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Profiling Immune Expression to Consider Repurposing Therapeutics for the Ichthyoses

  • Amy S. Paller
    Correspondence
    Correspondence: Amy S. Paller, Department of Dermatology, Northwestern University, Feinberg School of Medicine, 676 North St. Clair Street, Suite 1600, Chicago, Illinois 60611.
    Affiliations
    Departments of Dermatology and Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
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Open ArchivePublished:January 19, 2019DOI:https://doi.org/10.1016/j.jid.2018.08.027
      Despite extensive discovery about the mutations underlying genetic skin disorders, there have been few therapeutic advances. Better understanding of the molecular changes that may lead to the phenotypic manifestations of genetic disorders may lead to the discovery of new pharmacologic interventions. The ichthyoses are characterized by scaling, inflammation, and an impaired epidermal barrier. Recent studies have uncovered T helper type 17 skewing in ichthyotic skin, resembling psoriasis, and high frequencies of IL-17– and IL-22–expressing T cells in blood, correlating with severity and transepidermal water loss. Repurposing systemic T helper type 17/IL-23–inhibitory therapies for psoriasis may prove useful for patients with ichthyosis.

      Abbreviations:

      AD (atopic dermatitis), ARCI (autosomal recessive congenital ichthyosis), CIE (congenital ichthyosiform erythroderma), EI (epidermolytic ichthyosis), LI (lamellar ichthyosis), NS (Netherton syndrome), RT-PCR (reverse transcriptase-PCR), TEWL (transepidermal water loss), Th (T helper), TNF (tumor necrosis factor)
      Our understanding of the mutations underlying genetic disorders, including those with prominent skin manifestations, has tremendously increased during the past two decades because of the availability of new tools for discovery, in particular whole-exome and whole-genome sequencing. Linking a skin disease with a specific gene, especially a gene with a product not previously explored in skin, opens the door for studies that explore the localization and function of gene expression products in skin and how they are altered by disease. Similarly, finding that a disease phenotype can be replicated by mutations in several genes suggests that their gene products interact or share a pathway in tissue that leads to a similar clinical effect.
      Discoveries of underlying gene mutations have also led to great excitement about the potential to leverage this knowledge toward therapeutic benefit. New technologies to introduce a normal gene or functioning gene product are considered the ideal therapy, with gene therapy optimal for “cure” of genetic disease, including genetic disorders with skin manifestations. For dominant negative disorders, gene knockdown approaches have been shown to reverse disease manifestations, such as in the pilot human study with injected small interfering RNA targeting abnormal KRT6A, encoding keratin 6A, in a patient with pachyonychia congenita (
      • Leachman S.A.
      • Hickerson R.P.
      • Schwartz M.E.
      • Bullough E.E.
      • Hutcherson S.L.
      • Boucher K.M.
      • et al.
      First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder.
      ). Approaches for topical delivery enabling knockdown of gene expression are also in development for genetic (
      • Kaspar R.L.
      • Hickerson R.P.
      • González-González E.
      • Flores M.A.
      • Speaker T.P.
      • Rogers F.A.
      • et al.
      Imaging functional nucleic acid delivery to skin.
      ) and inflammatory (
      • Lewandowski K.T.
      • Thiede R.
      • Guido N.
      • Daniel W.L.
      • Kang R.
      • Guerrero-Zayas M.I.
      • et al.
      Topically delivered tumor necrosis factor-α-targeted gene regulation for psoriasis.
      ,
      • Randeria P.S.
      • Seeger M.A.
      • Wang X.Q.
      • Wilson H.
      • Shipp D.
      • Mirkin C.A.
      • et al.
      siRNA-based spherical nucleic acids reverse impaired wound healing in diabetic mice by ganglioside GM3 synthase knockdown.
      ) skin disorders. For disorders in which the mutation reduces gene and protein expression, progress has been made through introducing the normal gene and expanding corrected cells for skin transplantation for life-threatening junctional (
      • Hirsch T.
      • Rothoeft T.
      • Teig N.
      • Bauer J.W.
      • Pellegrini G.
      • De Rosa L.
      • et al.
      Regeneration of the entire human epidermis using transgenic stem cells.
