Roberta Di Caprio

Mechanistic target of rapamycin (mTOR) expression is increased in acne patients' skin.

Abbreviations: mTOR, Mechanistic target of rapamycin; mTORC, mTOR signalling complex; BCAA, branched-chain essential amino acid; 4E-BP1, the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1; SREBP-1, sterol response element-binding protein-1; S6K1, ribosomal protein S6 kinase 1; FOXO1, forkhead box protein O1; HOMAIR, homoeostatic model assessment of insulin resistance; GAGS, global acne grading system; HS, healthy skin; LS, lesional skin; NLS, non-lesional skin; RT-PCR, real-time polymerase chain reaction; IHC, immunohistochemistry; IF, immunofluorescence; ELISA, enzyme-linked immunosorbent assay; P-S6K1, phospho-S6-ribosomal protein.

Anti-inflammatory properties of low and high doxycycline doses: an in vitro study.

Doxycycline is used to treat infective diseases because of its broadspectrum efficacy. High dose administration (100 or 200 mg/day) is often responsible for development of bacterial resistances and endogenous flora alterations, whereas low doses (20–40 mg/day) do not alter bacteria susceptibility to antibiotics and exert anti-inflammatory activities. In this study, we wanted to assess the efficacy of both low and high doxycycline doses in modulating IL-8, TNF-α, and IL-6 gene expression in HaCaT cells stimulated with LPS. Three experimental settings were used, differing in the timing of doxycycline treatment in respect to the insult induced by LPS: pretreatment, concomitant, and posttreatment. Low doses were more effective than high doses in modulating gene expression of LPS-induced proinflammatory cytokines (IL-8, TNF-α, and IL-6), when added before (pretreatment) or after (posttreatment) LPS stimulation. This effect was not appreciated when LPS and doxycycline were simultaneously added to cell cultures: in this case high doses were more effective. In conclusion, our in vitro study suggests that low doxycycline doses could be safely used in chronic or acute skin diseases in which the inflammatory process, either constantly in progress or periodically recurring, has to be prevented or controlled.

IL-33 is regulated by TNF-α in normal and psoriatic skin

Interleukin-33 (IL-33) is the most recently discovered IL-1 family member. Considered an endogenous “alarmin” released by necrotic cells in response to tissue injury or damage, IL-33 is constitutively expressed in tissues exposed to the environment, where endothelial and epithelial cells constitute its major sources. Several findings reported that pro-inflammatory stimuli, such as IFN-γ and TNF-α, as well as IL-17, can induce IL-33 expression in normal human epidermal keratinocytes. In the present study, we deeply investigated the relation between IL-33 and TNF-α, by employing the whole skin as study model. TNF-α dose- and time-dependently induced IL-33 gene expression in ex vivo healthy skin organ culture. Similarly, TNF-α significantly increased IL-33 mRNA expression in normal human epidermal sheets. Moreover, IL-33 was enhanced in psoriatic skin and anti-TNF-α therapy was able to significantly reduce it. The biology of IL-33 is gaining in complexity, and this molecule is now known to have additional roles beyond its original description. In particular, we can assess that IL-33 is regulated by TNF-α in normal and psoriatic skin.

Biologics that inhibit the Th17 pathway and related cytokines to treat inflammatory disorders

ABSTRACT Introduction: Advances in the understanding of TNF-α and IL-17 synergistic functions have recently led to the concept that patients who do not respond or who respond inadequately to TNF-α inhibitors may have IL-17-driven diseases, opening up the way for a new class of therapeutic development: Th17-inhibitors. Areas covered: In this review, the authors discuss the central role that the IL-23/Th17 axis plays in the pathogenesis of several inflammatory diseases, such as psoriasis, highlighting its position as a relevant therapeutic target. In particular, the authors start by giving a brief historical excursus on biologic agent development up until the success of TNF-α inhibitors, and continue with an overview of IL12/23 pathway inhibition. Next, they describe Th17 cell biology, focusing on the role of IL-17 in host defense and in human immune-inflammatory diseases, discussing the use and side effects of IL-17 inhibitors. Expert opinion: The IL-23/Th17 signaling pathway plays a central role in the pathogenesis of several inflammatory diseases, such as psoriasis. Recent data has demonstrated that biologics neutralizing IL-17 (ixekizumab, secukinumab) or its receptor (brodalumab) are highly effective with a positive safety profile in treating moderate to severe psoriasis, offering new treatment possibilities, especially for patients who do not respond adequately to anti-TNF-α therapies.

