IL-36γ/IL-1F9, an Innate T-bet Target in Myeloid Cells*

Background: The transcription factor T-bet is pivotal for initiation of Th1-related immunoactivation. Identification of novel genes directly regulated by T-bet is crucial. Results: Genome-wide analysis and subsequent experiments revealed that T-bet up-regulates IL-36γ/IL-1F9 in myeloid cells. Conclusion: IL-1-related IL-36γ is a direct T-bet target in myeloid cells. Significance: Observations suggest that IL-36γ, besides IFNγ, contributes to T-bet functions in immunopathology. By concerted action in dendritic (DC) and T cells, T-box expressed in T cells (T-bet, Tbx21) is pivotal for initiation and perpetuation of Th1 immunity. Identification of novel T-bet-regulated genes is crucial for further understanding the biology of this transcription factor. By combining siRNA technology with genome-wide mRNA expression analysis, we sought to identify new T-bet-regulated genes in predendritic KG1 cells activated by IL-18. One gene robustly dependent on T-bet was IL-36γ, a recently described novel IL-1 family member. Promoter analysis revealed a T-bet binding site that, along with a κB site, enables efficient IL-36γ induction. Using knock-out animals, IL-36γ reliance on T-bet was extended to murine DC. IL-36γ expression by human myeloid cells was confirmed using monocyte-derived DC and M1 macrophages. The latter model was employed to substantiate dependence of IL-36γ on endogenous T-bet in human primary cells. Ectopic expression of T-bet likewise mediated IL-36γ production in HaCaT keratinocytes that otherwise lack this transcription factor. Additional experiments furthermore revealed that mature IL-36γ has the capability to establish an inflammatory gene expression profile in human primary keratinocytes that displays enhanced mRNA levels for TNFα, CCL20, S100A7, inducible NOS, and IL-36γ itself. Data presented herein shed further light on involvement of T-bet in innate immunity and suggest that IL-36γ, besides IFNγ, may contribute to functions of this transcription factor in immunopathology.

T-box expressed in T cells (T-bet, Tbx21) 2 is regarded as a transcription factor crucial for Th1 lineage commitment and subsequent Th1-like immunoactivation that culminates in ample production of the corresponding signature cytokine IFN␥ (1)(2)(3). The impressive potency of this transcription factor to determine T cell behavior is highlighted by enforced IFN␥ production in committed Th2 cells that underwent ectopic expression of T-bet (3). Increased susceptibility of T-bet knockout mice for Salmonella (4) or Mycobacterium tuberculosis (5) infections is manifest and in accordance with the pivotal role of Th1-like immunoactivation for anti-bacterial host defense. However, T-bet expression is not confined to T cells but has been detected likewise in additional hematopoietic cell types, among others, IFN␥-activated human monocytes and monocyte-derived dendritic cells (DC) (6). DC derived from T-betdeficient mice actually display diminished capability to produce IFN␥ and to install Th1 differentiation (7,8). Accordingly, Th1 priming is enhanced in the context of T-bet-overexpressing human DC acting on naïve T cells (9). Altogether, T-bet promotes Th1 immune responses by apparently acting in a coordinated manner on the level of DC and T cells. The regulatory role of T-bet concerning Th17 development has been recently evaluated. Notably, T-bet has the capability to directly restrain Th17-mediated immunopathology (10) likely by decreasing expression of the Th17 signature transcription factor retinoic acid-binding receptor ␥ (11). Moreover, initiation of T-bet expression in Th17 cells may also favor transdifferentiation into Th1 cells, again strengthening the latter path of T cell polarization under the influence of T-bet (12). Current data furthermore indicate that T-bet expression is up-regulated in human inflammatory/M1 macrophages (13,14).
On the whole, T-bet knock-out mice display reduced disease severity in models of autoimmunity. Specifically, T-bet deficiency has been associated with amelioration in experimental autoimmune encephalitis (15) and collagen-induced arthritis (CIA) (16). Because T-bet has been associated foremost with IFN␥ bioactivity, those observations at first sight do not concur with seemingly protective properties of IFN␥ in those aforementioned models of autoimmunity (17,18) and beyond that with some context-specific anti-inflammatory characteristics of this cytokine (19). Thus, it is tempting to speculate that T-bet-inducible genes apart from IFN␥ may play a significant role in the pathophysiology of autoimmune inflammation.
Recently, we reported on robust induction of T-bet mRNA, protein, and biological activity (20) by IL-18 (IL-1F4) (21,22) in the predendritic acute myelogenous leukemia-derived cell line KG1 (23). Based on this experimental system we set out to identify novel T-bet-inducible genes by combining siRNA technology with genome-wide mRNA expression analysis. We herein introduce the recently described proinflammatory IL-1 cytokine family member IL-36␥ (IL-1F9) (24) as a novel T-bet-regulated gene in cells of myeloid origin.
