Cytokines as therapeutic targets in skin inflammation.

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Contents lists available at ScienceDirect

Cytokine & Growth Factor Reviews journal homepage: www.elsevier.com/locate/cytogfr

Mini review

Cytokines as therapeutic targets in skin inflammation Miriam Wittmann a,b,c,*, Dennis McGonagle a,b, Thomas Werfel d a

Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds LS7 4SA, UK NIHR Leeds Musculoskeletal Biomedical Research Unit, Chapel Allerton Hospital, Leeds LS7 4SA, UK c Centre for Skin Sciences, University of Bradford, Bradford BD7 1DP, UK d Department for Dermatology and Allergy, Hannover Medical School, Carl-Neuberg-Str 1, 30625 Hannover, Germany b

A R T I C L E I N F O

A B S T R A C T

Article history: Available online xxx

This review focuses on treatment targets for the most common inflammatory skin diseases, eczema and psoriasis with an emphasis on cytokines expressed in the uppermost layer of the skin which is easily accessible for diagnostic and therapeutic approaches. Recently, a significant body of research has highlighted the influence of the skin barrier and the patients’ microbiome on skin inflammatory responses and we will comment on their impact on mediator regulation. Itch is a prominent dermatology symptom which is influenced by cytokines and can via itch–scratch cycle impact on the skin barrier and mediator expression associated with damage. Taking the contribution of pruritus and superficial skin damage into account, we address cytokines as targets for stratified treatment approaches in subgroups of eczema and psoriasis. ß 2014 Elsevier Ltd. All rights reserved.

Keywords: Psoriasis Atopic dermatitis Antimicrobial peptides Microbiome Pruritus Keratinocytes

1. Introduction Non-infectious, chronic inflammatory skin diseases such as eczema (up to 20% of the population in certain age groups) and psoriasis (2–4%) are very common. These are chronic, non curable conditions which often show a relapsing course. Inflammatory skin diseases have considerable socioeconomic impact and can significantly impair the patients’ quality of life. Itch, which still lacks effective therapy, is the most disturbing symptom for the patient with eczema and is also a considerable problem for many psoriasis sufferers. Furthermore, the quality of life and self-esteem of patients are affected in particular when the inflammatory condition presents in visible body parts including hair loss, scars and red (erythema) and flaky lesions. Despite advances, a need to understand the underlying immunopathogenesis of ezema and psoriasis exists, as treatment options can be limited due to insufficient efficacy of existing drugs in subgroups of patients or contraindications for/side-effects of systemic or ultraviolet light treatment. Many of the available

Abbreviations: AMP, antimicrobial peptides; AD, atopic dermatitis; HF, hair follicle; FLG, filaggrin; ESC, epidermal stem cells; CE, cornified envelope. * Corresponding author at: University of Leeds, Chapel Allerton Hospital, LMBRU, Leeds LS7 4SA, UK. Tel.: +44 113 392 44 83. E-mail addresses: [email protected], [email protected] (M. Wittmann), [email protected] (D. McGonagle), [email protected] (T. Werfel).

topical treatment options are time consuming to apply, perceived as unpleasant (in particular if in a ‘‘greasy’’ formulation) or complicated for the patient (e.g. if different topicals have to be applied to different body sites or disease ‘‘stages’’). As a consequence, adherence to topical treatment is, for a substantial number of patients, very poor (outlined in [1]) and usually better for systemic than topical medication. The recent addition of anti-cytokine biologics to our therapeutic portfolio has largely increased the spectrum of available treatments. New drugs interfering with cytokine and cytokine signalling have shed light on mediator regulation and expression in healthy and inflamed skin. Therapeutics which target a specific mediator in the skin, in particular when delivered topically, hold the promise of less systemic side effects than widely used immunosuppressants such as cyclosporine A or metothrexate. Inflammatory skin diseases including psoriasis and eczema have the following in common. 1. There is a great heterogeneity in how patients respond to a given topical or systemic therapy. Clinical evidence as well as genetic and molecular findings suggest that these inflammatory skin diseases are composed of different subgroups. Understanding distinct ‘‘inflammatory’’ subtypes (or, as previously referred to, ‘‘endotypes’’/‘‘molecular’’ subtypes) and drug responses (pharmacogenetic) will be key for successfully treating all patients affected by a phenotypically similar disease. For the common inflammatory skin diseases research into and approaches for stratified/personal medicine are underway [2–6].

http://dx.doi.org/10.1016/j.cytogfr.2014.07.008 1359-6101/ß 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Wittmann M, et al. Cytokines as therapeutic targets in skin inflammation. Cytokine Growth Factor Rev (2014), http://dx.doi.org/10.1016/j.cytogfr.2014.07.008

