Mast cells express IL17A, IL17F and RORC, are activated and persist with IL-17 production in resolved skin of patients with chronic plaque-type psoriasis.

Little is known about IL-17 expression in psoriasis and the actual cellular source of IL-17 remains incompletely defined. We show that high numbers of IL-17 + mast cells persisted in resolved lesions after treatment (anti-IL-17A, anti-IL-23, UVB or topical dithranol) and correlated inversely with the time span in remission. IL-17 + mast cells were found in T cell-rich areas and often close to resident memory T cells (Trm) in active psoriasis and resolved lesional skin. Digital cytometry by deconvolution of RNA-seq data showed that activated mast cells were increased in psoriatic skin, while resting mast cells were almost absent and both returned to normal levels after treatment. When primary human skin mast cells were stimulated with T cell cytokines (TNFα, IL-22 and IFNγ), they responded by releasing more IL-17A, as measured by ELISA. In situ mRNA detection using padlock probes specific for transcript variants of IL17A, IL17F, and RORC (encoding the Th17 transcription factor RORγt) revealed positive mRNA signals for IL17A, IL17F, and RORCin tryptase + cells, demonstrating that mast cells have the transcriptional machinery to actively produce IL-17. Mast cells thus belong to the center of the IL-23/IL-17 axis and high numbers of IL-17 + mast cells predict an earlier disease recurrence.

Phototherapy Restores Deficient Type I IFN Production and Enhances Antitumor Responses in Mycosis Fungoides.

Transcriptional profiling demonstrated markedly reduced type I IFN gene expression in untreated mycosis fungoides (MF) skin lesions compared with that in healthy skin. Type I IFN expression in MF correlated with antigen-presenting cell-associated IRF5 before psoralen plus UVA therapy and epithelial ULBP2 after therapy, suggesting an enhancement of epithelial type I IFN. Immunostains confirmed reduced baseline type I IFN production in MF and increased levels after psoralen plus UVA treatment in responding patients. Effective tumor clearance was associated with increased type I IFN expression, enhanced recruitment of CD8+ T cells into skin lesions, and expression of genes associated with antigen-specific T-cell activation. IFNk, a keratinocyte-derived inducer of type I IFNs, was increased by psoralen plus UVA therapy and expression correlated with upregulation of other type I IFNs. In vitro, deletion of keratinocyte IFNk decreased baseline and UVA-induced expression of type I IFN and IFN response genes. In summary, we find a baseline deficit in type I IFN production in MF that is restored by psoralen plus UVA therapy and correlates with enhanced antitumor responses. This may explain why MF generally develops in sun-protected skin and suggests that drugs that increase epithelial type I IFNs, including topical MEK and EGFR inhibitors, may be effective therapies for MF.

Eukaryotic Initiation Factor 4E (eIF4E) as a Target of Anti-Psoriatic Treatment.

Eukaryotic initiation factor 4E (eIF4E) has been known to play a critical role in the regulation of gene expression and essential cellular processes, such as proliferation, apoptosis and differentiation. In this study, we explored its role in the pathophysiology of psoriasis. The inhibition of eIF4E by small interfering RNA or briciclib, an eIF4E small molecule inhibitor, downregulated the expression of eIF4E itself and its two complex partners eIF4A and G, as well as other eIFs (eg, eIF1A, eIF2α, eIF3A, eIF3B, eIF5, and eIF6). This inhibition also abolished psoriatic inflammation in both the imiquimod and TGFß mouse model, as well as in a human 3 dimensional-psoriasis tissue model. Downregulation of eIF4E and the other eIFs by application of briciclib (particularly when given topically) was linked to the normalization of cellular proliferation, epidermal hyperplasia, levels of proinflammatory cytokines (eg, TNFα, IL-1b, IL-17, and IL-22), and keratinocyte differentiation markers (eg, KRT16 and FLG). These results demonstrate translational imbalance and underline the crucial role played by eIF4E and other eIFs in the pathophysiology of psoriasis. This work opens up avenues for the development of novel topical antipsoriatic treatment strategies by targeting eIF4E.

