ORIGINAL ARTICLE

The Double-Stranded RNA Analogue Polyinosinic-Polycytidylic Acid Induces Pyroptosis and Release of IL-36c Li-Hua Lian1,2, Katelynn A. Milora1,2, Katherine K. Manupipatpong1 and Liselotte E. Jensen1

IL-36 is the common name for the three IL-1 family members IL-36a, IL-36b, and IL-36g, formerly known as IL-1F6, IL-1F8, and IL-1F9, respectively. IL-36 appears to have pro-inflammatory activities; however, the physiological function of these remains unknown. Expression of IL-36 by implies its possible involvement in innate immune responses in the skin. We observed that, of the three IL-36 isoforms, human keratinocytes express high levels of IL-36g. IL-36g mRNA expression was dramatically induced by the Toll-like ligands polyinosinic-polycytidylic acid (poly(I:C)) and flagellin. Surprisingly, the IL-36g was released by cells treated with poly(I:C), but remained intracellular in cells treated with flagellin only. poly(I:C), but not flagellin, induced cell death and caspase-3/7 activation. Inhibition of caspase-3/7 and caspase-1 blocked extracellular release of IL-36g from poly(I:C)-treated cells. Furthermore, caspase-1 inhibition prevented poly(I:C)-induced caspase-3/7 activation. Interestingly, transcription of the IL36G was dependent on caspase-1, but not caspase-3/7, activation. This demonstrates that the pathways leading to IL36G transcription and caspase-3/7 activation branch after caspase-1. This divergence of the pathways allows the cells to enter a state of de novo protein synthesis before committing to pyroptosis. Overall, our observations suggest that IL- 36g may be an alarmin that signals the cause, e.g., viral infection, of cell death. Journal of Investigative Dermatology (2012) 132, 1346–1353; doi:10.1038/jid.2011.482; published online 9 February 2012

INTRODUCTION Release of these cytokines from keratinocytes subsequently The skin is continuously exposed to potential pathogens and orchestrates the recruitment and activation of immune cells. plays an active role in initiating immune responses to IL-1 and IL-18 are among the many pro-inflammatory eliminate these microorganisms. Keratinocytes express func- cytokines that are expressed following TLR and/or retinoic tionally active pathogen-associated molecular pattern recog- acid–inducible gene-I–like receptor engagement. IL-18 is nition receptors such as the Toll-like receptors (TLRs) and considered to be an IL-1 family member due to sequence retinoic acid–inducible gene-I–like receptors (Kalali et al. and structural similarity to IL-1. There are two forms of IL-1: (2008) and references therein). Different TLRs bind a diverse IL-1a is the foremost form produced by keratinocytes, whereas range of structural motifs associated with bacteria, fungi, and IL-1b is primarily expressed by myeloid cells (Dinarello, 2009). viruses. In contrast, retinoic acid–inducible gene-I–like With the recent discovery of the , which are receptors appear to be specific for viral double-stranded intracellular protein complexes involved in the initiation of RNA (Takeuchi and Akira, 2010). Engagement of TLRs and inflammation (van de Veerdonk et al., 2011), the process retinoic acid–inducible gene-I–like receptors activates intra- whereby myeloid cells secrete IL-1b and IL-18 has gained cellular signaling pathways, which regulate the expression of renewed interest. Both cytokines lack signal peptides and are pro-inflammatory cytokines (Takeuchi and Akira, 2010). initially produced as pro-. Assembly of the inflamma- somes activates the protease caspase-1, which in turn cleaves the pro-IL-1b and pro-IL-18 (van de Veerdonk et al., 2011). This