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2. Skin barrier properties are closely involved in the pathogenesis and course of the disease. Barrier changes influence cytokine expression and vice versa. 3. The skin microbiome and antimicrobial molecules influence cytokine mediators and vice versa. 2. Skin barrier and cytokines The skin is an important barrier organ and protects the body from environmental harm and from water loss. The top epithelial layer of the skin, the epidermis, is composed of keratinocytes which undergo proliferation and differentiation processes resulting in constant renewal of the upper skin layers with an average turnover time of about 3 weeks. Keratinocytes derived from epidermal stem cells (ESC) in the basal layer and the reservoir of the hair follicle (HF) undergo differentiation processes resulting in their ‘‘cornification’’ (e.g. living cells turn into ‘‘scales’’ which are ultimately shedded (=desquamation) from the surface). A wide range of extracellular molecules, melanocytes, tissue resident leukocytes (dendritic cell (DC) subtypes, skin resident lymphocytes), soluble mediators (antimicrobial peptides (AMP), lipid mediators, cytokines) and skin commensals contribute to the epidermal barrier. Barrier alterations are probably best studied in atopic dermatitis (AD) [7] which is associated with very dry skin due to loss of function mutation in filaggrin in a substantial number of cases [8,9]. Filaggrin (FLG) is expressed in the upper layer of the epidermis and has multiple properties leading to strengthening of the outermost skin barrier. FLG breakdown products add to formation of natural moisturising factors and decrease of pH. Lack of FLG breakdown products favours transepidermal water loss, allergen penetration and skin colonisation with Staphylococcus S. aureus. FLG seems also involved in sphingomyelinase secretion. This enzymes cleaves sphingomyelin which is a receptor for alpha toxin secreted by some S. aureus strains (see below). The skin pH influences the activity of proteases (serine proteases such as kallikrein 5 and 7) involved in desquamation but also activation of cytokines. Unleashed skin protease activity is known to lead to skin inflammation (as seen in Netherton Syndrome which presents with a genetically determined deficiency in the protease inhibitor Lympho-epithelial Kazal-type-related inhibitor (LEKTI) which is encoded by the SPINK5 gene). All IL-1 family members expressed in the skin are processed by proteases and cleavage is required to result in bioactive IL-1b, IL-18 and IL36. The inflammasome dependent caspase 1 processing is well described for IL-1b and IL-18. However, there is good evidence for pro-forms of IL-1 family members to be present outside the cell in particular in ‘‘damaged’’ tissue (e.g. by scratching) and skin proteases can process IL-1 and IL-18 into active forms [10]. The link between low FLG expression and increased pH influencing proteases and inflammatory mediators has been highlighted by Elias et al. [11] and the notion that breakdown of barrier properties could lead to eczema symptoms has been described in the context of the ‘‘outside-in’’ hypothesis. A surprisingly large number of cytokines have been described to reduce the expression, production and function of FLG. Strong inhibitors of FLG expression are the Stat6 signalling cytokines IL-4 and IL-13 [12] which also reduce the expression of other late cornified envelope (LCE) molecules hornerin, involucrin and loricrin and the production of lamellar bodies [13,14]. Lamellar bodies are secretory organelles responsible for secretion of lipids, protease inhibitors, and AMP, in the upper keratinocyte layers. IL4/IL-13 also impair caspase-14 synthesis required for the breakdown of FLG into natural moisturising factors [15]. They can induce kallikrein 7 production by keratinocytes which promotes skin desquamation by degrading corneodesmosomal

proteins. Apart from IL-4 and IL-13, we [16] found IL-33, an IL-1 family member with type 2 response properties, to reduce FLG expression in the epidermis. Of interest, thymic stromal lymphopoietin (TSLP), a keratinocyte derived chemokine involved in DC mediated type 2 immune responses [17] seems increased in the context of FLG knockdown. It remains to be shown to which extent T cell derived cytokines IL-31, OSM, IL-22 [18] and IL-17 act via secondary, keratinocyte produced, molecules for their recognised downregulatory action on FLG expression. These could include IL33 but also TNFa [19] and other c-Jun N-terminal kinase (JNK) activators. For IL-31, which significantly influences keratinocyte proliferation and differentiation including abrogation of FLG expression, Cornelissen et al. [20] have shown that endogenous IL-20 in combination with IL-24 mediate some of the epidermal IL31 effects. The functional effect of IL-22 on FLG may as well involve IL-20, known to be induced by IL-22 in keratinocytes [18]. While a number of different mediators impact on FLG expression, the quality of their response regarding the overall epidermal barrier property is certainly different as e.g. Kim et al. [19] have shown that TNF does not act on involucrin reduction as observed by Stat6 signalling cytokines. Important differences also include proliferation/epidermal thickness which is reduced in the context of IL-31 but enhanced by OSM [21] and IL-22. FLG mutations are common in the Western European population with about 10% carrying a heterozygous loss-of-function mutation. Many of those affected show ‘‘only’’ dry skin (xerosis) as symptoms but may be more prone to skin inflammatory responses upon exposure to additional trigger factors such as exposure to skin irritants (soap, water), contact allergens, protein allergens in individuals prone to allergic reactions (atopy) or mechanical stress/scratching. However, among patients with AD up to 50% carry FLG loss-of-function mutations [22]. In addition to mutation in FLG, functional alterations in tight junction proteins, which regulate paracellular permeability in the skin layers below the outermost stratum corneum, have been found in AD skin [23]. Antimicrobial peptides (AMP) [24] are important for the immunological barrier and antimicrobial defence and significant differences regarding AMP production exist between AD and psoriasis ([25] and references therein) AMPs show in many cases also chemokine [26] and/or damage associated molecular pattern (DAMP) and thus proinflammatory properties (reviewed in [27]). Keratinocyte produce a range of AMP including S100 molecules (S100A7 – psoriasin; S100A8/S100A9 – calprotectin), RNase7, free fatty acides and beta defensins (hBD) (reviewed in [28]). Many AMPs, including hBD2/3 and LL37 are expressed upon disruption of the barrier and/or pathogen invasion and are involved in the onset of inflammation e.g. in AD [29] and rosacea [30]. LL37 gains activity by processing of the cathelicidin precursor molecule by kallikrein 5 [30]. Constitutively expressed S100A7 and RNase7 can be further upregulated upon exposure to pathogens or cytokines. IL-22, IL-17 and TNF induce the production of AMPs in keratinocytes. These cytokines are overexpressed in psoriasis [18,31] which – although clearly showing an impaired skin barrier – almost never shows super-infection or clinical problems resulting from skin colonisation with S. aureus which is a very frequent problem in AD. Indeed, AD is characterised by complications due to S. aureus which plays a role in maintaining the skin inflammatory response. AD skin preferentially harbours exotoxin and biofilm-producing [32] S. aureus strains. Alpha toxin and enterotoxins with superantigens properties (e.g. TSST, SEB, SEA) have been shown to reach lower layers of the epidermis and even the dermal compartment. These potent toxins contribute to T cell activation and cytokine production including the itch promoting IL-31 (and upregulation of its receptor in the epidermis) [33,34] and IFNg which supports the development of chronic eczema [34–36]. Alpha toxin can also increase cell death in non-differentiated keratinocytes in the


