Topical Plant Polyphenols Prevent Type I Interferon Signaling in the Skin and Suppress Contact Hypersensitivity.

Human keratinocytes were recently shown to respond to anti-EGFR (epidermal growth factor receptor) drugs with activation of an interferon-κ-driven autocrine loop, leading to enhanced expression of innate antiviral effectors and of the pro-inflammatory chemokines CXCL10 (C-X-C motif chemokine 10) and CCL2 (C-C motif ligand 2). Here we showed active type I interferon signaling in the skin lesions of cancer patients undergoing treatment with the anti-EGFR drug cetuximab. Strong nuclear positivity for Interferon Regulatory Factor 1 and phosphorylated Signal Transducer and Activator of Transcription 1, enhanced interferon-κ expression and CXCL10 was associated to the epidermal compartment. Notably, 50 micromolar resveratrol and quercetin fully suppressed the low constitutive levels of type I interferon signaling and prevented its activation by the anti-EGFR cetuximab or gefitinib in cultured keratinocytes. In sensitized mice undergoing DNFB (2,4-dinitro-1-fluorobenzene)-induced contact hypersensitivity, local administration of gefitinib prior to elicitation further amplified hapten-induced type I interferon activation, tissue edema, and infiltration by T cells, whereas resveratrol or quercetin suppressed this inflammatory cascade. Overall, these data suggest that topical application of resveratrol or quercetin could be potentially effective in preventing pathological conditions due to overactivation of type I IFN (interferon)-driven circuits in the skin, including the inflammatory manifestations of anti-EGFR drug-induced skin-targeted toxicity.

The MEK Inhibitors Trametinib and Cobimetinib Induce a Type I Interferon Response in Human Keratinocytes.

Mitogen-activated protein kinase kinases (MEK) 1 and 2 have crucial roles in tumorigenesis, cell proliferation, and protection from apoptosis, and their inhibition is therefore an attractive therapeutic strategy in cancer. Orally available and highly selective MEK inhibitors have been developed and assessed in numerous clinical trials, either alone or in combination with cytotoxic chemotherapy and/or other targeted agents. Of note, a complex picture of class-specific adverse effects associates with these drugs, frequently including inflammatory skin rash. Here, we investigated the response of normal human keratinocytes to the MEK inhibitors trametinib and cobimetinib, alone and in combination with the v-Raf murine sarcoma viral oncogene homolog B (BRAF) inhibitors dabrafenib and vemurafenib, in terms of signal transduction and de novo gene expression. MEK inhibitors triggered enhanced expression of interferon regulatory factor 1 (IRF1) and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and up-regulated the keratinocyte-specific type I interferon κ (IFN-κ), the anti-viral effectors interferon-induced tetratricopeptide repeats (IFIT) 1 and 2, and the pro-inflammatory chemokine (C-C motif) ligand 2 (CCL2) and the C-X-C motif chemokine 10 (CXCL10), both at the mRNA and protein level. Impairment of IRF1 expression, or abrogation of STAT1 phosphorylation due to IFN-κ gene silencing, suppressed anti-viral and pro-inflammatory gene expression. These data suggest that, similar to what we observed for epidermal growth factor receptor (EGFR) blockade, MEK inhibition activates a type I interferon response, which is now recognized as an effective anti-cancer response, in human epidermal keratinocytes.

Epidermal growth factor receptor signalling in keratinocyte biology: implications for skin toxicity of tyrosine kinase inhibitors.

The epidermal growth factor receptor (EGFR) and its ligands have been long recognized as centrally involved in the growth and repair process of epithelia, as well as in carcinogenesis. In addition, the EGFR has been demonstrated to be importantly involved in the control of inflammatory responses. During this last decade, a number of highly specific agents targeting this system have become an integral component of pharmacologic strategies against many solid malignancies. These drugs have led to increased patient survival and made therapy more tolerant when compared to conventional cytotoxic drugs. Nonetheless, their use is associated with a constellation of toxic effects on the skin, including follicular pustules, persistent inflammation, xerosis and pruritus, and enhanced susceptibility to infections. This dramatic impairment of skin homoeostasis underscores the centrality of the EGFR-ligand system in the whole skin immune system. So far, no mechanism-based approaches are available to specifically counteract the adverse effects of anti-EGFR drugs or any other class of tyrosine kinase inhibitors. Only the knowledge of the cellular and molecular events underlying these adverse effects in humans, combined with in vitro/in vivo models able to mimic these toxic responses, may guide the development of mechanism-based treatment or prevention strategies.

