Keratinocyte-derived IκBζ drives psoriasis and associated systemic inflammation.

The transcriptional activator IκBζ is a key regulator of psoriasis, but which cells mediate its pathogenic effect remains unknown. Here we found that IκBζ expression in keratinocytes triggers not only skin lesions but also systemic inflammation in mouse psoriasis models. Specific depletion of IκBζ in keratinocytes was sufficient to suppress the induction of imiquimod- or IL-36-mediated psoriasis. Moreover, IκBζ ablation in keratinocytes prevented the onset of psoriatic lesions and systemic inflammation in keratinocyte-specific IL-17A-transgenic mice. Mechanistically, this psoriasis protection was mediated by IκBζ deficiency in keratinocytes abrogating the induction of specific proinflammatory target genes, including Cxcl5, Cxcl2, Csf2, and Csf3, in response to IL-17A or IL-36. These IκBζ-dependent genes trigger the generation and recruitment of neutrophils and monocytes that are needed for skin inflammation. Consequently, our data uncover a surprisingly pivotal role of keratinocytes and keratinocyte-derived IκBζ as key mediators of psoriasis and psoriasis-related systemic inflammation.

IκBζ is a key transcriptional regulator of IL-36-driven psoriasis-related gene expression in keratinocytes.

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.

Simultaneous quantification of DNA damage and mitochondrial copy number by long-run DNA-damage quantification (LORD-Q).

DNA damage and changes in the mitochondrial DNA content have been implicated in ageing and cancer development. To prevent genomic instability and tumorigenesis, cells must maintain the integrity of their nuclear and mitochondrial DNA. Advances in the research of DNA damage protection and genomic stability, however, also depend on the availability of techniques that can reliably quantify alterations of mitochondrial DNA copy numbers and DNA lesions in an accurate high-throughput manner. Unfortunately, no such method has been established yet. Here, we describe the high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) and its suitability to simultaneously measure DNA damage rates and mitochondrial DNA copy numbers in cultured cells and tissue samples. Using the LORD-Q multiplex assay, we exemplarily show that the mitochondrial DNA content does not directly affect DNA damage susceptibility, but influences the efficacy of certain anticancer drugs. Hence, LORD-Q provides a fast and precise method to assess DNA lesions, DNA repair and mtDNA replication as well as their role in a variety of pathological settings.

The Atypical Inhibitor of NF-κB, IκBζ, Controls Macrophage Interleukin-10 Expression.

Macrophages constitute a first line of pathogen defense by triggering a number of inflammatory responses and the secretion of various pro-inflammatory cytokines. Recently, we and others found that IκBζ, an atypical IκB family member and transcriptional coactivator of selected NF-κB target genes, is essential for macrophage expression of a subset of pro-inflammatory cytokines, such as IL-6, IL-12, and CCL2. Despite defective pro-inflammatory cytokine expression, however, IκBζ-deficient mice develop symptoms of chronic inflammation. To elucidate this discrepancy, we analyzed a regulatory role of IκBζ for the expression of anti-inflammatory cytokines and identified IκBζ as an essential activator of IL-10 expression. LPS-challenged peritoneal and bone marrow-derived macrophages from IκBζ-deficient mice revealed strongly decreased transcription and secretion of IL-10 compared with wild-type mice. Moreover, ectopic expression of IκBζ was sufficient to stimulate Il10 transcription. On the molecular level, IκBζ directly activated the Il10 promoter at a proximal κB site and was required for the transcription-enhancing trimethylation of histone 3 at lysine 4. Together, our findings show for the first time the IκBζ-dependent expression of an anti-inflammatory cytokine that is crucial in controlling immune responses.

IκBζ is a key driver in the development of psoriasis.

Psoriasis is a common immune-mediated, chronic, inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes and infiltration of inflammatory cells. Although TNFα- and IL-17A-targeting drugs have recently proven to be highly effective, the molecular mechanism underlying the pathogenesis of psoriasis remains poorly understood. We found that expression of the atypical IκB member IκB (inhibitor of NF-κB) ζ, a selective coactivator of particular NF-κB target genes, was strongly increased in skin of patients with psoriasis. Moreover, in human keratinocytes IκBζ was identified as a direct transcriptional activator of TNFα/IL-17A-inducible psoriasis-associated proteins. Using genetically modified mice, we found that imiquimod-induced psoriasis-like skin inflammation was completely absent in IκBζ-deficient mice, whereas skin inflammation was still inducible in IL-17A- and TNFα-deficient mice. IκBζ deficiency also conferred resistance against IL-23-induced psoriasis. In addition, local abrogation of IκBζ function by intradermal injection of IκBζ siRNA abolished psoriasis-like skin inflammation. Taken together, we identify IκBζ as a hitherto unknown key regulator of IL-17A-driven effects in psoriasis. Thus, targeting IκBζ could be a future strategy for treatment of psoriasis, and other inflammatory diseases for which IL-17 antagonists are currently tested in clinical trials.