RAS-activated PI3K/AKT signaling sustains cellular senescence via P53/P21 axis in experimental models of psoriasis.

BACKGROUND: Psoriasis is a chronic immune-mediated skin disease in which upper epidermal keratinocytes exhibit a senescent-like phenotype. In psoriatic skin, a variety of inflammatory cytokines can activate intracellular pathways including phosphatidylinositol 3-kinase (PI3K)/AKT signaling and RAS effectors. AKT and RAS participate to cellular senescence, but currently their role in senescence responses occurring in psoriasis have not yet been investigated. OBJECTIVE: The role of AKT molecular axis and RAS activation was evaluated in the context of cellular senescence in psoriasis disease. METHODS: RAS/AKT involvement in senescence was analyzed in psoriatic keratinocytes cultures subjected to multiple passages to promote senescence in vitro, as well as in skin lesions of patients affected by psoriasis. The impact of pharmacological inhibition of PI3K/AKT pathway on senescence and inflammation responses was tested in senescent psoriatic keratinocytes and in a psoriasiform dermatitis murine model induced by RAS overexpression in the upper epidermis of mice. RESULTS: We found AKT hyperactivation associated to the upregulation of senescence markers, in senescent psoriatic keratinocyte cultures, as well as in skin lesions of psoriatic patients. AKT-induced senescence was sustained by constitutive RAS activation, and down-stream responses were mediated by P53/P21 axis. PI3K/AKT inhibition contrasted senescence processes induced by cytokines in psoriatic keratinocytes. Additionally, RAS-induced psoriasis-like dermatitis in mice was accompanied by AKT upregulation, increase of senescence marker expression and by skin inflammation. In this model, both senescence and inflammation were significantly reduced by selective AKT inhibition. CONCLUSION: Therefore, targeting RAS-AKT pathway could be a promising novel strategy to counteract multiple psoriasis symptoms.

Identification of immunological patterns characterizing immune-related psoriasis reactions in oncological patients in therapy with anti-PD-1 checkpoint inhibitors.

Introduction: Immunotherapy with biologics targeting programmed cell death protein-1 (PD-1) is highly effective in the treatment of various malignancies. Nevertheless, it is frequently responsible for unexpected cutaneous manifestations, including psoriasis-like dermatitis. The pathogenesis of anti-PD-1-induced psoriasis has yet to be clarified, even though it is plausible that some innate and adaptive immunity processes are in common with canonical psoriasis. The genetic predisposition to psoriasis of patients could also be a contributing factor. Here, we investigated the immunological and genetic profiles of two patients with metastatic melanoma and one patient affected by lung cancer, who developed severe psoriasis after receiving anti-PD-1 nivolumab therapy. Methods: The immune patterns of the three patients were compared with those detectable in classical, chronic plaque-type psoriasis or paradoxical psoriasis induced by anti-TNF-α therapy, mostly sustained by adaptive and innate immunity processes, respectively. Therefore, immunohistochemistry and mRNA analyses of innate and adaptive immunity molecules were conducted on skin biopsy of patients. Genetic analysis of polymorphisms predisposing to psoriasis was carried out by NGS technology. Results: We found that anti-PD-1-induced psoriasis showed immunological features similar to chronic psoriasis, characterized by the presence of cellular players of adaptive immunity, with abundant CD3+, CD8+ T cells and CD11c+ dendritic cells infiltrating skin lesions, and producing IL-23, IL-6, TNF-α, IFN-γ and IL-17. On the contrary, a lower number of innate immunity cells (BDCA2+ plasmacytoid dendritic cells, CD15+ neutrophils, CD117+ mast cells) and reduced IFN-α/ß, lymphotoxin (LT)-α/ß, were observed in anti-PD-1-induced psoriasis lesions, as compared with anti-TNF-α-induced paradoxical psoriasis. Importantly, the disintegrin and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5) psoriasis autoantigen was significantly upregulated in psoriasis lesions of anti-PD-1-treated patients, at levels comparable with chronic plaque-type psoriasis. Finally, NGS analysis revealed that all patients carried several allelic variants in psoriasis susceptibility genes, such as HLA-C, ERAP1 and other genes of the major psoriasis susceptibility PSORS1 locus. Discussion: Our study showed that adaptive immunity predominates over innate immunity in anti-PD-1-induced psoriasis lesions, consistently with the local ADAMTSL5 overexpression. The presence of numerous SNPs in psoriasis susceptibility genes of the three patients also suggested their strong predisposition to the disease.