Insight into immune profile associated with vitiligo onset and anti-tumoral response in melanoma patients receiving anti-PD-1 immunotherapy.

Introduction: Immunotherapy with checkpoint inhibitors is an efficient treatment for metastatic melanoma. Development of vitiligo upon immunotherapy represents a specific immune-related adverse event (irAE) diagnosed in 15% of patients and associated with a positive clinical response. Therefore, a detailed characterization of immune cells during vitiligo onset in melanoma patients would give insight into the immune mechanisms mediating both the irAE and the anti-tumor response. Methods: To better understand these aspects, we analyzed T cell subsets from peripheral blood of metastatic melanoma patients undergoing treatment with anti-programmed cell death protein (PD)-1 antibodies. To deeply characterize the antitumoral T cell response concomitant to vitiligo onset, we analyzed T cell content in skin biopsies collected from melanoma patients who developed vitiligo. Moreover, to further characterize T cells in vitiligo skin lesion of melanoma patients, we sequenced T cell receptor (TCR) of cells derived from biopsies of vitiligo and primary melanoma of the same patient. Results and discussion: Stratification of patients for developing or not developing vitiligo during anti-PD-1 therapy revealed an association between blood reduction of CD8-mucosal associated invariant T (MAIT), T helper (h) 17, natural killer (NK) CD56bright, and T regulatory (T-reg) cells and vitiligo onset. Consistently with the observed blood reduction of Th17 cells in melanoma patients developing vitiligo during immunotherapy, we found high amount of IL-17A expressing cells in the vitiligo skin biopsy, suggesting a possible migration of Th17 cells from the blood into the autoimmune lesion. Interestingly, except for a few cases, we found different TCR sequences between vitiligo and primary melanoma lesions. In contrast, shared TCR sequences were identified between vitiligo and metastatic tissues of the same patient. These data indicate that T cell response against normal melanocytes, which is involved in vitiligo onset, is not typically mediated by reactivation of specific T cell clones infiltrating primary melanoma but may be elicited by T cell clones targeting metastatic tissues. Altogether, our data indicate that anti-PD-1 therapy induces a de novo immune response, stimulated by the presence of metastatic cells, and composed of different T cell subtypes, which may trigger the development of vitiligo and the response against metastatic tumor.

Safe-Shields: Basal and Anti-UV Protection of Human Keratinocytes by Redox-Active Cerium Oxide Nanoparticles Prevents UVB-Induced Mutagenesis.

Cerium oxide nanoparticles (nanoceria), biocompatible multifunctional nanozymes exerting unique biomimetic activities, mimic superoxide-dismutase and catalase through a self-regenerating, energy-free redox cycle driven by Ce3+/4+ valence switch. Additional redox-independent UV-filter properties render nanoceria ideal multitask solar screens, shielding from UV exposure, simultaneously protecting tissues from UV-oxidative damage. Here, we report that nanoceria favour basal proliferation of primary normal keratinocytes, and protects them from UVB-induced DNA damage, mutagenesis, and apoptosis, minimizing cell loss and accelerating recovery with flawless cells. Similar cell-protective effects were found on irradiated noncancerous, but immortalized, p53-null HaCaT keratinocytes, with the notable exception that here, nanoceria do not accelerate basal HaCaT proliferation. Notably, nanoceria protect HaCaT from oxidative stress induced by irradiated titanium dioxide nanoparticles, a major active principle of commercial UV-shielding lotions, thus neutralizing their most critical side effects. The intriguing combination of nanoceria multiple beneficial properties opens the way for smart and safer containment measures of UV-induced skin damage and carcinogenesis.

Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases.

