G. Caiazzo

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.

Mammalian target of rapamycin, insulin resistance and hidradenitis suppurativa: a possible metabolic loop

mosomal STS was detectable by fluorescent in situ hybridization analysis, suggesting no gross deletion of the STS locus. Suspecting the possible involvement of minor mutation(s), all the 10 exons of STS were sequenced, revealing that each patient had different single nucleotide substitutions in close proximity within exon 7. Patient 1 had a T>G substitution at nucleotide 1049, changing a valine to a glycine (c.1049T>G, p.V350G; Fig. 1e), whereas patient 2 had a G>A substitution at nucleotide 1075, changing a glycine to an arginine (c.1075G>A, p.G359R; Fig. 1f). The possibility of polymorphism was excluded using a world-wide SNPs database. To date, 20 point mutations of STS, including those reported here, have been identified in XLRI (Fig. 2b). Of these, 15 (75%) were missense mutations and the remaining 5 (25%) were nonsense mutations. The majority of these mutations (18/20, 90%) were localized exclusively within the downstream of exon 7, supporting the hypothesis that this region is essential for in vivo enzyme activity. More precisely, STS protein has 26 substrate binding and 10 catalytic sites that are highly homologous to those in other sulfatase family members, aryl sulfatase A and B. These characteristic amino acids are indispensable for maintenance of the tertiary structure, substrate binding/scaffolding and catalytic activity. Nevertheless, none of these functional amino acids, except two substrate binding sites (T165 and E349), relate to the 20 STS point mutations. Combining these assumptions with the considerably high incidence of complete STS deletion in XLRI, it is conceivable that STS point mutations sufficiently affect the baseline enzymatic activity to elicit the development of the disease. To look for mutation-specific conformational alterations, we aligned the secondary and tertiary structures of STS (Fig. 2c,d). It comprises tandem transmembrane domains across the cytoplasmic domain within exon 5, two post-translational glycosylation sites, five Ca binding sites and six disulphide bonds, thereby forming the ‘mushroom-like’ configuration embedded into the lumenal membrane of the endoplasmic reticulum. Interestingly, all mutations were clustered within the lumen side, but not within the transmembrane or cytoplasmic domains (Fig. 2d), indicating that the lumenal portion of STS is highly sensitive to the transversion of a single amino acid, albeit to a lesser extent with basic secondary structures (e.g. a-helices; Fig. 2b). Collectively, STS point mutations demonstrate restricted localization, causing efficient impairment of the corresponding enzyme activity, and are more unlikely to be responsible for the phenotypic heterogeneity in XLRI subjects.

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.

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.

The effects of etanercept on replication, proliferation, survival, and apoptosis markers in moderate to severe psoriasis

References 1 Bhushan P, Manjul P, Baliyan V. Flagellate dermatoses. Indian J Dermatol Venereol Leprol 2014; 80: 149–152. 2 Schulman JM, McCalmont TH, Shinkai K. Fulminant dermatomyositis with flagellate erythema. J Drugs Dermatol 2011; 10: 902–904. 3 Dupre A, Viraben R, Bonafe JL, Touron P, Lamon P. Zebra-like dermatomyositis. Arch Dermatol 1981; 117: 4. 4 Jara M, Amerigo J, Duce S, Borbujo J. Dermatomyositis and flagellate erythema. Clin Exp Dermatol 1996; 21: 440–441. 5 Nousari HC, Ha VT, Laman SD, Provost TT, Tausk FA. “Centripetal flagellate erythema”: a cutaneous manifestation associated with dermatomyositis. J Rheumatol 1999; 26: 692–695. 6 Ferrer M, Herranz P, Manzano R, Fernandez-Diaz ML, de Lucas R, Casado M. Dermatomyositis with linear lesions. Br J Dermatol 1996; 134: 600–601. 7 Kimyai-Asadi A, Tausk FA, Nousari HC. A patient with dermatomyositis and linear streaks on the back. Centripetal flagellate erythema (CFE) associated with dermatomyositis. Arch Dermatol 2000; 136: 667. 8 Gomez CP, Sanchez-Aguilar D, Pereiro M Jr, Toribio J Flagellate erythema and dermatomyositis Clin Exp Dermatol 1998; 23: 239–240 9 Molina-Ruiz AM, Romero F, Carrasco L, Feltes F, Haro R, Requena L Amyophatic dermatomyositis presenting as a flagellated skin eruption with positive MDA5 antibodies and thyroid cancer: a real association? Clin Exp Dermatol 2015; 40: 887–890. 10 Watanabe T, Tsuchida T ‘Flagellate’ erythema in dermatomyositis Dermatology 1995; 190: 230–231 11 Requena C, Alfaro A, Traves V, Nagore E, Llombart B, Serra C, et al. Paraneoplastic dermatomyositis: a study of 12 cases Actas Dermosifiliogr 2014; 105: 675–682 12 Ito K, Imafuku S, Hamaguchi Y, Fujimoto M, Nakayama J Case report of anti-transcription intermediary factor-1-gamma/alpha antibody-positive dermatomyositis associated with gastric cancer and immunoglobulin G4positive pulmonary inflammatory pseudotumor J Dermatol 2013; 40: 567–569 13 Parodi A, Caproni M, Marzano AV, De Simone C, La Placa M, Quaglino P, et al. Dermatomyositis in 132 patients with different clinical subtypes: cutaneous signs, constitutional symptoms and circulating antibodies Acta Derm Venereol 2002; 82: 48–51 14 Loo HV, Oon HH Flagellate dermatitis following consumption of shiitake mushroom Dermatol Reports 2011; 3: e21 15 Dalakas MC Inflammatory muscle diseases N Engl J Med 2015; 372: 1734–1747

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.