Involvement of transcribed lncRNA uc.291 in hyperproliferative skin disorders.

The uc.291 transcript controls keratinocytes differentiation by physical interaction with ACTL6A and subsequent induction of transcription of the genes belonging to the epidermal differentiation complex (EDC). Uc.291 is also implicated in the dedifferentiation phenotype seen in poorly differentiated cutaneous squamous cell carcinomas. Here, we would like to investigate the contribution of uc.291 to the unbalanced differentiation state of keratinocytes observed in hyperproliferative skin disorders, e. g., psoriasis. Psoriasis is a multifactorial inflammatory disease, caused by alteration of keratinocytes homeostasis. The imbalanced differentiation state, triggered by the infiltration of immune cells, represents one of the events responsible for this pathology. In the present work, we explore the role of uc.291 and its interactor ACTL6A in psoriasis skin, using quantitative real-time PCR (RT-qPCR), immunohistochemistry and bioinformatic analysis of publicly available datasets. Our data suggest that the expression of the uc.291 and of EDC genes loricrin and filaggrin (LOR, FLG) is reduced in lesional skin compared to nonlesional skin of psoriatic patients; conversely, the mRNA and protein level of ACTL6A are up-regulated. Furthermore, we provide evidence that the expression of uc.291, FLG and LOR is reduced, while ACTL6A mRNA is up-regulated, in an in vitro psoriasis-like model obtained by treating differentiated keratinocytes with interleukin 22 (IL-22). Furthermore, analysis of a publicly available dataset of human epidermal keratinocytes treated with IL-22 (GSE7216) confirmed our in vitro results. Taken together, our data reveal a novel role of uc.291 and its functional axis with ACTL6A in psoriasis disorder and a proof of concept that biological inhibition of this molecular axis could have a potential pharmacological effect against psoriasis and, in general, in skin diseases with a suppressed differentiation programme.

Extracellular serine empowers epidermal proliferation and psoriasis-like symptoms.

The contribution of nutrient availability to control epidermal cell proliferation, inflammation, and hyperproliferative diseases remains unknown. Here, we studied extracellular serine and serine/glycine metabolism using human keratinocytes, human skin biopsies, and a mouse model of psoriasis-like disease. We focused on a metabolic enzyme, serine hydroxymethyltransferase (SHMT), that converts serine into glycine and tetrahydrofolate-bound one­carbon units to support cell growth. We found that keratinocytes are both serine and glycine auxotrophs. Metabolomic profiling and hypoxanthine supplementation indicated that SHMT silencing/inhibition reduced cell growth through purine depletion, leading to nucleotide loss. In addition, topical application of an SHMT inhibitor suppressed both keratinocyte proliferation and inflammation in the imiquimod model and resulted in a decrease in psoriasis-associated gene expression. In conclusion, our study highlights SHMT2 activity and serine/glycine availability as an important metabolic hub controlling both keratinocyte proliferation and inflammatory cell expansion in psoriasis and holds promise for additional approaches to treat skin diseases.

Allelic Variants of HLA-C Upstream Region, PSORS1C3, MICA, TNFA and Genes Involved in Epidermal Homeostasis and Barrier Function Influence the Clinical Response to Anti-IL-12/IL-23 Treatment of Patients with Psoriasis.