We previously showed that genotoxic stress induced an active extracellular release of nucleophosmin (NPM) in human cardiac mesenchymal progenitor cells, and that serum deprivation provokes NPM secretion from human endothelial cells, eliciting inflammation via nuclear factor kappa B (NF-kB) transcriptional activation. In this study, we wanted to determine whether NPM was similarly modulated in the skin and plasma of psoriatic patients (Pso). We found that NPM was induced in 6 skin biopsies compared to 6 normal skin biopsies and was markedly increased in lesional (LS) vs. non-lesional skin (NLS) biopsies. Moreover, NPM was also increased at the transcriptional levels in LS vs. NLS. Both the innate stimuli, such as lipopolysaccharides and Poly inositol-cytosine and adaptive stimuli, that is, cytokine mix, were able to induce the extracellular release of NPM in immortalized keratinocytes and human skin fibroblasts in the absence of cytotoxicity. Interestingly, NPM interacts with Toll-like receptor (TLR)4 in these cells and activates an NF-kB-dependent inflammatory pathway upregulating interleukin IL-6 and COX-2 gene expression. Finally, circulating NPM was increased in the plasma of 29 Pso compared to 29 healthy controls, and positively correlates with psoriasis area severity index (PASI) and with determinants of cardiovascular diseases (CVDs), such as pulse wave velocity, systolic pressure, and left ventricular mass. Furthermore, NPM positively correlates with miR-200c circulating levels, which we previously showed to increase in Pso and correlate with CVD progression. Our data show that circulating miR-200c is physically associated with extracellular NPM, which most probably is responsible for its extracellular release and protection upon cytokine mix via a TLR4-mechanism. In conclusion, NPM is increased in psoriasis both in the skin and plasma and might be considered a novel biologic target to counteract chronic inflammation associated with CVD risk.

Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NFκB and AhR and EGFR-ERK pathway.

Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA+UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure, the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems. The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50µM resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR.

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.

Epidermal growth factor receptor inhibitors trigger a type I interferon response in human skin.

The Epidermal Growth Factor Receptor (EGFR) is centrally involved in the regulation of key processes of the epithelia, including cell proliferation, survival, differentiation, and also tumorigenesis. Humanized antibodies and small-molecule inhibitors targeting EGFR were developed to disrupt these functions in cancer cells and are currently used in the treatment of diverse metastatic epithelial cancers. By contrast, these drugs possess significant skin-specific toxic effects, comprising the establishment of a persistent inflammatory milieu. So far, the molecular mechanisms underlying these epiphenomena have been investigated rather poorly. Here we showed that keratinocytes respond to anti-EGFR drugs with the development of a type I interferon molecular signature. Upregulation of the transcription factor IRF1 is early implicated in the enhanced expression of interferon-kappa, leading to persistent activation of STAT1 and further amplification of downstream interferon-induced genes, including anti-viral effectors and chemokines. When anti-EGFR drugs are associated to TNF-α, whose expression is enhanced by the drugs themselves, all these molecular events undergo a dramatic enhancement by synergy mechanisms. Finally, high levels of interferon-kappa can be observed in epidermal keratinocytes and also in leukocytes infiltrating the upper dermis of cetuximab-driven skin lesions. Our data suggest that dysregulated activation of type I interferon innate immunity is implicated in the molecular processes triggered by anti-EGFR drugs and leading to persistent skin inflammation.

Resveratrol induces long-lasting IL-8 expression and peculiar EGFR activation/distribution in human keratinocytes: mechanisms and implications for skin administration.

UNLABELLED: Anti-inflammatory and skin tumour preventing effects of resveratrol have been extensively studied pre-clinically and resveratrol has been proposed for clinical investigations. To provide a basis or/and limitations for topical administration to human skin, molecular mechanisms underlying resveratrol effects towards normal human epidermal keratinocytes (NHEK) were evaluated. NHEK were challenged by either resveratrol alone or by its combination with TNFalpha or TGFalpha, and time-dependent molecular events were monitored. Interleukin 8 (IL-8) expression and its mRNA stability, ERK1/2, p65/RelA, and EGFR phosphorylation were determined. Intracellular distribution of EGFR/P-EGFR was measured in the membrane, cytoplasmic, and nuclear fractions. Specific DNA binding activity of NFκB (p65/RelA) and AP-1(c-Fos), NHEK proliferation, and molecular markers of apoptosis/cell cycle were detected. Resveratrol induced delayed, long-lasting and steadily growing IL-8 gene and protein over-expression as well as enhanced EGFR phosphorylation, both abrogated by the EGFR kinase inhibitor PD168393. However, resveratrol did not act as a phosphatase inhibitor. ERK phosphorylation was transiently inhibited at early time-points and activated at 6-24 h. Accordingly, c-Fos-specific DNA binding was increased by resveratrol. Cellular distribution of EGFR/P-EGFR was shifted to membrane and nucleus while cytosolic levels were reduced concomitant with enhanced degradation. Notwithstanding high nuclear levels of EGFR/P-EGFR, spontaneous and TGFalpha-triggered cell proliferation was strongly suppressed by resveratrol mainly through cell cycle arrest. CONCLUSIONS/SIGNIFICANCE: Resveratrol synergized with TNFα in the induction of delayed, long-lasting IL-8 expression through sustained EGFR-ERK axis activation. The time course indicates that resveratrol metabolites could be implicated. Topical administration of Resv to psoriatic patients over-expressing TNFα, IL-8 and EGFR-ERK in the skin should be cautiously considered. Since high nuclear levels of EGFR correspond to increased risk of tumorigenesis, chronic resveratrol application to the skin may be potentially dangerous. Wound healing acceleration by resveratrol could not be envisaged due to its anti-proliferative effects towards normal keratinocytes.