1 process permits extracellular release of the mature cytokines Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA through a process called pyroptosis (Franchi et al., 2009). 2These two authors contributed equally to this work. Pyroptosis is a highly inflammatory form of programmed cell Correspondence: Liselotte E. Jensen, Department of Microbiology and death. The process is dependent on caspase-1 activation and Immunology, Temple University School of Medicine, 1158 MERB, has so far only been described in myeloid cells (Franchi et al., 3500 North Broad Street, Philadelphia, Pennsylvania 19140, USA. 2009). Although it is known that keratinocytes have inflamma- E-mail: [email protected] somes (Feldmeyer et al., 2010), how IL-1 cytokines are released Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; from keratinocytes is poorly understood. IL-1RL, IL-1 receptor–like; PBS, phosphate-buffered saline; PGN, peptidoglycan; poly(I:C), polyinosinic-polycytidylic acid; TLR, Toll-like receptor IL-36a (formerly known as IL-1F6), IL-36b (IL-1F8), and Received 15 July 2011; revised 20 November 2011; accepted 5 December IL-36g (IL-1F9) are three recently discovered new members of 2011; published online 9 February 2012 the IL-1 family (Dinarello et al., 2010). IL-36 binds to IL-1

1346 Journal of Investigative Dermatology (2012), Volume 132 & 2012 The Society for Investigative Dermatology L-H Lian et al. Pyroptosis and IL-36g in Keratinocytes

receptor–like 2 (IL-1RL2) to initiate intracellular activation of RESULTS mitogen-activated protein kinase and NF-kB signal transduction TLR ligands induce dramatic changes in IL-36c expression (Debets et al., 2001; Towne et al., 2004). IL-1RL2 is primarily Since IL-36 is expressed in the skin (Jensen, 2010), we expressed in epithelial cells of tissues, such as the skin, lungs, speculated that these cytokines play a role in the homeostatic and digestive tract. Recent studies have demonstrated that processes regulating the continuous interaction between the transgenic overexpression of IL-36a in basal keratinocytes leads skin and colonizing or invading microorganisms. We first to a psoriasis-like phenotype (Blumberg et al., 2007), and compared expression of the new IL-36 cytokines to the intratracheal administration of recombinant IL-36g in mice classical IL-1 family members IL-1a and IL-1b in unstimulated results in increased neutrophil influx (Ramadas et al., 2011). cells. Levels of the IL-36a mRNA were the lowest observed. These studies suggest that IL-36 has pro-inflammatory activities. Relative to the IL-36a mRNA, levels of IL-36b, IL-36g, IL-1b, Epidermal expression of the IL-36 and IL-1RL2 mRNAs is and IL-1a mRNAs were 3,100-, 25,000-, 190,000-, and upregulated in many inflammatory skin conditions, such as 1,800,000 times higher, respectively (Figure 1a). No discern- psoriasis and eczema (reviewed in Jensen, 2010). However, the able differences were noticed between adult and neonatal specific physiological functions of the three IL-36 isoforms and keratinocytes (data not shown). how they may contribute to disease remain unclear. Keratinocytes were challenged with a panel of TLR ligands The high degree of IL-36 and IL-1RL2 expression in tissues to examine their capacity to stimulate IL-1 and IL-36 mRNA in direct contact with the environment may suggest that these expression (Figure 1b). While the IL-1a and IL-1b mRNA proteins are involved in first-line defenses against micro- levels were modestly increased (1.5- to 4-fold, Po0.05) by organisms colonizing these areas. To initiate studies of this most TLR ligands, the IL-36b and IL-36g mRNAs were most hypothesis, we explored the expression of IL-36 cytokines in significantly (Po0.05) regulated by flagellin (4- and 24-fold, keratinocytes. Our investigations presented here establish respectively) and poly(I:C) (8- and 143-fold, respectively). that keratinocytes respond differently depending on how they The dramatic induction of the IL-36g mRNA by poly(I:C) was are challenged. Furthermore, we demonstrate that the concentration dependent (Figure 1c) and resulted in mRNA double-stranded RNA analogue poly(I:C) induces pyroptosis levels similar to that of the IL-1a mRNA (compare Figure 1a in keratinocytes, thereby facilitating the extracellular release and b). The very clear differential expression patterns of IL-1 of IL-36g. To our knowledge this is previously unreported. versus IL-36 suggest that these proteins have distinct functions.

ab7 1 Hour 6Hours 24 Hours 10 *** 4.0 106 *** * IL-1α * IL-36β * 8 * 105 *** *** 3.0 ** 104 * 6 * 103 * ** * 2.0 ** * 102 * * * 4 1 10 Fold change Relative expression