Central memory T cells are the most effective precursors of resident memory T cells in human skin.

The circulating precursor cells that give rise to human resident memory T cells (TRM) are poorly characterized. We used an in vitro differentiation system and human skin-grafted mice to study TRM generation from circulating human memory T cell subsets. In vitro TRM differentiation was associated with functional changes, including enhanced IL-17A production and FOXP3 expression in CD4+ T cells and granzyme B production in CD8+ T cells, changes that mirrored the phenotype of T cells in healthy human skin. Effector memory T cells (TEM) had the highest conversion rate to TRM in vitro and in vivo, but central memory T cells (TCM) persisted longer in the circulation, entered the skin in larger numbers, and generated increased numbers of TRM. In summary, TCM are highly efficient precursors of human skin TRM, a feature that may underlie their known association with effective long-term immunity.

Furin Expression in Patients With Psoriasis-A Patient Cohort Endangered to SARS-COV2?

Background: SARS-Cov2 has raised concerns among dermatologists regarding psoriasis and its respective treatments. Comorbidities, which induce the expression of the proprotease furin have been associated with severe course of COVID-19. Furin and angiotensin converting enzyme 2 (ACE2) play a major role in viral host cell entry of SARS-Cov2. Objective: To evaluate mRNA expression of Furin and ACE2 from blood cells in psoriasis patients, and whether systemic or topical treatment reduces expression levels. Methods: This observational translational study analyzed blood samples from patients from a clinical trial and samples retrieved from the biobank of the Psoriasis Registry Austria (PsoRA). Furin and ACE2 expression levels were analyzed prior to as well as 3 and 12-24 months after start of biologic treatment with either ustekinumab or secukinumab. Additionally, the study analyzed expression levels prior to, 6 days after start of dithranol treatment and 4-6 weeks after end of dithranol treatment. Results: Furin mRNA expression was significantly increased at baseline in the biologic (4.9 ± 2.6 fold, p < 0.0001) and in the dithranol group (2.7 ± 1.4 fold, p < 0.001) compared to controls. There was a trend for arthritis patients to express more furin than patients with psoriatic skin involvement only (5.26 ± 2.30 vs. 3.48 ± 2.27, p = 0.078). Analyzing furin mRNA expression after treatment initiation with secukinumab or ustekinumab revealed a normalization of levels after 3 and 12 to 24 months. Similar findings were obtained for patients treated with dithranol, with significantly decreased expression levels 6 days after start of dithranol treatment and also at follow-up, (4-6 weeks after dithranol treatment had been terminated). ACE2 expression levels did not differ from controls at any timepoint, regardless of biologic or topical treatment. Conclusion: Significantly overexpressed levels of furin were observed in untreated patients, and, thus, these patients may be at risk for infection and a severe course of COVID-19. However, the data indicate that successful therapeutic intervention in psoriasis, by systemic biologic or topical treatment, can efficiently reduce furin levels in blood cells, possibly limiting the risk of psoriasis patients for a severe COVID-19 course. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02752672.

Induction of IL-1ß and antimicrobial peptides as a potential mechanism for topical dithranol.

Topical dithranol is effective in autoimmune conditions like alopecia areata, inducing hair regrowth in a high percentage of cases. Exact mechanisms of dithranol in alopecia areata, with seemingly healthy epidermis besides altered hair follicles, are not well understood. To better understand dithranol's mechanisms on healthy skin, we analysed its effect on normal murine as well as xenografted human skin. We found a strong increase in mRNA expression of anti-microbial peptides (AMPs) (eg Lcn2, Defb1, Defb3, S100a8, S100a9), keratinocyte differentiation markers (eg Serpinb3a, Flg, Krt16, Lce3e) and inflammatory cytokines (eg Il1b and Il17) in healthy murine skin. This effect was paralleled by inflammation and disturbed skin barrier, as well as an injury response resulting in epidermal hyperproliferation, as observed in murine and xenografted adult human skin. This contact response and disturbed barrier induced by dithranol might lead via a vicious loop between AMPs such as S100a8/a9 (that led to skin swelling itself after topical application) and cytokines such as IL-1ß to an immune suppressive environment in the skin. A better understanding of the skin's physiologic response to dithranol may open up new avenues for the establishment of novel therapeutics (including AMP-related/interfering molecules) for certain skin conditions, such as alopecia areata.