      ,
      • Mavilio F.
      • Pellegrini G.
      • Ferrari S.
      • Di Nunzio F.
      • Di Iorio E.
      • Recchia A.
      • et al.
      Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
      ) and dystrophic forms (
      • Siprashvili Z.
      • Nguyen N.T.
      • Gorell E.S.
      • Loutit K.
      • Khuu P.
      • Furukawa L.K.
      • et al.
      Safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with recessive dystrophic epidermolysis bullosa.
      ) of epidermolysis bullosa. Furthermore, the ability to introduce corrected genes into stem cells promises to both improve transplantation of corrected cells and potentially direct introduction systemically to reach target sites (
      • Vanden Oever M.
      • Twaroski K.
      • Osborn M.J.
      • Wagner J.E.
      • Tolar J.
      Inside out: regenerative medicine for recessive dystrophic epidermolysis bullosa.
      ). Newer technology using gene editing has yet to reach human trials for skin disease, but it is showing great potential in vitro and in early animal models.

      Can Targeted Pharmacologic Therapy for Psoriasis and Atopic Dermatitis be Repurposed for Inflammatory Diseases With a Monogenic Basis?

      Although these curative gene-based therapies are in development, there remains a significant need for new approaches to treating genetic skin disorders. Pharmacologic approaches require an improved understanding of the changes in mRNA and protein expression that result from gene alteration. Fortunately, technological developments have also allowed scientists to uncover alterations in expression patterns in disease using relatively small skin biopsy samples or blood samples and to correlate these alterations in expression with phenotypic manifestations of skin disease. These technologies have been applied to understand immune system skewing in inflammatory skin disorders, such as psoriasis (
      • Kim J.
      • Krueger J.G.
      Highly effective new treatments for psoriasis target the IL-23/type 17 T cell autoimmune axis.
      ) and atopic dermatitis (AD) (
      • Brunner P.M.
      • Guttman-Yassky E.
      • Leung D.Y.
      The immunology of atopic dermatitis and its reversibility with broad-spectrum and targeted therapies.
      ) and have contributed greatly to the ongoing emergence of new targeted therapeutic options for treating these inflammatory skin disorders. In the case of psoriasis, these studies led to the availability of highly successful biologics that inhibit tumor necrosis factor-α (TNF-α). More recent discoveries showing the importance of activation of T-helper (Th) type 17/IL)-23 signaling pathways (with TNF-α activation as a synergistic pathway) have led to the commercial availability of even more effective systemically administered monoclonal antibodies that target IL-23, IL-17A, or IL17RA (the IL-17A/F receptor). In AD, mRNA expression studies in both adult and early pediatric skin have shown that Th2/Th22 pathways are activated (
      • Esaki H.
      • Brunner P.M.
      • Renert-Yuval Y.
      • Czarnowicki T.
      • Huynh T.
      • Tran G.
      • et al.
      Early-onset pediatric atopic dermatitis is TH2 but also TH17 polarized in skin.
      ,
      • Mansouri Y.
      • Guttman-Yassky E.
      Immune pathways in atopic dermatitis, and definition of biomarkers through broad and targeted therapeutics.
      ), and clinical trials have led to the recent commercial availability of the first Th2-targeting monoclonal antibody (
      • Simpson E.L.
      • Bieber T.
      • Guttman-Yassky E.
      • Beck L.A.
      • Blauvelt A.
      • Cork M.J.
      • et al.
      Two phase 3 trials of dupilumab versus placebo in atopic dermatitis.
      ).
      Inflammation is a common phenotypic manifestations of several inherited disorders of skin, raising the possibility that commercially available and pipeline therapeutics that target common inflammatory diseases could be harnessed toward genetic skin disorders. The possibility of “repurposing” drugs that are existent or already in clinical trials allows for the very rapid transition from laboratory-based discovery to clinical trials and use by patients, in contrast to the long delay from discovery to commercial availability if trials show achievement of primary endpoint(s). An additional benefit of treatment based on discovery of the molecular expression patterns, downstream of genetic alteration, is the ability to find shared molecular disease characteristics that correlate with phenotypic manifestations, enabling treatment of groups of patients who share molecular characteristics, rather than the potentially more restrictive personalization at the gene mutation level.