(compared to IL-36 α ) 1.0 2 * 1

0 0 IL-1 β IL-1 α IL-36 γ IL-36 β IL-36 α c 5.0 160 IL-1β *** IL-36γ * 140 ** 4.0 * 1,000 * 120 ** * ** * 100 3.0 * * ** 100 24 Hours * * ** 80 ** * 2.0 60 10 *

*** Fold change 6 Hours 40 * ** 1.0 ** 20 * *** 1.0 * * * ** * 0 0 * * * * *** 0.1 LTA LTA LPS LPS

Fold change in IL-36 γ mRNA 0.25 2.5 10025 Medium Medium Flagellin Flagellin –1 poly(I:C) poly(I:C) poly(I:C) concentration (μgml ) Zymosan Zymosan E.coli DNA E.coli DNA

Figure 1. Expression of IL-1 and IL-36 in human primary keratinocytes. Keratinocytes were left (a) untreated or (b) treated with medium only, polyinosinic- polycytidylic acid (poly(I:C)) (25 mgml1), flagellin (1 mgml1), Escherichia coli DNA (10 mgml1), lipoteichoic acid (LTA) (5 mgml1), lipopolysaccharide (LPS) (5 mgml1), or zymosan (25 mgml1), or (c) treated with medium only or poly(I:C) at the indicated concentrations. Total RNA was isolated after 1, 6, or 24 hours and reverse transcription and real-time PCR performed. IL-1 family mRNA levels were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fold differences (mean±standard deviation (SD), n ¼ 2 per independent timepoint and treatment) calculated relative to the (a) IL-36a mRNA or (b and c) medium-only treated samples. Only IL-36g mRNA expression was examined in c.*Po0.05; **Po0.01; ***Po0.001 (comparing treatment with medium only at the (b and c) same timepoint or (a and c) as indicated). Data shown are from one representative experiment of at least three independent experiments.

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Similar to previous reports (Kumar et al., 2000; Debets Extracellular et al., 2001), we also observed increased IL-36b and IL-36g mRNA expression in response to IL-1 and TNFa (data not shown). As observed with the TLR ligands (Figure 1b), IL-36g Medium DNA LPS LTA Flagellin Zymosan poly(I:C) was the most noticeably regulated (approx. 25-fold increases IL-36γ vs. maximum 12-fold increases in IL-36b mRNA levels (data 6 Hours not shown)). Other cytokines such as IFN-g and IL-6 did not IL-36γ affect the degree of IL-36 mRNA expression (data not shown). 24 Hours Several additional cytokines have been shown to regulate IL- 36 expression, including IL-17A, IL-22, and IL-36 itself 48 Hours IL-36γ (Carrier et al., 2011; Muhr et al., 2011). Although we observed no indication of a biphasic expression profile in the γ dose–response curves or time courses (Figure 1b and c), it is 72 Hours IL-36 plausible that the dramatic induction of the IL-36g mRNA is, in part, driven by an autocrine mechanism involving IL-36 5 * ** (see below with regard to extracellular localization of IL-36). ** ** 4 ** IL-36c is only secreted in response to poly(I:C) 24 Hours Similar to IL-1b, the IL-36 cytokines lack signal peptides 3 ** ** required for conventional protein secretion ((Martin et al. 2 (2009) and references therein). Interestingly, unlike IL-1b, 6 Hours

Fold change **

which is synthesized as a pro-protein that is cleaved before IL-36 γ secretion extracellular release, the IL-36 proteins appear to lack pro- 1 domains. Hence, they are predicted to be transcribed directly ** ** into mature forms (Martin et al., 2009). We therefore 0 examined whether IL-36b and IL-36g were present in the 0.25 2.5 25 100 medium or cellular fractions of cells treated with TLR ligands. poly(I:C) concentration (μgml–1) Since both poly(I:C) and flagellin significantly increased IL- 36b and IL-36g mRNA levels (Figure 1b), we predicted that Intracellular these TLR ligands would trigger a corresponding rise in IL- 36b and IL-36g production. Furthermore, cellular localization