Verbascum xanthophoeniceum-derived phenylethanoid glycosides are potent inhibitors of inflammatory chemokines in dormant and interferon-gamma-stimulated human keratinocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Verbascum xanthophoeniceum is a representative of mullein species with a strong tradition of use in folk medicine as a remedy in inflammatory and infectious contexts. This plant accumulates phenylethanoid and iridoid metabolites with a partially characterized bioactivity. MATERIALS AND METHODS: Here, we compared anti-inflammatory effects of Verbascum xanthophoeniceum crude extract, its fractions, isolated iridoid glycosides, including aucubin, ajugol, harpagide, harpagoside, nigroside III and nigroside VI, and phenylethanoid glycosides verbascoside and forsythoside B in primary cultures of normal human keratinocytes (NHK). The gene expression, synthesis, and release of soluble pro-inflammatory chemokines, such as IL-8, MCP-1 and IP-10, in dormant and IFN-γ-activated NHK were investigated, and IC(50) for each extract/individual substance was determined. RESULTS: We found that the phenylethanoid glycosides verbascoside and forsythoside B were effective, dose-dependent inhibitors of gene expression and de novo synthesis of all the chemokines, whereas the iridoid glycosides and phenylpropanoid aglycone rosmarinic acid displayed a poor and selective inhibition. Accordingly, the fraction of the crude extract containing verbascoside effectively impaired both spontaneous and induced chemokine expression in NHK. CONCLUSION: This is the first report on the identification of active constituents of Verbascum xanthophoeniceum possessing anti-inflammatory properties towards human keratinocytes. Phenylethanoid glycosides exerted exquisite corticosteroid-like inhibition of pro-inflammatory chemokines at transcriptional and translational levels.

Resveratrol enhances solar UV-induced responses in normal human epidermal keratinocytes.

Resveratrol (RV) differentially affects UV-induced death/pro-survival pathways in normal and tumor cells. On these grounds, RV-containing topical products have been developed to prevent UV-associated tumorigenesis/damage to human skin. In this study, we evaluated mechanisms of combined effects of RV and low-dose solar simulated UVA+UVB or 6-formylindo[3,2-b]carbazole (FICZ), a product of tryptophan photo-oxidation known to mediate UV effects, on the inflammatory, metabolic and proliferative responses of cultured normal human epidermal keratinocytes (HEK). Applied alone, RV, UV and FICZ induced time- and dose-dependent activation of aryl hydrocarbon receptor (AhR) pathway followed by over-expression of Cyp1A1 (metabolic response), UV and RV induced IL-8 expression (inflammatory response), while RV enhanced also HEK proliferation revealed by MTT assay and (3)H-thymidine incorporation. In the combined treatment, RV synergized with both UV and FICZ, leading to further activation of AhR machine, Cyp1A1 transcription and IL-8 expression, the latter partly AhR-dependent as assessed by AhR silencing. RV enhanced UV-induced NFkappaB activation and nuclear translocation of epidermal growth factor receptor. By contrast, proliferative effect of RV was abolished in the presence of UV, whereas synergic anti-proliferative action of RV+UV was observed in the Nrf2-silenced HEK. Our data suggest cooperative effects of RV-specific and UV-/FICZ-activated transcription factors leading to deregulated inflammatory, metabolic and proliferative responses of HEK.

Plant polyphenols regulate chemokine expression and tissue repair in human keratinocytes through interaction with cytoplasmic and nuclear components of epidermal growth factor receptor system.

