Intracellular IL-1 Receptor Antagonist Isoform 1 Released from upon Cell Death Acts as an Inhibitor for the Alarmin IL-1 α This information is current as of September 27, 2021. Praxedis Martin, Gaby Palmer, Emiliana Rodriguez, Jennifer Palomo, Sylvain Lemeille, Jérémie Goldstein and Cem Gabay J Immunol 2020; 204:967-979; Prepublished online 13 January 2020; Downloaded from doi: 10.4049/jimmunol.1901074 http://www.jimmunol.org/content/204/4/967 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2020/01/10/jimmunol.190107 Material 4.DCSupplemental References This article cites 68 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/204/4/967.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1a

Praxedis Martin,*,† Gaby Palmer,*,† Emiliana Rodriguez,*,† Jennifer Palomo,*,†,1 Sylvain Lemeille,* Je´re´mie Goldstein,*,† and Cem Gabay*,†

The inflammatory effects of IL-1a/b are controlled by IL-1R antagonist (IL-1Ra). One IL-1Ra isoform is secreted, whereas three other isoforms (intracellular IL-1Ra [icIL-1Ra] 1, 2, and 3) are supposed to remain intracellular because of the absence of a signal peptide. In contrast to the well-characterized function of the secreted isoform, the biological role of the intracellular isoforms remains largely unclear. icIL-1Ra1 represents the major isoform in keratinocytes. We created icIL-1Ra12/2 mice and investigated 2/2 the role of icIL-1Ra1 in Aldara (5% imiquimod)-induced psoriasis-like inflammation. Naive icIL-1Ra1 mice bred habit- Downloaded from ually and exhibited a normal phenotype. icIL-1Ra1 deficiency aggravated Aldara-induced skin inflammation, as demonstrated by increased ear thickness and increased mRNA levels of key proinflammatory . No intracellular effect of icIL-1Ra1 could be detected in isolated keratinocytes using RNA-sequencing analysis; however, Aldara treatment led to caspase 1/11-, caspase 8-, and RIPK3-independent cell death accompanied by the release of both icIL-1Ra1 and IL-1a. Furthermore, blocking IL-1a attenuated the clinical severity of Aldara-induced ear thickening in icIL-1Ra12/2 mice. Our data suggest that upon keratinocyte damage icIL-1Ra1 acts extracellularly as an antagonist of the alarmin IL-1a to immediately counteract its inflam- http://www.jimmunol.org/ matory effects. The Journal of Immunology, 2020, 204: 967–979.

soriasis is a common chronic inflammatory skin disorder vehicle, have been previously described to contribute to the affecting ∼2% of the world’s population. Its pathophysi- observed pathological effects (3). The IL-23/IL-17/IL-22 axis is P ology is characterized by epidermal hyperplasia (acanthosis) key for the development of this skin immunopathology (2, 4), because of altered keratinocyte proliferation and differentiation, but members of the IL-1 family of cytokines are also implicated. scaling, erythematous plaque formation, and immune cell infiltration Indeed, Aldara-induced skin inflammation is markedly attenu- (see review in Ref. 1). In mice, the repeated topical application of ated in IL-36R–deficient mice. In addition, mice deficient in the Aldara (5% imiquimod [IMQ]), an FDA-approved drug for the inflammatory cytokines IL-1a and IL-1b or in their signaling by guest on September 27, 2021 treatment of genital and perianal warts, actinic keratosis, and basal receptor IL-1R1 showed reduced infiltration and cell carcinoma, induces skin lesions that resemble human psoriasis acanthosis (5–7). lesions (2). The principal active ingredient of Aldara, the TLR 7 IL-1 (referring to both IL-1a and IL-1b) is a master proin- ligand IMQ, but also isostearic acid, a major component of the flammatory . However, IL-1a and IL-1b differ in their regulation. Although both cytokines are produced as 31-kDa peptides, pro–IL-1b is biologically inert and requires enzy- *Department of Pathology and Immunology, School of Medicine, University of Geneva, matic processing to become active, whereas pro–IL-1a is able 1211 Geneva 4, Switzerland; and †Division of Rheumatology, Department of Internal to bind to IL-1R1 and stimulate inflammatory responses. Pro– Medicine Specialties, University Hospitals, 1211 Geneva 4, Switzerland IL-1a is a cell-associated that can be released following 1 Current address: Micalis Institute, Institut National de la Recherche Agronomique, various forms of cell death. Therefore, IL-1a is able to trigger AgroParis Tech, Universite´ Paris-Saclay, Jouy-en-Josas, France. early inflammatory responses upon cell or tissue damage, ORCIDs: 0000-0001-7567-1798 (G.P.); 0000-0003-4197-6483 (S.L.); 0000-0001- 6853-3063 (C.G.). thereby acting as an alarmin. The inflammatory effects of IL-1 are tightly controlled by IL-1R Received for publication September 3, 2019. Accepted for publication November 25, 2019. antagonist (IL-1Ra), which competitively blocks the binding of IL- This work was supported by Swiss National Science Foundation Grant 310030_172674 1 to IL-1R1. Four isoforms of IL-1Ra exist that are produced from (to C.G.), the Institute of Rheumatology Research, and the RHEUMA SEARCH the same Il1rn (see review in Ref. 8). The original and best Foundation. described isoform is secreted IL-1Ra (sIL-1Ra). It is mainly Address correspondence and reprint requests to Prof. Cem Gabay, Division of Rheu- expressed in myeloid cells and synthesized as proprotein sIL- matology, University Hospitals of Geneva, 26 Avenue de Beau-Se´jour, CH-1211 Geneva 14, Switzerland. E-mail address: [email protected] 1RA (prosIL-1Ra), containing a signal peptide (9–11). The three The online version of this article contains supplemental material. other isoforms lack a signal peptide and thus are considered to stay intracellular (intracellular IL-1Ra [icIL-1Ra] 1, icIL-1Ra2, Abbreviations used in this article: BMDC, bone marrow–derived ; BMDM, bone marrow–derived ; EGFP, enhanced green fluorescence icIL-1Ra3). The icIL-1Ra1 isoform is generated by alternative protein; ES, embryonic stem; icIL-1Ra, intracellular IL-1Ra; IL-1Ra, IL-1R antag- splicing of an icIL-1Ra1–specific first exon upstream of the se- onist; IMQ, imiquimod; iTL, InGenious Targeting Laboratory; K-SFM, keratinocyte serum-free medium; LDH, lactate dehydrogenase; prosIL-1Ra, proprotein sIL-1RA; quence for sIL-1Ra into an internal splice-acceptor site within qRT-PCR, quantitative RT-PCR; rec. m, recombinant mouse; RIPK3, receptor- the exon coding for the signal peptide area of sIL-1Ra (12, 13). interacting protein kinase 3; RNA-Seq, RNA-sequencing; sIL-1Ra, secreted IL- The cDNA for a second intracellular isoform (icIL-1Ra2) was 1Ra; WT, wild-type. cloned from human and is coded by an extra exon Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 located downstream of the first icIL-1Ra1–specific exon. A www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901074 968 KERATINOCYTE-DERIVED icIL-1Ra1 ANTAGONIZES IL-1a corresponding protein has not yet been described (14). icIL- has a beneficial effect on the severity of Aldara (5% IMQ)-induced 1Ra3 is produced by alternative translation initiation from sIL- skin inflammation. No intracellular effect of icIL-1Ra1 could be 1Ra mRNA (15). Despite the lack of a signal peptide, the three detected using isolated keratinocytes; however, addition of Aldara intracellular isoforms can be passively released by dying cells or led to keratinocyte cell death, accompanied by the release of both actively released by a yet unknown leaderless pathway (16–19). icIL-1Ra1 and IL-1a. Furthermore, blocking IL-1a attenuated the In vitro assays using recombinant corresponding to the clinical severity of Aldara-induced ear thickening in icIL-1Ra12/2 intracellular isoforms demonstrated that they can compete ex- mice. Taken together, our results suggest that icIL-1Ra1 is stored tracellularly for IL-1R1 binding in a manner similar to sIL-1Ra in keratinocytes and that upon cell death, icIL-1Ra1 is coreleased (13, 20, 21). with the alarmin IL-1a and immediately counteracts its inflam- Although the collective role of all four IL-1Ra isoforms has been matory potential, thus acting as a natural anti-alarmin. well characterized using Il1rn knockout mice and in humans de- ficient in IL-1Ra (22, 23), the specific function(s) of the intra- Materials and Methods cellular isoforms in vivo remains unknown. We thus generated a Mice knock-in mouse line by insertion of an enhanced green fluores- 2 2 Mice specifically deficient for the icIL-1Ra isoform 1 (icIL-1Ra1 / ; cence protein (EGFP) cassette into the icIL-1Ra1–specific exon, B6.129-Il1rntm(GFP)CemG) were generated by InGenious Targeting Labo- which resulted in the deletion of icIL-1Ra1 without affecting the ratory (iTL; Stony Brook, NY). An EGFP knock-in targeting vector was other IL-1Ra isoforms. designed (iTL) to delete the icIL-1Ra1–specific first exon of the Il1rn gene Because icIL-1Ra1 is constitutively expressed and represents the and replace it by an EGFP reporter gene (Fig. 1A). A 6.14-kb region used to construct the targeting vector was first subcloned from a C57BL/6 BAC Downloaded from major IL-1Ra isoform in both mouse and human keratinocytes clone (RP23: 159M23) into a 2.4-kb backbone vector (pSP72; Promega, (12, 24, 25), we investigated the role of icIL-1Ra1 in an experimental Madison, WI) prior to electroporation into iTL BA1 (C57BL/6 3 129/ model of skin inflammation. We show in this study that icIL-1Ra1 SvEv) hybrid embryonic stem (ES) cells. The cassette consisting of the http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Generation of icIL-1Ra12/2 mice. (A) Targeting strategy used for the introduction of an EGFP–Neo cassette into the icIL-1Ra1–specific first exon (icIl1rn1) of the mouse Il1rn gene. This approach does not interfere with the expression of the other transcript variants. Shown from top to bottom are the WT icIl1rn1 genomic locus (WT allele), the targeting vector and the targeted genomic locus (Mutant allele). Bold lines indicate arms of homology for recombination. Exon 1 is represented as a gray box and numbered. The neomycin resistance gene (Neor) is flanked by two loxP sites (triangles). The primers used to identify recombinant ES cells are indicated as S1 and S2. (B) Southern blot analysis of WT (+/+) and heterozygous (+/2) (one WT allele, one mutant allele) ES cells using a 59 external probe (left panel) or a 39 internal probe (right panel). The WT (6.4-kb [BglII digestion] or 8.9-kb [HindIII digestion] bands) and mutant alleles (7.4-kb [BglII digestion] or 6.2-kb [HindIII digestion] bands) were detected in BglII- or HindIII-digested ES cell genomic DNA, using, respectively, the 59 or 39 probes shown in (A). (C) PCR genotyping to distinguish WT (+/+), heterozygous (+/2) and icIL-1Ra1(2/2)mice.The primers used are indicated as P1, P2, and P3 in (A). The sizes of the WT and icIL-1Ra12/2 PCR products are 300 and 842 bp, respectively. (D)Male (left panel) and female (right panel) WT (male: n =13/n = 6 [10/17 wk]; female: n = 11/n = 6 [10/17 wk]) and icIL-1Ra12/2 (male: n =13/n =13[10/17 wk]; female: n =11/n = 9 [10/17 wk]) mice were weighed at 10 and 17 wk of age. Data are shown as the mean 6 SEM. No significant differences in body weight were observed between the genotypes (unpaired two-tailed Student t test). (E) Analysis of IL-1Ra isoforms in lysates of BMDC generated from naive WT and icIL-1Ra12/2 (KO) mice. IL-1Ra was detected using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms. Protein loading was assessed by the determination of GAPDH expression. One representative sample per genotype out of n = 3 per genotype is shown. Arrows indicate different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms are as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa (13, 69). The Journal of Immunology 969