      The Ichthyoses: A Group of Rare Disorders Without Satisfactory Therapy

      Ichthyosis is a group of genetic disorders resulting from variants in more than 50 genes. The clinical feature that characterizes ichthyosis, regardless of underlying genetic basis, are scaling, usually generalized and in association with inflammation (
      • Mazereeuw-Hautier J.
      • Hernandez-Martin A.
      • O’Toole E.A.
      • Bygum A.
      • Amaro C.
      • Aldwin M.
      • et al.
      Management of congenital ichthyoses: European guidelines of care: part two.
      ,
      • Takeichi T.
      • Akiyama M.
      Inherited ichthyosis: non-syndromic forms.
      ,
      • Vahlquist A.
      • Fischer J.
      • Törmä H.
      Inherited nonsyndromic ichthyoses: an update on pathophysiology, diagnosis and treatment.
      ). However, all forms of ichthyosis have been associated with protein and lipid abnormalities that lead to a defective epidermal barrier, manifesting functionally as increased transepidermal water loss (TEWL). Ichthyosis vulgaris typically results from heterozygous or compound heterozygous mutations in FLG, the gene encoding the barrier protein filaggrin, and is a common disorder (approximately 10% of individuals) that is associated with an increased risk of developing AD (
      • Baurecht H.
      • Irvine A.D.
      • Novak N.
      • Illig T.
      • Bühler B.
      • Ring J.
      • et al.
      Toward a major risk factor for atopic eczema: meta-analysis of filaggrin polymorphism data.
      ). Although less common (up to 1:1,500 males), recessive X-linked ichthyosis results from mutations in STS, encoding steroid sulfatase, in which accumulation of cholesterol sulfate promotes corneocyte retention (
      • Elias P.M.
      • Crumrine D.
      • Rassner U.
      • Hachem J.P.
      • Menon G.K.
      • Man W.
      • et al.
      Basis for abnormal desquamation and permeability barrier dysfunction in RXLI.
      ). Ichthyosis vulgaris and recessive X-linked ichthyosis usually respond well to topically applied emollients and keratolytic agents.
      In contrast, there are dozens of rarer (“orphan”) forms of syndromic and nonsyndromic ichthyosis (
      • Oji V.
      • Tadini G.
      • Akiyama M.
      • Blanchet Bardon C.
      • Bodemer C.
      • Bourrat E.
      • et al.
      Revised nomenclature and classification of inherited ichthyoses: results of the First Ichthyosis Consensus Conference in Sorèze 2009.
      ). The most common among these orphan forms of ichthyosis are autosomal recessive congenital ichthyosis (ARCI), with its phenotypic spectrum from congenital ichthosiform erythroderma (CIE) to lamellar ichthyosis (LI), Netherton syndrome (NS) (also autosomal recessive), and epidermolytic ichthyosis (EI) (autosomal dominant) (
      • Vahlquist A.
      • Fischer J.
      • Törmä H.
      Inherited nonsyndromic ichthyoses: an update on pathophysiology, diagnosis and treatment.
      ) (Figure 1). Biallelic mutations in TGM1, encoding transglutaminase 1, cause most cases of LI, whereas mutations in 10 other genes, many of which are involved in the lipoxygenase pathway or lipid metabolism (ABCA12, ALOX12B, ALOXE3, CERS3, CYP4F22, LIPN, NIPAL4, PNPLA1, SDR9C7, and SLC27A4/FATP4), more commonly lead to the CIE or an intermediate phenotype of variable severity (
      • Richard G.
      Autosomal Recessive Congenital Ichthyosis.
      • Shigehara Y.
      • Okuda S.
      • Nemer G.
      • Chedraoui A.
      • Hayashi R.
      • Bitar F.
      • et al.
      Mutations in SDR9C7 gene encoding an enzyme for vitamin A metabolism underlie autosomal recessive congenital ichthyosis.
      ,
      • Vahlquist A.
      • Fischer J.
      • Törmä H.
      Inherited nonsyndromic ichthyoses: an update on pathophysiology, diagnosis and treatment.