was anticipated to be similar. Surprisingly, we found that IL- Medium poly(I:C) Flagellin DNA LTA LPS Zymosan 36g could only be detected in the medium from cells 1 Hour stimulated with poly(I:C) (Figure 2a). The level of IL-36g secretion was dependent on the poly(I:C) concentration 6 Hours IL-36γ b IL-36γ (Figure 2b). We were unable to detect IL-36 in the same 24 Hours samples (data not shown). In contrast, flagellin-activated cells contained increased levels of intracellular IL-36g (Figure 2c). 1 Hour A low level of intracellular IL-36g could also be detected in GAPDH poly(I:C)-treated cell fractions. No discernable size differ- 6 Hours GAPDH ences between the intracellular and extracellular IL-36g were detected. 24 Hours GAPDH It is well known that ATP stimulates the release of Figure 2. Extracellular and intracellular localization of IL-36c from Toll-like intracellular IL-1b by (Franchi et al., 2009). receptor (TLR) ligand–activated keratinocytes. Cells were treated as Overexpression of a murine green fluorescence protein–IL- described in Figure 1b and c. The medium and cells were collected at the 36a fusion protein in mouse macrophages results in indicated timepoints and (a and b) extracellular (medium) and (c) intracellular intracellular protein buildup. This cytokine fusion protein is (cells) IL-36g levels examined by (a and c) western blotting or (b) ELISA. secreted when cells are treated with ATP (Martin et al., 2009). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading Similar to this, we found that a 20-minute pulse with 5 mM control for intracellular protein fractions. *Po0.05; **Po0.01 (comparing ATP stimulated the extracellular release of IL-36g (data not treatment with medium only at the same timepoint or as indicated). LPS, lipopolysaccharide; LTA, lipoteichoic acid; poly(I:C), polyinosinic- shown), which accumulated in cells treated with flagellin. polycytidylic acid. Similar observations have recently been reported by Johnston et al. (2011).

poly(I:C) causes cell death and caspase-3/7 activation (Figure 3a). Staining with Trypan blue indicated that 40–60% During our analyses of IL-36 expression in response to TLR of the cells remained alive 24 hours after poly(I:C) addition ligands, we observed lower cell density 24 hours after (data not shown). The poly(I:C)-treated cultures further poly(I:C) treatments compared with other agent applications revealed cell morphologies suggestive of cells at different

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Medium Flagellin poly(I:C)

1,000 1,400 * 1,200 800 1,000 ** 600 800 600 400

400 Caspase-3/7 activity (RLU) activity 200

Cell viability (RLU) 200 0 0 Medium Medium Flagellin Flagellin poly(I:C) poly(I:C)

Figure 3. Polyinosinic-polycytidylic acid (poly(I:C)) induces cell death and pro-apoptotic caspase-3/7 activation. Keratinocytes were treated with medium, poly(I:C), or flagellin for (b, c) 6 or (a) 24 hours. (a) Arrowheads in phase-contrast images indicate cells at different stages of cell death. Bar ¼ 0.05 mm. (b) The number of viable cells and (c) levels of active caspase-3/7 were determined using luminescence assays as described in Materials and Methods. Relative luminescence units (RLU) are represented as means±standard deviation (SD). *Po0.05; **Po0.01 (compared to medium only).