EGFP coding sequence, followed by a loxP-flanked Neo sequence in was collected and pooled from the three cultures for each ear. Cytokine reverse orientation, was inserted immediately upstream of the ATG start levels were analyzed by ELISA. Cytotoxicity was determined by mea- codon of the Il1rn gene, thereby replacing the coding sequence and suring the release of lactate dehydrogenase (LDH; Cytotoxicity Detection splice donor site of the icIL-1Ra1–specific first exon. The long homology Kit; Roche or Sigma-Aldrich) according to manufacturer’s instructions. arm extends 3.94 kb 59 of EGFP. The short homology arm extends 1.95 kb Lysis of parallel control cultures with 1% Triton X-100 in assay medium 39 of the Neo cassette. The deletion of exon 1 does not interfere with the was used to determine the maximum amount of releasable LDH enzyme expression of the other isoforms with the transcription start site in exon 2, activity. located 10 kb downstream of exon 1 (Fig. 1E). To identify recombinant cell clones, neomycin-resistant ES cells were screened with primers S1 Generation and stimulation of bone marrow–derived dendritic (59-CCAGAGGCCACTTGTGTAGC-39) and S2 (59-CTGTGCTCCTGG- cells and bone marrow–derived CTCTTTCTACC-39) to confirm the integration of the short homology arm and the EFGP-loxP–Neo-loxP cassette. Positive ES cells were confirmed Bone marrow–derived dendritic cells (BMDC) and bone marrow–derived macrophages (BMDM) were generated as previously described (34). A by digesting DNA with Bgl II or HindIII for Southern blot hybridization 3 6 analysis with a probe against the 59 external or 39 internal regions, total of 1 10 BMDC or BMDM per well were seeded in medium respectively (Fig. 1A, 1B). A correctly targeted ES cell clone was containing GM-SCF or L929 cell-derived M-CSF, respectively, into a microinjected into C57BL/6 blastocysts. Resulting chimeras with a high 24-well plate and left to adhere overnight. The medium was discarded, the percentage of agouti coat color were mated to wild-type (WT) C57BL/6 to cells were rinsed with PBS, and fresh medium without growth factors was generate F1 heterozygous offspring, which were then backcrossed onto the added to the cells. Cells were stimulated with 100 ng/ml LPS (Escherichia C57BL/6 background for six generations using a marker-assisted selection coli purified by phenol extraction [serotype O55:B5)]: Sigma-Aldrich) for protocol (26). Genotyping for the WT or icIl1rn1-deleted allele was per- 24 h. Supernatant was collected, and cytokine production was analyzed by formed by a three-primer PCR combining a common forward primer (P1: 59- ELISA or Western blot. Cells were lysed for analysis by quantitative real- CTGCTAGACTGAGTCACGCCTC-39) with reverse primers specific for the time PCR or Western blot. Downloaded from WT (P2: 59-GACCTCAAAGATCTTACCTGAAGCC-39)ortheicIl1rn1- Culture and stimulation of primary mouse keratinocytes deleted (P3: 59-CCGATGGGGGTGTTCTGCTGG-39) alleles, resulting in a 300- or 842-bp band for the WT or icIl1rn1-deleted allele, respectively Primary keratinocytes were isolated from tails of adult mice. Tails were 2/2 (Fig. 1C). The icIL-1Ra1 mouse line was maintained by inter- incubated in keratinocyte serum-free medium (K-SFM) without CaCl2 crossing icIL-1Ra1+/2 mice. Male and female mice were weighed at the (Thermo Fisher Scientific) with penicillin/streptomycin/10 mg/ml Dispase age of 10 and 17 wk (Fig. 1D). The deletion of exon 1 does not interfere II (Thermo Fisher Scientific) at 4˚C overnight. The was detached with the expression of other IL-1Ra isoforms (sIL-1Ra, icIL-1Ra3) from the dermis and gently mixed three times for 1 min in 0.05% Trypsin/ 3 5 (Fig. 1E) for which the transcription start site in exon 2 is located 0.02% EDTA (PAN Biotech, Aidenbach, Germany). A total of 3 10 http://www.jimmunol.org/ 10 kb downstream of the icIL-1Ra1–specific exon 1. isolated cells were cultured in collagen type IV (Sigma-Aldrich, Buchs, Mice lacking all isoforms of IL-1Ra in myeloid cells were generated Switzerland)–coated 48-well plates in 500 ml of complete K-SFM flox/flox tmCemG by crossing homozygous IL-1Ra mice [B6.129-Il1rn (27)], (without CaCl2, supplemented with human recombinant epidermal backcrossed onto the C57BL/6J background for six generations using a and bovine pituitary extract [Thermo Fisher Scientific] and marker-assisted selection protocol (28), with mice expressing the Cre- penicillin/streptomycin) for 5 d. Medium was replaced with fresh com- recombinase under the transcriptional control of the endogenous lysozyme plete K-SFM on days 1 and 4. On day 5, keratinocytes were rinsed with M promotors (LysMcre; B6.129P2-Lyz2tm1(cre)Ifo/J), which specifically PBS and treated with different concentrations of Aldara, IMQ (R837; drives Cre-recombinase expression in myeloid cells (29). PCR genotyping of Invivogen, Toulouse, France), 100 ng/ml recombinant mouse (rec. m) IL- the IL-1Raflox/flox mice was previously described (27). Genotyping for the 1a (PeproTech, London, U.K.), 100 ng/ml rec. m IL-1b (PeproTech), or Cre-recombinase transgene was performed by PCR following The Jackson 100 ng/ml rec. m IL-36b [Ser4–Lys156, kindly provided by Amgen (35)], by guest on September 27, 2021 Laboratory protocols. This mouse line was maintained by intercrossing ho- as indicated in the figures, in 500 ml K-SFM without CaCl2 with mozygous IL-1Raflox/flox mice negative for the Cre-recombinase (control lit- penicillin/streptomycin. One pack of Aldara (250 mg containing 5% termates) with homozygous IL-1Raflox/flox mice expressing one allele of the IMQ) was either dissolved in 1.5 ml DMSO and further diluted 1:1,000, Cre-recombinase transgene (IL-1RaDM). All mice were maintained under 1:3,000, or 1:10,000, as previously described (3), in K-SFM without conventional conditions in the animal facility of the Geneva University School CaCl2, with penicillin/streptomycin. IMQ was diluted in K-SFM without of Medicine, and water and food were provided ad libitum. CaCl2 with penicillin/streptomycin at a concentration of 8.4 mg/ml Tails of casp1/112/2 (B6N.129S2-Casp1tmFlv) (30) and casp1/11 3 (equivalent to the amount of IMQ contained in Aldara diluted 1:1000). casp8 3 receptor-interacting protein kinase 3 (RIPK3)2/2 [Casp8tm1Raz Keratinocytes were stimulated for 6 h at 37˚C. Supernatant was col- (31); Ripk3tm1Vmd (32)] mice were kindly provided by Dr. Mohamed lected, and cytokine production was analyzed by ELISA. Cytotoxicity Lamkanfi (Janssen Pharmaceutica, Beerse, Belgium) and Dr. Andy Wullaert was determined by measuring the release of LDH into the culture me- (University of Ghent) to isolate primary keratinocytes. Genotyping was dium according to manufacturers’ instructions. Lysis of parallel control performed by PCR following The Jackson Laboratory protocols or as cultures with 1% Triton X-100 in assay medium was used to determine described previously (30–32), respectively. the maximum amount of releasable LDH enzyme activity. TRIzol re- Animal studies were approved by the Animal Ethics Committee of the agent was added to keratinocytes, and levels or differ- Geneva Veterinarian Office (license numbers GE/43/15, GE/113/18, and ential gene expression was analyzed by quantitative real-time PCR or GE/77/19) and performed according to the appropriate codes of practice. RNA-sequencing (RNA-Seq), respectively. Induction of psoriasis-like dermatitis in mice Histology, immunohistochemistry, and immunofluorescence Sex- and age-matched (8–12-wk-old) mice received a daily topical dose of Ears were fixed in 4% buffered formaldehyde and embedded in paraffin. Ear 12.5 mg of Aldara Cream (MEDA Pharma, Wangen, Switzerland) con- sections (4 mm) were deparaffinized and stained with H&E (Diapath taining 5% IMQ on the right ear for eight consecutive days. The equivalent S.p.A., Milano, Italy). Slides were scanned with an Axio Scan.Z1 (Zeiss, volume of Vaseline cream applied on the left ear served as control. In some Jena, Germany) and the ZEN lite software (Zeiss) was used for image experiments, mice were injected i.p. on days 1, 3, 5, and 7 of the Aldara acquisition and measurements. The average epidermal thickness and in- experiment with a neutralizing Ab directed against IL-1a (functional filtration of inflammatory cells were evaluated as previously described grade, hamster anti-mouse IL-1a, clone ALF-161, 200 mg/mouse; Thermo (36). Local expression of IL-1Ra was studied by immunohistochemistry Fisher Scientific, Zug, Switzerland) or the appropriate isotype control and immunofluorescence on paraffin-embedded ear sections as previously (functional grade, Armenian hamster IgG, clone eBio299Arm, 200 mg/mouse; described (28) with slight modifications. In brief, tissue sections were Thermo Fisher Scientific) (33). Ear thickness was measured in tripli- incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture cates daily using a pocket thickness gauge (Mitutoyo Europe GmbH, Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, Du¨sseldorf, Germany). Mice were euthanized on day 8, and ears were MN) overnight at 4˚C. For immunohistochemistry, the sections were rinsed collected for histological analysis and RNA and protein extraction or and incubated with HRP-labeled donkey anti-goat IgG (Santa Cruz Bio- explant cultures. For the latter, ear skin was collected as described with technology, Heidelberg, Germany) for 1 h at room temperature. Color was slight modifications (6). In brief, three pieces of skin were excised from developed with diaminobenzidine, and sections were counterstained with each control and Aldara-treated ear using a circular punch biopsy tool, hematoxylin. Slides were scanned with an Axio Scan.Z1, and pictures generating full thickness skin samples of identical surface area. Each were taken using the ZEN lite software. For immunofluorescence, the explant (diameter: 2 mm) was incubated in 200 mlassaymedium sections were rinsed, incubated with donkey anti-goat IgG Rhodamine (DMEM/5% FCS/penicillin/streptomycin) for 24 h at 37˚C. Supernatant (Jackson ImmunoResearch, Cambridgeshire, U.K.) and counterstained 970 KERATINOCYTE-DERIVED icIL-1Ra1 ANTAGONIZES IL-1a with DAPI (Thermo Fisher Scientific). Confocal fluorescent images were Systems]) served as positive control. Samples were separated by electro- acquired on a Zeiss LSM 700 microscope (Zeiss), and pictures were taken phoresis in homemade 17% SDS-PAGE and blotted onto a PVDF mem- using ZEN lite software. brane. For Western blot analyses of IL-1Ra expression in cytosolic and nuclear fractions, 4–12% gradient gels (NuPAGE; Thermo Fisher Scien- RNA extraction and quantitative RT-PCR tific) were used. Membranes were blocked with 5% horse serum in TBS/ 0.1% Triton X-100 and then incubated with polyclonal goat anti-mouse IL- Total RNA was extracted from BMDC, BMDM, mouse primary kerati- 1Ra (capture Ab of mouse IL-1Ra; DuoSet ELISA Kit; R&D Systems) at nocytes, or mouse ears using TRIzol reagent, followed by a cleanup of the 0.14 mg/ml, or with polyclonal rabbit anti-human KDM1/LSD1 (Abcam, last two using RNeasy Micro Kit columns (QIAGEN). RNA was reverse Cambridge, U.K.) diluted 1:2.000 in TBS/0.1% Triton X-100/1% horse se- transcribed, and mRNA levels were examined as previously described rum. Upon incubation with a donkey anti-goat IgG HRP-conjugated Ab (37). The primer sequences (Eurofins Scientific, Ebersberg, Germany) (Santa Cruz Biotechnology) or goat anti-rabbit IgG HRP-conjugated Ab are shown in Table I. mRNA expression levels were determined by (Santa Cruz Biotechnology) membranes were developed using Clarity quantitative RT-PCR (qRT-PCR) using iQ SYBR Green Supermix (Bio- Western ECL Substrate (Bio-Rad Laboratories AG) and by using the Rad Laboratories, Cressier, Switzerland) on a CFX Connect Real-Time imager LAS 4000 (Fujifilm, Du¨sseldorf, Germany). After stripping of the PCR Detection System (Bio-Rad Laboratories). Gene expression levels 2 membranes with ReBlot Plus Strong Stripping Solution (Merck were calculated using the comparative method (2 DCt) for relative Millipore, Schaffhausen, Switzerland) according to the manufacturer’s quantification by normalization to Mrpl32 gene expression. Non– instructions, the membranes were probed with polyclonal rabbit anti- reverse-transcribed RNA samples were included as negative controls. human GAPDH (FL-335; Santa Cruz Biotechnology) at 0.1 mg/ml in RNA-Seq analysis TBS/0.1% Triton X-100/1% horse serum, followed by incubation with a goat anti-rabbit IgG HRP-conjugatedAb(SantaCruzBiotechnology). Library preparation, sequencing, and read mapping to the reference genome. Total RNA was isolated from mouse primary keratinocytes us- Measurement of cytokine levels

