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, 115 (40), 10088-10093

IκBζ Is a Key Transcriptional Regulator of IL-36-driven Psoriasis-Related Gene Expression in Keratinocytes


IκBζ Is a Key Transcriptional Regulator of IL-36-driven Psoriasis-Related Gene Expression in Keratinocytes

Anne Müller et al. Proc Natl Acad Sci U S A.


Proinflammatory cytokine signaling in keratinocytes plays a crucial role in the pathogenesis of psoriasis, a skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes and infiltration of inflammatory cells. Although IL-17A and TNFα are effective therapeutic targets in psoriasis, IL-36 has recently emerged as a proinflammatory cytokine. However, little is known about IL-36 signaling and its downstream transcriptional responses. Here, we found that exposure of keratinocytes to IL-36 induced the expression of IκBζ, an atypical IκB member and a specific transcriptional regulator of selective NF-κB target genes. Induction of IκBζ by IL-36 was mediated by NF-κB and STAT3. In agreement, IL-36-mediated induction of IκBζ was found to be required for the expression of various psoriasis-related genes involved in inflammatory signaling, neutrophil chemotaxis, and leukocyte activation. Importantly, IκBζ-knockout mice were protected against IL-36-mediated dermatitis, accompanied by reduced proinflammatory gene expression, decreased immune cell infiltration, and a lack of keratinocyte hyperproliferation. Moreover, expression of IκBζ mRNA was highly up-regulated in biopsies of psoriasis patients where it coincided with IL36G levels. Thus our results uncover an important role for IκBζ in IL-36 signaling and validate IκBζ as an attractive target for psoriasis therapy.

Keywords: IL-36; IκBζ; NFKBIZ; keratinocytes; psoriasis.

Conflict of interest statement

The authors declare no conflict of interest.