      ). NS results from mutations in SPINK5, leading to deficiency of the proteinase inhibitor LEKTI (lympho-epithelial Kazal-type-related inhibitor), whereas EI most commonly occurs from heterozygous mutations in KRT10 or KRT1. In general, these orphan forms cause a much greater burden, including on quality of life, and are a greater treatment challenge. Available treatments, primarily systemic or topical retinoids, promote keratinocyte differentiation and shedding, thereby reducing scaling and skin thickening. Retinoids sometimes improve, but more often increase, skin inflammation.
      Figure thumbnail gr1
      Figure 1Major orphan forms of ichthyosis. Representative images showing the scaling and variable erythema of (a) congenital ichthyosiform erythroderma, (b) lamellar ichthyosis, (c) epidermolyic ichthyosis, and (d) Netherton syndrome.
      Although the inflammation, cutaneous thickening, and scaling that characterize the ichthyoses are traditionally attributed to the cardinal barrier defect, few studies have addressed the molecular basis for these phenotypic features. However, understanding the pathway to phenotypic features could lead to more effective, targeted therapeutic approaches. For example, absence in CHILD syndrome (i.e., congenital hemidysplasia, ichthyosiform erythroderma, and limb defects) of the NSDHL gene, encoding nicotinamide adenine dinucleotide phosphate–dependent steroid dehydrogenase-like, was recognized to lead to both cutaneous deficiency of cholesterol and accumulation of toxic early pathway components toward cholesterol biosynthesis. By both blocking the pathway upstream of NSHDL and correcting the deficiency of the cholesterol end product with a topically applied compounded medication containing lovastatin and cholesterol, clinical, histological, and ultrastructural cutaneous changes of CHILD syndrome were markedly improved within 6 weeks (
      • Paller A.S.
      • van Steensel M.A.
      • Rodriguez-Martín M.
      • Sorrell J.
      • Heath C.
      • Crumrine D.
      • et al.
      Pathogenesis-based therapy reverses cutaneous abnormalities in an inherited disorder of distal cholesterol metabolism.
      ).

      Few Investigations of Biomarkers in Human and Mouse Model Ichthyotic Skin and Blood

      Most investigations with human ichthyotic skin have focused on barrier alterations, such as epidermal hyperplasia/increases in KRT16 and KRT6B, and abnormalities in differentiation (LOR, FLG) and lipid metabolism genes (
      • Descargues P.
      • Deraison C.
      • Prost C.
      • Fraitag S.
      • Mazereeuw-Hautier J.
      • D’Alessio M.
      • et al.
      Corneodesmosomal cadherins are preferential targets of stratum corneum trypsin- and chymotrypsin-like hyperactivity in Netherton syndrome.
      ,
      • Pavez Lorie E.
      • Ganemo A.
      • Borgers M.
      • Wouters L.
      • Blockhuys S.
      • van de Plassche L.
      • et al.
      Expression of retinoid-regulated genes in lamellar ichthyosis vs. healthy control epidermis: changes after oral treatment with liarozole.
      ). Few have studied the potential role of inflammatory dysregulation in disease development, and most of these have focused on NS; more comprehensive analysis of skin from patients with ichthyosis has only recently been performed. Nevertheless, these early studies suggest an interplay among expression of proinflammatory cytokines and chemokines, epidermal hyperkeratosis, and barrier abnormalities. For example, human NS keratinocytes, human keratinocytes incubated with kallikrein 5, and skin from Spink5–/– mice have shown increases in PAR2, TSLP, IL1b, IL8, and TNF mRNA expression (
      • Briot A.
      • Deraison C.
      • Lacroix M.
      • Bonnart C.
      • Robin A.
      • Besson C.
      • et al.
      Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome.
      ); however, inactivation of PAR2 (protease-activated receptor 2) did not reduce the cutaneous inflammation in an adult NS mouse model, despite inhibiting TSLP expression (
      • Briot A.
      • Lacroix M.
      • Robin A.
      • Steinhoff M.
      • Deraison C.
      • Hovnanian A.
      Par2 inactivation inhibits early production of TSLP, but not cutaneous inflammation, in Netherton syndrome adult mouse model.