ing TRIzol reagent, followed by a cleanup using RNeasy Micro Kit columns Downloaded from (QIAGEN). cDNA libraries were constructed by the Genomics Platform of In cell culture supernatants, IL-1Ra, IL-1a, CXCL1, or IL-1b protein levels the University of Geneva, using the Illumina TruSeq Stranded RNA were determined using DuoSet ELISA (R&D Systems) or ELISA Ready Library Preparation Kit according to the manufacturer’s protocol. Li- Set Go kits (eBioscience), respectively. The IL-1Ra DuoSet ELISA detects braries were sequenced using single-end (50 nt-long) reads on Illumina all isoforms of IL-1Ra. HiSeq 4000. FastQ reads were mapped to the ENSEMBL reference ge- Statistical analysis nome (GRCm38.89) using STAR version 2.4.0j (38) with standard set- tings, except that any reads mapping to more than one location in the Significant variations were calculated using the unpaired or paired two- genome (ambiguous reads) were discarded (m =1). tailed Student t test or two-way repeated measures ANOVA, as indicated http://www.jimmunol.org/ Unique gene model construction and gene coverage reporting. A unique in the figure legends. A p value ,0.05 was considered significant. Results gene model was used to quantify reads per gene. Briefly, the model considers are expressed as the mean 6 SEM. all annotated exons of all annotated protein-coding isoforms of a gene to create a unique gene, in which the genomic region of all exons is considered Results coming from the same RNA molecule and merged together. Generation and characterization of icIL-1Ra12/2 mice RNA-Seq analysis. All reads overlapping the exons of each unique gene model were reported using featureCounts version 1.4.6-p1 (39). Gene Homologous recombination of the WT allele with the targeting expressions were reported as raw counts and are in parallel normalized vector in ES cells (Fig. 1A) was confirmed by Southern blot in reads per kb million to filter out with low expression value analysis (Fig. 1B). A correctly targeted ES cell clone was used (1 read per kb million) before calling for differentially expressed genes. to generate icIL-1Ra1–deficient mice, which were genotyped by guest on September 27, 2021 Library size normalizations and differential gene expression calcula- tions were performed using the package edgeR (40) designed for the R by PCR to distinguish WT and/or targeted alleles (Fig. 1C). icIL- 2/2 software (41). Only genes having a significant fold change (Benjamini– 1Ra1 mice were fertile, showed no phenotypic abnormalities Hochberg corrected p value , 0.05) were considered for the rest of the (data not shown), and showed no difference in body weight RNA-Seq analysis. (Fig. 1D), in contrast to mice deficient in all four IL-1Ra iso- Subcellular fractionation forms (42). The insertion of the EGFP–Neo cassette at the ATG start codon in the icIL-1Ra1–specific first exon of the mouse All steps were performed on ice. Mouse primary keratinocytes were washed two times with ice-cold PBS, and the pellet was lysed in ice-cold hypotonic Il1rn gene deleted only the expression of icIL-1Ra1 but did not buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mm EDTA, 0.1 mm interfere with the expression of the isoforms icIL-1Ra3 and sIL- EGTA, 1 mm DTT, 13 EDTA-free , and phosphatase inhibitor 1Ra (shown is the immature proform of sIL-1Ra [pro–IL-1Ra]; mixture) 15 min on ice. 0.125% v/v NP40 were added, and the lysate was Fig. 1E). No EGFP signal could be detected by immunofluorescence vortexed 10 s. After centrifugation for 5 min at maximal speed, the su- microscopy on skin sections (data not shown). pernatant containing the cytosolic fraction was collected. The pellet was washed once with ice-cold hypotonic buffer supplemented with 0.1% Characterization of BMDC and BMDM from WT and NP40 and lysed in ice-cold nuclear protein extraction buffer (20 mM icIL-1Ra12/2 mice HEPES [pH 7.9], 0.4 M NaCl, 1 mm EDTA, 1 mM EGTA, 1 mM DTT, 13 EDTA-free protease and phosphatase inhibitor mixture), vortexed 10 s and It is known that upon stimulation myeloid cells express both sIL- incubated 10 min on ice. This procedure was repeated for a total of five 1Ra and the intracellular isoforms icIL-1Ra1 and icIL-1Ra3. We times. After centrifugation for 5 min at maximal speed, the supernatant containing the soluble nuclear protein fraction was collected. Protein generated BMDC and BMDM to further investigate the deletion of concentration in cytosolic and nuclear fractions was determined using the icIL-1Ra1. The presence of icIL-1Ra1 mRNA was only detected in DC Protein Assay Kit (Bio-Rad Laboratories AG). BMDC and BMDM from WT mice but was absent in BMDC and BMDM from icIL-1Ra12/2 mice, both at baseline and upon LPS Western blot analyses treatment (Fig. 2A). Expression levels of mRNA encoding both Total proteins from BMDC, BMDM, or mouse dorsal skin or mouse ears sIL-1Ra and icIL-1Ra3 (referred to as sIl1rn) were significantly were extracted in RIPA buffer (150 mM NaCl, 50 mM Tris/HCl [pH 8], 1% increased in BMDC or similar in BMDM of icIL-1Ra12/2 com- Triton X-100, 0.5% sodium deoxycholate. 0.1% SDS, 5 mM EDTA, and 13 EDTA-free protease inhibitor mixture) for 30 min on ice or by ho- pared with WT mice upon LPS stimulation (Fig. 2A). However, by mogenizing in 700 ml of RIPA buffer using a POLYTRON homogenizer using an ELISA that detects all IL-1Ra isoforms, no differences in followed by 30-min incubation on ice, respectively. The lysates were IL-1Ra protein levels between both genotypes were detected in cleared by centrifugation. Protein concentration in tissue lysates was de- the supernatants of control and LPS-treated BMDC and BMDM termined using the DC Protein Assay Kit (Bio-Rad Laboratories AG). Culture supernatants of BMDC and BMDM were collected and directly (Fig. 2B). Western blot analysis revealed that the only isoform used for Western blotting without further concentration. The rec. m IL-1Ra detected in the supernatants of control and LPS-treated BMDC secreted isoform (standard of mouse IL-1Ra DuoSet ELISA Kit [R&D and BMDM was sIL-1Ra (Fig. 2C). Furthermore, Western blot The Journal of Immunology 971