Several biologic therapies have been developed to treat moderate-to-severe psoriasis, with patients exhibiting different clinical benefits, possibly due to the heterogeneity of pathogenic processes underlying their conditions. Ustekinumab targets the IL-12/IL-23-p40 subunit and inhibits type-1 and type-17 T-cell responses. Although ustekinumab is effective as both short- and long-term treatment, therapeutic response varies considerably among patients. Ustekinumab biosimilars will be commercialized in the very next future, likely broadening the use of this drug in the treatment of psoriasis patients. Our pharmacogenomic study evaluated the influence of 417 single-nucleotide polymorphisms (SNPs) in psoriasis-risk alleles on the clinical response to ustekinumab in a cohort of 152 patients affected by moderate-to-severe plaque-type psoriasis. Differences in SNP pattern characterizing HLA-Cw6+ or HLA-Cw6- patients, showing high or low responses to ustekinumab, were also analysed. We identified twelve SNPs in HLA-C upstream region (rs12189871, rs4406273, rs9348862 and rs9368670), PSORS1C3 (rs1265181), MICA (rs2523497), LCE3A-B intergenic region (rs12030223, rs6701730), CDSN (rs1042127, rs4713436), CCHCR1 (rs2073719) and in TNFA (rs1800610) genes associated with excellent response to ustekinumab. We also found that HLA-Cw6+ and HLA-Cw6- patients carried out distinct patterns of SNPs associated with different clinical responses. The assessment of HLA-C alleles, together with other genetic variants, could be helpful for defining patients who better benefit from anti-IL-12/IL-23 therapy.

PI3Kδ Sustains Keratinocyte Hyperproliferation and Epithelial Inflammation: Implications for a Topically Druggable Target in Psoriasis.

The phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway is aberrantly activated in psoriatic lesions and contributes to disease pathogenesis. Among PI3Ks enzymes, PI3Kα, ß, and δ isoforms are known to bind the p85 regulatory subunit and mediate activation of AKT and other downstream effectors. In this study, we deepened our understanding of the expression and function of PI3Kδ in skin lesions of patients affected by psoriasis. For the first time, we found that PI3Kδ is overexpressed in psoriatic plaques, and its expression is not only confined to infiltrating immune cells but also accumulates in proliferating keratinocytes of the epidermal basal layer. We investigated the function of PI3Kδ in psoriatic skin by evaluating the impact of seletalisib, a newly developed selective PI3Kδ inhibitor, in both in vitro and in vivo experimental models of psoriasis. Of note, we found that PI3Kδ sustains keratinocyte hyperproliferation and impaired terminal differentiation induced by IL-22, as well as induces epithelial inflammation and resistance to apoptosis mediated by TNF-α in human keratinocytes. Mechanistically, PI3Kδ promotes PDK1 phosphorylation and signals through AKT-dependent or -independent pathways. It is worth mentioning that PI3Kδ inhibition by seletalisib attenuates the severity of psoriasiform phenotype induced in the Imiquimod-induced mouse model of psoriasis by restoring the physiological proliferation and differentiation programs in epidermal keratinocytes and contrasting the cutaneous inflammatory responses. Therefore, we suggest PI3Kδ as a potential topically druggable target in psoriasis and skin diseases characterized by epidermal hyperproliferation and skin inflammation.

Enhanced NAMPT-Mediated NAD Salvage Pathway Contributes to Psoriasis Pathogenesis by Amplifying Epithelial Auto-Inflammatory Circuits.

Dysregulated cross-talk between immune cells and epithelial compartments is responsible for the onset and amplification of pathogenic auto-inflammatory circuits occurring in psoriasis. NAMPT-mediated NAD salvage pathway has been recently described as an immunometabolic route having inflammatory function in several disorders, including arthritis and inflammatory bowel diseases. To date, the role of NAD salvage pathway has not been explored in the skin of patients affected by psoriasis. Here, we show that NAD content is enhanced in lesional skin of psoriatic patients and is associated to high NAMPT transcriptional levels. The latter are drastically reduced in psoriatic skin following treatment with the anti-IL-17A biologics secukinumab. We provide evidence that NAMPT-mediated NAD+ metabolism fuels the immune responses executed by resident skin cells in psoriatic skin. In particular, intracellular NAMPT, strongly induced by Th1/Th17-cytokines, acts on keratinocytes by inducing hyper-proliferation and impairing their terminal differentiation. Furthermore, NAMPT-mediated NAD+ boosting synergizes with psoriasis-related cytokines in the upregulation of inflammatory chemokines important for neutrophil and Th1/Th17 cell recruitment. In addition, extracellular NAMPT, abundantly released by keratinocytes and dermal fibroblasts, acts in a paracrine manner on endothelial cells by inducing their proliferation and migration, as well as the expression of ICAM-1 membrane molecule and chemokines important for leukocyte recruitment into inflamed skin. In conclusion, our results showed that NAMPT-mediated NAD salvage pathway contributes to psoriasis pathogenic processes by amplifying epithelial auto-inflammatory responses in psoriasis.