Affymetrix Oligonucleotide Microarray Analysis-For affymetrix microarray analysis, KG1 cells were transfected by electroporation (Bio-Rad) (20) either with 2.0 g of siRNA-Tbet targeting T-bet (5Ј-GGAAGUUUCAUUUGGGAAA-3Ј, nt 916-934, NM_013351; Eurofins MWG Operon, Ebersberg, Germany) or with 2.0 g of siRNAc, a negative control (Silencer Negative Control siRNA, #4611, Applied Biosystems, Weiterstadt, Germany). After 5 h of rest, transfected cells were stimulated with IL-18 (10 ng/ml) for 12 h, which mediates robust T-bet expression in KG1 cells (20). Total RNA from five independently performed experiments was isolated (TriReagent, Sigma) according to the manufacturer's instructions and pooled in equal shares. Analysis of differential gene expression using the GeneChip HG-U133A 2.0 Array (Affymetrix, Santa Clara, CA) was performed by the BioChip laboratory of the University Duisburg-Essen (Germany). Mock transfection denotes an electroporation/transfection procedure without siRNA/nucleic acid. The microarray analysis that initiated the studies presented herein has been submitted to the NCBI GEO data repository and obtained the GEO accession number GSE37595.
Cultivation of KG1 Cells and HaCaT Keratinocytes-The human acute myelogenous leukemia-DC cell line KG1 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were cultivated in RPMI 1640 supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated FCS (Invitrogen). For experiments, cells were seeded at a density of 3 ϫ 10 6 cells/ml in 6-well polystyrene plates (Greiner, Frickenhausen, Germany) using the aforementioned medium. HaCaT keratinocytes (Prof. Stefan Frank, University Hospital Goethe-University Frankfurt) were maintained in DMEM supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 10% FCS. For experiments, HaCaT keratinocytes were seeded on 6-well polystyrene plates in the aforementioned culture medium. All incubations were performed at 37°C and 5% CO 2 .
Ectopic Expression of T-bet and Overexpression of IL-36␥ in HaCaT Keratinocytes-Full-length IL-36␥ was cloned in pCDNA3-vector (HindIII, EcoRI) and sequenced (Seqlab, Göttingen, Germany). Overexpression of IL-36␥ in HaCaT keratinocytes was used herein as a positive control for detection of the cytokine by Western blot analysis. For transfection experi-ments 2 g of the CMV6-XL4-Tbet vector (Origene Technologies, Rockville, MD), of control vector (CMV6-XL4), or of pCDNA3-IL-36␥ vector were transfected using Nucleofector Technology according to the manufacturer's instructions (Lonza, Cologne, Germany). After a 20-h incubation upon the indicated conditions, total RNA (T-bet-transfection) and cellular lysates (T-bet and IL-36␥ transfection) were obtained as previously described (25).
Generation of Human DC, Inflammatory Macrophages, and Th1-primed T Cells-For isolation of peripheral blood mononuclear cells, written informed consent was obtained from healthy donors, and blood was taken. The procedure was approved by the "Ethik Kommission" of the University Hospital Goethe-University Frankfurt (Geschäfts-Nr.: 170/1998). Healthy donors had abstained from taking drugs for 2 weeks before the study. Peripheral blood mononuclear cells were isolated from peripheral blood using Histopaque-1077 (Sigma) according to the manufacturer's instructions. The CD14 ϩ cell population of peripheral blood mononuclear cells was isolated by using CD14 antibody-conjugated magnetic microbeads (Miltenyi, Bergisch-Gladbach, Germany). After isolation, CD14 ϩ cell purity was determined by FACS analysis (FACS Canto, BD Biosciences) using mouse monoclonal anti-human CD14-phycoerythrin (PE) (eBioscience, Frankfurt, Germany). Enrichment of CD14 ϩ cells was Ͼ94.5%. Cells were resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS and seeded at a density of 8 ϫ 10 5 cells/0.75 ml on 12-well polystyrene plates (Greiner). CD14 ϩ cells were further used to generate monocyte-derived DC or macrophages, respectively. For generation of inflammatory macrophages (26), CD14 ϩ cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (10 ng/ml)/TNF␣ (10 ng/ml) for 7 days and LPS (10 g/ml) during a final maturation period of 3 days (26). Medium was changed on days 3 and 5. For differentiation into DC, CD14 ϩ cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (50 ng/ml)/IL-4 (20 ng/ml) for 7 days (27). 75% of spent medium was exchanged for fresh medium and fresh cytokines on days 3 and 5. T-bet-siRNA or siRNAc was transfected into macrophages using Lipofectamine 2000 (Invitrogen). For each reaction 20 nM concentrations of indicated siRNA was transfected into cells according to the manufacturer's instructions. The transfection was stopped after 4 h. After 3h of rest, cells were used as unstimulated control or were further stimulated as indicated.
For Th1 differentiation, CD4 ϩ T cells were isolated using the Naïve CD4 ϩ T cell isolation kit II according to the manufacturer's instructions (Miltenyi). To assess successful CD4 ϩ isolation, FACS analysis was performed with the following antibodies: mouse monoclonal anti-human CD4-PE-Cy7 (BD Biosciences) and mouse monoclonal anti-human CD45RA-FITC (Biolegend, Fell, Germany). Enrichment of CD4 ϩ cells was Ͼ97.2%. To induce Th1 cell differentiation, cells were resuspended in RPMI 1640 supplemented with 1% human serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 50 M 2-mercaptoethanol and seeded onto 6-well plates coated with anti-CD3 or anti-CD3/anti-CD28 antibodies (coating with 4 g/ml of each antibody). Cells were kept in the presence of IL-2 (20 ng/ml), IL-12 (20 ng/ml), and anti-IL-4 (10 ng/ml) for 2 days. After this time period cells were transferred to new culture plates without medium change. After 3 more days, cells were harvested, and T-bet expression was analyzed by Western blot analysis. To confirm successful Th1 differentiation, IFN␥ expression was evaluated by intracellular FACS-analysis. To that end, cells were stimulated on day 5 with PMA (50 g/ml), ionomycin (1 g/ml), and monensin (2 M) for 5 h. Thereafter, analysis was performed using the following antibody: mouse monoclonal anti-human IFN␥-FITC (BD Biosciences). Percentage of IFN␥-positive cells after treatment was 37.8 Ϯ 3.9%. Th0 denotes T cells immediately harvested for Western blot analysis after isolation by the Naïve CD4 ϩ T cell isolation kit II.