The modulatory effect of ellagic acid and rosmarinic acid on ultraviolet-B-induced cytokine/chemokine gene expression in skin keratinocyte (HaCaT) cells

Ultraviolet radiation (UV) induces an increase in multiple cutaneous inflammatory mediators. Ellagic acid (EA) and rosmarinic acid (RA) are natural anti-inflammatory and immunomodulatory compounds found in many plants, fruits, and nuts. We assessed the ability of EA and RA to modulate IL-1β, IL-6, IL-8, IL-10, MCP-1, and TNF-α gene expression in HaCaT cells after UVB irradiation. Cells were treated with UVB (100 mJ/cm2) and simultaneously with EA (5 μM in 0.1% DMSO) or RA (2.7 μM in 0.5% DMSO). Moreover, these substances were added to the UVB-irradiated cells 1 h or 6 h before harvesting, depending on the established UVB-induced cytokine expression peak. Cytokine gene expression was examined using quantitative real time polymerase chain reaction. RA produced a significant reduction in UVB-induced expression of IL-6, IL-8, MCP-1, and TNF-α when applied at the same time as irradiation. EA showed milder effects compared with RA, except for TNF-α. Both substances decreased IL-6 expression, also when applied 5 h after irradiation, and always produced a significant increase in UVB-induced IL-10 expression. Our findings suggest that EA and RA are able to prevent and/or limit the UVB-induced inflammatory cascade, through a reduction in proinflammatory mediators and the enhancement of IL-10, with its protective function.

IL-36 cytokines are increased in acne and hidradenitis suppurativa

Interleukin (IL)-36 cytokines are new members of the IL-1 family, which include pro-inflammatory factors, IL-36α, IL-36β and IL-36γ, and a natural receptor antagonist IL-36Ra. Over recent years, much has been learned on their important functions in the regulation of immune response and, especially, on their role in many inflammatory skin diseases. However, to date, no data have been reported on their possible involvement in acne and hidradenitis suppurativa (HS). Here, we have shown that IL-36α, IL-36β, and IL-36γ are increased in lesional skin of acne and HS, highlighting their possible pathogenetic contribution to these two skin conditions. In contrast, IL-36Ra (the anti-inflammatory member of IL-36 sub-family) was increased just in psoriasis, suggesting that an imbalance in IL-36/IL36Ra functions could play a role in the phenotype of skin damage. One of the consequences of this imbalance may be the increased induction of IL-8 that we found higher in acne, HS, and ACD respect to psoriasis.

Mechanistic target of rapamycin complex 1 is involved in psoriasis and regulated by anti-TNF-α treatment