Table I. Sequences of primers used for qRT-PCR

Gene Sequence Forward Primer Sequence Reverse Primer icIl1rn1 59-AGTGAGACGTTGGAAGGCAG-39 59-TGAAGGCTTGCATCTTGCAG-39 sIl1rn 59-TGTGGCCTCGGGATGGAAAT-39 59-ATGAGCTGGTTGTTTCTCAGGT-39 Il1a 59-GGGAAGATTCTGAAGAAGAG-39 59-GAGTAACAGGATATTTAGAGTCG-39 Il1b 59-TGTGAAATGCCACCTTTTGA-39 59-GTGCTCATGTCCTCATCCTG-39 Il1r1 59-GAGTTACCCGAGGTCCAGTGG-39 59-GAGGGCTCAGGATAACAGG-39 Il17a 59-ATCCCTCAAAGCTCAGCGTGTC-39 59-GGGTCTTCATTGCGGTGGAGAG-39 Il22 59-AGCGGTGACGACCAGAACATCC-39 59-TCCCCAATCGCCTTGATCTCT-39 Il23a 59-CCAGCGGGACATATGAATCTACT-39 59-CTTGTGGGTCACAACCATCTTC-39 Il36b 59-GCTAGCAACAATGTCAAGCCT-39 59-TTGCCCTCCATCTCAACACA-39 Il1f5 59-TGGAGCTCATGATGGTTCTG-39 59-TAATGACCTTCTCTGCGTGC-39 Cxcl1 59-ACTCAAGAATGGTCGCGAGG-39 59-GTGCCATCAGAGCAGTCTGT-39 Cxcl2 59-AGGGCGGTCAAAAAGTTTGC-39 59-CGAGGCACATCAGGTACGAT-39 Csf3 59-TGTCCTGGCCATTTCGTACC-39 59-AAGGGCTTTCTGCTCAGGTC-39 S100a9 59-CACCCTGAGCAAGAAGGAAT-39 59-TGTCATTTATGAGGGCTTCATTT-39 Ivl 59-CCTGTGAGTTTGTTTGGTCTACA-39 59-GCTGGATATGATCTGGAGAA-39 Defb4 59-ACCTTCTCTTCACATTTCTCCTG-39 59-CCACAATTGCCAATCTGTCG-39 Mrpl32 59-CACCAGTCAGACCGATATGTGAAAA-39 59-TGTTGTCAATGCCTCTGGGTTT-39 Downloaded from analysis of cell lysates demonstrated the presence of all IL-1Ra iso- naive or control conditions, icIL-1Ra1 was mainly expressed in the forms in WT BMDC and BMDM, whereas icIL-1Ra1 was absent cytoplasm of keratinocytes, whereas Aldara treatment led to de- in BMDC and BMDM from icIL-1Ra12/2 mice (Fig. 2C). tection of icIL-1Ra1, additionally, in the nucleus (Supplemental Fig. 1A, 1B). Western blot analysis identified icIL-1Ra1 as the Increased severity of Aldara (5% IMQ)-induced ear thickness 2/2 principal isoform in whole ear lysates of control and Aldara-treated

in icIL-1Ra1 mice http://www.jimmunol.org/ WT mice, whereas icIL-1Ra1 was absent in ear lysates of icIL- icIL-1Ra1 is the major IL-1Ra isoform expressed in naive mouse 1Ra12/2 mice. Aldara treatment also led to the presence of IL-1Ra+ dorsal skin (Fig. 3A). Therefore, we investigated the functional cells in the dermis (Fig. 4A) as well as to production of immature role of icIL-1Ra1 in Aldara (5% IMQ)-induced skin inflammation, prosIL-1Ra and icIL-1Ra3 in ears of both genotypes (Fig. 4B), a widely used mouse model for human psoriasis. Clinical probably originating from infiltrating inflammatory myeloid cells. scoring showed a significantly higher increase in ear thickness Therefore, to investigate the impact of myeloid cell–derived IL-1Ra 2/2 in Aldara-treated icIL-1Ra1 compared with WT mice during Aldara-induced skin inflammation, we used mice with (Fig. 3B). Consistent with the clinical picture, the histopatho- LysMCre-mediated deletion of IL-1Ra in myeloid cells (IL-1RaDM). logical analysis showed a trend for higher epidermal thickness Clinical scoring showed no difference in ear thickening between by guest on September 27, 2021 (acanthosis) (p = 0.086) and more infiltration of inflammatory IL-1RaDM mice and littermate controls (IL-1Raflox/flox;Fig.4C). 2/2 cells (p = 0.086) into ears of icIL-1Ra1 as compared with Furthermore, there was no difference in mRNA expression levels for WT mice (Fig. 3C, 3D). In support of the clinical and histopath- icIL-1Ra1inearsofbothgenotypes.Incontrast,expressionofmRNA ological data, mRNA expression levels for the proinflammatory encoding sIL-1Ra and icIL-1Ra3 (sIl1rn) was almost completely ab- cytokines IL-1a, IL-1b, IL-36b, IL-17A, IL-22, and IL-23p19, rogateduponAldaratreatment in IL-1RaDM mice (Fig. 4D). which are all implicated in Aldara-induced skin inflammation (43), were significantly increased in ears of icIL-1Ra12/2 as No intracellular effect of icIL-1Ra1 on gene regulation compared with WT mice upon Aldara treatment (Fig. 3E). in keratinocytes Furthermore, expression levels of mRNA encoding sIL-1Ra and Intracellular effects of icIL-1Ra1 were previously reported in vitro icIL-1Ra3 (sIl1rn) and IL-36R antagonist (IL-36Ra; gene Il1f5) using icIL-1Ra1 genetically modified cells (44–46). To examine a were significantly increased in ears of icIL-1Ra12/2 as compared possible intracellular role of icIL-1Ra1 in skin inflammation, we with WT mice upon Aldara treatment, whereas there was no dif- stimulated primary keratinocytes isolated from tails of naive WT ference in mRNA expression levels for the decoy IL-1R2. Transcript or icIL-1Ra12/2 mice with IL-1a or with IL-36b. In addition to levels for the signaling receptor IL-1R1 were downregulated upon Il1rn, only three genes were differentially expressed in control treatment with Aldara but did not differ between the genotypes. There keratinocytes (Thbs2, Defb4, and Cyp1A1), whereas there were no was no difference between genotypes in control ears for all tested differentially expressed genes, except for Il1rn, in WT as com- mRNA expression levels except for icIL-1Ra1. pared with icIL-1Ra12/2 keratinocytes upon stimulation with IL-1a or IL-36b (Fig. 5A). Additionally, there was no difference icIL-1Ra1 is the main IL-1Ra isoform regulating in expression levels of selected mRNAs in primary keratinocytes Aldara-induced skin inflammation of WT as compared with icIL-1Ra12/2 mice after in vitro stim- icIL-1Ra1 is constitutively expressed and represents the major IL- ulation with Aldara or with the equivalent amount of IMQ 1Ra isoform in mouse keratinocytes (12, 13). Indeed, by using an (Supplemental Fig. 2). Of note, transcripts for IL-1b could not be anti–IL-1Ra Ab that recognizes all IL-1Ra isoforms, ear sections detected in primary keratinocytes (data not shown). Interestingly, from control and Aldara-treated WT mice but not from icIL- RNA-Seq analysis demonstrated that IL-1a– and IL-36b–induced 2/2 + 1Ra1 mice showed IL-1Ra keratinocytes (Fig. 4A). Of note, responses were similar in primary keratinocytes from both WT upon Aldara treatment, icIL-1Ra1 staining was barely detectable and icIL-1Ra12/2 mice (Supplemental Fig. 3A, 3B). in basal layers but rather was restricted to keratinocytes in the middle and outer layers of the epidermis. Staining with Ki67, a Aldara treatment leads to keratinocyte cell death accompanied a proliferation-associated protein, showed rare Ki67+ cells in control by the release of icIL-1Ra1 and IL-1 ears and an increased Ki67 expression restricted to the basal Despite the lack of a signal peptide, the three icIL-1Ra isoforms can keratinocyte layer upon Aldara treatment (data not shown). Under be passively released from dying cells or actively released by a yet 972 KERATINOCYTE-DERIVED icIL-1Ra1 ANTAGONIZES IL-1a Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Characterization of BMDC and BMDM from WT and icIL-1Ra12/2 mice. (A) Determination of mRNA levels for icIL-1Ra1 (icIl1rn1) and for sIL-1Ra and icIL-1Ra3 (sIl1rn) in BMDC (left panel) and BMDM (right panel). BMDC and BMDM from naive WT (n = 5) and icIL-1Ra12/2 (n =5) mice were left untreated (control) or stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated for qRT-PCR analysis. Results represent icIl1rn1 and sIl1rn mRNA expression levels relative to Mrpl32 mRNA levels. (B) Release of IL-1Ra protein (all isoforms) into the supernatant. BMDC (left panel) and BMDM (right panel) from naive WT (n = 5) and icIL-1Ra12/2 (n = 5) mice were left untreated (control) or stimulated with LPS for 24 h. IL-1Ra in the supernatant was measured using an ELISA that recognizes all isoforms of IL-1Ra. (A and B) Data are shown as the mean 6 SEM of values obtained from three (BMDC) or two (BMDM) independent experiments. The unpaired or paired two-tailed Student t test was used to test statistical significance. All p values ,0.05 were considered to be significant. (C) Analysis of IL-1Ra isoforms in lysates and cell supernatants of BMDC (left panel) and BMDM (right panel) by Western blot. BMDC and BMDM from naive WT and icIL-1Ra12/2 (KO) mice were left untreated (control) or stimulated with LPS for 24 h. IL-1Ra isoforms were analyzed in cell lysates or collected supernatants (30 ml) using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms. Protein loading was assessed by the determination of GAPDH expression in lysates. One nanogram of rsIL-1Ra served as control. Two rep- resentative samples out of n = 5 per genotype are shown. Arrows indicate different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms is as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa. n.d., not detected. SN, supernatant. unknown leaderless pathway (16–19) and can then compete ex- icIL-1Ra12/2 mice. The levels of IL-1Ra detected in superna- tracellularly with IL-1 for IL-1R1 binding, similar to sIL-1Ra (13, tants of icIL-1Ra12/2 explants are likely to represent sIL-1Ra 20, 21). To investigate whether icIL-1Ra1 can be released during and icIL-1Ra3 isoforms expressed by infiltrating myeloid cells. Aldara-induced skin inflammation, we examined the presence of IL-1a is another member of the IL-1 family expressed in kera- IL-1Ra in conditioned media of skin explants collected from tinocytes that does not have a signal peptide and can be released control and Aldara-treated ears of WT and icIL-1Ra12/2 mice, upon cell damage. IL-1a levels were significantly increased in using an ELISA that recognizes all isoforms of IL-1Ra. Levels of supernatants of ear explants upon Aldara treatment as compared IL-1Ra released from Aldara-treated explants of both WT and with control but without any difference between genotypes icIL-1Ra2/2 mice were significantly higher than from control (Fig.5B).AmodestsecretionofIL-1b wasdetectedinsome explants (Fig. 5B). Furthermore, IL-1Ra levels were significantly explant cultures of icIL-1Ra12/2 mice after Aldara treatment higher in supernatants of ear explants from WT as compared with (Fig. 5B). Of note, in addition to the increase of icIL-1Ra1 and The Journal of Immunology 973