      ). In another study, NS mouse models showed diverse cytokine activation with increases in mRNA levels of genes encoding innate, Th2, Th17, and Th22 cytokines (IL-1β, TNF-α, IL-4, IL-13, IL-17, and IL-22) and corresponding chemokines (TSLP, CCL17, CXCL1, CCL20, and S100A8/9) (
      • Furio L.
      • de Veer S.
      • Jaillet M.
      • Briot A.
      • Robin A.
      • Deraison C.
      • et al.
      Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome.
      ), and increases in IL-33 were uncovered in the skin of two patients with NS (
      • Konishi T.
      • Tsuda T.
      • Sakaguchi Y.
      • Imai Y.
      • Ito T.
      • Hirota S.
      • et al.
      Upregulation of interleukin-33 in the epidermis of two Japanese patients with Netherton syndrome.
      ). In LI organotypic cultures, IL-1α receptor antagonists block hyperkeratosis (
      • O’Shaughnessy R.F.
      • Choudhary I.
      • Harper J.I.
      Interleukin-1 alpha blockade prevents hyperkeratosis in an in vitro model of lamellar ichthyosis.
      ), whereas IL-37β partially suppressed the cutaneous phenotype in Abca12–/– mouse models of harlequin ichthyosis through reduction of increased chemokine expression (
      • Cottle D.L.
      • Ursino G.M.
      • Ip S.C.
      • Jones L.K.
      • Ditommaso T.
      • Hacking D.F.
      • et al.
      Fetal inhibition of inflammation improves disease phenotypes in harlequin ichthyosis.
      ). In addition, Krt1-knockout mice (EI) have increased IL-18, and depleting IL-18 partially rescues the EI skin phenotype (
      • Roth W.
      • Kumar V.
      • Beer H.D.
      • Richter M.
      • Wohlenberg C.
      • Reuter U.
      • et al.
      Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18.
      ). In the few studies of blood from patients with NS, levels of selected polar cytokines were increased (
      • Akagi A.
      • Kitoh A.
      • Moniaga C.S.
      • Fujimoto A.
      • Fujikawa H.
      • Shimomura Y.
      • et al.
      Case of Netherton syndrome with an elevated serum thymus and activation-regulated chemokine level.
      ,
      • Hosomi N.
      • Fukai K.
      • Nakanishi T.
      • Funaki S.
      • Ishii M.
      Caspase-1 activity of stratum corneum and serum interleukin-18 level are increased in patients with Netherton syndrome.
      ,
      • Renner E.D.
      • Hartl D.
      • Rylaarsdam S.
      • Young M.L.
      • Monaco-Shawver L.
      • Kleiner G.
      • et al.
      Comel-Netherton syndrome defined as primary immunodeficiency.
      ,
      • Van Gysel D.
      • Koning H.
      • Baert M.R.
      • Savelkoul H.F.
      • Neijens H.J.
      • Oranje A.P.
      Clinico-immunological heterogeneity in Comel-Netherton syndrome.
      ), but there was inconsistent evidence of Th2 skewing.
      Therapeutic interventional studies, coupled with investigation of cytokine alterations, have provided further evidence of cytokine alteration with improvement, although expression of only selected cytokines has been evaluated. In a patient with NS,
      • Fontao L.
      • Laffitte E.
      • Briot A.
      • Kaya G.
      • Roux-Lombard P.
      • Fraitag S.
      • et al.
      Infliximab infusions for Netherton syndrome: sustained clinical improvement correlates with a reduction of thymic stromal lymphopoietin levels in the skin.
      found an increase in mRNA expression of several cytokines in lesional skin (IFN-γ, IL-6, IL-8, IL-10, IL-17, and IL-23, but not IL-4, IL-5, or IL-31) and nonlesional skin (TSLP, TNF-α, and TGF-β). Infliximab therapy led to clearance of inflammation (but not xerosis, scaling, and serum IgE levels) by 1 year with reduction in all cytokines in lesional skin, except in IL10 and TNFA, both of which remained increased. In another NS patient, 4 months of omalizumab therapy (with pulsed steroids in the first month) led to marked clinical improvement in association, with more than 50% reduction in blood levels of IL-1β, IL-5, and IL-17 (TNF was not assessed) (
      • Yalcin A.D.