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context of Stat6 signalling cytokines by decreasing levels of sphingomyelinase [37]. This enzyme reduces the binding sites for alpha toxin on the cell surface and increased sphinomyelinase activity can protect keratinocytes against alpha toxin mediated death. Type 2 associated cytokines IL-4, IL-13 are overexpressed in the blood compartment and in acute skin lesions in AD individuals and negatively influence the expression and induction of some AMPs and in particular hBD2/3 (reviewed in [11]). Thus Stat6 signalling cytokines and to some degree the type 2 response associated cytokine IL-33 [38] reduce both, the expression of FLG and inducible AMPs which in combination may favour S. aureus superinfection/colonisation. Of note, an increase in AMP expression is clearly seen in inflamed eczema skin, however this may be insufficient to prevent infection with gram-positive bacteria [39]. The AMP inducing IL-22 drives hallmark features of psoriasis including disturbed, premature differentiation of keratinocytes and defective keratinisation with altered formation of the cornified envelope (CE) resulting in impaired skin barrier. There is an emerging role for IL-22 (and related IL-20, IL-19, IL-24) in wound healing, tissue regeneration and repair processes (reviewed in [18]) in both the skin and other epithelial barrier organs (gut, lung) and controlling endogenous AMP expression seem to be part of this function. Accordingly, it has been suggested that hidradenitis suppurativa (acne inversa), a common disease with purulent inflammation in skin fold areas, was associated with low expression of IL-22 and the IL-22/IL-20 inducible hBDs [40]. There is good evidence to propose that the commensal S. epidermidis contributes to the skin barrier by acting against colonisation of potentially pathogenic microbes and overgrowth of opportunistic pathogens (reviewed in [41]). S. epidermidis derived molecules upregulate keratinocyte AMP (hBD2/3, RNAse7) production and prime epidermal cells for responses against S. aureus [41]. Staphylococcal TLR2 stimulation may be involved in the steady state production of AMPs in keratinocytes and enhance resistance to infection. It is noteworthy in this context that weaker response to TLR2 stimulation is found in AD derived leukocytes and keratinocytes [42,43] and one could speculate that there is a failure of the commensal flora to properly ‘‘prime’’ the innate skin immune surveillance in this condition. Thus resident commensals are necessary for optimal skin immune fitness for which IL-1 pathway activation seems to play a role [44]. It is now recognised that the cutaneous microbiota shows a wide interpersonal variability. Only few data are yet available regarding how different skin microbioms may be linked to inflammatory diseases. However, with the wider availability of DNA sequencing techniques this is a rapidly developing field. Sequencing approaches are needed as most cutaneous microbial species are difficult or impossible to culture in vitro. The presence of up to 300 different bacteria species on our skin depends on age, sex [45] and environmental factors (lifestyle, geographical location). Skin bacteria differ between different body sites [46] and change over time [47]. Aberrant microbial compositions have been linked to inflammatory skin diseases including psoriasis, AD, rosacea and acne [48]. It is interesting to note that in healthy individuals skin sites typically affected by AD such as the popliteal and antecubital fossa are particularly rich in Staphylococci. For children suffering from AD Kong et al. found [49] that changes in the microbiota was seen before an eczema flare. While the overall microbial diversity was reduced, Staphylococci increased proportionally and in particular S. aureus. Significant microbiome differences between psoriatic, rosacea, AD and healthy epidermis [41,48] have been reported. However, the functional consequences of diminished or altered species diversity for the skin immune response in these conditions remain to be clarified. Recently, Di Meglio et al. [50] have shown that ligands of the Aryl Hydrocarbon

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Receptor (AhR) reduce inflammation in psoriasis models and specifically downregulate type I and type II IFN pathways in human skin. It is interesting to note that AhR does not only detect environmental toxins but also tryptophan metabolites generated by exposure to UV light or by bacteria of the intestinal microflora. Products of the skin microflora such as tryptophan metabolites are likely to also activate the AhR which is expressed on Th17, Treg and innate lymphoid cells (ILC) subtypes. It remains to be determined if changes in the skin microbiota lead to altered AhR activation and thus altered regulation in inflammatory skin responses. It is worth mentioning that the anti-inflammatory property of coal tar has recently also been linked to AhR activation [51]. 2.1. Therapeutic approaches There is clear evidence for barrier impairment to be involved in inflammatory skin pathology (for review: [13,23,52]). The notion that treatment of all inflammatory diseases with epidermal contribution should involve skin barrier strengthening is shared by many experts in the field. Suggested approaches include the prolonged reduction of skin surface pH, the topical application of serine protease inhibitors (or kallikrein inhibitors) or recombinant AMP, lipid replacement therapy and strategies supporting S. epidermidis colonisation. Preliminary results from a pilot study aiming to prevent eczema by early use of topical barrier enhancement [53] were presented at the 2014 ISAD meeting and showed favourable results regarding starting topical moisturiser treatment early in life. Regarding downregulation of FLG, AMP and susceptibility to alpha toxin in eczema conditions – reduction of Stat6 signalling in the epidermal compartment seems of benefit. Targeting Gata3, Stat6 activating pathway, IL-4/IL-13 and their receptors may all be of therapeutic value. The most promising currently recruiting clinical trial aims to block IL-4Ra, thus targeting both IL-4 and IL13 function. Preliminary results for this monoclonal antibody (Dupilumab, NCT01979016) in AD seem promising. A phase II clinical trial targeting CRTH2 in AD is currently ongoing and awaits first results. CRTH2 is a cell surface molecule which binds to prostaglandin D2. The CRTH2 antagonist may act on mast cells, Th2 cells, type 2 ILCs and eosinophils. 3. Cutaneous response to barrier disruption All chronic inflammatory skin diseases are known to have genetic and environmental components contributing to their phenotype. Among the environmental factors which can contribute to flare ups are psychological stress and events with further disrupt the skin barrier. Tissue damage by scratching, exposure to irritants (detergents) or physical barrier disruption lead to increased expression of AMPs [45] and DAMPs of which IL-1a is best known for the epidermal compartment. Other DAMPs, which have been linked to inflammatory skin pathology often form complexes with nucleic acids or other proinflammatory molecules such as IL-1b (shown for both LL37 and HMGB1). In lesional skin (e.g. cutaneous lupus, psoriasis, eczema) increased expression of HMGB1, LL37, heat shock protein 70 (HSP70) and S100 proteins has been described which all exert pro-inflammatory functions. S100 proteins are overexpressed in all inflammatory skin diseases with epidermal involvement and impact on keratinocyte proliferation, differentiation, danger signalling and antimicrobial defence. S100A8 and -A9 form heterodimers and are stress inducible in particular after superficial injuries [54,55]. Both HMGB1 and S100 proteins bind to the receptor for advanced glycation end products (RAGE) [56]. The stress inducible HSP70 can bind peptide-antigens. Our data show high release of HSP70 from tissue resident cells [57] and pronounced functional effects on DC maturation and T cell