Isolation of Murine Splenic DC-Isolation of splenocytes from wild-type (C57BL/6J) and T-bet knock-out mice (B6.129S6-Tbx21 tm1Glm /J (stock number 004648; The Jackson Laboratory) and further DC isolation was performed according to the protocol from Miltenyi Biotec. For each experiment (n ϭ 3), splenocytes of 2 wild-type and 2 T-bet knock-out mice were pooled for further analysis. 0.5 ϫ 10 6 DC were seeded on 12-well polystyrene plates in RPMI 1640 culture medium supplemented with 10% FCS. CD11c ϩ cell isolation was evaluated by FACS analysis (FACS Canto) with the following antibodies: hamster monoclonal anti-mouse CD11c-FITC (Miltenyi) and rat monoclonal anti-mouse MHC-II (I-A/I-E)-PE (eBioscience). Enrichment of CD11c ϩ cells was 91.7 Ϯ 2.8% and 92.9 Ϯ 1.2% for wild-type and T-bet knock-out mice, respectively.
Preparation of Human Primary Keratinocytes-Human foreskin specimens were obtained from a resident physician (board-certified specialist for urology) located in metropolitan Frankfurt. This procedure was approved by the Ethik Kommission of the University Hospital Goethe-University Frankfurt (Geschäfts-Nr.: 162/12). Foreskin tissue was cut in small pieces and incubated with dispase (PromoCell, Heidelberg, Germany) overnight at 4°C. Thereafter, the epidermis was separated from the dermis, incubated in Hepes, and trypsinized for 30 min at 37°C. The reaction was stopped by adding trypsin inhibitor. To prepare a single cell suspension, a nylon cell strainer (70 m; BD Biosciences) was used. Keratinocytes were resuspended in primary keratinocyte cell culture medium (PromoCell) supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 50 g/ml gentamycin and seeded at a density of 6 ϫ 10 5 cells/ml in 6-well polystyrene plates (Greiner). For gene expression studies, cells were stimulated with cytokines at a density of 80 -90%.
Evaluation of Human IL-36␥ mRNA by RNase Protection Assay-Total RNA isolated using Tri-Reagent (Sigma) was used for RNase protection assay, performed as previously described (28). Briefly, IL-36␥ DNA probe was cloned into the transcription vector pBluescript II KS (ϩ) (Stratagene, Heidelberg, Germany). After linearization, an antisense transcript was synthesized in vitro with T3 RNA polymerase (Roche Diagnostics) and [␣-32 P]UTP (800 Ci/mmol; PerkinElmer Life Sciences. RNA samples were hybridized at 42°C overnight with 100,000 cpm of the labeled antisense transcript. Hybrids were digested with RNase A and T1 (both from Roche Diagnostics) for 1 h at 30°C. Under these conditions every single mismatch was recognized by the RNases. Protected fragments were separated on 5% polyacrylamide, 8 M urea gels and analyzed using phospho-imaging (Fuji, Straubenhardt, Germany). Individual gene expression of IL-36␥ was evaluated on the basis of GAPDH housekeeping gene expression. The cDNAs correspond to nt 112-380 of IL-36␥ (NM_019618) and nt 961-1071 of GAPDH (M33197) of the published sequences.
Determination of the Human IL-36␥ Transcriptional Start Site by RNA Ligase-mediated Rapid Amplification of 5Ј-cDNA Ends-To identify the IL-36␥ transcriptional start site in KG1 cells, Gene Racer-and TOPO TA cloning kits (Invitrogen) were used according to the manufacturer's instructions. For analysis, 12 clones were sequenced that were obtained from three independent rapid amplification of 5Ј-cDNA ends experiments. All 12 clones gave an identical result. We identified the transcriptional start site at nucleotide 113,452,054 (denoted ϩ1, Fig. 3A) within the sequence given in NC_000002.10 (chromosome 2). All topographic information concerning the human IL-36␥ gene locus refers to this transcriptional start site.