Experimental Methods for the Immunological Characterization of Paradoxical Psoriasis Reactions Induced by TNF-α Biologics.

Immunomodulation with anti-TNFα biologics is highly effective in the treatment of various immune-mediated inflammatory diseases, even though 2-5% of patients treated can develop paradoxical psoriasiform skin lesions. We recently analyzed three patients affected by severe hidradenite suppurativa (HS), and who developed paradoxical psoriasiform reactions following treatment with the TNF-α blockers. Psoriasiform skin reactions showed immunological and immunohistochemical features common to acute psoriasis, characterized by cellular players of innate immunity, such as plasmacytoid dendritic cells (pDC), neutrophils, mast cells, macrophages, and monocytes. In addition, IFN-ß and IFN-α2a, two type I IFNs typical of early psoriasis, were highly expressed in paradoxical skin reactions. Concomitantly, the lymphotoxin (LT)-α and LT-ß were overproduced. Detection of innate immunity cells was carried out on skin sections from HS patients, by immunohistochemistry (IHC) by using antibodies (Abs) against markers identifying specific leukocyte subpopulations. Anti-BDCA2, anti-CD15, anti-CD117, anti-CD68, anti-CD11c, and anti-CD3 Abs were employed to detect pDC, neutrophils, mast cells, macrophages, monocytes/dendritic cells, and T lymphocytes, respectively. In parallel, skin expression of the innate immunity soluble mediators IL-36γ, IFN-ß, IFN-κ, LT-α and LT-ß was also evaluated by IHC by using specific Abs. In this chapter, we describe the methods and protocols to detect the in situ expression and localization of innate immunity molecules and leukocyte subpopulations in skin lesions where inflammatory and psoriasiform reactions are evoked by anti-TNF- α biological therapy.

HLA-Cw6 and other HLA-C alleles, as well as MICB-DT, DDX58, and TYK2 genetic variants associate with optimal response to anti-IL-17A treatment in patients with psoriasis.

Objective: Our pharmacogenomic study evaluated the influence of the presence/absence of genetic variants of psoriasis-risk loci on the clinical response to secukinumab. Differences in the single-nucleotide polymorphism (SNP) pattern characterizing HLA-Cw6+ or HLA-Cw6- patient subpopulations, showing high or low responses to secukinumab, were also analyzed. Methods: 417 SNPs were analyzed by Next-Generation Sequencing technology, in a cohort of 62 psoriatic patients and undergone secukinumab treatment. Univariate regression analysis was employed to examine the association between SNP and clinical response to secukinumab. Multivariate analysis was also performed to assess multivariate differences in SNP pattern of HLA-Cw6+ or HLA-Cw6- patients showing high or low responses to secukinumab. Results: Eight SNPs in HLA-C and upstream region (rs13207315, rs6900444, rs12189871, rs12191877, rs4406273, and rs10484554), including HLA-Cw6 classical allele (rs1131118), and three in MICB-DT (rs9267325), DDX58 (rs34085293) and TYK2 (rs2304255) genes, associating with excellent response to secukinumab were identified. Importantly, rs34085293 or rs2304255 SNP status defined a subgroup of super-responder patients. We also found that HLA-Cw6+ and HLA-Cw6- patients carried out specific patterns of SNPs associating with different responses to secukinumab. Conclusion: Assessment of HLA-Cw6, together with other allelic variants of genes, could be helpful to define patients which better benefit from anti-IL-17 therapy. Abbreviations: PASI: Psoriasis Area and Severity Index; SNP: Single-Nucleotide Polymorphism Rs: Reference SNP; PASI75: 75% reduction in Psoriasis Area and Severity Index; PASI90: 90% reduction in Psoriasis Area and Severity Index; PASI100: 100% reduction in Psoriasis Area and Severity Index; NGS: Next-Generation Sequencing; OR: Odds Ratio; CAP: Canonical Analysis of Principal coordinates; BMI: Body Mass Index; LD: Linkage Disequilibrium.

