo- programmed lysate. (B) Pull-down of endogenous IL-33 with ST2-Fc fusion thelial cells mechanically wounded by cell scraping (Fig. 5B). protein. Endothelial cell freeze-thaw extracts were incubated with ST2-Fc and HMGB1 was similarly released under these conditions. Scratch- the precipitates were analyzed by Western blot with IL-33 mAb 305B. Asterisk ing of endothelial monolayers by tracing lines with a surgical indicates non-specific band. (C and D) Full-length IL-331–270 activates an ST2- dependent NF␬B-GFP reporter gene. Assays were performed in HEK293T cells scalpel, a needle, or a pipette tip has been widely used as an in transfected with plasmids pNF-␬B-hrGFP and pEF-BOS-hST2, using in vitro vitro model of wound healing. Using this model, we confirmed translated IL-331–270, IL-33112–270, and IL-331–111 proteins, as described in Ma- that IL-33, like HMGB1, is released into the supernatant after terials and Methods. Cells were analyzed for GFP expression by fluorescence mechanical injury of endothelial cells. In contrast, IL-33 and microscopy (C) and flow cytometry (D). The percentage increase in GFPϩ cells HMGB1 were not released extracellularly in the absence of is shown (Below). Results are shown as means and SDs of 3 independent endothelial cell damage (Fig. 5B). We then tested the effect of transfection experiments. necrosis induced by several cycles of freezing and thawing (26), and found IL-33 was released by necrotic cells and its levels in the supernatant increased with the number of freezing-thawing cluded that, unlike full-length IL-331–270, the 2 caspase-1 cleav- cycles (Fig. 5C). Finally, full-length IL-33 and HMGB1 were also age products, IL-331–178 and IL-33179–270, do not possess biolog- detected in the medium of endothelial cells treated with non- ical activity, indicating that caspase-1 processing inactivates ionic detergents Nonidet P-40 and Triton X-100, which provoke IL-33. damage to the cell membrane (Fig. 5D). Together, these data demonstrate that IL-33, like HMGB1, can be released in the IL-33 Is Released Extracellularly After Endothelial Cell Damage or extracellular space after endothelial cell damage or mechanical Injury. IL-33 and the alarmin HMGB1 are both chromatin- injury. associated cytokines, and we then studied the possibility that full-length IL-33, similarly to HMGB1 (26), may be released Discussion after cell damage or necrosis. We first tested the effect of In the present study, we demonstrate that IL-33 does not require mechanical injury on primary human endothelial cells that maturation for binding and activation of the IL-33 receptor ST2, express high levels of endogenous IL-33 both in vivo (20, 21) and and that, contrary to the current belief, processing by caspase-1 in culture (Fig. 5A). Mechanical wounding of endothelial cells by results in IL-33 inactivation, rather than activation. Indeed, we cell scraping, a process that mimics the transient sublethal unexpectedly discovered that cleavage of IL-33 by caspase-1 does

9024 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812690106 Cayrol and Girard Downloaded by guest on September 26, 2021 A B Supernatants Supernatants may constitute a major source of danger signals in Pellets ‘vivaspin’ ‘TCA’ vivo. In addition to endothelium, we also detected abundant