The mechanistic target of rapamycin (mTOR) acts through two distinct signalling complexes, known as mTOR complex (mTORC) 1 and mTORC2. One of the main upstream regulator of mTORC1 is tumour necrosis factor (TNF)-α, an important pro-inflammatory cytokine involved in psoriasis. When active, mTORC1 phosphorylates and activates its downstream effector ribosomal protein S6 kinase 1 (S6K1) which is also found increased in psoriasis. Thus, the aim of the study was to assess the involvement of mTORC1 signalling as well as to shed a light on the possible relationship between mTORC1 and TNF-α in psoriasis. Our results showed that mTOR, S6K1 and, in particular, its active form P-S6K1 were increased in psoriatic plaque, suggesting a specific involvement of mTORC1 pathway in the disease. Moreover, for the first time, we reported that P-S6K1 was completely abolished after antiTNF-α therapy, indicating a stronger action of anti-TNF-α agent on mTORC1. A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. Background Mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase, regulates a variety of cellular functions, including survival and cell proliferation as well as innate and adaptative immune response (1,s1). Interfering with other proteins it forms two complexes: mTOR signalling complex (mTORC)1, responsible for the increase in cell growth and proliferation, and mTORC2, involved in the regulation of cell polarity and phosphorilation of cytoskeleton actin fibers (1). These complexes recognize many upstream signals and downstream effectors. One of the main upstream modulator of mTORC1 is tumour necrosis factor (TNF)-α (1,2). In particular, TNF-α suppresses tuberous sclerosis (TSC)1 resulting in mTORC1 activation (3). When active, mTORC1 phosphorylates and activates its downstream effector ribosomal protein S6 kinase 1 (S6K1) involved in cell proliferation (1). Some inflammatory skin diseases are characterized by elevated levels of mTOR (4-6) and there are few reports indicating an increase of mTOR and S6K1 in psoriasis too (4, 7). Up to now, the link between mTORC1 and TNF-α in psoriasis has not been yet elucidated even though it is well known that TNFα is an upstream signal of mTORC1 and also one of main protagonists of the disease. Question addressed The aim of this study was to assess the involvement of mTORC1 signalling as well as to shed a light on the possible relationship between mTORC1 and TNF-α in psoriasis. Experimental design The study population comprised 28 psoriatic patients and 10 healthy controls. Out of 28 patients five underwent to anti-TNF-α therapy with adalimumab for 16 weeks, 6 (of which one non responder) with etanercept for 12 weeks, and 2 received acitretin for 12 weeks. Skin A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. biopsies were taken from lesional skin (LS) and non-lesional skin (NLS); real-time polymerase chain reaction (RT-PCR) (primers listed in ST-1) and immunohistochemistry was performed to assess mTOR gene and protein expression, respectively. Immunofluorescence was performed to assess S6K1 and its phosphorylated active form P-S6K1 protein expression. Moreover, gene expression of S6K1 and of the main psoriasis-signature cytokines was assessed through RT-PCR. Details in SM. Results mTOR gene expression was significantly increased in NLS as well as LS of psoriatic patients compared to control skin from healthy donors (Fig. 1a). Moreover, mTOR positive cells were only weakly expressed in the epidermis from healthy subjects and markedly expressed thorough all epidermal layers in psoriatic NLS (Fig. 1c) as well as LS (Fig. 1d). Thus, our results concur with previous data (4, 7), that activation of mTOR signalling might be involved in psoriasis pathogenesis and confirm its increasing relevance in skin inflammatory process (8). Furthermore, no differences were detected in gene expression of the downstream effector S6K1 between healthy and psoriatic skin (SF-1). Protein analysis, through immunofluorescence, revealed that S6K1 was present either in healthy skin (SF-2) or at psoriatic plaque level, even if with a stronger staining intensity for the last one (fig. 2a). This difference was much more evident regarding the activated form, P-S6K1 (fig. 2e) highlighting the specific involvement of mTORC1 complex in psoriasis. This data was confirmed also by western blot (SF-3). Next, we sought to analyse the relationship between this complex and TNF-α, so we looked at S6K1 as well as P-S6K1 before and after anti-TNFα treatment. S6K1 was strongly downregulated after 12 weeks (W12) of etanercept therapy, whereas P-S6K1 resulted completely absent (fig. 2b and f). Western blot analysis confirmed this result showing a strong reduction of P-S6K1 after treatment (SF-3). To notice, S6K1 and A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. mostly P-S6K1 were still significantly expressed in LS from a non responder patient (fig. 2c and g). Moreover, treatment with acitretin didn’t completely decrease S6K1 as well as PS6K1 compared to anti-TNF-α treatment (fig. 2d and h). Hence, to investigate how the antiTNF-α therapy influenced the inflammatory scenario in psoriasis, we performed gene expression of the main psoriasis-signature cytokines before and after 16 weeks (16W) of adalimumab treatment (ST-2). We reported that all cytokines were downregulated, except for IL-1Ra and IL-36β. Taken together these data suggest that the blockage of TNF-α improved psoriatic inflammatory milieu plus a more specific downregulation of mTORC1 than traditional systemic treatments. Conclusions In our study, mTOR was significantly increased in NLS as well as LS of psoriatic subjects, both at gene and protein level, indicating that it is involved in psoriasis pathogenesis. Our results are in line with previous studies, which found elevated mTOR levels in psoriatic LS and suggest a possible role of mTOR pathway in regulating keratinocytes immune response and cytokine production in psoriasis (2). We found that S6K1 and, in particular, its activated form P-S6K1, were slightly present in healthy skin but strongly at psoriatic plaque level in according with Burger et al. (7), suggesting the specific involvement of mTORC1 complex in psoriasis. The overexpression of the mTORC1 play an important role in the pathogenesis of this disease (s2), representing a potential target for the therapy. It is known that metformin is an inhibitor of mTORC1 complex and it represents a new approach to treat male subjects with resistant acne (s3, s4). However, recent evidences showed that metformin is a useful add-on drug also for treatment of psoriasis: indeed, it has been demonstrated that metformin inhibits in vitro the secretion of pro-inflammatory cytokines from human keratinocytes via mTOR signalling A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. (s5, s6). In this contex, psoriasis becomes part of the wide group of “diseases of civilization” related to mTORC1, such as acne and insulin resistance (9, s7-9). In this study, we showed, for the first time, that P-S6K1 was completely abolished after 12 weeks of etanercept therapy, whereas it was still expressed after systemic retinoid treatment, indicating a stronger action of anti-TNF-α agent on mTORC1. Previous evidence showed that silencing mTOR leads to the inhibition of TNF-α-induced P-S6K1 (s10). Our data reinforced the hypothesis that TNF-α exerts its effects acting on mTORC1 complex. In conclusion, mTORC1 pathway is modulated by anti-TNF-α treatment, highlighting a possible new mechanism by which TNF-α inhibition improves psoriasis. References 1. Laplante M, Sabatini DM. Cell 2012: 149: 274-293. 2. Young CN, Koepke JI, Terlecky LJ, et al. J Invest Dermatol 2008: 128: 2606-2614. 3. Lee DF, Kuo HP, Chen CT, et al. Int J Mol Med 2008: 22: 633-638. 4. Balato A, Di Caprio R, Lembo S, et al. Eur J Inflamm 2014; 12: 341-350. 5. Monfrecola G, Balato A, Caiazzo G, et al. J Eur Acad Dermatol Venereol 2015: doi: 10.1111/jdv.13233. 6. Monfrecola G, Lembo S, Caiazzo G et al. Exp Dermatol 2016: 25: 153-155. 7. Buerger C, Malisiewicz B, Eiser A, et al. Br J Dermatol 2013: 169: 156-159. 8. Leo MS, Sivamani RK. Arch Dermatol Res 2014: 306: 861-871. 9. Melnik BC, Zouboulis CC. Exp Dermatol 2013: 22: 311-315.