Cloning of the Human IL-36␥ Promoter, Transient Transfection of KG1 Cells, and Luciferase Reporter Assays-Using human genomic DNA (KG1 cells), we amplified the 5Ј-flanking region of IL-36␥ (NM_019618) using Pfu polymerase (Invitrogen) to generate IL-36␥ promoter fragments. For that purpose the following primers (excluding an additional flanking BglII cloning/restriction site) were used: Prom1 (2209 bp), forward 5Ј-AGCATTGACCAGACTGTACTC-3Ј; Prom2 (1630 bp), forward 5Ј-TAGGGTGAAAAGTAAGAGAC-3Ј. The reverse primer for both fragments (excluding an additional flanking HindIII cloning/restriction site) was 5Ј-AGTGTGGTTGTCT-CAGCACCT-3Ј. Both promoter fragments end 5Ј adjacent to the adenine nucleotide of the IL-36␥ translational start site. Fragments were cloned into pGL3-Basic (Promega, Mannheim, Germany) and sequenced (Seqlab). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, Amsterdam, Netherlands) to generate promoter fragments that show dysfunctional putative T-bet and nuclear factor-B (NF-B) binding sites. The following primers were used: pGL3-Prom2-TPM (Ϫ501/Ϫ464 nt relative to IL-36␥ transcriptional start site) forward 5Ј-CCTGGCTTTC-CATTCAGGAAAGGCCTTAGGTGGGGTAG-3Ј; pGL3-Prom2-NPM (Ϫ299/Ϫ255 nt) forward 5Ј-GGAGAGCTGA-ACTCTGGGAAATTAGCTTAGTTTCCCCTTTATGAG-3Ј; the identity of the mutants was confirmed by sequencing. Plasmids were transiently transfected into KG1 cells using DMRIE-C reagent (Invitrogen) according to the manufacturer's instructions. Those specifically recommend the addition of PMA (50 g/ml) as co-stimulus for efficient promoter activation in KG1 cells. Accordingly, PMA was added to all cultures including control cultures at the aforementioned concentration. 0.1 g of pRL-TK (Promega) coding for Renilla luciferase was cotransfected. For siRNA/Luciferase experiments, KG1 cells were additionally transfected (along with the plasmids) with 100 nM either siRNA-Tbet or siRNAc. After 2.5 h of rest, transfected cells were used as control or were stimulated as described in the figure legends. Thereafter, luciferase activity was determined using the dual reporter gene system (Promega) and an automated chemiluminescence detector (Berthold, Bad Wildbad, Germany).
ChIP-Chromatin immunoprecipitation was performed as described previously (25). For immunoprecipitation, 3 g of an IgG control or T-bet antibody (4B10, Santa Cruz Biotechnology) were used. To amplify the IL-36␥ promoter fragment containing the T-box site under investigation (Ϫ495/Ϫ475 nt relative to the IL-36␥ transcriptional start site), the following primers were used: forward, 5Ј-GAGGGTACCTGAAAC-TAGGATTGC-3Ј; reverse, 5Ј-GGCTAAAGGCATCCTG-TAAGAGC-3Ј. PCR conditions were: 95°C for 10 min (1 cycle); 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s for 40 cycles; and a final extension phase at 72°C for 7min. The identity of amplicon (216 nt) was confirmed by sequencing.

Genome-wide mRNA Expression Analysis during T-bet Silencing Identifies IL-36␥ as T-bet-regulated Gene in KG1 Cells-
Recently, we observed induction of T-bet expression and biological activity in predendritic KG1 cells under the influence of IL-18. In this cellular model, T-bet is activated independently from IFN␥ by concerted action of p38 MAPK and IB kinase (IKK)/NF-B pathways (20). To classify T-bet expression in activated KG1 cells, protein expression of this transcription factor was compared between IL-18-stimulated KG1 cells and human Th1 cells. We observed in 4 of 5 experiments that Th1 cells expressed higher levels of T-bet protein as compared with activated KG1 cells. Notably, T-bet protein was in all cases detectable by Western blot analysis in IL-18-stimulated KG1 cells and Th1 cells, respectively (Fig. 1A). Because T-bet is capable of mediating gene expression in KG1 cells (20), this experimental system was utilized herein to identify novel T-bet-inducible genes. To that end, KG1 cells were transfected with either control-siRNA (siRNAc) or siRNA targeting T-bet (siRNA-Tbet) in five independently performed experiments. Concurring with previous data (20), silencing of IL-18-induced T-bet was evident on mRNA ( Fig. 1B) and protein level. In fact, T-bet protein induction was essentially absent in cells transfected with siRNA-Tbet (Fig. 1C). In further accordance with previous work (20) we observed that T-bet silencing associated with impaired immediate IL-18-induced IFN␥ production (Fig.  1D). Pooled total RNA from this same set of experiments was evaluated by genome-wide analysis using the Affymetrix GeneChip Array System (GSE37595). Among the genes robustly regulated by T-bet was IL-36␥ (IL1F9), a previously described novel member of the IL-1 cytokine family (24, 29 -31). Because our group is particularly interested in the biology of the IL-1 cytokine family and information on T-bet regarding IL-36␥ is lacking, we subsequently focused on detailed analysis of mechanisms directing expression of this cytokine. Fig. 1E displays down-regulation of T-bet and IL-36␥ mRNA by siRNA-Tbet in the pooled total mRNA population as detected by gene chip analysis and real-time PCR, respectively. Down-regulation of IL-36␥ expression by siRNA-Tbet in unpooled mRNA of this particular set of experiments is shown in Fig. 1F.