Bone morphogenetic protein signaling regulates skin inflammation via modulating dendritic cell function.

BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the TGF-ß family that signal via the BMP receptor (BMPR) signaling cascade, distinct from canonical TGF-ß signaling. BMP downstream signaling is strongly induced within epidermal keratinocytes in cutaneous psoriatic lesions, and BMP7 instructs monocytic cells to acquire characteristics of psoriasis-associated Langerhans dendritic cells (DCs). Regulatory T (Treg)-cell numbers strongly increase during psoriatic skin inflammation and were recently shown to limit psoriatic skin inflammation. However, the factors mediating Treg-cell accumulation in psoriatic skin currently remain unknown. OBJECTIVE: We sought to investigate the role of BMP signaling in Treg-cell accumulation in psoriasis. METHODS: The following methods were used: immunohistology of patients and healthy controls; ex vivo models of Treg-cell generation in the presence or absence of Langerhans cells; analysis of BMP versus canonical TGF-ß signaling in DCs and Treg cells; and modeling of psoriatic skin inflammation in mice lacking the BMPR type 1a in CD11c+ cells. RESULTS: We here demonstrated a positive correlation between Treg-cell numbers and epidermal BMP7 expression in cutaneous psoriatic lesions and show that unlike Treg cells from healthy skin, a portion of inflammation-associated Treg cells exhibit constitutive-active BMP signaling. We further found that BMPR signaling licenses inflammation-associated Langerhans cell/DC to gain an enhanced capacity to promote Treg cells via BMPR-mediated CD25 induction and that this effect is associated with reduced skin inflammation. CONCLUSIONS: Psoriatic lesions are marked by constitutive high BMP7/BMPR signaling in keratinocytes, which instructs inflammatory DCs to gain enhanced Treg-cell-stimulatory activity. Locally secreted BMP7 can directly promote Treg-cell generation through the BMP signaling cascade.

Dithranol targets keratinocytes, their crosstalk with neutrophils and inhibits the IL-36 inflammatory loop in psoriasis.

Despite the introduction of biologics, topical dithranol (anthralin) has remained one of the most effective anti-psoriatic agents. Serial biopsies from human psoriatic lesions and both the c-Jun/JunB and imiquimod psoriasis mouse model allowed us to study the therapeutic mechanism of this drug. Top differentially expressed genes in the early response to dithranol belonged to keratinocyte and epidermal differentiation pathways and IL-1 family members (i.e. IL36RN) but not elements of the IL-17/IL-23 axis. In human psoriatic response to dithranol, rapid decrease in expression of keratinocyte differentiation regulators (e.g. involucrin, SERPINB7 and SERPINB13), antimicrobial peptides (e.g. ß-defensins like DEFB4A, DEFB4B, DEFB103A, S100 proteins like S100A7, S100A12), chemotactic factors for neutrophils (e.g. CXCL5, CXCL8) and neutrophilic infiltration was followed with much delay by reduction in T cell infiltration. Targeting keratinocytes rather than immune cells may be an alternative approach in particular for topical anti-psoriatic treatment, an area with high need for new drugs.

BMP7 aberrantly induced in the psoriatic epidermis instructs inflammation-associated Langerhans cells.