FIGURE 3. icIL-1Ra1 deficiency in- creases the clinical severity of Aldara- induced ear thickness and the expression of proinflammatory cytokines. (A) IL- 1Ra isoforms were analyzed in lysates of total dorsal skin from naive WT, hetero- zygous (HET) and icIL-1Ra12/2 (KO) mice using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra iso- forms. Protein loading was assessed by the determination of GAPDH expression. One representative sample out of n =3 per genotype is shown. Arrow indicates the icIL-1Ra1 isoform. Theoretical mo- lecular mass of icIL-1Ra1: 18 kDa. (B–E) Aldara, or Vaseline as a control, were applied on the right or left ear, respec- tively, of WT (n = 21) and icIL-1Ra12/2 (n = 21) littermates for eight consecutive days. Ear thickness was measured daily. Downloaded from (B) The increase in ear thickness (mi- crometer) compared with day 0 is shown. Data were pooled from three indepen- dent experiments. The two-way repeated measures ANOVA was used to test sta- tistical significance. All p values ,0.05 were considered to be significant. (C) http://www.jimmunol.org/ Representative H&E-stained ear sections of WT and icIL-1Ra12/2 mice on day 8 after daily application of Vaseline (con- trol) or Aldara, respectively. Scale bar, 100 mm. (D) Epidermal thickness and the area of infiltration (percentage of total earsurface)inearsofWT(n =9)and icIL-1Ra12/2 (n =9)miceonday8 after daily application of Aldara. Data by guest on September 27, 2021 are shown as the mean 6 SEM of values obtained from two independent experi- ments. The unpaired two-tailed Student t test was used to test statistical signi- ficance. All p values ,0.05 were consid- ered to be significant. (E) Determination of mRNA levels for icIL-1Ra1, sIL-1Ra, IL-1a, IL-1b, IL-1R1, IL-1R2, IL-36b, IL-36R agonist (Il-1f5), IL-17A, IL-22, and IL-23p19. Total RNA was isolated from ears of WT (n = 10 [control], n =13 [Aldara]) and icIL-1Ra12/2 (n =10 [control], n = 15 [Aldara]) mice on day 8 after daily application of Vaseline (con- trol) or Aldara for analysis by qRT-PCR. Results represent icIl1rn1, sIl1rn, Il1a, Il1b, Il1r1, Il1r2, Il36b, Il1f5, Il17a, Il22, and IL23a mRNA levels relative to Mrpl32 mRNA levels. Of note, mRNA for sIL-1Ra also encodes icIL-1Ra3 and is referred to as sIl1rn.Dataareshownasthe mean 6 SEM of values obtained from two independent experiments. The unpaired or paired two-tailed Student t test was used to test statistical significance. All p values ,0.05 were considered to be significant. n.d., not detected.

IL-1a, a higher percentage of cell death was detected in Aldara- and icIL-1Ra12/2 mice and stimulated them with different treated ear explants, as assessed by LDH measurement in cul- concentrations of Aldara in vitro. Aldara induced the release of ture supernatants (Fig. 5B). To mimic the results observed with IL-1Ra from keratinocytes of WT mice. Only marginal amounts ear explants, we isolated primary keratinocytes from naive WT of IL-1Ra were measured in the supernatants of icIL-1Ra12/2 974 KERATINOCYTE-DERIVED icIL-1Ra1 ANTAGONIZES IL-1a Downloaded from http://www.jimmunol.org/

FIGURE 4. icIL-1Ra1 is the main IL-1Ra isoform regulating Aldara-induced skin inflammation. (A) Representative immunohistochemical detection of IL-1Ra (brown staining) on ear sections of WT and icIL-1Ra12/2 mice on day 8 after daily application of Vaseline (control) or Aldara. Scale bar, 100 mm. (B) Analysis of IL-1Ra isoforms in lysate of Vaseline-treated (control) (upper panel) and Aldara-treated (lower panel) ears from WT and icIL-1Ra12/2 (KO) mice by Western blot. IL-1Ra isoforms were detected in lysate using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms.