Interleukin (IL)-17/IL-36 axis participates to the crosstalk between endothelial cells and keratinocytes during inflammatory skin responses.

In inflammatory skin conditions, such as psoriasis, vascular enlargement is associated with endothelial cell proliferation, release of cytokines and adhesion molecule expression. Interleukin (IL)-17A is a pro-inflammatory cytokine mainly secreted by T helper-17 cells that is critically involved in psoriasis pathogenesis. IL-36α, IL-36ß and IL-36γ are also inflammatory cytokines up-regulated in psoriasis and induced by various stimuli, including IL-17A. In this study, we found that human keratinocytes are the main source of IL-36, in particular of IL-36γ. This cytokine was strongly induced by IL-17A and, together with IL-17A, efficiently activated human dermal microvascular endothelial cells (HDMECs), which expressed both IL-17 and IL-36 receptors. Both IL-36γ and IL-17A induced cell proliferation through specific molecular cascades involving ERK1/2 only or ERK1/2, STAT3 and NF-κB, respectively. We highlighted the intense IL-17A- and IL-36γ -dependent interplay between keratinocytes and HDMECs, likely active in the psoriatic lesions and leading to the establishment of a cytokine network responsible for the development and maintenance of the inflamed state. IL-17A or IL-36γ showed in HDMECs a synergic activity with TNF-α by potently inducing inflammatory cytokine/chemokine release and ICAM-1 expression. We also investigated the involvement of IL-36γ and VEGF-A, substantially reduced in lesional skin of psoriatic patients pharmacologically treated with the anti-IL-17A antibody Secukinumab. Importantly, keratinocyte-derived IL-36γ represented an additional pro-angiogenic mediator of IL-17A. We observed that keratinocyte-derived VEGF-A influenced proliferation but did not act on expression of adhesion molecules in HDMECs. On the other hand, inhibition of IL-36γ released by IL-17A-treated keratinocytes impaired either proliferation or ICAM-1 expression both in HDMECs and in an in vivo murine model of psoriasis. Taken together, our data demonstrated that IL-17A and IL-36γ are highly involved in endothelial cells/keratinocytes crosstalk in inflammatory skin conditions.

Platelet lysate promotes the expansion of T regulatory cells that favours in vitro wound healing by increasing keratinocyte migration and fibroblast production of extracellular matrix components.

BACKGROUND: Platelet lysate (PL) contains a cocktail of growth factors and cytokines that promote tissue repair and regeneration. In vitro studies have shown that PL may affect the reparative function of keratinocytes and fibroblasts, but little is known about the effect of PL on immune cells involved in wound healing. OBJECTIVES: To analyse the effects of PL on T cells involved in the wound repair process. MATERIALS AND METHODS: The effect of PL on T cell proliferation, activation, and cytokine production was measured by ELISA and cytofluorometry and regulatory function based on cytofluorometry and Foxp3 RNA expression. Using an in vitro model of wound healing, we investigated the effect of PL-treated T cells on fibroblast proliferation and production of fibronectin and type-1 collagen as well as keratinocyte migration. RESULTS: PL induced T lymphocyte proliferation and CD69 expression, and promoted a transient upregulation of IFN-γ and TNF-α. However, later on, PL enhanced the number of CD25+ T cells releasing TGF-ß and expressing Foxp3 RNA, which was accompanied by a suppression in the level of type 1 cytokines. In the in vitro model, supernatants of PL-treated T cells positively affected the reparative capacity of human keratinocytes and induced fibroblast proliferation and production of fibronectin and type-1 collagen. CONCLUSION: These results indicate that PL temporally regulates T cells during the healing process, enhancing protective cytokines in the early phase, followed by a prominent expansion of TGF-ß+ T regulatory cells that promote tissue regeneration and dampen the inflammatory response to prevent excessive tissue damage.

Intracellular Insulin-like growth factor binding protein 2 (IGFBP2) contributes to the senescence of keratinocytes in psoriasis by stabilizing cytoplasmic p21.