BACKGROUND: Epidermal hyperplasia represents a morphologic hallmark of psoriatic skin lesions. Langerhans cells (LCs) in the psoriatic epidermis engage with keratinocytes (KCs) in tight physical interactions; moreover, they induce T-cell-mediated immune responses critical to psoriasis. OBJECTIVE: This study sought to improve the understanding of epidermal factors in psoriasis pathogenesis. METHODS: BMP7-LCs versus TGF-ß1-LCs were phenotypically characterized and their functional properties were analyzed using flow cytometry, cell kinetic studies, co-culture with CD4 T cells, and cytokine measurements. Furthermore, immunohistology of healthy and psoriatic skin was performed. Additionally, in vivo experiments with Junf/fJunBf/fK5cre-ERT mice were carried out to assess the role of bone morphogenetic protein (BMP) signaling in psoriatic skin inflammation. RESULTS: This study identified a KC-derived signal (ie, BMP signaling) to promote epidermal changes in psoriasis. Whereas BMP7 is strictly confined to the basal KC layer in the healthy skin, it is expressed at high levels throughout the lesional psoriatic epidermis. BMP7 instructs precursor cells to differentiate into LCs that phenotypically resemble psoriatic LCs. These BMP7-LCs exhibit proliferative activity and increased sensitivity to bacterial stimulation. Moreover, aberrant high BMP signaling in the lesional epidermis is mediated by a KC intrinsic mechanism, as suggested from murine data and clinical outcome after topical antipsoriatic treatment in human patients. CONCLUSIONS: These data indicate that available TGF-ß family members within the lesional psoriatic epidermis preferentially signal through the canonical BMP signaling cascade to instruct inflammatory-type LCs and to promote psoriatic epidermal changes. Targeting BMP signaling might allow to therapeutically interfere with cutaneous psoriatic manifestations.

Resolution of plaque-type psoriasis: what is left behind (and reinitiates the disease).

Psoriasis is a chronic inflammatory skin disease that involves numerous types of immune cells and cytokines resulting in an inflammatory feedback loop and hyperproliferation of the epidermis. A more detailed understanding of the underlying pathophysiology has revolutionized anti-psoriatic treatment and led to the development of various new drugs targeting key inflammatory cytokines such as IL-17A and IL-23. Successfully treated psoriatic lesions often resolve completely, leaving nothing visible to the naked eye. However, such lesions tend to recur within months at the exact same body sites. What is left behind at the cellular and molecular levels that potentially reinitiates psoriasis? Here, we elucidate the cellular and molecular "scar" and its imprints left after clinical resolution of psoriasis treated with anti-TNFα, anti-IL-17, or anti-IL-23 antibodies or phototherapy. Hidden cytokine stores and remaining tissue-resident memory T cells (TRMs) might hold the clue for disease recurrence.

Multigene methylation analysis of enriched circulating tumor cells associates with poor progression-free survival in metastatic breast cancer patients.

Blood-based biomarkers such as circulating tumor cells (CTCs) provide dynamic real-time assessment of molecular tumor characteristics beyond the primary tumor. The aim of this study was to evaluate the feasibility of a size-based microfilter to assess multigene methylation analysis of enriched CTCs in a prospective proof-of principle study. We examined the quantitative methylation status of nine genes (AKR1B1, BMP6, CST6, HOXB4, HIST1H3C, ITIH5, NEUROD1, RASSF1, SOX17) in enriched CTCs from metastatic breast cancer patients. Feasibility and clinical performance testing were assessed in a test set consisting of 37 patients and 25 healthy controls. With established cut-off values from the healthy control group, methylation of enriched CTCs was detected in at least one gene in 18/37 patients (48.6%), while 97.8% of all control samples were unmethylated. Patients with CTCs unmethylated for CST6, ITIH5, or RASSF1 showed significantly longer PFS compared to patients with corresponding enriched methylated CTCs. This proof-of-principle study shows the feasibility of a size-based microfilter to enrich and analyze multigene methylation profile of CTCs from metastatic breast cancer patients. For the first time, we report that multigene methylation analysis of enriched CTCs provides prognostic information in metastatic breast cancer patients.

Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing.

BACKGROUND: Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively. METHODS: We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC). RESULTS: The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone. CONCLUSIONS: The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'.