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cytotoxicity [58]. HSP70-peptide complexes are efficiently taken up by keratinocytes which then can act on increasing the production of IFN in autologous T cells which may point to their cross-presentation capacity (cross-presentation refers to the presentation of extracellular antigens to CD8 T cells). Interestingly, HMGB1 increases the uptake of HSP70 by cultured keratinocytes. LL37 acts in synergy with IL-1 or GM-CSF on the release of cytokines by neutrophils, tissue resident cells and APC including IL-8, IL-1 and IL-18 and can form complexes with self-DNA and self-RNA [59,60]. These nucleic acids are likely to originate from damaged cells. Lande et al. [60] and others put the concept forward that LL37–DNA complexes induce the production of type I IFNs by pDC which may be of significance for psoriasis and lupus erythematosus [60]. Surface injury and mechanical stress (Koebner phenomenon) are trigger factors in particular for psoriatic inflammation and DAMPs play a role in their cellular recognition. Psoriasis skin may be particularly prone to react to DAMPs many of which signal via NF-kB, a pathway overactive in psoriasis due to genetic predisposition (CARD14 gain of function mutations [61]; polymorphism, although not all functionally characterised yet, in genes linked to NF-kB activation including REL, TNIP1, TRAF3IP2, TNFAIP3 (A20), NFKBIA, FBXL19 [31,62,63]). Furthermore, epidermal disruption by proteases has been shown to lead to CCL20 production by keratinocytes in the absence of any further stimuli [64]. CCL20 is a key chemokine for attraction of CCR6+, IL-17/IL-22 producing lymphocytes which drive psoriasis pathology. Epithelial injury (also in epithelial airways [65]) by proteases or pathogens as well as tape stripping leads to epidermal TSLP and IL33 production along with other AMPs (see above), DAMPs [66–68] and cytokines such as IL-21 and IL-25. Thus, skin surface injuries may – via predominance of TSLP and IL-33 – favour a more type 2 like response in particular in those individuals prone for a Th2 shift in their immune response. The negative role of Th2 cytokines on FLG and AMP expression has been mentioned above and they may also impede barrier recovery [69]. The type 2 cytokine promoting properties of IL-33 seem not only to rely on its action on mast cells but also on the recently described role in polarising ILCs. ILCs which are closely linked to tissue responses at epithelial surfaces have been divided in different functional subsets [70]. Group 1 ILCs includes NK cells and other IFN producing ILCs, group 3 produce IL17 and/or IL-22. Group 2 ILCs produce type 2 cytokines (IL-4, IL-5, IL-9, IL-13), express the prostaglandin D(2) receptor CRTH2 and respond to IL-33 and IL-25 [71]. It is also interesting to note that large inter-individual variation in the antimicrobial protein expression and recolonisation patterns following superficial injury of human skin (tape stripping) have recently been reported [45].

expression. Currently, IL-33 is not being explored in clinical studies for skin diseases although ST2 is targeted in clinical trials for other conditions. 4. Itch and cytokines Itch (=pruritus), although intensively studied over the last decade, is still incompletely understood. Intense itch can significantly disturb the sleep pattern and daily activities resulting in sometimes severely impaired quality of life. Currently, we lack efficient anti-pruritus therapies. Pruritus, via itch–scratch cycles, also negatively impacts on skin barrier. It is widely accepted that subtypes of eczema exist where the primary cause of the disease is itch. Although almost all pruritic conditions are treated with antihistamins (H1 blockers) in the clinic, their therapeutic effect on the symptom itch seems limited in inflammatory conditions. Histamine 4 receptor blockade [73] may prove useful for treatment of scratch inducing itch but is currently not available in the clinic. Of interest, histamine has recently been described to act on keratinocyte differentiation and to impair skin barrier function [74] and may thus indirectly contribute to pro-pruritic xerosis. Many other mediators have been implicated in itch [75]. Substance P, Semaphorin 3A, IL-2 and endothelin have been proposed as peripheral mediators of pruritus. The molecules currently most studied and targeted by therapeutic approaches are NGF and IL-31. Almost all itch intensive diseases including AD, prurigo nodularis, and contact dermatitis show high levels of IL-31 [76–79]. IL-31 is significantly upregulated in itch intense AD skin as compared to only mildly pruritic psoriatic skin [80]. It can elicit an AD-like phenotype in mice, which present with scratching behaviour, skin lesion formation and dry, scaly skin [81]. As mentioned, IL-31 represses terminal keratinocyte differentiation with reduced expression of FLG and reduced epidermal thickening in skin equivalents [20]. It binds to the IL31 receptor A (IL-31RA) and the oncostatin M receptor (OSMR). Keratinocyte activation by a TLR2 ligand or IFNg upregulates both IL-31RA and OSMR [20,82,83]. Activated, predominantly Th2 T cells produce IL-31 [76,80] and production can be induced in skin homing, cutaneous lymphocyte antigen positive T cells [78]. In the context of bacterial toxins IL-31 induces proinflammatory cytokine production in antigen presenting cells and keratinocytes (CCL2) [33]. IL-31RA transcripts are expressed in dorsal root ganglia, where the cell bodies of the primary sensory neurons reside [76,80]. IL-31 may thus represent a key molecule in neuroimmune communication influencing itch and inflammation. 4.1. Therapeutic targets