Psoriasis is a chronic Th1/Th17 lymphocytes-mediated inflammatory skin disease, in which epidermal keratinocytes exhibit a peculiar senescent state, resistance to apoptosis and the acquisition of senescence-associated secretory phenotype (SASP). SASP consists of the release of soluble factors, including IGFBPs, that exert extracellular and intracellular functions in IGF-dependent or independent manner.In this report, we investigated the expression and function of IGFBP2 in senescent keratinocytes isolated from the skin of patients with plaque psoriasis. We found that IGFBP2 is aberrantly expressed and released by these cells in vivo, as well as in vitro in keratinocyte cultures undergoing progressive senescence, and it associates with the cyclin-dependent kinase inhibitors p21 and p16 expression. For the first time, we provide evidence for a dual action of IGFBP2 in psoriatic keratinocytes during growth and senescence processes. While extracellular IGFBP2 counter-regulates IGF-induced keratinocyte hyper-proliferation, intracellular IGFBP2 inhibits apoptosis by interacting with p21 and protecting it from ubiquitin-dependent degradation. Indeed, we found that cytoplasmic p21 sustains anti-apoptotic processes, by inhibiting pro-caspase 3 cleavage and JNK phosphorylation in senescent psoriatic keratinocytes. As a consequence, abrogation of p21, as well as that of IGFBP2, found to stabilize cytoplasmic p21 levels, lead to the restoration of apoptosis mechanisms in psoriatic keratinocytes, commonly observed in healthy cells.

Paradoxical psoriasis induced by TNF-α blockade shows immunological features typical of the early phase of psoriasis development.

Immunomodulation with anti-TNF-α is highly effective in the treatment of various immune-mediated inflammatory diseases, including hidradenitis suppurativa (HS). However, this may be responsible for unexpected paradoxical psoriasiform reactions. The pathogenic mechanisms underlying the induction of these events are not clear, even though the involvement of innate immune responses driven by plasmacytoid dendritic cells (pDC) has been described. In addition, the genetic predisposition to psoriasis of patients could be determinant. In this study, we investigated the immunological and genetic profiles of three HS patients without psoriasis who developed paradoxical psoriasiform reactions following anti-TNF-α therapy with adalimumab. We found that paradoxical psoriasiform skin reactions show immunological features common to the early phases of psoriasis development, characterized by cellular players of innate immunity, such as pDC, neutrophils, mast cells, macrophages, and monocytes. In addition, IFN-ß and IFN-α2a, two type I IFNs typical of early psoriasis, were highly expressed in paradoxical skin reactions. Concomitantly, other innate immunity molecules, such as the catheledicin LL37 and lymphotoxin (LT)-α and LT-ß were overproduced. Interestingly, these innate immunity molecules were abundantly expressed by keratinocytes, in addition to the inflammatory infiltrate. In contrast to classical psoriasis, psoriasiform lesions of HS patients showed a reduced number of IFN-γ and TNF-α-releasing T lymphocytes. On the contrary, IL-22 immunoreactivity was significantly augmented together with the IL-36γ staining in leukocytes infiltrating the dermis. Finally, we found that all HS patients with paradoxical reactions carried allelic variants in genes predisposing to psoriasis. Among them, SNPs in ERAP1, NFKBIZ, and TNFAIP genes and in the HLA-C genomic region were found.

Selective Immunomodulation of Inflammatory Pathways in Keratinocytes by the Janus Kinase (JAK) Inhibitor Tofacitinib: Implications for the Employment of JAK-Targeting Drugs in Psoriasis.