AIMS: To evaluate mechanisms underlying modulation of inflammatory chemokines in primary human keratinocytes (normal human epidermal keratinocytes) and repair-related processes in wound models by plant polyphenols (PPs) with antioxidant and superoxide scavenging properties (verbascoside [Vb], resveratrol [Rv], polydatin [Pd], quercetin [Qr], and rutin). RESULTS: Epidermal growth factor receptor (EGFR)-controlled chemokines CXCL8/interleukin 8 (IL-8), CCL2/monocyte chemotactic protein-1 (MCP-1), and CXCL10/interferon gamma-produced protein of 10 kDa (IP-10) were modulated by transforming growth factor alpha (TGF-α) and by the tumor necrosis factor alpha/interferon gamma combination (T/I). EGFR phosphorylation, nuclear translocation, and downstream cytoplasmic signaling pathways (extracellular regulation kinase [ERK]1/2, p38, STAT3, and PI-3K) were studied. All PPs did not affect TGF-α-induced STAT3 phosphorylation, whereas they suppressed T/I-activated NFkappaB and constitutive and T/I-induced but not TGF-α-induced ERK1/2 phosphorylation. Vb and Qr suppressed total EGFR phosphorylation, but they synergized with TGF-α to enhance nuclear accumulation of phosphorylated EGFR. Vb strongly inhibited TGF-α-induced p38 phosphorylation and T/I-induced NFkappaB and activator protein-1 (AP-1) binding to DNA. Vb was an effective inhibitor of T/I-stimulated chemokine synthesis, and it accelerated scratch wound healing in vitro. Anti-inflammatory and wound healing activities of Vb were confirmed in vivo in the full-thickness excision wound. Although Pd and Rv did not affect EGFR activation/translocation, they and Qr synergized with TGF-α and T/I in the induction of IL-8 transcription/synthesis while opposing enhanced MCP-1 and IP-10 transcription/synthesis connected with pharmacologically impaired EGFR functioning. INNOVATION: PPs perturb the EGFR system in human keratinocytes, and this effect may be implicated in the regulation of inflammatory and repair-related processes in the skin. CONCLUSION: Anti-inflammatory and wound healing effects of PPs depend on their interaction with EGFR-controlled cytoplasmic and nuclear pathways rather than on their direct redox properties.

Photo-oxidation products of skin surface squalene mediate metabolic and inflammatory responses to solar UV in human keratinocytes.

UNLABELLED: The study aimed to identify endogenous lipid mediators of metabolic and inflammatory responses of human keratinocytes to solar UV irradiation. Physiologically relevant doses of solar simulated UVA+UVB were applied to human skin surface lipids (SSL) or to primary cultures of normal human epidermal keratinocytes (NHEK). The decay of photo-sensitive lipid-soluble components, alpha-tocopherol, squalene (Sq), and cholesterol in SSL was analysed and products of squalene photo-oxidation (SqPx) were quantitatively isolated from irradiated SSL. When administered directly to NHEK, low-dose solar UVA+UVB induced time-dependent inflammatory and metabolic responses. To mimic UVA+UVB action, NHEK were exposed to intact or photo-oxidised SSL, Sq or SqPx, 4-hydroxy-2-nonenal (4-HNE), and the product of tryptophan photo-oxidation 6-formylindolo[3,2-b]carbazole (FICZ). FICZ activated exclusively metabolic responses characteristic for UV, i.e. the aryl hydrocarbon receptor (AhR) machinery and downstream CYP1A1/CYP1B1 gene expression, while 4-HNE slightly stimulated inflammatory UV markers IL-6, COX-2, and iNOS genes. On contrast, SqPx induced the majority of metabolic and inflammatory responses characteristic for UVA+UVB, acting via AhR, EGFR, and G-protein-coupled arachidonic acid receptor (G2A). CONCLUSIONS/SIGNIFICANCE: Our findings indicate that Sq could be a primary sensor of solar UV irradiation in human SSL, and products of its photo-oxidation mediate/induce metabolic and inflammatory responses of keratinocytes to UVA+UVB, which could be relevant for skin inflammation in the sun-exposed oily skin.

Differential modulation of stress-inflammation responses by plant polyphenols in cultured normal human keratinocytes and immortalized HaCaT cells.