stages of cell death (Figure 3a, arrowheads). Activation of Caspase-1 is an upstream activator of caspase-3/7 caspase-3 and -7 commits cells to undergo programmed cell In myeloid cells, the pyroptosis-associated release of IL-1b death such as apoptosis (Lakhani et al., 2006). We therefore and IL-18 requires activation of caspase-1 (Franchi et al., speculated that pro-apoptotic caspase activity would be 2009). Furthermore, caspase-1 can activate caspase-7 (Lam- present at earlier timepoints. Preliminary microscopic kanfi et al., 2008). To examine whether caspase-1 is an observations of cultures revealed that around 6 hours post- upstream activator of caspase-3/7 in keratinocytes, we treated treatment, a few single dispersed cells, which appeared to be cells with poly(I:C) in the presence and absence of the dying, could be observed only in cultures treated with caspase-1 inhibitor VI and then examined caspase-3/7 poly(I:C) (data not shown). Measurements of intracellular ATP activity. Inclusion of the caspase-1 inhibitor prevented levels revealed significant reduction in the number of viable caspase-3/7 activation (Figure 5a; Po0.05), demonstrating cells when cultures were treated with poly(I:C) compared that poly(I:C)-mediated caspase-3/7 activation requires prior with those treated with medium only or flagellin (Figure 3b). caspase-1 activation. Further measurements of active caspase-3/7 demonstrated a significant 8-fold (Po0.05) increase in caspase activity IL-36c expression is caspase-1 dependent compared with cells treated with medium only (Figure 3c). In analogy with the above studies on the role of caspase-3/7 In comparison, caspase-3/7 was not significantly activated in in the release of IL-36g, we next examined whether IL-36g cells treated with flagellin (Figure 3c). secretion was caspase-1 dependent. Inclusion of the target- specific caspase-1 inhibitor blocked (Po0.05) extracellular poly(I:C)-induced IL-36c extracellular release is caspase-3 accumulation of IL-36g (Figure 5b) in a manner similar to that dependent observed when caspase-3/7 activation was prevented (Figure Since poly(I:C), but not flagellin, caused cell death, caspase-3/7 4a). However, while caspase-3/7 inhibition resulted in activation, and extracellular release of IL-36g, we speculated intracellular IL-36g accumulation (Figure 4b), IL-36g did not that the poly(I:C)-induced release of IL-36g was dependent build up in the cells when caspase-1 was specifically on the caspase-3/7-mediated cell death. Inclusion of inhibited (Figure 5c). We therefore examined IL-36g mRNA the target-specific caspase-3/7 inhibitor III significantly expression in the presence and absence of the caspase-1 (Po0.01) blocked the extracellular release of IL-36g inhibitor. Interestingly, we found that the poly(I:C)-mediated (Figure 4a). Furthermore, in the presence of the caspase-3/7 increase in IL-36g mRNA expression was prevented (Po0.01) inhibitor, the de novo synthesized IL-36g instead accumu- by blocking caspase-1 activity (Figure 5d). This demonstrates lated inside the cells (Figure 4b). The caspase-3/7 inhibitor that the poly(I:C)-induced IL-36g gene expression is caspase- had no effect on the poly(I:C)-induced IL-36g mRNA 1 dependent (Figure 6). Since expression of the IL-36g- expression (Figure 4c). encoding gene (IL36G) is not caspase-3/7 dependent

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12 * 300 ** * * 10 250 200 8 150 6 Caspase-3/7

activity (RLU) 100

Fold change 4 IL-36 γ secretion 50 2 0 0 12 * * 10 IL-36γ 8

6 GAPDH

Fold change Fold 4 IL-36 γ secretion 40 * P > 0.05 2

0 30 IL-36γ

20 GAPDH Fold change IL-36 γ mRNA 10 15 *** ** 0 12

9 Medium Medium poly(I:C) poly(I:C)

6 Vehicle Caspase-3/7 Fold change Fold inhibitor IL-36 γ mRNA 3 Figure 4. Extracellular release of IL-36c is caspase-3/7 dependent. Cells were treated with medium only or polyinosinic-polycytidylic acid (poly(I:C)) 0 in the presence or absence of the caspase-3/7 inhibitor III. (a) Extracellular and (b) intracellular (b) levels of IL-36g protein were determined using ELISA and western blotting, respectively. (c) IL-36g mRNA levels were determined after Medium Medium 3 hours as described in Figure 1. ELISA and real-time PCR results are poly(I:C) poly(I:C) represented as fold changes (mean±standard deviation (SD)) compared to Vehicle Caspase-1 medium-only samples treated with the equivalent vehicle or caspase-3/7 inhibitor inhibitor. *Po0.05; **Po0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Figure 5. Caspase-1 activation is required for caspase-3/7 activation and IL-36c expression. Cells were treated with medium only or polyinosinic- polycytidylic acid (poly(I:C)) in the presence or absence of the caspase-1 inhibitor VI. Caspase-3/7 activity (a) was determined using a luminescence assay as described in Materials and Methods and relative luminescence units (Figure 4c), our data further establish that the pathways (RLU) are represented as means±standard deviation (SD). (b) Extracellular leading to IL36G transcription and activation of cell death IL-36g,(c) intracellular protein, and (d) IL-36g mRNA levels were determined pathways branches after caspase-1 (Figure 6). as described in Figure 4. ELISA and real-time PCR results are represented as fold changes (mean±SD) compared to medium-only samples treated with the DISCUSSION equivalent vehicle or caspase-1 inhibitor. *Po0.05; **Po0.01; ***Po0.001. Preliminary studies indicate that IL-36 has pro-inflammatory GAPDH, glyceraldehyde-3-phosphate dehydrogenase. properties (Debets et al., 2001; Towne et al., 2004; Blumberg et al., 2007; Ramadas et al., 2011); however, the actual skin, we examined IL-36 expression in keratinocytes chal- physiological function(s) remain unknown. To explore the lenged with a panel of TLR ligands. poly(I:C), a double- possibility that IL-36 plays a role in immune responses in the stranded RNA analogue, proved to be a very potent activator