IFN-γ and IL-22 are deeply involved in the pathogenesis of psoriasis, as they boost the expression of inflammatory genes and alter proliferative and differentiative programs in keratinocytes. The JAK1/JAK2/STAT1 and JAK1/TYK2/STAT3 pathways triggered by IFN-γ and IL-22, respectively, are aberrantly activated in psoriasis, as highlighted by the peculiar STAT1 and STAT3 signatures in psoriatic skin lesions. To limit the detrimental consequences of IFN-γ and IL-22 excessive stimulation, psoriatic keratinocytes activate suppressor of cytokine signaling (SOCS)1 and SOCS3, which in turn dampen molecular signaling by inhibiting JAK1 and JAK2. Thus, JAK targeting appears to be a reasonable strategy to treat psoriasis. Tofacitinib is an inhibitor of JAK proteins, which, similarly to SOCS, impedes JAK phosphorylation. In this study, we evaluated the immunomodulatory effects of tofacitinib on epidermal keratinocytes in in vitro and in vivo models of psoriasis. We demonstrated the selectivity of tofacitinib inhibitory action on IFN-γ and IL-22, but not on TNF-γ or IL-17 proinflammatory signaling, with suppressed expression of IFN-γ-dependent inflammatory genes, and restoration of normal proliferative and differentiative programs altered by IL-22 in psoriatic keratinocyte cultures. Tofacitinib also potently reduced the psoriasiform phenotype in the imiquimod-induced murine model of psoriasis. Finally, we found that tofacitinib mimics SOCS1 or SOCS3 activities, as it impaired the same molecular pathways in IFN-γ or IL-22-activated keratinocytes.

IL-38 has an anti-inflammatory action in psoriasis and its expression correlates with disease severity and therapeutic response to anti-IL-17A treatment.

IL-36 cytokines, a subgroup of IL-1 family, comprise IL-36α, IL-36ß, and IL-36γ agonists, abundantly expressed in psoriatic skin, and IL-36RA and IL-38 antagonists. In psoriatic skin, IL-36 cytokines interfere with keratinocyte cornification programs and induce the release of antimicrobial peptides and chemokines active on neutrophils and Th17 lymphocytes. To date, the role of IL-38 antagonist in psoriasis remains to be defined. Here, we demonstrate that skin and circulating IL-38 levels are reduced in psoriatic patients and in other skin diseases characterized by neutrophilic infiltrate. In psoriasis, the balance of IL-36γ agonist/IL-38 antagonist serum levels is in favor of agonists and is closely associated with disease severity. Interestingly, IL-38 is upregulated by anti-IL-17A biological treatment and positively correlates with the therapeutic efficacy of secukinumab in psoriatic patients. The downregulation of IL-38 expression is strictly related to keratinocyte de-differentiation triggered by the inflammatory cytokines IL-36γ, IL-17, and IL-22. Finally, we demonstrate that administration of recombinant full-length IL-38 counteracts in vitro the biological processes induced by IL-36γ in human keratinocytes and endothelial cells and attenuates in vivo the severity of the psoriasiform phenotype induced by IMQ in mice. Such effects are achieved by restoring the physiological programs of keratinocyte proliferation and differentiation, and reducing the immune cell infiltrates.

Eosin treatment for psoriasis reduces skin leukocyte infiltration and secretion of inflammatory chemokines and angiogenic factors.

BACKGROUND: Eosin has been traditionally employed as a topical treatment for psoriasis, but the biological mechanism of its therapeutic action has not been fully elucidated. OBJECTIVES: To analyse eosin effects on psoriatic skin in vivo and keratinocytes and endothelial cells in vitro. MATERIALS & METHODS: Skin biopsies were taken from psoriatic plaques before and after a three-day eosin treatment and processed for histological analysis. Cultured human psoriatic keratinocytes and dermal endothelial cells were treated with eosin, and release of inflammatory chemokines was analysed by multiplexed bead-based immunoassay and ELISA. RESULTS: In patients, the three-day eosin treatment significantly reduced the number of infiltrating T lymphocytes, neutrophilic granulocytes, and dermal dendritic cells. A reduction in VEGF-A expression was also observed. In vitro, eosin treatment significantly decreased the release of CCL2, CCL5, and VEGF-A by keratinocytes and angiopoietin-2 by endothelial cells. CONCLUSIONS: Eosin treatment impacts on psoriatic inflammatory infiltrates and dampens the release of proinflammatory chemokines and angiogenic factors.

Knockout of the Arp2/3 complex in epidermis causes a psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2.