Protein loading was assessed by the determination of GAPDH expression. Two representative samples out of n = 6 per genotype are shown. Arrows indicate by guest on September 27, 2021 different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms is as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa. (C and D) Aldara or Vaseline as a control was applied on the right or left ear, respectively, of control IL-1Raflox/flox (n = 6) and IL-1RaDM (n = 6) littermates for eight consecutive days. Ear thickness was measured daily. (C) The increase in ear thickness (micrometer) compared with day 0 is shown. Data were obtained from one experiment. The two-way repeated measures ANOVAwas used to test statistical significance. All p values ,0.05 were considered to be significant. (D) Determination of mRNA levels for icIL-1Ra1 (icIl1rn1) and for sIL-1Ra and icIL-1Ra3 (sIl1rn). Total RNA was isolated from ears of control IL-1Raflox/flox (n = 6 [control], n = 6 [Aldara]) and IL-1RaDM (n = 6 [control], n = 6 [Aldara]) mice on day 8 after daily application of Vaseline (control) or Aldara for analysis by qRT-PCR. Results represent icIl1rn1 and sIl1rn mRNA expression levels relative to Mrpl32 mRNA levels. Data are shown as the mean 6 SEM of values obtained from one experiment. The unpaired or paired two-tailed Student t test was used to test statistical significance. All p values ,0.05 were considered to be significant. keratinocytes (Fig. 5C). Furthermore, Aldara induced the release of with additional deficiency in caspase 8, which blocks extrinsically IL-1a and a dose-dependent cell lysis in both WT and icIL-1Ra12/2 induced apoptosis, and RIPK3, which blocks necroptosis. We ob- keratinocytes (Fig. 5C). IMQ alone, the main active component of served that deficiencies in these three different cell death pathways Aldara, did not have any of the above-mentioned effects. The were devoid of any impact on the release of IL-1Ra, IL-1a,andcell proinflammatory cytokines IL-1a,IL-1b, and IL-36b did not induce lysis upon incubation of keratinocytes with Aldara in vitro (Fig. 5E). the release of icIL-1Ra1, IL-1a, or cell lysis. A dose of CXCL1 IL-1a blockade attenuates the clinical severity of served as positive control for cell stimulation (Fig. 5C). 2 2 Aldara-induced ear thickening in icIL-1Ra1 / mice a The release of icIL-1Ra1 and IL-1 is independent of caspase We demonstrated that IL-1a is released by mouse keratinocytes 1/11, caspase 8, and RIPK3 cultured with Aldara. We next determined whether excessive It has previously been shown that Aldara induces pyroptosis, a ear inflammation was because of the unbalanced effect of IL-1a in caspase 1–dependent cell death, which results in the release of icIL-1Ra12/2 mice. In agreement with this hypothesis, in IL-1b, in human keratinocytes (3). We therefore decided to de- comparison with its isotype control, the administration of a termine whether the release of icIL-1Ra1 and IL-1a from mouse neutralizing monoclonal anti–IL-1a Ab during Aldara treat- keratinocytes was also related to Aldara-induced caspase-1 acti- ment significantly attenuated the increase in ear thickness in vation and pyroptosis. We observed that levels of IL-1Ra, IL-1a, icIL-1Ra12/2 but not in WT mice (Fig. 6). and LDH did not differ in the supernatants of WT and casp1/112/2 keratinocytes cultured in the presence of Aldara, thus ruling out Discussion the involvement of the inflammasome–caspase-1 pathway in Using recently generated knock-in mice, in which the expression Aldara-induced cell death (Fig. 5D). To further examine the role of the icIL-1Ra1 isoform was specifically deleted, we showed for of other cell death pathways, we used keratinocytes from mice the first time, to our knowledge, that icIL-1Ra1 exerts specific The Journal of Immunology 975 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. icIL-1Ra1 plays no intracellular role in keratinocytes but is released together with IL-1a upon Aldara treatment. (A) Differential gene expression analysis based on RNA-Seq data of primary keratinocytes from icIL-1Ra12/2 versus WT mice. Primary keratinocytes were isolated from WT (n = 4) and icIL-1Ra12/2 (n = 4) mouse tails and left untreated as a control (left panel) or stimulated with 100 ng/ml rec. m IL-1a (middle panel) or 100 ng/ml rec. m IL-36b (right panel) for 6 h. Total RNA was then isolated from keratinocytes for RNA-Seq analysis. A total of 10,750, 10,709, or 10,727 genes were tested for control, IL-1a–, or IL-36b–treated cells, respectively. The fold-change threshold and Benjamini–Hochberg corrected p value threshold were set to 2 and 0.05, respectively. (B) Ear explants taken from WT (n = 6) and icIL-1Ra12/2 (n = 6) mice at day 8 after daily treatment with Vaseline (control) or Aldara were cultured for 24 h. Levels of IL-1Ra, IL-1a,andIL-1b in the supernatant or the percentage of cytotoxicity were assessed by ELISA or LDH assay, respectively. The IL-1Ra ELISA recognizes all isoforms of IL-1Ra. Data are shown as the mean 6 SEM from one (Figure legend continues) 976 KERATINOCYTE-DERIVED icIL-1Ra1 ANTAGONIZES IL-1a

FIGURE 6. Blocking IL-1a attenuates the clinical severity of Aldara-induced ear thickening in icIL-1Ra12/2 mice. WT and icIL-1Ra12/2 mice were injected i.p. with 200 mg neutralizing hamster anti-mouse IL-1a Ab (WT: n = 5; icIL-1Ra12/2: n = 7) or Armenian hamster IgG isotype control (WT: n =6; icIL-1Ra12/2: n = 7) on days 1, 3, 5, and 7 during the application of Aldara or Vaseline. Aldara or Vaseline as a control were applied on the right or left ear, respectively, of WT (left panel) and icIL-1Ra12/2 (right panel) mice for eight consecutive days. Ear thickness was measured daily. The increase in ear thickness (micrometer) compared with day 0 is shown. Data were obtained from two independent experiments. The two-way repeated measures ANOVA was used to test statistical significance. All p values ,0.05 were considered to be significant. Downloaded from anti-inflammatory effects in vivo. Indeed, icIL-1Ra1 deficiency One key question is how icIL-1Ra1 controls the severity of ear led to aggravated Aldara (5% IMQ)-induced skin inflammation, a inflammation. Consistent with previous reports (13, 24, 51, 52), we mouse model for human psoriasis. We could not detect an intra- demonstrated that icIL-1Ra1 is constitutively expressed in kera- cellular effect of icIL-1Ra1 using isolated keratinocytes. However, tinocytes and the main isoform found in mouse skin. An intra- http://www.jimmunol.org/ Aldara led to keratinocyte cell death, accompanied by the release cellular role for icIL-1Ra1 has been reported in vitro. Human of both icIL-1Ra1 and IL-1a from keratinocytes. Furthermore, ovarian cancer cells expressing icIL-1Ra1 showed a shorter in- blocking IL-1a attenuated the clinical severity of Aldara-induced duction of GRO and IL-8 mRNA in response to IL-1 as compared ear thickening in icIL-1Ra12/2 mice (Fig. 7). with cells lacking icIL-1Ra1 (46). Furthermore, icIL-1Ra1 Patients with a deficiency in all IL-1Ra isoforms (DIRA syn- inhibited IL-1–induced IL-6 and IL-8 production in icIL-1Ra1– drome) present sterile osteomyelitis and periostitis but also present transfected Caco-2 intestinal epithelial cells through the inhibition clinical features resembling human psoriasis (22, 23, 47). In dis- of p38 MAPK and NF-kB signal transduction pathways (45). tinction from mice deficient in all four IL-1Ra isoforms, which Moreover, icIL-1Ra1 overexpressed in a mouse skin carcinoma exhibit reduced body weight as well as other specific tissue- cell line appeared to have antiproliferative effects in vitro and by guest on September 27, 2021 inflammatory responses, depending on their genetic background in vivo (44). In contrast to these findings, we did not find differ- (42, 48, 49), icIL-1Ra12/2 mice bred normally and revealed a entially expressed genes, except for Il1rn in isolated keratinocytes normal phenotype. This points to either distinct roles of the dif- from icIL-1Ra12/2 compared with WT mice upon in vitro stim- ferent IL-1Ra isoforms or a cumulative effect in case of the de- ulation with either IL-1a or IL-36b, which argues against an in- ficiency in all four isoforms. tracellular role of icIL-1Ra1 in keratinocytes. We did not follow We observed a higher icIL-1Ra1 expression in keratinocytes of up on the three differentially expressed genes (Thbs2, Defb4, and the middle and outer layers of the epidermis upon Aldara treatment. Cyp1A1), found in keratinocytes from icIL-1Ra12/2 compared The activation state of keratinocytes can alter the protein ex- with WT mice under control conditions because we aimed to find pression. Lee et al. (50) showed a stronger IL-1Ra staining in- a role for icIL-1Ra1 under inflammatory, not naive conditions. tensity in irritated than in naive mouse epidermis. In line with Furthermore, the expression levels for b-defensin 4 (gene: Defb4) Hammerberg et al. (51), who described a cytosolic expression of tested by qRT-PCR were not different in keratinocytes of icIL-1Ra1 in normal skin of healthy individuals, our findings WT compared with icIL-1Ra12/2 mice (Supplemental Fig. 2). It demonstrate the expression of icIL-1Ra1 only in the cytoplasm cannot be excluded that icIL-1Ra1 could have intracellular effects of naive keratinocytes, whereas Aldara causes expression also in under different physiological or pathological conditions or in other the nucleus. Why icIL-1Ra1 shuttles into the nucleus upon cell types. Aldara treatment and why its expression is increased in kera- Our findings that keratinocyte cell death is accompanied by the tinocytes of the middle and outer layers of the epidermis in release of icIL-1Ra1 and IL-1a are in line with previous reports (50, inflamed ears remains unknown and is to be addressed in further 53, 54). The release of icIL-1Ra1 from keratinocytes has been sug- studies. gested to serve as a compensatory mechanism to counter-regulate