Psoriatic cutaneous inflammation promotes human monocyte differentiation into active osteoclasts, facilitating bone damage

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathy that can be associated with focal bone erosions. Psoriasis usually precedes the psoriatic arthritis onset by an average of 10 years, but this relation is not yet fully elucidated. Pro‐inflammatory cytokines, such as IL‐33, OPN, IL‐17, and TNF‐α are involved in both psoriasis and PsA pathogenesis as well as in bone homeostasis. In this study, we have demonstrated that IL‐33, OPN, IL‐17, and TNF‐α induced the release of a wide range of pro‐osteoclastogenic factors from the skin, such as RANKL, that promote monocyte differentiation in osteoclasts. The addition of osteoprotegerin, a RANKL inhibitor, to monocyte cultures treated with supernatant from stimulated skin did not completely deplete osteoclast formation, suggesting that skin produced several additional pro‐osteoclastogenic mediators, which could act in a RANKL‐independent manner. Moreover, we have found that RANKL serum levels as well as osteoclast number and activity in psoriatic patients with and without arthritis, was influenced by severity of cutaneous disease. Our data demonstrate that psoriatic cutaneous inflammation contributes to bone damage.

Psoriasis in children with type 1 diabetes: A new comorbidity to be considered?

Type 1 diabetes mellitus (T1DM) is the most common endocrine disease of childhood, due to an immune-mediated destruction of insulin-producing pancreatic b-cells. Comorbidities are frequent in T1DM and can severely affect clinical management [1]. To date, vitiligo and alopecia areata are the only described cutaneous comorbidities associated with T1DM, while psoriasis is usually associated with type 2 diabetes mellitus [2]. Psoriasis is an inflammatory skin disease affecting up to 2% of children in Europe and, in particular, 2.1% in Italy [3, 4]. To date, psoriasis prevalence in T1DM is not well known. Aim of our study was to investigate prevalence of psoriasis in T1DM patients in pediatric age.

IL-26 in allergic contact dermatitis: Resource in a state of readiness

In this study, we investigated the role of IL‐26 in allergic contact dermatitis (ACD), highlighting its’ contribute in the cytotoxic mechanism responsible for the tissue injury. IL‐26 is a signature Th17 cytokine, and immune cells are its predominant sources. Recently, it has shown that Th17 cell‐derived‐IL‐26 functions like an antimicrobial peptide. Here, we hypothesized that IL‐26 could be involved in cytotoxicity mechanism that underlies ACD. Indeed, we have attributed a role to IL‐26 in this context, through PBMC cytotoxicity assays vs HaCat. To demonstrate that IL‐26 was effectively involved in this activity, we performed the assay using transfected ACD PBMCs by siRNA for IL‐26. Indeed, we demonstrated that these cells were less able to kill keratinocytes compared with ACD PBMCs (P < .01). In conclusion, our findings support the idea that this emergent cytokine, IL‐26, is implicated in the killing mechanisms of KC observed during ACD.