3.1. Therapeutic targets Damage to the skin surface by mechanical stress, scratching or exposure to irritants plays a role in both psoriasis and eczema. Apart from the neutralisation of IL-1a (use of IL-1R antagonist IL-1RA) specific, locally applied anti-DAMP strategies have not been addressed in clinical studies. RAGE, the receptor shared by several DAMP molecules may be an interesting therapeutic target for topical approaches. Further research should also aim to improve our knowledge on how the expression endogenous ‘‘neutralising’’ molecules such as soluble RAGE could be induced in skin exposed to exogenous ‘‘stress’’. Blockade of IL-1a/b by the recombinant IL-1RA anakinra is in clinical trial for AD and psoriasis. IL-33, due to its described role as inducible by mechanical stress [72], and its receptor ST2 are therapeutic target for those disease which also show a bias towards type 2 responses. IL-33 activates mast cells, polarises (IL-4/IL-13 producing) ILC2 and has been shown to downregulate filaggrin as well as hBD2

IL-31 and its receptor seem very promising targets to treat itchy skin conditions such as prurigo, AD and other eczema conditions. A phase I study on safety and tolerability (NCT01614756) with an anti-IL31 monoclonal antibody is currently being performed. OSM and IL-31 share the OSMR; Venereau et al. [84] have developed a fusion protein with good neutralising properties for IL-31 effects without affecting OSM functions. However, the significance of known mediators involved in pruritus may be different for different diseases or disease subtypes. Combination with NGF neutralising approaches and anti-histamines which cover H4 receptor signalling may prove useful. Several anti-NGF drugs are under investigation (tanezumab, fulranumab, REGN475) for nondermatology condition (e.g. osteoarthritis, lower back pain). As pruritus, xerosis and scratch induced barrier damage are closely linked and most pro-pruritic mediators also have barrier impairing properties all anti-itch measures should go hand in hand with barrier strengthening approaches.


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5. Inhibitory cytokines Endogenous pathways exist on multiple pre- and posttranscriptional levels to ultimately reduce the expression and limit the activity of all secreted cytokines. These include induction of microRNAs, expression of receptor antagonists, decoy and soluble receptors, binding proteins and production of antiinflammatory and inhibitory cytokines. Surprisingly little effort has so far focused on upregulation of endogenous inhibitory molecules and pathways. Recent research focus has been on members of the IL-1 family IL-37 and IL-38 as well as the inhibitory receptor SIGIRR. IL-37 seems to act as a fundamental inhibitor of inflammatory responses [85]. The anti-inflammatory cytokine TGFb and to a lesser extent TLR ligands and proinflammatory cytokines (IL-1b, TNF, IL-18) can induce IL-37 production (reviewed in [86]). IL-37 shares the nuclear localisation property with other members of the IL-1 family (IL-1a, IL-33) and seems to suppress gene expression. Despite binding to IL-18Ra, IL-37 does not act as classical receptor antagonist. IL-37, binding to IL-18R, may deliver an inhibitory signal by using SIGIRR. SIGIRR (also known as TIR8) inhibits NF-kB and JNK activation by interfering with the recruitment of TIR-containing adaptor molecules. Interestingly, SIGIRR is one of the most downregulated genes in the peripheral blood in patients suffering from psoriatic arthritis and IL-37 has been shown to be underexpressed in skin psoriasis [4]. The disease limiting activity of IL-37 has been clearly shown in mouse models of psoriasis [87]. IL-38 binds to IL-36R and is a partial receptor antagonist of the IL-36R. IL-38 may be involved in mTOR regulation and in the regulation of IL-17/IL-22 production by human memory T cells [88]. 5.1. Therapeutic implication Studies regarding the significance of the IL-1 family members IL-37 and IL-38 for human inflammatory skin diseases are outstanding. Of note, inhibitors need to be present locally as systemic application may not reach the necessary concentration in the affected tissue. With the skin being an easily accessible organ, the approach to upregulate endogenous cytokine inhibitors or anti-inflammatory cytokines such as IL-37, IL-38, IL-10, IL-18BP, IL36RA or IL-1RA should be feasible. 6. Cytokines and disease subgroups Mostly, the diagnosis of chronic inflammatory skin diseases can be and is performed based on clinical symptoms and appearance. For example psoriasis and AD show symptoms in typical anatomical locations such as extensor sites for psoriasis and the skin lesions differ in shape and appearance. However there are cases of overlap or uncertainty, and usually skin biopsies and histology analysis aids differential diagnosis in these cases. Currently, skin expressed mediators are not used in routine clinical diagnostic. A number of studies investigated the value of potential specific biomarkers for inflammatory skin diseases [2,6,89]. Epidermally expressed molecules are of high interest for diagnosis of different diseases and disease subtypes as this skin compartment is easily accessible by non-invasive approaches (e.g. tape stripping, washing fluid). These include all IL-1 family members, chemokines, DAMPs, serine proteases and their inhibitors, AMP, barrier molecules and microbiota. Keratinocytes produce a wide range of chemokines and thus influence the nature of the inflammatory infiltrate. However, none of these chemokines is exclusive for any given disease or subtype and their relative expression compared to other mediator may be of greatest value. While CCL20 (psoriasis) and TSLP, CCL17/CCL22 (AD) are associated with different diseases, they are expressed to some level