d d d expression of IL-33 in the nucleus of epithelial cells of tissues in e ’ e ’ e ’ SiRNA 0 t t t g 7 g ’ g ’ a ’ 2 a l a l n l contact with the environment, including the skin and gastroin- - in in e i e 1 e e e e r 3 r p p r p p t p p l 3 t l t l a l r 3 3 t a t a t r a t - r a r a o testinal tract, where pathogens, allergens, and other environ- L- o c c o c c c c C I L N S I N ‘S ‘S N ‘S ‘S ‘ ‘S mental agents are frequently encountered (20). The constitutive IL-33 * expression of IL-33 in epithelial barriers of the human body thus supports the possibility that IL-33 may also play important roles HMGB-1 as an epithelial alarmin. IL-33 is likely to be a very good alarm n o 0 it 0 signal because it has the capacity to activate many actors of the C Freeze/Thaw D 4 r 0 P T 1 1x 3x 6x 9x N X innate immune system, including mast cells, basophils, eosino- phils, NK cells, and invariant NK T cells (4, 10–13, 15–19). P S P S P S P S P S P S Among these, mast cells, which are strategically positioned close IL-33 to vessel walls and epithelial surfaces exposed to the environ- HMGB-1 ment (36), the major sites of IL-33 expression in vivo (20) may play a major role in the response to the IL-33 danger signal. IL-33 Fig. 5. Full-length IL-33 is released by damaged endothelial cells. (A-D) is also able to activate Th2 cells (4, 14, 17, 18) and it may Western blot analysis of confluent primary human endothelial cells lysates or therefore play a role not only in the innate response, but also in supernatants was performed using antibodies against IL-33 (AT-110) and the adaptive immune response, after tissue damage. Another HMGB1. (A) Similar amounts of HMGB1 were observed in the presence or property of IL-33 that qualifies it as a bona fide danger signal is absence of siRNA to IL-33. (B) IL-33 and HMGB1 were released in the super- the fact IL-33 signals through the TLR/IL-1R/MyD88 signaling natants after scraping of the cells from the substratum (followed by 20 min pathway (4, 13, 17), like IL-1␣ (27) and the exogenous danger incubation at 37 °C) or scratching the endothelial monolayer with a surgical scalpel. Supernatants were collected from wounded cells and the presence of signals from microorganisms, the so-called pathogen-associated IL-33 and HMGB1 was assayed in both pellets and supernatants concentrated molecular patterns. IL-33 may therefore represents a novel by TCA precipitation or filtration on Vivaspin columns. Asterisk indicates member of the damage-associated molecular pattern family, non-specific band. (C) Higher amounts of IL-33 and HMGB1 were released in which regroups both endogenous (ie, alarmins) and exogenous the supernatants after endothelial cell damage induced by repeated cycles of (ie, pathogen-associated molecular patterns) danger signals (30). freezing and thawing. (D) IL-33 and HMGB1 were also released in the super- In summary, the results presented here are important because natants after treatment of the endothelial cells for 5 min at 37 °C with they demonstrate that IL-33 does not require maturation for non-ionic detergents 0.2% Nonidet P-40 and 0.2% Triton X-100. biological activity and that, contrary to the current view, pro- cessing by caspase-1 results in IL-33 inactivation, rather than not occur at the site initially proposed (Ser ), but rather at a activation. In addition, the data reveal that full-length IL-33 can 111 be released after endothelial cell damage or injury, suggesting site located in the middle of the IL-1-like domain (DGVD G). 178 IL-33 may function as a novel endogenous danger signal. Future To the best of our knowledge, IL-33 is the first member of the studies will aim at defining the precise functions of IL-33 in IL-1 family shown to be inactivated after maturation by human health and disease, including its roles in the tissue caspase-1. IL-33 thus differs from IL-1␤ and IL-18, which response to trauma or infection. require maturation by caspase-1 for liberation of their mature biologically active forms (1, 2); from IL-1 family member IL-1F7, Materials and Methods which requires caspase-1 processing for translocation to the ␣ Plasmid Constructions and Protein Production. IL-33 deletion mutants were nucleus (34); and from IL-1 , which is not a substrate for amplified by PCR using the human IL-33/NF-HEV cDNA (NM࿝033439) (3, 5) as a caspase-1 but is cleaved by calpain to release a C-terminal 17 kDa template. The PCR fragments thereby obtained were cloned into plasmid form with biological activity (1). IL-33 is therefore very unique pcDNA3.1A/myc-his (Invitrogen). The IL-33D178A mutant was generated by PCR in the IL-1 family, in terms of processing and biologically active and cloned into the same expression vector. IL-331–270 and IL-331–178 deletion domains. As our data convincingly demonstrate caspase-1 mutant were also cloned into plasmid pcDNA3 (Invitrogen) for use in exper- MEDICAL SCIENCES iments presented in Figs. 2, 3B, and 5B. Human IL-1␤ (NM࿝000576.2) was cleaves IL-33 at position D178 but not at position S111, and no amplified by PCR and cloned into vector pcDNA3.1A/myc-his. All primer protease is known to cleave after S111, there is no evidence left sequences are available upon request. Pro-IL1␤, full-length IL-331–270, IL- for the existence of IL-33112–270 either in vitro or in vivo. 33D178A, and IL-33 deletion mutants were synthesized in vitro in rabbit reticu- The caspase-1 processing site within IL-33 is very similar to a locyte lysate using the TNT-T7 kit with (fluorescent protein) or without (un- consensus site for caspase-3, the pro-apoptotic caspase, and we labeled protein) the FluoroTect GreenLys labeling system according to the found that IL-33 is cleaved at this site by caspase-1 and caspase-3 manufacturer’s instructions (Promega). in vitro and by endogenous caspases in cells undergoing apo- In Vitro Caspase Cleavage Assays. In vitro translated fluorescent or unla- ptosis. IL-33 is a potent stimulator of pro-inflammatory cytokine beled proteins (5 ␮L lysate) were incubated with various amounts of recom- production by mast cells and other cells of the innate immune binant caspase-1 (Sigma) or caspase-3 (Calbiochem) in 14 ␮L assay buffer system (10, 11, 15, 18, 19). Processing of IL-33 by caspases may (Calbiochem) for 2 h at 37 °C. The resulting cleavage products were analyzed by SDS/PAGE and fluorography (Typhoon 9400 fluoroimager; GE Healthcare) therefore provide a mechanism to inactivate IL-33 pro- or Western blot. In some experiments, caspases 1 and 3 were preincubated for inflammatory cytokine activities during apoptosis, a process that 20 min at 37 °C with 100 ␮M of their respective inhibitors, Ac-YVAD-CHO and does not trigger inflammation in vivo. Ac-DEVD-CHO (Calbiochem). Similarly to IL-1␣ (1, 27–29), IL-33 may be released by damaged cells in vivo and function as an endogenous danger Western Blot and Pull-Down Assays. Proteins were fractionated by SDS/PAGE, signal (35), or alarmin (30), to alert cells of the innate immune electroblotted, and detected with mAbs to myc-epitope tag (9E10, 1:1,000; system of tissue injury. This possibility is supported by our data Sigma), IL-33 (Nessy-1, 1:1,000; Alexis Biochemicals; 305B, 1:1,000; Alexis Bio- indicating that IL-33, which is constitutively expressed to high chemicals), or PARP (1:2,000; BD PharMingen), or rabbit antiserum to IL-33- levels by endothelial cells in most normal human tissues (20, 21), Nter (IL-331–15, 1:400) (3, 5), IL-33-Cter (AT-110, no. 210–447, 1:1,000; Alexis Biochemicals), or HMGB1 (Ab18256, 1:200; Abcam), followed by HRP- can be released in the extracellular space after endothelial cell ␣ conjugated goat anti-mouse or anti-rabbit Ig (1:10,000; Promega), and finally damage or mechanical injury. Interestingly, IL-1 and the an enhanced chemiluminescence kit (GE Healthcare). Pull-down assays with prototypical alarmin HMGB1 have also been shown to be human ST2-Fc chimera protein (1 ␮g; R&D Systems) were performed as pre- released from damaged endothelial cells (28), suggesting the viously described (4), using in vitro translated IL-33 proteins (25 ␮L lysate) or