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from dying cells therefore may not only alert surrounding poly(I:C) γ cells about the associated tissue damage, but also about the dsRNA IL-36 cause (double-stranded RNA/viral infection) of the cell death. This may further ensure that a specific immune response Cytoplasm Pyroptosis towards viruses is initiated. Cell death can occur through necrosis or apoptosis. While Caspase-1 Caspase-7 IL-36γ the latter does not cause inflammation, the former is highly inflammatory because the ruptured cell releases cellular components. Research in recent years have revealed that Nucleus there are many other forms of cell death mechanisms, one of IL36G which is pyroptosis (Fink and Cookson, 2005). Pyroptosis is defined as a form of programmed cell death that is caspase-1 Figure 6. Schematic representation of pathways whereby polyinosinic- polycytidylic acid (poly(I:C)) and double-stranded RNA may activate IL-36c dependent and pro-inflammatory. The latter is mediated expression and secretion. Dashed and dotted line indicates the disintegration through IL-1b and IL-18. Pyroptosis has so far only been of the cell during pyroptosis. Dashed arrow lines indicate pathways that may described in myeloid cells such as macrophages, monocytes, involve several intermediates. and dendritic cells (van de Veerdonk et al., 2011). We observed that poly(I:C) induced keratinocyte cell death and caspase-3/7 activation (Figure 3). Furthermore, the caspase-3/ 7 activation was caspase-1 dependent (Figure 5a). By of IL-36g expression and extracellular release. Double- definition, the observed keratinocyte cell death should stranded RNA is a molecular pattern associated with viral therefore be described as pyroptosis. This nomenclature is infections, and IL-36g mRNA expression is known to be further supported by the anticipated pro-inflammatory actions upregulated in proliferating keratinocytes during in vivo skin of IL-36 (Debets et al., 2001; Towne et al., 2004; Blumberg infections with herpes simplex virus-1 (Kumar et al., 2000). et al., 2007; Ramadas et al., 2011) and our data demonstrat- The direct implication of our observations is that IL-36g ing extracellular release of IL-36g. may be involved in regulating innate or adaptive Dermatological studies often examine cell death, e.g., immune responses aimed at eliminating viral infections in when studying the effects of UV light on the skin (Weather- keratinocytes. head et al., 2011). UV light has been demonstrated to activate Alarmins are endogenous danger signals released by the inflammasomes and caspase-1 in keratinocytes (Feld- pathogen-activated and/or injured cells (Yang et al., 2004; meyer et al., 2007). Whether the UV-induced apoptosis Oppenheim and Yang, 2005). The functions of alarmins are reported by Weatherhead et al. (2011) is caspase-1 depen- to promote both chemotaxis and activation of antigen- dent, and hence could be characterized as pyroptosis, is yet presenting cells (Yang et al., 2004; Oppenheim and Yang, to be determined. However, distinguishing the different types 2005). IL-1a and IL-33, an additional member of the IL-1 of cell death is important, as these have not only distinct family, have previously been characterized as alarmins mechanisms of activation and execution, but also have largely due to their extracellular release by damaged cells important differential physiological consequences, e.g., (Bianchi, 2007; Haraldsen et al., 2009). The observed release inflammation versus no inflammation. Adaptation of the of IL-36g only by dying cells (Figures 2-4) suggests that pyroptosis terminology in the dermatological field is in our extracellular IL-36g may also act as a danger signal to alert opinion warranted and timely. surrounding cells about the cell death. Combined with the Caspase-1 can directly cleave and activate caspase-7, but reported involvement of IL-36g in promoting inflammation not caspase-3 (Lamkanfi et al., 2008). Caspase-3 and -7 have (Debets et al., 2001; Towne et al., 2004; Blumberg et al., redundant functions in the execution of apoptosis, i.e. both 2007; Ramadas et al., 2011) and activation of dendritic and T must be deleted to prevent apoptosis (Lakhani et al., 2006). cells (Vigne et al., 2011), our observations support the notion During myeloid pyroptosis caspase-7 but not caspase-3 of IL-36g as an alarmin involved in activating the adaptive activation has been observed; however, cell lysis appears to immune system in response to pathogen challenges such as be independent, at least in some situations, of caspase-7 viruses infecting the skin. (Lamkanfi, 2011). Owing to the redundancies between Interestingly, both IL36G transcription (Figure 5d) and caspase-3 and -7, the substrate and the inhibitor used in caspase-3/7 activation (Figure 5a) are dependent on caspase-1 our studies cannot distinguish caspase-3 and -7 activities. activation; however, only the secretion of IL-36g is caspase- However, given the previously reported activation of 3/7 dependent (Figure 4). This demonstrates that the two caspase-7 only in myeloid pyroptosis, our data (Figures 3-5) pathways branch after caspase-1 (Figure 6). The branching of likely reflect caspase-7 activity (Figure 6). Interestingly, the pathways appears to allow the cells to enter a stage of although myeloid pyroptotic cell lysis has been reported to de novo protein synthesis before committing to self-termina- be independent of caspase-7 activation (Lamkanfi, 2011), our tion. This protein synthesis stage may last more than 6 hours data demonstrate that in keratinocytes the extracellular as we observed increased mRNA expression and protein release of IL-36g is dependent on caspase-3/7 activity release between the 6 and 24 hours timepoints (Figures 1 and (Figure 5). This could suggest that the process of pyroptosis 2). The subsequent extracellular release of the alarmin IL-36g is regulated slightly differently in keratinocytes than in