Next, regulation of IL-36␥ in KG1 cells was investigated on mRNA and protein levels in additional independently performed experiments. First, induction of IL-36␥ mRNA in IL-18-stimulated KG1 cells was confirmed using a standard DECEMBER 7, 2012 • VOLUME 287 • NUMBER 50 biochemical methodology different from real-time PCR and not depending on additional amplification steps, namely RNase protection assay. Using this technique we confirm IL-36␥ mRNA expression by KG1 cells under the influence of IL-18 ( Fig. 2A). Further experiments revealed that IL-18, but not IFN␥, mediates IL-36␥ mRNA expression in KG1 cells as detected by real-time (Fig. 2B) and standard PCR (Fig. 2B,  inset), respectively. mRNA induction by IL-18 translated into IL-36␥ protein, which was detectable by Western blot analysis in cell lysates (Fig. 2C, upper panel) and culture supernatants (Fig. 2C, lower panel), respectively. To validate KG1 cell-associated IL-36␥ immunoreactivity detected by Western blot analysis, recombinant pro-IL-36␥ and lysates from IL-1␤/TNF␣stimulated HaCaT keratinocytes as well as those from HaCaT keratinocytes transfected with the pCDNA3-IL-36␥ expression plasmid were run on the same gel. Keratinocytes activated by IL-1␤/TNF␣ are supposed to express IL-36␥ (31). In fact, immunoreactivity induced by IL-18 in KG1 cells and detected by the polyclonal anti-IL-36␥ antibody (R&D Systems) in use co-migrated with immunoreactivity associated with IL-1␤/ TNF␣ stimulation or pCDNA3-IL-36␥ transfection of HaCaT keratinocytes and, most importantly, with that of recombinant pro-IL-36␥ (at ϳ18.7 kDa, R&D Systems) (Fig. 2D). Induction of IL-36␥ was not detected in KG1 cells exposed to IFN␥ (Fig.  2B), an observation concurring with insufficient up-regulation of T-bet by IFN␥ in this cell type (Fig. 2E).
Promoter Studies Reveal a Crucial Function of Specific T-box and B Sites for Induction of Human IL-36␥-To further delineate molecular mechanisms driving IL-36␥ in KG1 cells, a human IL-36␥ promoter fragment was cloned that spans from Ϫ1576 to ϩ633 nt (immediately adjacent to the translational start site at ϩ634 nt) relative to the transcriptional start site (at ϩ1). Sequence analysis revealed the presence of a non-canonical TATA box, recently entitled GATA box at position Ϫ29/ Ϫ24 nt (5Ј-GATAAA-3Ј) in this fragment. Based on this pri- mary fragment (Prom1), a deletion mutant (Prom2) was generated (Fig. 3A), and fragments were analyzed in luciferase reporter assays performed in the context of IL-18-activated KG1 cells. Robust IL-36␥ promoter activation was achieved by IL-18 with no significant differences between Prom1 and Prom2 (Fig. 3B). Accordingly, all further experiments were performed using Prom2. Sequence analysis of Prom2 by MatInspector (Genomatix 7.4.4., Munich, Germany) brought

. IL-18 up-regulates IL-36␥ mRNA and protein in KG1 cells; contribution of p38 MAPK and NF-B.
A, KG1 cells were either kept as unstimulated control (C) or stimulated with IL-18 (10 ng/ml). After 12 h, IL-36␥ mRNA was analyzed by RNase protection assay (10 g total RNA/condition). Three independently performed experiments are shown. tR and FP denote tRNA and free probe, respectively. B, KG1 cells were either kept as unstimulated control or stimulated with IL-18 (10 ng/ml) or IFN␥ (20 ng/ml) for 12 h. IL-36␥ mRNA was assessed by real-time PCR. Target mRNA was normalized to that of GAPDH and is shown as -fold induction compared with unstimulated control Ϯ S.D. (n ϭ 6 for control and IL-18, n ϭ 4 for IFN␥); *, p Ͻ 0.05 compared with unstimulated control; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. B, inset, additionally, data from one representative experiment was analyzed by standard PCR.  4); ***, p Ͻ 0.001 compared with unstimulated control; ### , p Ͻ 0.001; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. F, cells were either kept as unstimulated control or stimulated with IL-18 (10 ng/ml). Where indicated, cells were preincubated with SB203580 (10 M) or IKK-VII (10 M) for 1 h before IL-18 addition. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for SB203580, IKK-VII). After 12 h, IL-36␥ was assessed by real-time PCR analysis. IL-36␥ mRNA was normalized to that of GAPDH and is shown as -fold induction compared with unstimulated control Ϯ S.D. (n ϭ 5); **, p Ͻ 0.01 compared with unstimulated control; #, p Ͻ 0.05; ##, p Ͻ 0.01 compared with stimulation with IL-18 alone; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. F, inset, cellular IL-36␥ protein (of cultures treated as described in F) was determined by Western blot analysis. One experiment representative of three independently performed experiments is shown. SB, SB203580. DECEMBER 7, 2012 • VOLUME 287 • NUMBER 50 to light a potential T-box (T) site (Ϫ495/Ϫ475 nt) with a monomeric binding mode. This element (Fig. 3A) displayed significant similarity to monomeric binding mode T-box sites of transcription factors related to T-bet, among others Brachyury and Tbx2 (32)(33)(34)(35). To further substantiate a role for T-bet in IL-36␥ promoter activation, two strategies were pursued. First, luciferase reporter assays were performed upon silencing of T-bet by siRNA. Second, the aforementioned T-box site (Ϫ495/Ϫ475 nt) was mutated (Prom2-TPM) as outlined under "Experimental Procedures." Both strategies in fact resulted in significant reduction of IL-36␥ promoter activity detected in IL-18-stimulated cells by 55.4% (siRNA-Tbet) and 56.9% (Prom2-TPM) (Fig. 3C). Likewise, enhanced binding of T-bet to this specific promoter site (Ϫ495/Ϫ475 nt) was proven by ChIP analysis (Fig. 3D). Thus, data presented herein demonstrate that activation of T-bet significantly contributes to IL-36␥ induction as detected in IL-18-activated KG1 cells.