Cayrol and Girard PNAS ͉ June 2, 2009 ͉ vol. 106 ͉ no. 22 ͉ 9025 Downloaded by guest on September 26, 2021 endothelial cell freeze-thaw extracts (3.5 ϫ 106 cells) containing endogenous plemented with 20% fetal calf serum (Invitrogen) and heparin (Sigma), and native IL-33. used at confluence in all experiments. Knockdown of IL-33 expression with ON-TARGET plus SMARTpool siRNA duplexes (Dharmacon) was performed as Reporter Gene Assays and ELISA. HEK293T cells (1.5 ϫ 105 cells/well in 12-well described (22). For induction of apoptosis, cells were incubated with 1 ␮M plates) were transfected with 2 ␮g pNF-␬B-hrGFP (Stratagene) reporter plas- staurosporine (Sigma) for 3 h (endothelial cells) or 2 ␮M doxorubicin (Sigma) mid and 1 ␮g of pEF-BOS-hST2 (provided by S. Tominaga, Tochigi, Japan), for 24 h (U2OS cells), with or without pretreatment with 50 ␮M pan-caspase using a phosphate calcium precipitation method. One day after transfection, inhibitor Z-VAD-fmk (Calbiochem) for 16 h (endothelial cells) or 24 h (U2OS cells were stimulated for 16 h with in vitro translated full-length IL-33 or cells). In other experiments, cell damage was induced by repeated cycles of deletion mutants (12.5 ␮L lysate/well). Cells were then analyzed for GFP freezing and thawing (26), mechanical scraping from the substratum (33), or expression by fluorescence microscopy (Eclipse TE300 microscope; Nikon) and mechanical wounding of monolayers by tracing lines with a surgical scalpel. flow cytometry (FACScan, Cellquest Software; Becton Dickinson). DuoSet IL-6 Supernatants were prepared from treated and untreated cells in Opti-MEM ELISA assays (R&D Systems) were performed as described (32) using MC/9 mast serum-free media (Invitrogen), by spinning the cells or cell lysates at 16000ϫ ϫ 5 cells (ATCC; 4 10 cells/well in 96-well plates) stimulated for 40 h with in vitro g for 5 min. In some cases, supernatants were concentrated by TCA or filtration ␮ translated full-length IL-33 or deletion mutants (10 L lysate/well). using Vivaspin columns (Sartorius).

Mammalian Cell Culture and Induction of Apoptosis or Cell Damage. Human ACKNOWLEDGMENTS. We thank Drs. Tominaga and Yanagisawa (Tochigi, HEK293T and U2OS epithelial cells were grown in Dulbecco modified Eagle Japan) for the gift of hST2 expression vector. This work was supported by medium supplemented with 10% fetal calf serum and 1% penicillin- grants from Ligue Nationale contre le Cancer (Equipe labellise´e Ligue 2009), streptomycin (all from Invitrogen). Human umbilical vein endothelial cells ANR-Program Blanc ‘‘Cuboïdale,’’ and MAIN European Network of Excellence (Praxcell) were cultured in endothelial cell growth medium (Promocell), sup- (FP6–502935).

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