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in all inflammatory skin conditions. Another example is S100 proteins which are highly expressed in psoriasis but also in eczema. Thus, the quantitative expression of a number of molecules and their relative hierarchy of expression may be needed for attempts to diagnose inflammatory subgroups based on epidermal cytokine expression. There are, however, a number of challenges when measuring tissue expressed mediator patterns. Firstly, skin microanatomy, microbiota and immune competence differ at different body sites. E.g., dermal fibroblasts possess an epigenetically determined positional memory [90]. Secondly, the inflammatory response is dynamic. Inflammatory patterns can differ significantly during the course of the same disease when comparing acute or chronic lesions. The best known example is eczema, where acute reactions show a more Th2 dominated inflammatory patterns (TSLP, IL-4, MDC, TARC, IL-13), while chronic lesions show a shift towards a more Th1 influenced pattern (IFNg, CXCL10, IL-18, IL-27) [91]. When it comes to therapeutic approaches, differences with regard to pharmacogenetic, disease subtype and location of the lesion have to be considered (Fig. 1). The largest proportion of patients show mild to moderate eczema or psoriasis disease activity. Only a minority of very severely affected patients receive therapy with biologics due to their high costs to the health systems. For those with limited extent of their disease a topically deliverable treatment approach would be preferable regarding costs and potential side effects. Topical therapies will work best when epidermal molecules are targeted as delivery into deeper skin compartments is still a challenge. 7. Psoriasis Targeting cytokines has fundamentally improved our understanding of the genetically complex, chronic inflammatory disease psoriasis and its treatment. The clinical success of targeting TNF, IL12/IL-23p40 and more recently also IL-23 and IL-17 left no doubt on the significance of IL-17 pathway activation and TNFa in the disease process. Johnson-Huang et al. [31] comprehensively summarised biologics/anti-cytokine therapy for psoriasis currently in clinical practise or under development. Among those the main therapeutic targets are TNFa, IL-12, IL-23 and the T cell mediators dependent on these IL-12 family members for polarisation, namely IL-22 and IL-17. Why do many different patients with severe psoriasis and presumably different genetic and inflammatory subtypes respond (almost equally well) to targeting TNF, IL-17 and IL-12/IL-23? One important explanation is that molecules directly or indirectly targeted by these treatments exert synergistic actions on tissue and immune cells. For keratinocyte responses that is in particular true for any combination of IL-17 or IFNg with TNFa. IFNg [92] – which is undoubtedly involved in psoriasis inflammation – is a key priming factor for keratinocytes making them dramatically more responsive to second signals including CD40 ligation or TNF. Despite the cardinal involvement of IL-23 in Th17 responses one should not oversee its initially described impact on IFNg secretion by memory T cells. Thus clinical manifestion of psoriasis can be controlled if important amplifiers of the mediator network response are neutralised. This suggests that amplifiers critically influence the severity of psoriatic inflammation and that endogenous counterregulatory mechanisms are overridden at a certain ‘‘threshold’’. The above mentioned decreased expression of regulators (IL-38 and IL-37) may explain why the ‘‘inflammation’’ threshold is low in psoriais resulting in high susceptibility to innocuous triggers for inflammation onset. Another molecule which also works in synergy with IL-17 and TNF (e.g. for IL-8 production) and is greatly induced by their synergistic action [93] is the IL-1 family member IL-36 for which no specific therapeutic antagonist exists so far. IL-36 has been identified as


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Fig. 1. Examples of disease subtypes and factors influencing personalised therapy approaches. SIT = specific immunotherapy

one of the most highly expressed genes in lesional compared to non-lesional psoriasis [94]. Bioactive IL-36 induces the secretion of IL-8 (attracts neutrophils) and hBD2. We have previously shown that cultured keratinocytes derived from psoriasis patients show increased expression of IL-36 upon cytokine stimulation (either with IL-17 and or TNF) [95]. The functional activity of IL-36 is regulated by protease cleavage [96] and the IL-36 receptor antagonist (IL-36RA). Loss-of-function mutations of the IL-36RA have been associated with the clinical subtype pustular psoriasis [97,98]. Thus, the emerging view is that IL-36 is a very important amplifier of psoriatic inflammation and this is also true for nonpustular psoriasis forms where IL-36RA is functional but may either be impaired in its neutralising capacity or overruled by high levels of bioactive IL-36. Psoriatic tissue cells seem particularly susceptibility to the action of IL-36 and this could be the results of decreased endogenous IL-36RA secretion, altered receptor expression/affinity or altered signalling pathway. Most of the morphologic alterations seen in the epidermal compartment of psoriasis are believed to be mediated by IL-22 and related molecules (IL-19, IL-20, IL-24) [99]. As IL-22 has overlapping functional properties on keratinocytes with IL-19 and IL24, receptor blockade of their shared IL-22R has been proposed as a promising therapeutic target [18]. As IL-22 is intimately involved in repair processes at epithelial surfaces (gut, lung, skin) potential side effects of IL-22 neutralisation may affect wound healing. Clinical trials targeting IL-22 are currently recruiting. Over 80% of patients respond to the currently available anticytokine therapies which target TNF, IL-12/IL-23p40 and IL-17 and the treatment goal for the latest biologics is not only to control disease activity but to clear the patient from any symptoms. So why do we still need information on disease subgroups (Table 1)? This is due to the fact that the majority of patients show mild to moderate disease activity and thus do not quality for therapy with very costly biologics in most health systems and we urgently need more information on response to conventional therapies (cyclosporine A, UV therapy, acitretin, fumaric acid, metothrexate). There are also primary or secondary non-responders and patients

