Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes.DOI: http://dx.doi.org/10.7554/eLife.03422.001

Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10 7 /ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.

BackgroundPsoriasis is one of the most frequent skin diseases world-wide. The disease impacts enormously on affected patients and poses a huge financial burden on health care providers. Several lines of evidence suggest that the nuclear hormone receptor peroxisome proliferator activator (PPAR) β/δ, known to regulate epithelial differentiation and wound healing, contributes to psoriasis pathogenesis. It is unclear, however, whether activation of PPARβ/δ is sufficient to trigger psoriasis-like changes in vivo.Methodology/Principal FindingsUsing immunohistochemistry, we define the distribution of PPARβ/δ in the skin lesions of psoriasis. By expression profiling, we confirm that PPARβ/δ is overexpressed in the vast majority of psoriasis patients. We further establish a transgenic model allowing inducible activation of PPARβ/δ in murine epidermis mimicking its distribution in psoriasis lesions. Upon activation of PPARβ/δ, transgenic mice sustain an inflammatory skin disease strikingly similar to psoriasis, featuring hyperproliferation of keratinocytes, dendritic cell accumulation, and endothelial activation. Development of this phenotype requires the activation of the Th17 subset of T cells, shown previously to be central to psoriasis. Moreover, gene dysregulation in the transgenic mice is highly similar to that in psoriasis. Key transcriptional programs activated in psoriasis, including IL1-related signalling and cholesterol biosynthesis, are replicated in the mouse model, suggesting that PPARβ/δ regulates these transcriptional changes in psoriasis. Finally, we identify phosphorylation of STAT3 as a novel pathway activated by PPARβ/δ and show that inhibition of STAT3 phosphorylation blocks disease development.ConclusionsActivation of PPARβ/δ in the epidermis is sufficient to trigger inflammatory changes, immune activation, and signalling, and gene dysregulation characteristic of psoriasis.

Psoriasis is a common skin disease involving keratinocyte proliferation and altered differentiation, as well as T-cell activation. Here, we show that altered gene transcription in psoriatic skin lesions is highly reproducible between independent data sets. Analysis of gene expression confirmed dysregulation in all expected functional categories, such as IFN signaling and keratinocyte differentiation, and allowed molecular fingerprinting of a previously characterized dendritic cell subset associated with psoriasis tumor necrosis factor alpha (TNF-alpha)- and inducible nitric oxide synthase (iNOS)-producing CD11b(INT) DC (Tip-DC). Unexpectedly, a large group of dysregulated transcripts was related to fatty acid signaling and adipocyte differentiation, exhibiting a pattern consistent with the activation of peroxisome proliferator-activated receptor delta (PPARdelta). PPARdelta itself was strongly induced in psoriasis in vivo. In primary keratinocytes, PPARdelta was induced by the transcription factor activator protein 1, in particular by junB, but not by canonical WNT signaling, in contrast to its regulation in colon carcinoma cells. Activation of PPARdelta enhanced proliferation of keratinocytes, while this was inhibited by knockdown of PPARdelta. Finally, heparin-binding EGF-like growth factor (HB-EGF), known to induce epidermal hyperplasia and itself overexpressed in psoriasis, was identified as a direct target gene of PPARdelta. The present data suggest that activation of PPARdelta is a major event in psoriasis, contributing to the hyperproliferative phenotype by induction of HB-EGF.