BACKGROUND: Environmental and endogenous stresses to skin are considered causative reasons for skin cancers, premature ageing, and chronic inflammation. Screening of substances with preventive and/or curative properties is currently based on mechanistic studies of their effects towards stress-induced responses in skin cell cultures. OBJECTIVE: We compared effects of plant polyphenols (PPs) on the constitutive, UVA-, LPS-, or TNF-alpha-induced inflammatory responses in cultured normal human epidermal keratinocytes (NHEK) and immortalized HaCaT cells. METHODS: Representatives of three classes of PPs, flavonoids, stilbenoids, and phenylpropanoids were studied. Their effects on mRNA were determined by qRT-PCR; protein expression was assayed by Western blot and bioplexed ELISA; phosphorylation of Akt1, ERK1/2, EGFR, and NFkappaB was quantified by intracellular ELISA or Western blot. RESULTS: PPs or their combination with UVA or LPS induced strong up-regulation of stress responses in HaCaT but not in NHEK. In addition, compared to NHEK, HaCaT responded to TNF-alpha with higher synthesis of MCP-1, IP-10 and IL-8, concomitant with stronger NFkappaB activation. PPs down-regulated the chemokine release from both cell types, although with distinct effects on NFkappaB, Akt1, ERK, and EGFR activation. CONCLUSION: Results of pharmacological screenings obtained by using HaCaT should be cautiously considered while extending them to primary keratinocytes from human epidermis.

Glutathione peroxidase activity in the blood cells of psoriatic patients correlates with their responsiveness to Efalizumab.

Biological treatment of psoriasis, a chronic inflammatory immune-mediated pathology of huge social impact, has become a recent revolutionizing breakthrough in the management of the disease. Apart from anti-TNF-alpha biologics, recombinant proteins-inhibitors of the T lymphocytes-antigen presenting cells interaction, Efalizumab among them, have been successfully used in the therapy of psoriasis. Serious concern regarding safety and efficacy of biologics remains because they induce numerous adverse effects and a significant number of patients are non-responders. Up-to-now, there are no biochemical or/and immunological markers of the clinical efficacy of these drugs. This study searches for immunological and redox markers of the clinical response in the group of psoriatic patients treated with Efalizumab. Clinical response to Efalizumab was assessed by Psoriasis Area and Severity Index and correlated with suppression of T-cell functions, plasma cytokines, membrane-associated polyunsaturated fatty acids (PUFAs), antioxidant enzymes and markers of oxidative stress. A 12-week Efalizumab therapy did not affect abnormal plasma levels of pro-inflammatory cytokines and lower-than-normal content of PUFAs esterified in phospholipids of red cell membranes. It did, however, suppress T-cell-mediated functions and decrease nitrites/nitrates and malonyl dialdehyde levels independently on the clinical outcome. On contrast, activities of glutathione peroxidase (GPx) and glutathione S-transferase in granulocytes were remarkably increased and catalase decreased exclusively in non-responders vs complete or partial responders. High baseline GPx in erythrocytes decreased in responders. It is concluded that clinical response to Efalizumab correlates with GPx activity in the blood cells, suggesting that high hydroperoxide levels are involved in psoriasis persistence.

Plant polyphenols effectively protect HaCaT cells from ultraviolet C-triggered necrosis and suppress inflammatory chemokine expression.

Oxidative stress is a common response of epidermal cells to a variety of noxious stimuli such as ultraviolet (UV) radiation from solar light and proinflammatory cytokines from skin-infiltrating leukocytes. Here, we report that two types of plant-derived antioxidants, the phenylpropanoid glycoside verbascoside as well as the flavonoids rutin and quercetin possess protective effects against UVC-induced cell damage and proinflammatory activation. The molecules under investigation were effective against the loss of cell integrity associated with necrosis in doses consistent with their antioxidant activity, whereas they did not significantly oppose UVC-induced proliferation arrest and apoptosis. By contrast, only verbascoside effectively inhibited cytokine-induced release of proinflammatory mediators in a dose-dependent fashion. Verbascoside and its homologue teupolioside dramatically impaired NF-kappaB and AP-1 DNA binding activity. These results suggest that plant polyphenols with antioxidant properties have distinct mechanisms in the suppression of oxidative stress induced in keratinocytes by different stimuli. Verbascoside and teupolioside are hence of potential interest in the protection of the skin from both environmental and inflammatory insults.