showing side effects or having contraindication to therapy with biologics. Choosing the best suited and efficient therapy for a given patient will also help to save costs as lower drug levels may be needed and better clinical responses may be achieved including remission of the disease. Furthermore, novel epidermal targets suitable for topical treatment approaches could largely benefit mild to moderate disease sufferers if therapy responders can be identified. Based on the genetic background, subgroups may exist which show mainly alterations in skin barrier function, T cell signalling, antigen presentation or NF-kB pathway. Optimised, individualised therapy should also consider important associated co-morbidities and their prevention. 8. Eczema Compared with psoriasis, treatment success for eczema with currently available therapies is less efficient and in many cases acceptable control of the disease is not achieved. Eczema is a complex disease with many different environmental factors involved. There is a pressing need for better therapeutic efficacy, and stratification of therapeutic approaches according to disease subgroups could result in significant advances. The traditional dermatology ‘‘nomenclature’’ uses both morphological criteria and assumed ‘‘aetiology’’ to classify different eczema types. The most common ones are AD, contact dermatitis and irritant dermatitis. While we have mainly referred to AD in this review, AD and contact allergy can co-exist. Contact dermatitis is caused by a classical hypersensitivity reaction to a contact allergen. The pathways leading to sensitisation and elicitation phase of the contact allergy have been studied in great detail. It is now clear that contact sensitisers in susceptible individuals mimic infection pathways by triggering innate immune responses via pattern recognition receptors (PRRs) and endogenous danger signals [100]. Therapeutic strategies targeting DAMPs, chemokines and cytokines have been proposed based on these findings [38,100]. AD is part of the atopic diseases going along with high IgE levels in about 80% of cases. As outlined above and proposed previously


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7

Table 1 Hypothesised cytokine patterns for different ‘‘inflammatory’’ types of eczema and psoriasis. Predominant change/cause Eczema (all subtypes can occur with or without atopy (e.g. specific IgE responses to protein antigens) Mainly barrier defect/xerosis Mainly itch–scratch cycle Mainly TH2 polarisation Mainly irritant/mechanical injury response Mainly infection Mainly chronic Mainly protease activity Psoriasis Increased response to barrier disruption Mainly antimicrobial Mainly neutrophilic Mainly Th22 Mainly type I and II IFNs Mainly Th17 pathway Mainly overactive NFkB pathway

Key (epidermal) inflammatory molecules

Examples for involved genes (mutations/SNP)

Therapy/targets for topical approaches

FLG

Barrier strengthening IL-31, H4R, TSLP barrier strengthening CRTH2, H4R, IL-4R

IL-31, NGF, histamine, TSLP, AMP TSLP, CRTH2, IL-4, IL-13, CCL26, CCL22, CCL17, histamine AMP, IL-33, TSLP, CCL20, S100, IL-1a

CRTH2, IL-4

AMPs, IL-22, IL-1 IFN, IL-18, IL-27, CXCR3 LL37, IL-18, IL-1b, IL-36

IL-4 IL-18 SPINK5

topical AMPs CXCR3, IL-18BP Protease inhibition

IL-1a, AMP, TNFa, IL-36, TSLP, IL-33, HMGB1, S100 hBD2, LL37 IL-36, IL-8

LCE3b,c,d

TNFa, RAGE, S100

IL-33, TSLP

hBD2 copy number variation IL-36RA

IL-19, 20, 24, 22, hBD2 IFNalpha, beta; CXCL10, LL37, IL-12, IFN, CXCR3 ligands CCL20, IL-22, IL-17; IL-23 TNF, IL-18, IL-36, low IL-37, low SIGIRR, low IL-38

[2,3,5] subgroups of AD have been proposed based on atopy status, barrier properties, lymphocyte infiltrate, pruritogenic mediators and cytokine expression (Table 1). A number of clinical trials have been undertaken aiming to neutralise Th2 molecules (IL-4, IL-5), IgE, TNF or IL-23/IL-12p40 [5] but results have not been overly convincing when ‘‘unselected’’ patient groups have been treated. A phase I study with TSLP-R binding protein has been performed, however current clinical trials with this agent focus on allergic asthma. None of the studies in AD reaches clinical responses seen in psoriasis studies. However, the preliminary data for IL-4R blockers look very promising and results from the trials targeting IL-31 and CRTH2 may hopefully result in new therapies to be added to our clinical portfolio. It is likely that for some disease subgroups complementary measures regarding skin barrier and skin microbiota are necessary in addition to neutralisation of soluble mediators to achieve a satisfactory clinical response. 9. Concluding remarks Cytokines and their (soluble) receptors act in a complex network which makes it difficult to predict the outcome of blocking a single component. In psoriasis and eczema, mutations in cytokine, transcription factor, barrier or adhesion molecule genes determine the individual pattern of up- or downregulated mediators. Owing to the heterogeneous nature and the complex genetic background the most suitable mediator to target may differ between individual patients and disease stages. A number of biologic agents have been developed or are under development, which target a specific cytokine or its receptor but we clearly need deeper knowledge in the field of pharmacogenetics, skin microbiome but also epigenetic alterations to allow truly individualised therapy aiming for minimal disease activity or remission. Furthermore, for the large group of patients not eligible to therapy with biologics showing mild to moderate disease activity a wider range of topical therapies, which target different, subgroup relevant epidermal molecules, would be of great benefit.