Fig. 1.
Fig. 1.
IL-36 induces IκBζ expression in KCs. (A and B) HaCaT cells (Left) or human primary KCs (Right) were treated with 100 ng/mL IL-36α (amino acids 6158) (A) or 100 ng/mL IL-36γ (amino acids 18–169) (B) for the indicated times. IκBζ protein was analyzed by Western blotting. Relative mRNA levels of NFKBIZ were measured in parallel and normalized to the reference RPL37A. (C) HaCaT cells (Upper) and primary KCs (Lower) were treated for 2 h with 100 ng/mL IL-36α alone or in combination with 100 ng/mL IL-17A, 10 ng/mL TNFα, 100 ng/mL IFNγ, or 100 ng/mL IL-1β. IκBζ was detected by Western blotting. HSC70 or β-actin served as loading controls.
Fig. 2.
Fig. 2.
Molecular dissection of IκBζ induction by IL-36. HaCaT cells were stimulated for the indicated times with 100 ng/mL IL-36α. (A) Cells stably expressing a control (sh ctrl) or two different shRNAs targeting MyD88 were treated for 2 h with IL-36α and were analyzed by Western blotting. (B) Analysis of NFKBIZ promoter accessibility and structure. The genomic region around NFKBIZ was analyzed from a published DNase I dataset and a polymerase II ChIP-seq track (33). Exon reads of NFKBIZ were derived from our own RNA-seq data of HaCaT cells stimulated for 1.5 h with IL-36α. (C) Analysis of the NFKBIZ promoter 2 region in IL-36α–stimulated HaCaT cells using luciferase reporter constructs harboring deletions of transcription factor-binding sites. (D) P65, STAT3, and RNA polymerase II (Pol II) bind to NFKBIZ promoter region 2 in IL-36α–treated cells. ChIP was performed from HaCaT cells treated for 30 min with IL-36α. The promoter region of the muscle-specific gene MYOD1 represents a negative control. (E) Immunoblot analysis of IL-36α–induced signaling pathways. Active NF-κB and STAT3 were detected by the phosphorylated forms of IκBα (p-IκBα at Ser32), p65 (p-p65 at Ser536), and STAT3 (p-STAT3 at Tyr705). MAPK activation was detected by phosphorylated JNK (p-JNK at Thr183/Tyr185) and ERK (p-p44/42 at Thr202/Thr204). (F) Cells were treated for 1 h with IL-36α in the presence or absence of the vehicle DMSO or 10 µM of the IKK inhibitors BMS-345541 or IMD0354. NFKBIZ mRNA and IκBζ protein levels were measured after 2 h of IL-36α stimulation. Detection of p-IκBα served as a control for NF-κB inhibition. (G and H) Gene expression and Western blot analysis of IκBζ in control and RELA (p65)-knockdown (G) or STAT3-knockdown (H) cells after 1 h of IL-36α treatment. Knockdown was controlled by detection of p65 or STAT3. ***P < 0.001.
Fig. 3.
Fig. 3.
IκBζ regulates a subset of psoriasis-related IL-36 target genes. Primary KCs or HaCaT cells were transduced with a control or NFKBIZ-specific shRNA. Triplicates of each time point and shRNA were analyzed by RNA-seq or qPCR and were normalized to the reference gene RPL37A. (A) Control of NFKBIZ-knockdown efficiency. (Upper) IκBζ protein was detected in primary KCs treated for 1 h with 100 ng/mL IL-36α. (Lower) NFKBIZ mRNA levels were measured after 1.5 h and 24 h of IL-36α stimulation. (B) After library preparation from total RNA, primary KC samples were sequenced, and reads were aligned to the human genome hg19. Depicted are two separate heatmaps with normalized z-scores of IκBζ target genes after 1.5 h and 24 h of IL-36α treatment. As a cutoff, genes with a minimum fold change of 1 and a P value < 0.05 were considered. (C) Venn diagrams showing the fraction of IκBζ target genes among IL-36α–regulated genes 1.5 and 24 h after stimulation of primary KCs. (D) GO term analysis of significantly enriched IκBζ-dependent gene sets after 1.5 and 24 h of IL-36α treatment. (E) Validation of selected IκBζ target genes by qPCR in primary KCs after 1.5 and 24 h of incubation with 100 ng/mL IL-36α. (F) Gene-expression analysis of IκBζ target genes in primary KCs stimulated with 100 ng/mL IL-17A and 10 ng/mL TNFα for 1.5 and 24 h. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4.
Fig. 4.
Characterization of the IL-36/IκBζ axis in vivo. (A, Upper) Scheme of tamoxifen and IL-36α treatment of control and inducible Nfkbiz-KO mice. (Lower) Verification of Nfkbiz deletion at the protein and mRNA level. For induction of IκBζ KO, Nfkbiz flox/flox (Ctrl) and Rosa-creERT2 Nfkbiz flox/flox (KO) mice received i.p. injections of tamoxifen (75 mg/kg) for four consecutive days to induce activation of Cre recombinase. Afterward, 1 µg murine IL-36α or PBS control was intradermally injected into one ear of the mice for five consecutive days. (B) Ear and epidermal thickness (± SEM) of PBS- and IL-36α–treated mice at day 5 from two (for PBS) or six (for IL-36α) animals per group. Pictures were taken at day 5 to show scaling at the treatment area. (C) H&E staining of ears from PBS- and IL-36α–treated control and KO mice. (Scale bars: 140 µM.) (D) Immunohistochemistry for the macrophage marker F4/80 and the neutrophil marker MPO. (Scale bars: 80 µM) (E) Characterization of CD45+ immune cell infiltrates by flow cytometry. Neutrophils were characterized as CD45+ Ly6G+ and macrophages as CD45+, CD11bhi and F4/80+. Error bars indicate results from two independent experiments. (F) Psoriasis-related gene expression in ears from IL-36α–treated mice. Results are shown as means ± SEM; n = 6 animals per group. (G) Expression data from skin biopsies of 64 healthy individuals and 58 psoriasis patients were analyzed from the Gene Expression Omnibus profile dataset GDS4602. Shown are normalized expression values of NFKBIZ and IL17A or NFKBIZ and IL36G, which were plotted against each other in every single nonlesional and lesional biopsy. Depicted is the regression coefficient (R2) from the expression values of the psoriatic skin biopsies. *P < 0.05; **P < 0.01; ***P < 0.001.

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