T-bet Induces IL-36␥ in Myeloid Cells
Because inhibition of IKK impaired IL-36␥ mRNA and protein expression (Fig. 2F), promoter activation was investigated in the presence of IKK-VII. As expected, this inhibitor likewise suppressed IL-36␥ promoter activation (Fig. 3E). Sequence analysis (MatInspector) of IL-36␥ Prom2 actually revealed a B site (Ϫ286/Ϫ274 nt) that was further examined by mutational analysis. Deactivation of this specific B binding site by mutation (Prom2-NPM) resulted in a significant 79.6% reduction of IL-36␥ promoter activity detected in IL-18-stimulated cells. Notably, a Prom2 construct holding a B (Ϫ286/Ϫ274 nt)/Tbox (Ϫ495/Ϫ475 nt) double-mutation (Prom2-TNPM) displayed nearly complete loss of inducibility by IL-18 (Fig. 3F). Altogether, data indicate that those two specific B and T-box sites identified in the present study are crucial for efficient induction of human IL-36␥ gene expression.
Analysis of IL-36␥ Expression as Detected in Splenic DC Obtained from Wild-type or T-bet-deficient Mice-To further the assumption of T-bet being crucial for IL-36␥ regulation in myeloid cells, experiments were performed using splenic DC obtained from wild-type or T-bet knock-out mice, respectively. Cells were stimulated with the Toll-like receptor (TLR)-4 ligand LPS, a prototypic activator of myeloid cells including murine splenic DC (36). Experiments revealed that purified splenic DC activated by LPS displayed a significant 73.1% reduction of IL-36␥ mRNA expression upon lack of T-bet (Fig.  4A). Activation of splenic DC by LPS mediated robust T-bet induction in wild-type cells but, as expected, not in cells obtained from T-bet knock-out mice (Fig. 4B).
T-bet Mediates Expression of IL-36␥ in Human Inflammatory M1 Macrophages-T-bet expression has been detected on mRNA or mRNA/protein level in activated murine DC (7), human monocytes or monocyte-derived DC (6), and in human predendritic KG1 cells (20) as well as human inflammatory or M1 macrophages (13,14) but not in murine macrophages (7). To extend these current observations to human primary cells, experiments were performed using monocyte-derived DC and activated primary macrophages. Fig. 5A demonstrates IL-36␥ mRNA induction in both human cell types as detected by RNase protection assay. We furthermore confirm by real-time PCR previous observations (6) on robust T-bet mRNA induction in monocyte-derived DC under the influence of IFN␥, which was used as a single stimulus or in combination with IL-1␤ plus TNF␣. Up-regulation of T-bet mRNA in fact associated with enhanced expression of IL-36␥ mRNA in these same experiments (Fig. 5B). However, we were unable to reliably detect T-bet protein in human monocyte-derived DC. Therefore, we focused on inflammatory/activated human macrophages (26) in subsequent experiments. Exposure of these macrophages to IFN␥ alone or together with IL-1␤/TNF␣ mediated strong gene induction of TNF␣, IL-12-p35, and IL-23-p19 (Table 1) echoing an inflammatory M1 macrophage activation status (37). Under those conditions, T-bet expression was likewise induced (Fig. 5C). Thus, we confirm previous observations (14) indicating that M1 macrophages display increased T-bet mRNA. Notably, robust up-regulation of T-bet protein became likewise apparent under those conditions (Fig.  5D). T-bet protein was accompanied by significant induction of IL-36␥ mRNA under the influence of IFN␥ as a single stimulus or in combination with IL-1␤/TNF␣ (Fig. 5E). siRNA technology was used to verify the function of endogenous T-bet for IL-36␥ regulation in M1 macrophages. Macrophages of four healthy donors were either mock-treated or transfected with siRNAc or siRNA-Tbet, respectively. Thereafter, cellular T-bet and IL-36␥ expression was verified in the context of exposure to IL-1␤/TNF␣/IFN␥. Analysis revealed that silencing of T-bet as shown by mRNA expression (Fig. 5F, left panel) was in all experiments performed associated with suppression of IL-36␥ mRNA induction (Fig. 5F, right panel).