      A case of Netherton syndrome: successful treatment with omalizumab and pulse prednisolone and its effects on cytokines and immunoglobulin levels.
      ). Although retinoids are not thought to primarily reduce skin inflammation, reduced TNFA expression was noted in a patient with LI treated with oral liazarole, which increases endogenous retinoid levels (
      • Pavez Lorie E.
      • Ganemo A.
      • Borgers M.
      • Wouters L.
      • Blockhuys S.
      • van de Plassche L.
      • et al.
      Expression of retinoid-regulated genes in lamellar ichthyosis vs. healthy control epidermis: changes after oral treatment with liarozole.
      ).

      Demonstration of Th17 Skewing in Ichthyotic Skin and Blood

      The first more comprehensive assessment of mRNA expression of proinflammatory cytokines and chemokines as a function of ichthyosis disease severity was recently published (
      • Paller A.S.
      • Renert-Yuval Y.
      • Suprun M.
      • Esaki H.
      • Oliva M.
      • Huynh T.N.
      • et al.
      An IL-17-dominant immune profile is shared across the major orphan forms of ichthyosis.
      ). mRNA expression patterns for 54 genes that encode proteins involved in skin barrier formation and T-cell differentiation were assessed by reverse transcriptase-PCR from the lesional skin of 21 patients, 10 years of age and older, with the most common subtypes of orphan ichthyosis (ARCI-LI, ARCI-CIE, EI, and NS) of variable severity. Data were compared with mRNA in skin from age-matched healthy control individuals and adult patients with AD or psoriasis. TEWL measurements from the upper arm were all high, with the NS and CIE subtypes significantly higher than LI and EI subtypes. Compared with age-matched controls, all subtypes of ichthyosis had strong Th17 skewing (e.g., significant increases in mRNA econding IL-17A, peptidase inhibitor 3/PI3, CCL20, DEFB4B, and the S100As). IL-23p19 was increased only for CIE and NS, IL-12/IL-23p40 only for CIE, and IL-22 for both CIE and NS. Some Th1 markers were increased in some patients, without correlation with disease severity; markers of general inflammation, innate immunity, and Th2 markers were minimally or not increased (for representative examples, see Figure 2). The Th17 skewing for several markers was comparable or greater to that seen in the skin of adults with psoriasis. IL-17A expression correlated well with the disease severity (r = 0.57) and even more strongly with the erythema subscore severity (r = 0.74).
      Figure thumbnail gr2
      Figure 2IL17/TNFA synergistic/additive gene expression is increased in ichthyosis. Expression of 42 immune genes was evaluated using reverse transcriptase-PCR from ichthyotic skin and compared with gene expression in control samples and the nonlesional (NL) and lesional skin (LS) from individuals with atopic dermatitis (AD) or psoriasis (Pso). mRNA log2 values were adjusted to hARP expression levels. Graphs shown represent (a, b) innate immunity, (c, d) Th1 cytokines, (e, f) Th2 cytokines, and (g–j) Th17/Th17-related cytokines. Asterisks directly over bars are P-values compared with control samples. Lines with associated asterisks define the comparators at either end of the lines. LSmean (log2 expression/hARP) ± SEM. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. TEWL, transepidermal water loss; Th, T helper; TNF, tumor necrosis factor.
      Subsequent analysis by gene array of skin from 29 patients with these forms of ichthyosis, including 21 from the RT-PCR study, showed 132 differentially expressed genes that were shared among the ichthyoses compared with age-matched healthy control skin (
      • Malik K.
      • He H.
      • Huynh T.
      • Tran G.
      • Mueller K.
      • Doytcheva K.
      • et al.
      Ichthyosis molecular fingerprinting shows profound Th17-skewing and a unique barrier genomic signature.