Anti-TNF-α therapy modulates mTORC1 signalling in hidradenitis suppurativa

Hidradenitis Suppurativa (HS) is a chronic, inflammatory, recurrent and debilitating skin disease of the hair follicle, which usually occurs after puberty with painful, deep-seated, inflammatory lesions in the apocrine gland-bearing areas of the body, most commonly in axillary, inguinal and anogenital regions This article is protected by copyright. All rights reserved.

Psoriasis, Cardiovascular Events, and Biologics: Lights and Shadows

Nowadays, it is well established a link between psoriasis and cardiovascular (CV) diseases. A series of different overlapping mechanisms including inflammation, homeostasis dysregulation, and genetic susceptibility are thought to underlie this association. Advances in understanding the molecular patterns involved in the complex scenario of psoriasis have highlighted a tight correlation with atherosclerosis. Indeed, common profiles are shared in term of inflammatory cytokines and cell types. In the last decade, the management of psoriasis patients has been revolutionized with the introduction of biological therapies, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-12/23, and IL-17 inhibitors. In clinical setting, the effectiveness of these therapies as well as the incidence of CV events is related to the type of biologics. In particular, anti-TNF-α agents seem to reduce these events in psoriasis patients whereas anti-IL-12/23 agents related CV events reduction still remain to clarify. It has to be taken into account that IL-12/23 inhibitors have a shorter post-marketing surveillance period. An even more restricted observational time is available for anti-IL-17 agents. IL-17 is associated with psoriasis, vascular disease, and inflammation. However, IL-17 role in atherosclerosis is still debated, exerting both pro-atherogenic and anti-atherogenic effects depending on the specific context. In this review, we will discuss the differences between the onset of CV events in psoriasis patients, referred to specific biological therapy and the underlying immunological mechanism. Given the development of new therapeutic strategies, the investigation of these inhibitors impact on heart failure outcome is extremely important.

Mesenchymal stem cells participate to inflammatory skin process of atopic dermatitis

An important new involvement of mesenchymal stem cells (MSCs) in the complex scenario of atopic dermatitis (AD) is addressed by Orciani et al. in the current issue of the BJD. In the last decade, researchers have investigated deeply on possible MSC involvement in the onset of different diseases. Among different types of adult stem cells, MSCs have been studied extensively and applied in several scientific and clinical fields, including dermatology. MSCs are undifferentiated, multipotent and self-renewing cells that reside in many adult tissues. They have several major characteristics including (i) the ability of homing to areas of inflammation and tissue damage, such as wounds or tumours; and (ii) the capacity to modulate innate and acquired immune responses. Interestingly, Orciani et al. have previously hypothesized that the psoriatic microenvironment may induce resident skin MSCs to produce angiogenic and proinflammatory mediators, contributing to the development of skin lesions. Moreover, it has been shown by Liu et al. that MSCs obtained from psoriatic skin lesions decreased the inhibitory effects on T-cell proliferation. An aberrant immune response, particularly by T cells, is also typical of AD, which is characterized by a marked imbalance of T-helper (Th)2 vs. Th1/Th17, especially in the early phase whereas a mixed Th1/Th2 pattern is found at the chronic stage. Phenotypic analysis of peripheral blood mononuclear cells derived from patients with AD demonstrated a marked increase in the interleukin (IL)-17CD4 T-cell population compared with healthy controls. The highest percentage of IL17-producing cells was found in severe AD, suggesting a direct correlation between the presence of Th17 cells and severity of the disease. In their original article in the current issue of the BJD, Orciani et al. convey their valuable experience with MSCs, evaluating whether a Th1/Th2 imbalance could already be detected in undifferentiated cells of AD. They have analysed the expression profile of 22 genes and proteins related to Th1, Th2 and Th17 cytokines/chemokines in MSCs from patients with AD (AD-MSCs) compared with healthy controls (C-MSCs). They have shown that 14 of 22 genes related to the Th1/Th17 pathway were upregulated whereas three of 22 genes related to the Th2 pathway were downregulated in AD-MSCs compared with C-MSCs. Moreover, they showed in this paper that the profile of AD-MSCs retraces the Th1/Th17 cell environment observed in differentiated cells of chronic AD. Overall, the data from this manuscript provide novel insights into AD pathogenesis, suggesting that the inflammatory process detected in the MSCs has a determining role in the typical Th1/Th2 imbalance. In summary, the findings reported by Orciani et al. in the current issue of the BJD have highlighted the current understanding of the interaction between MSCs and the inflammatory response. Finally, we would like to emphasize that although there is much evidence supporting the effectiveness of MSCs in clinical dermatological therapy, such as wound healing, it is important to underline that the inflammatory microenvironment might also induce transplanted MSCs to behave abnormally, modulating any possible beneficial effects. Future studies will need to elucidate the role of stem cells in the control of inflammatory skin diseases.