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myeloid cells. The pharmaceutical implication of this may be IL1F6-R, 50-CTTTAGCACACATCAGGCAG-30; IL1F8-F2C, 50-CAG that pyroptosis can be specifically inhibited in keratinocytes CATTAAGCCTGTCACTC-30; IL1F8-R2C, 50-GCACAGAAGAGACA without affecting the functions of myeloid cells. GAGATC-30; IL1F9-F2, 50-GGGCCGTCTATCAATCAATG-30; IL1F9- The observed differential accumulation of IL-36g inside R2, 50-TGATAACAGCAACAGTGACTG-30. and outside of the cells in response to flagellin and poly(I:C) (Figure 2) may indicate that IL-36g has both intra- and Western blotting extracellular functions, depending on the context in which it The medium was collected from treated cultures. Cells were rinsed is expressed. Other IL-1 family members such as IL-1a, IL-33, with PBS and lysed in mammalian protein extraction reagent and IL-37 have been reported to localize in the nucleus. (Thermo Fisher Scientific, Rockford, IL) supplemented with 10 mM While the nuclear function of IL-1a is unclear, the presence EDTA and Mammalian Cells Protease Inhibitor Cocktail (Sigma- of IL-33 or IL-37 in the nucleus appears to suppress pro- Aldrich). Proteins were separated by SDS-PAGE and transferred to inflammatory gene expression (reviewed in Luheshi et al., polyvinylidene difluoride membranes. Levels of IL-36b, IL-36g, and 2009). IL-37 mediates, at least in part, this effect by GAPDH were examined using goat anti-IL1F8, goat anti-IL1F9 (R&D interacting with the signaling factor Smad3 (Nold et al., Systems, Minneapolis, MN), and rabbit anti-GAPDH (FL-335) (Santa 2010). Further studies of the intracellular localization of Cruz Biotechnology, Santa Cruz, CA) as primary antibodies, IL-36g, using e.g., confocal microscopy, could provide respectively, appropriate horseradish peroxidase secondary anti- valuable support for, or against, the hypothesis that IL-36g bodies (Santa Cruz Biotechnology), and enhanced chemilumines- may have an intracellular function. cence reagents (GE Healthcare, Piscataway, NJ). Our studies have demonstrated that expression of IL-36g is highly complex. Strong induction of IL-36g by TLR ligands ELISA suggests that IL-36g plays a role in immune responses against Levels of extracellular IL-36g protein were determined using a direct microorganisms. Furthermore, TLR-specific differential cellu- ELISA. The medium from treated cells was collected and centrifuged lar localization indicates that IL-36g may have distinct at 200g for 5 minutes. Supernatants were diluted in PBS and coated overnight onto high-binding ELISA plates. Remaining protein- functions dependent on the context in which it is expressed. 1 Extracellular release of IL-36g during pyroptosis may imply binding sites on the ELISA plates were blocked with 1% (wt vol ) g BSA in PBS. IL-36g was detected using sequential incubations with that IL-36 acts as an alarmin. The specific function of this 1 1 danger signal activity may be to inform surround cells about 1 mgml rat anti-IL-36g (R&D Systems), 250 ng ml goat anti-rat the nature of the pathogen-causing cell damage and thereby IgG-horseradish peroxidase (Santa Cruz), and ABTS (2,2-azino-di-(3- ensuring engagement of an appropriately specific immune ethylbenzthiazoline-6-sulfonic acid) chromogen (Invitrogen). The response. Further studies of the role(s) this exciting cytokine direct ELISA was confirmed to be specific for IL-36g using may play in skin infections and chronic inflammatory recombinant IL-1a, IL-1b, IL-36a, and IL-36b (PeproTech, Rocky conditions will advance our knowledge of innate and Hill, NJ and R&D Systems). None of these IL-1 family members were adaptive immune responses in the skin. detected.