IL23R

IL23R, IL12B, IL23A CARD14, REL, TNIP1, TRAF3IP2, TNFAIP3 (A20), NFKBIA, FBXL19

Protease inhibition, IL-36RA inducers IL-22R CXCR3 CCL20, IL-23 IL-37/IL-38 inducers

All cytokines have endogenous antagonists. Although imbalances in the cytokine – cytokine antagonist expression have been described, our picture in this respect is incomplete. Ideally, therapeutic approaches should aim to re-balance the local endogenous antagonist expression in inflammatory skin diseases. Acknowledgments MW’s current research on skin inflammation is supported by the British Skin Foundation (5035s) TW is supported by the Deutsche Forschungsgemeinschaft (DFG) and the German Ministry for Research and Education (BMBF). MW and DMG are supported by the National Institute for Health Research (NIHR) funding. References [1] Reich K, Mrowietz U, Karakasili E, Zschocke I. Development of an adherenceenhancing intervention in topical treatment termed the topical treatment optimization program (TTOP). Arch Dermatol Res 2014. [2] Bieber T. Atopic dermatitis 2.0: from the clinical phenotype to the molecular taxonomy and stratified medicine. Allergy 2012;67:1475–82. [3] Novak N, Simon D. Atopic dermatitis – from new pathophysiologic insights to individualized therapy. Allergy 2011;66:830–9. [4] Ainali C, Valeyev N, Perera G, Williams A, Gudjonsson JE, Ouzounis CA, et al. Transcriptome classification reveals molecular subtypes in psoriasis. BMC Genomics 2012;13:472. [5] Eyerich K, Novak N. Immunology of atopic eczema: overcoming the Th1/Th2 paradigm. Allergy 2013;68:974–82. [6] Kamsteeg M, Jansen PA, van Vlijmen-Willems IM, van Erp PE, Rodijk-Olthuis D, van der Valk PG, et al. Molecular diagnostics of psoriasis, atopic dermatitis, allergic contact dermatitis and irritant contact dermatitis. Br J Dermatol 2010;162:568–78. [7] Cork MJ, Danby SG, Vasilopoulos Y, Hadgraft J, Lane ME, Moustafa M, et al. Epidermal barrier dysfunction in atopic dermatitis. J Invest Dermatol 2009;129:1892–908. [8] McAleer MA, Irvine AD. The multifunctional role of filaggrin in allergic skin disease. J Allergy Clin Immunol 2013;131:280–91. [9] Weidinger S, Illig T, Baurecht H, Irvine AD, Rodriguez E, Diaz-Lacava A, et al. Loss-of-function variations within the filaggrin gene predispose for atopic dermatitis with allergic sensitizations. J Allergy Clin Immunol 2006;118: 214–9. [10] Wittmann M, Kingsbury SR, McDermott MF. Is caspase 1 central to activation of interleukin-1? Joint Bone Spine: revue du rhumatisme 2011;78:327–30.


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and promotes keratinocyte antimicrobial peptide expression. J Immunol 2011;186:2613–22. Muhr P, Zeitvogel J, Heitland I, Werfel T, Wittmann M. Expression of interleukin (IL)-1 family members upon stimulation with IL-17 differs in keratinocytes derived from patients with psoriasis and healthy donors. Br J Dermatol 2011;165:189–93. Towne JE, Renshaw BR, Douangpanya J, Lipsky BP, Shen M, Gabel CA, et al. Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36alpha, IL-36beta, and IL-36gamma) or antagonist (IL-36Ra) activity. J Biol Chem 2011;286:42594–602. Marrakchi S, Guigue P, Renshaw BR, Puel A, Pei XY, Fraitag S, et al. Interleukin36-receptor antagonist deficiency and generalized pustular psoriasis. N Engl J Med 2011;365:620–8. Onoufriadis A, Simpson MA, Pink AE, Di Meglio P, Smith CH, Pullabhatla V, et al. Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis. Am J Hum Genet 2011;89:432–7. Wolk K, Haugen HS, Xu W, Witte E, Waggie K, Anderson M, et al. IL-22 and IL20 are key mediators of the epidermal alterations in psoriasis while IL-17 and IFN-gamma are not. J Mol Med (Berl) 2009;87:523–36. Martin SF. Contact dermatitis: from pathomechanisms to immunotoxicology. Exp Dermatol 2012;21:382–9. Miriam Wittmann worked as Lecturer in Immunology for 3 years before she was appointed as Senior Lecturer in Translational Research in Dermato-Rheumatology at the University of Leeds where she works in close interaction with dermatologists, immunologists and rheumatologists. Miriam spends part of her week as a specialist dermatologist at Bradford Teaching Hospital and she holds the position of Associate Director (clinical) at the Centre for Skin Sciences (University of Bradford), UK. She has a research interest in eczema, psoriasis and cutaneous lupus erythematosus. She currently works on the significance of protease cleavage of IL-1 family members for psoriatic inflammation. Recently, her work has also focused on hand eczema and scarring versus non scarring outcome of inflammatory skin responses. Before moving to Leeds she has been working with Thomas Werfel for more than 10 years on cytokine regulation in eczema at Hannover Medical School, Germany. Dennis McGonagle is Professor for Rheumatology in Leeds. He is a leading expert in psoriasis and psoriatic arthritis and particularly interested in the link of mechanical stress and inflammation. Together with Michael McDermott he has challenged the traditional view on autoinflammatory versus autoimmune conditions and devised an ‘‘immunological disease continuum’’ of inflammation against self rather than mutually exclusive processes. Dennis is a leading researcher into the micro-anatomical basis for psoriasis, nail disease and psoriatic arthritis disease localisation and how anticytokine therapy not only treat patients but also unravel underlying human disease immunopathogenesis. Thomas Werfel has a special interest in AD and allergic skin inflammation. He is head of research for the Hannover Department of Dermatology and Allergy Research. For 2 decades, he has been leading research projects on allergen specific T cell responses and cytokine regulation in eczema. He was lead of a graduate school on allergic responses and was instrumental in building up patient support training for patients and parents affected by AD (AGNES, ARNE). Thomas has a special interest in the role of autoallergens in the pathogenesis of AD, on immunologic effects of histamine, therapeutic potential of antimicrobial peptides and is a recognised specialist for food allergies. He is Deputy Editor for the Journal of Investigative Dermatology.


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