Ectopic Expression of T-bet Enforces IL-36␥ Production in HaCaT Keratinocytes-To further broaden the connection between T-bet and IL-36␥, T-bet was transiently expressed in HaCaT keratinocytes. Notably, keratinocytes usually do not display production of T-bet but are capable of producing IL-36␥ in response to proinflammatory cytokines (29, 31, 38 -40). Compared with transfection with control plasmid (CMV6-XL4), ectopic expression of T-bet achieved by transfection of CMV6-XL4-Tbet enhanced IL-36␥ mRNA (Fig. 6A) and protein (Fig. 6B) accumulation, which became apparent in otherwise unstimulated keratinocytes or those under the influence of IL-1␤/TNF␣. As anticipated, T-bet expression in HaCaT keratinocytes was only observed upon transfection FIGURE 4. Impaired IL-36␥ production in splenic DC obtained from T-bet knock-out mice. A and B, splenic DC isolated from wild-type or T-bet knockout mice were kept as unstimulated control or stimulated with LPS (1 g/ml). A, after 14 h IL-36␥ mRNA expression was assessed by real-time PCR. mRNA was normalized to that of GAPDH and is shown as -fold induction compared with unstimulated control (C, of the respective genotype) Ϯ S.E. (n ϭ 3); p Ͻ 0.05 compared with LPS-stimulated wild-type cells; statistics were performed on -fold induction by Student's t test. B, T-bet mRNA content was evaluated in these same experiments by standard PCR analysis. One representative of the three independently performed experiments is shown. DECEMBER 7, 2012 • VOLUME 287 • NUMBER 50

JOURNAL OF BIOLOGICAL CHEMISTRY 41691
by CMV6-XL4-Tbet (Fig. 6B) but remained undetectable even in cells activated by IFN␥ (data not shown). Neither did enforced T-bet initiate IFN␥ production by HaCaT keratinocytes nor was constitutive secretion of IL-8 enhanced by transfection with CMV6-XL4-Tbet in this same set of experiments (data not shown). This observation exposes discrete specificity of misplaced T-bet action in HaCaT keratinocytes. Altogether, those data further substantiate the potential of T-bet to mediate IL-36␥ production and beyond that indicate the notable capability of this transcription factor to unfold this activity in a rather broad or even misplaced cellular context. . Unstimulated and IL-18 (10 ng/ml) stimulated KG1 cells were used as the positive control for IL-36␥ mRNA induction as detected by RNase protection assay (8 or 5 g of total RNA/condition for analysis of DC/macrophages or KG1 cells, respectively). tR and FP denote tRNA and free probe, respectively. B, DC were either kept as unstimulated control or stimulated with IFN␥ (50 ng/ml) alone or in combination with TNF␣ (50 ng/ml) and IL-1␤ (20 ng/ml). After 14 h, T-bet and IL-36␥ mRNA was assessed by real-time PCR analysis. Target mRNA was normalized to that of GAPDH and is shown as -fold induction compared with unstimulated control Ϯ S.E. (n ϭ 7); *, p Ͻ 0.05; ***, p Ͻ 0.001 compared with unstimulated control; raw data were analyzed by one-way ANOVA with post-hoc Bonferroni correction. C-F, macrophages were either kept as control or stimulated with IFN␥ (50 ng/ml) alone or in combination with TNF␣ (50 ng/ml) and IL-1␤ (20 ng/ml) for 14 h. C and E, T-bet (C) and IL-36␥ (E) mRNA was assessed by real-time PCR. Target mRNA was normalized to that of GAPDH and is shown as -fold induction compared with control Ϯ S.E. (n ϭ 3-5); *, p Ͻ 0.05; **, p Ͻ 0.01, ***, p Ͻ 0.001 compared with control; raw data were analyzed by Student's t test. D, T-bet protein content of cellular lysates was evaluated by Western blot analysis. One experiment representative of three independently performed experiments is shown. F, macrophages were mock-transfected or either transfected with siRNA-Tbet or siRNAc as described under "Experimental Procedures." Thereafter, cells were further used as control or were stimulated with IFN␥ (50 ng/ml), TNF␣ (50 ng/ml), and IL-1␤ (20 ng/ml) for another 14 h. T-bet (left panel) and IL-36␥ (right panel) mRNA was assessed by real-time PCR analysis. Target mRNA was normalized to that of GAPDH and is shown as -fold induction compared with mock-transfected control (cultivation without IFN␥/TNF␣/IL-1␤). Four different donors were investigated, and individual results are shown. The different colors refer to individual donors.

DISCUSSION
Identification of novel genes susceptible to direct activation by T-bet is pivotal for further understanding pathophysiological functions of this crucial transcription factor. Previous studies following this route focused on lymphocytes, particularly T cells, and brought to light novel T-bet-inducible genes that in part visibly connected to Th1 functions. Exemplarily, CXCR3, CCL4, and NKG7 shall be quoted herein (41)(42)(43). However, expression of T-bet in activated DC (6, 7) and human monocytes/macrophages (13,14) likewise suggests a vital role for this transcription factor in myeloid cells and beyond that in innate immunity.
Herein, IL-36␥, a recently described member of the IL-1 cytokine family (24,30), is introduced as a novel direct target of T-bet action in myeloid cells. By using human predendritic acute myelogenous leukemia-derived KG1 cells, we present for the first time analysis of molecular mechanisms driving IL-36␥ gene activation. We demonstrate direct binding of T-bet to a specific T-box site at the IL-36␥ promoter that coupled to promoter activation and expression of the cytokine. Moreover, a B binding site is identified that allowed for T-bet independent IL-36␥ induction but likewise cooperated with the T-box site for most efficient cytokine expression. Observations were supported by using primary human DC and inflammatory M1 macrophages as well as splenic DC obtained from wild-type or T-bet knock-out mice. Notably, ectopic expression of T-bet was sufficient to drive IL-36␥ in HaCaT keratinocytes as a single stimulus, although low-level basal NF-B activity operating in this cell line (44) possibly synergized with T-bet for cytokine induction.