MATERIALS AND METHODS Caspase activation and inhibition Cell cultures and cell viability Activity of caspase-3/7 was measured using the Caspase-Glo 3/7 Pooled human neonatal primary keratinocytes (Invitrogen, Carlsbad, Assay (Promega) as described above for the CellTiter-Glo assay. CA) were maintained in defined keratinocyte serum-free medium Caspase-1 and -3/7 were inhibited with caspase-1 inhibitor VI and supplemented with 20 mgml1 gentamicin (Invitrogen). Cells were caspase-3 inhibitor III (EMD Biosciences, La Jolla, CA). Inhibitors treated with medium only or a panel of TLR ligands as described were solubilized in DMSO (vehicle control) and added to cultures previously (Olaru and Jensen, 2010a). poly(I:C) (Sigma-Aldrich, 30 minutes before stimulation with medium only or poly(I:C). Final St Louis, MO) and recombinant flagellin (Invivogen, San Diego, CA) concentrations were 20–50 mM (caspase-1 inhibitor) and 100–250 mM were used at 25 and 1 mgml1, respectively. All treatments were (caspase-3 inhibitor). Timepoints for examining caspase activation performed in duplicate. and inhibition were designed to evaluate activity in the early Cell viability was evaluated by measuring intracellular levels induction phases and further optimization performed empirically. of ATP. The medium was removed from treated cells. Cells were rinsed with phosphate-buffered saline (PBS) and then lysed in Statistical analyses Data are shown as arithmetic mean values and standard deviations CellTiter-Glo (Promega, Madison, WI) diluted 1/4 in PBS. The from one representative experiment of at least three independent luminescent signal was allowed to stabilize for 10 minutes before experiments (n ¼ 2 per independent timepoint and treatment within measurement. each individual experiment). Data were analyzed using the Student’s t-test when appropriate. RNA isolation and real-time reverse transcriptase–PCR RNA isolation and real-time reverse transcriptase–PCR was per- CONFLICT OF INTEREST formed as described previously (Sanmiguel et al., 2009). IL-1a, IL- The authors state no conflict of interest. 1b, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA-specific primers are described elsewhere ACKNOWLEDGMENTS (Olaru and Jensen, 2010b). The following additional gene-specific This work was supported by National Institutes of Health grant AR053672 to primers were used: IL1F6-F2, 50-GACCGTATGTCTCCAGTCAC-30; Jensen.

1352 Journal of Investigative Dermatology (2012), Volume 132 L-H Lian et al. Pyroptosis and IL-36g in Keratinocytes

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