The Arp2/3 complex assembles branched actin filaments, which are key to many cellular processes, but its organismal roles remain poorly understood. Here, we employed conditional Arpc4 knockout mice to study the function of the Arp2/3 complex in the epidermis. We found that depletion of the Arp2/3 complex by knockout of Arpc4 results in skin abnormalities at birth that evolve into a severe psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2. Knockout of Arpc4 in cultured keratinocytes was sufficient to induce nuclear accumulation of Nrf2, upregulation of Nrf2 target genes and decreased filamentous actin levels. Furthermore, pharmacological inhibition of the Arp2/3 complex unmasked the role of branched actin filaments in Nrf2 regulation. Consistent with this, we revealed that Nrf2 associates with the actin cytoskeleton in cells and binds to filamentous actin in vitro Finally, we discovered that Arpc4 is downregulated in both human and mouse psoriatic epidermis. Thus, the Arp2/3 complex affects keratinocyte shape and transcriptome through an actin-based cell-autonomous mechanism that influences epidermal morphogenesis and homeostasis.

SOCS3 inhibits the pathological effects of IL-22 in non-melanoma skin tumor-derived keratinocytes.

Basal cell carcinomas (BCC) and squamous-cell carcinomas (SCC) are common malignancies in humans, caused by neoplastic transformation of keratinocytes of the basal or suprabasal layers of epidermis, respectively. Tumor-infiltrating lymphocytes (TILs) are frequently found in BCC and SCC, and functionally promote epithelial carcinogenesis. TILs secreting IL-22, in particular, participate to BCC and SCC growth by inducing keratinocyte proliferation and migration, as well as the expression of inflammatory, anti-apoptotic and pro-angiogenic genes.In this study, we identified SOCS3 as a valid candidate to be manipulated for suppressing tumorigenic functions in BCC and SCC. We found that SOCS3 and SOCS1 expression was reduced in vivo, in tumor lesions of BCC and SCC, as compared to other skin inflammatory conditions such as psoriasis, despite the high number of IL-22-secreting TILs. Moreover, IL-22 was not able to induce in vitro the transcriptional expression of SOCS3 in BCC-or SCC-derived keratinocytes, contrarily to healthy cells. Aimed at rescuing SOCS3 activity in these tumor contexts, a SOCS3-derived peptide, named KIR-ESS, was synthesized, and its ability in suppressing IL-22-induced responses was evaluated in healthy and transformed keratinocytes. We found that KIR-ESS peptide efficiently suppressed the IL-22 molecular signaling in keratinocytes, by acting on STAT3 and Erk1/2 cascade, as well as on the expression of STAT3-dependent downstream genes. Interestingly, after treatment with peptide, both healthy and transformed keratinocytes could no longer aberrantly proliferate and migrate in response to IL-22. Finally, treatment of athymic nude mice bearing SCC xenografts with KIR-ESS peptide concomitantly reduced tumor growth and activated STAT3 levels. As a whole, these data provides the rationale for the use in BCC and SCC skin tumors of SOCS3 mimetics, being able to inhibit the deleterious effects of IL-22 in these contexts.

The role of oncogenic Ras in human skin tumorigenesis depends on the clonogenic potential of the founding keratinocytes.

The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras-V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying- or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis.

Interleukin-17 and interleukin-22 promote tumor progression in human nonmelanoma skin cancer.

Interleukin-17 (IL-17) and IL-22 have been reported to play critical roles in autoimmunity and inflammation but information about their role in cancer is limited. In this study, we investigated the role of IL-17 and IL-22 in the progression of human skin basal-cell carcinoma (BCC) and squamous-cell carcinoma (SCC). We found that both tumor types are infiltrated with an high number of IL-17(+) and IL-22(+) T lymphocytes, as demonstrated by immunohistochemistry and by FACS analysis performed on peritumoral T-cell lines isolated from skin biopsies. In vitro studies demonstrated that proliferation and migration of the BCC- and SCC-cell lines M77015 and CAL27 were increased by IL-17 and IL-22. Moreover, IL-17, alone or in combination with TNF-α, was able to induce the production of two cytokines important for tumor progression, IL-6 and IL-8, in CAL27. We also showed that IL-17 upregulated NF-κB signaling, while IL-22 activated the STAT3 pathway and the antiapoptotic AKT protein in M77015 and CAL27. Finally, in vivo experiments demonstrated that IL-17 and IL-22 enhanced tumor growth in nude mice injected with CAL27. Altogether, our findings indicate that high levels of IL-22 and IL-17 in the BCC and SCC microenvironment promote tumor progression.