experiment. The unpaired or paired two-tailed Student t test was used to test statistical significance, and p values , 0.05 were considered to be significant. n.d., not detected. (C–E) Primary keratinocytes isolated from tails of WT [(C): n =5;(D): n =5;(E): n = 4], icIL-1Ra12/2 [(C): n = 5], casp1/112/2 [(D): n = 5], and casp1/11 3 casp8 3 RIPK32/2 [(E): n = 4] mice were left untreated or stimulated with DMSO as control or with different dilutions of Aldara, IMQ, 100 ng/ml rec. m IL-1a, 100 ng/ml rec. m IL-1b, or 100 ng/ml rec. m IL-36b for 6 h. Levels of IL-1Ra, IL-1a, and CXCL1 in the supernatant or the percentage of cytotoxicity were assessed by ELISA or LDH assay, respectively. The IL-1Ra ELISA recognizes all isoforms of IL-1Ra. Data are shown as the mean 6 SEM from three (C and D) or two (E) independent experiments. The unpaired two-tailed Student t test was used to test statistical significance. All p values ,0.05 were considered to be significant. *p , 0.05, *p = 0.034, and NS with line spanning the different conditions used for stimulation: WT versus icIL-1Ra12/2 (C), WT versus casp1/112/2 (D), WT versus casp1/11 3 casp8 3 RIPK32/2.#p , 0.05, ##p , 0.01, ###p , 0.001 dilutions of Aldara versus DMSO or IMQ, IL-1 family cytokines versus control. n/a, not applicable. The Journal of Immunology 977 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. Balance between the antagonist icIL-1Ra1 and the alarmin IL-1a. icIL-1Ra1 and IL-1a are constitutively expressed in mouse keratinocytes. Topical application of Aldara induces release of icIL-1Ra1 and IL-1a independent of pyroptosis, apoptosis, or necroptosis. LDH is found in the supernatant of treated keratinocytes, which is indicative of cellular damage because of loss of plasma membrane integrity. The biological activity of IL-1a is mediated by IL-1R1 expressed on target cells in the skin, such as keratinocytes in the epidermis or fibroblasts in the dermis. IL-1a binding induces a conformational change in the extracellular domain of IL-1R1, enabling its interaction with IL-1R accessory protein (IL-1RAcP), which is required for MyD88-mediated intracellular signaling and induction of an inflammatory response. In WT mice, icIL-1Ra1 controls the IL-1a–mediated inflammation by competing with IL-1a for binding to IL-1R1. In contrast to IL-1a, binding of icIL-1Ra1 does not recruit IL-1RAcP and thus no inflammatory response is induced. De- ficiency of icIL-1Ra1 leads to enhanced binding of IL-1a to IL-1R1 and excessive inflammation. Neutralizing anti–IL-1a Abs attenuate this imbalance in icIL-1Ra1–deficient mice. Taken together, our results suggest that icIL-1Ra1 is stored in keratinocytes and coreleased with the alarmin IL-1a upon cell death, thereby counteracting its inflammatory potential and acting as a natural anti-alarmin. 978 KERATINOCYTE-DERIVED icIL-1Ra1 ANTAGONIZES IL-1a inflammation in the skin provoked by keratinocyte-derived IL-1a (50, was not neutralized and thus biologically active, argues for a 55). Because of the lack of a signal peptide, icIL-1Ra1 and IL-1a are predominant role of IL-1a in this particular experimental setting believed to require a cell death program with loss of membrane in- and thus a regulatory effect of icIL-1Ra1 directed mostly against tegrity to get released, as shown for IL-1b (30, 56). Furthermore, IL-1a. previous data (13) and our finding in BMDC and BMDM showing Like heat shock proteins, defensins, S100 proteins, or the cytokines that LPS stimulation led to the release of sIL-1Ra but not of the two IL-33 and high-mobility group ox 1 (HMGB1), IL-1a is considered an intracellular isoforms suggest that an inflammatory response is not alarmin. Alarmins are endogenous, constitutively expressed mole- sufficient to release icIL-1Ra1. Disruption of the plasma membrane cules that are released after necrotic cell damage to rapidly mobilize occurs during pyroptosis, apoptosis, necroptosis, and necrosis (see and activate immune cells (67). Because of their prominent role in review in Ref. 57). Release of icIL-1Ra1 and IL-1a,aswellasdeath immune responses, the activity of alarmins must be strictly regulated. of primary keratinocytes, were not reduced in absence of caspase 1/11, Tetramerization of the alarmins S100A8/S100A9, for instance, is a caspase 8, and RIPK3, which excludes pyroptosis, caspase 8–mediated mechanism of autoinhibition and regulation of alarmin activity apoptosis, and necroptosis as forms of Aldara-induced keratinocyte (68). icIL-1Ra1 is present in keratinocytes as a reservoir of bio- cell death and points to necrosis or another type of lytic cell death. logically active IL-1Ra that can immediately counteract the in- However, it cannot be excluded that icIL-1Ra1 and IL-1a are actively flammatory potential of the alarmin IL-1a, which is released upon released by an unknown mechanism also leading to loss of membrane lytic cell death of keratinocytes. Our findings give rise to the integrity. Supporting our data, Cohen et al. (53) observed that, together hypothesis that icIL-1Ra1 acts as a natural inhibitor for the alar- with other components, such as b-actin, IL-1a was present in the min IL-1a in the skin. supernatant of necrotic but not apoptotic mouse B16 skin melanoma Downloaded from cells. However, our findings differ from recent data that identified Acknowledgments pyroptosis as the type of cell death of human keratinocytes upon We thank Andy Wullaert (University of Ghent, Ghent, Belgium) and Mohamed Aldara treatment (3). The lack of inflammasome proteins and IL-1b in Lamkanfi (Janssen Pharmaceutica, Beerse, Belgium) for providing tails 2 2 2 2 2 2 2 2 mouse but not human keratinocytes could explain these different from casp1/11 / and casp1/11 / 3 casp8 / 3 RIPK3 / mice. We thank findings (58–64). Additionally, and in divergence from the findings of Mathilde Harel for help with the in vivo mouse experiments.

Walter et al. (3), we could not detect transcripts or protein for IL-1b in http://www.jimmunol.org/ isolated primary mouse keratinocytes. The modest levels of IL-1b Disclosures protein, which we found in supernatant of ear explants, were probably The authors have no financial conflicts of interest. coming from infiltrating myeloid cells, which are known to express and release IL-1b during Aldara-induced skin inflammation (2, 65). References Our in vitro data suggest that icIL-1Ra1 is passively released by 1. Boehncke, W. H., and M. P. Scho¨n. 2015. Psoriasis. Lancet 386: 983–994. 2. van der Fits, L., S. Mourits, J. S. Voerman, M. Kant, L. Boon, J. D. Laman, dying keratinocytes. We tried to make cell death of keratinocytes F. Cornelissen, A. M. Mus, E. Florencia, E. P. Prens, and E. 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