IL-36␥ displays decisive characteristics of an IL-1 cytokine family member, including a lack of a signal peptide and proteolytic processing as a prerequisite for efficient bioactivity. Notably, the IL-36␥ promoter contains at position Ϫ29/Ϫ24 nt a non-canonical TATA box, recently coined GATA box (5Ј-GATAAA-3Ј). Because this element can mediate TATA boxlike regulatory activity (45,46), IL-36 ␥ in this regard can be grouped together with IL-1␤/IL-1F2 that actually displays a canonical TATA box (47). A functional TATA box apparently determines the quantity of IL-1␤ expression (48). Moreover, IL-36␥ is part of the IL1 cluster at the human chromosome 2 (24, 29 -31, 49 -52). Binding of IL-36␥ to heterodimeric IL-36R (IL-1 receptor like 2/IL-1 receptor accessory protein), the common receptor for IL-36␣, IL-36␤, and IL-36␥, mediates activation of NF-B, MAPKs, and the IL-8 promoter (29,50). Altogether this concurs with a proinflammatory cytokine function. Biological activity of IL-36R ligands is controlled by action of IL-36 receptor antagonist (IL-36Ra) and IL-38 (52)(53)(54). Studies on IL-36␥ production hitherto focused largely on keratinocytes and the skin compartment (29,31,39,40,52,53,55). In fact, IL-36R ligands, including IL-36␥, emerge as crucial component of innate immune responses at host/environment interfaces. Inflammatory stimuli such as IL-1, TNF␣, and IL-17 as well as the TLR5 ligand flagellin increase IL-36␥ gene expression in primary human keratinocytes (29,39,40,55), an observation in accord with involvement of NF-B in IL-36␥ regulation as detected herein. In contrast, IFN␥ as single stimulus fails to mediate IL-36␥ in keratinocytes (38,55). This incapability of IFN␥ to induce IL-36␥ in this cell type reflects the incapability of IFN␥ to efficiently activate NF-B and moreover the universal lack of T-bet in keratinocytes. Proinflammatory activation of keratinocytes by IL-36␥ indicates its functional role in dermal inflammation (52). In fact, IL-36␥ is up-regulated in the lesional skin of psoriatic patients (29,38,39) and in murine models of this disease (38,39). A crucial role of IL-36R ligands in dermal pathology is furthermore suggested by psoriasis-like symptoms observed in skin-specific IL-36␣ transgenic mice (56). Notably, a deficiency of the IL-36Ra results in severe generalized pustular psoriasis in humans (53).
Induction of genes different from IFN␥ by T-bet is of particular interest under conditions where both parameters display discordant pathophysiological functions. As already alluded to, this applies exemplarily to murine CIA, a common in vivo model for rheumatoid arthritis where IFN␥ shows some protective (18), but T-bet pathogenic action (16). Notably, T-bet especially in DC is crucial for the pathogenesis of CIA (16). Data presented herein thus point to IL-36␥ as T-bet-dependent/DCderived pathogenic mediator of experimental arthritis. In fact, IL-36␥ is expressed in joints of mice undergoing CIA, and as detected by exposure of cells to IL-36␤, IL-36R activation upregulates inflammatory parameters in human synoviocytes and articular chondrocytes, respectively (57). To investigate CIA along with IL-36␥ deficiency is thus an important matter of future investigation.
Detailed reports on IL-36␥ production by myeloid cells are missing, although one initial study in passing mentioned expression of the cytokine by LPS-stimulated monocytes (31). Moreover, one most recent report demonstrates up-regulation

induction of signature cytokine genes by human inflammatory macrophages under the influence of IFN␥ (50 ng/ml) with/without IL-1␤ (20 ng/ml)/TNF␣ (50 ng/ml)
After a 14-h stimulation period, cytokine mRNA, analyzed by real time PCR, was normalized to that of GAPDH and is shown as -fold induction compared to control. Data are expressed as the means ϮS.E. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction.  DECEMBER 7, 2012 • VOLUME 287 • NUMBER 50

T-bet Induces IL-36␥ in Myeloid Cells
of IL-36␥ mRNA by TLR7 ligation in cultured murine bone marrow-derived DC. However, likely due to short term activation for 6 h, induction remained modest, and molecular mechanisms activating the IL-36␥ promoter were not investigated (58). Interestingly, expression of IL-36␥ is detectable in mononuclear infiltrates of lesional psoriatic skin and actually correlates with that of IFN␥ (38). It is tempting to speculate in this context that early IL-36␥ derived from macrophages/DC may have the potential to efficiently perpetuate local production of inflammatory parameters by autocrine action on DC (59,60) and paracrine action on adjacent cells such as CD4 ϩ T cells and keratinocytes (53,60). In fact, herein we confirm proinflammatory action of IL-36␥ on primary human keratinocytes. Specifically, was mature IL-36␥, but not pro-IL-36␥, able to induce itself and beyond that crucial parameters of cutaneous inflammation such as TNF␣, CCL20 (61), S100A7 (62), and iNOS (63). Data altogether reflect the inherent potential of IL-36␥ to initiate a broad keratinocyte-driven inflammatory cascade. Induction of IL-36␥ by T-bet in the partly overlapping macrophage/DC cell compartment as described herein likewise functionally relates IL-36␥ to initiation of Th1-like immune responses. In fact, administration of the IL-36R ligand IL-36␤ to mice is associated with enhanced ex vivo Th1 activity (60) and with differentiation of naïve CD4 ϩ T cells toward Th1 by IL-36R ligands in vitro (64). IL-36R ligands, including IL-36␥, moreover amplify DC maturation (59,60). Current data thus altogether imply that IL-36␥ derived from the macrophages/DC compartment is part of a self-amplifying loop (52) in which NF-B, activated by IL-1/TNF␣ or TLR ligands (55), and T-bet, potentially activated by early innate IFN␥ (65), may contribute to rapid implementation of strong immunoactivation either to install efficient host defense or as crucial component of tissue-destructive inflammatory processes.