      ). In addition to those found to be up-regulated in the initial RT-PCR study, this microarray analysis showed additional IL17 and TNFA co-regulated genes recognized to be markers of psoriasis (e.g., IL17F, IL36B/G, IL36R, KYNU, and VNN3). The increases in Th17 expression correlated with TEWL as well as severity. Although the ichthyoses lacked the reduction in expression of genes encoding proteins of differentiation and tight junctions (e.g., LOR, FLG, and CLDN1) seen in AD, several alterations in lipid metabolism genes were notable among the ichthyoses, including encoding an enzyme that promotes chain elongation in the synthesis of very-long-chain ceramides and fatty acids. These gene expression changes were accompanied by reduction and abnormal distribution of lipids in frozen biopsy sections. Although IL-17 has been shown to reduce lipid-related gene expression in preadipocytes (
      • Lee S.H.
      • Jhun J.
      • Byun J.K.
      • Kim E.K.
      • Jung K.
      • Lee J.E.
      • et al.
      IL-17 axis accelerates the inflammatory progression of obese in mice via TBK1 and IKBKE pathway.
      ,
      • Zuniga L.A.
      • Shen W.J.
      • Joyce-Shaikh B.
      • Pyatnova E.A.
      • Richards A.G.
      • Thom C.
      • et al.
      IL-17 regulates adipogenesis, glucose homeostasis, and obesity.
      ), the potential relationship between Th17 cytokines and epidermal lipid expression is poorly understood and deserves investigation.
      Gene array analyses also showed some distinct expression patterns among the ichthyoses, which may contribute to their differences in clinical manifestations (
      • Malik K.
      • He H.
      • Huynh T.
      • Tran G.
      • Mueller K.
      • Doytcheva K.
      • et al.
      Ichthyosis molecular fingerprinting shows profound Th17-skewing and a unique barrier genomic signature.
      ). For example, NS showed the greatest T-cell activation and a broader immune phenotype than other subtypes. EI, in contrast to the other three subtypes, did not show significant lipid abnormalities. Patients with LI had the most severe reduction in lipid metabolism genes and the most atypical Nile red lipid staining pattern, possibly because transglutaminase 1, which was deficient in LI patients, crosslinks the epidermal ω-hydroxyceramides to terminal differentiation proteins. Future studies with larger numbers of ichthyosis patients will be required to further delineate the differences among ichthyosis subtypes and even individuals with the same clinical phenotype and gene alteration.
      Given the growing recognition that inflammatory skin disorders can be associated with systemic immune system activation, blood from patients with these orphan forms of ichthyosis was also analyzed. Serum samples from the ichthyoses had increased IL-17A protein expression, as shown by Erenna immunoassays (
      • Malik K.
      • He H.
      • Huynh T.
      • Tran G.
      • Mueller K.
      • Doytcheva K.
      • et al.
      Ichthyosis molecular fingerprinting shows profound Th17-skewing and a unique barrier genomic signature.
      ). Furthermore, there was a high frequency of activated cutaneous lymphocyte antigen-positive and cutaneous lymphocyte antigen-negative T cells that expressed IL-17 (P < 0.05) and IL-22 (P < 0.01) from 47 patients with ichthyosis, particularly the NS and CIE subtypes, compared with with blood cells from healthy control individuals (
      • Czarnowicki T.
      • He H.
      • Leonard A.
      • Malik K.
      • Magidi S.
      • Rangel S.
      • et al.
      The major orphan forms of ichthyosis are characterized by systemic T-cell activation and Th-17/Tc-17/Th-22/Tc22 polarization in blood.
      ). These high frequencies clustered with severity and TEWL. The peripheral blood lymphocyte skewing toward Th17/IL-22 polarity raises the question of risk of systemic comorbidities, as occurs in psoriasis, particularly cardiovascular and joint inflammation, which have received little attention in ichthyosis beyond the association of arthritis as a comorbidity of harlequin ichthyosis, a severe subset of ARCI (
      • Beazley J.C.
      • Ho K.
      • Ilchyshyn A.
      • Foguet P.
      Total hip replacement in an adolescent patient with harlequin ichthyosis; a case report.
      ,
      • Chan Y.C.
      • Tay Y.K.
      • Tan L.K.
      • Happle R.
      • Giam Y.C.
      Harlequin ichthyosis in association with hypothyroidism and juvenile rheumatoid arthritis.
      ,
      • Clement S.A.
      • Burrows N.P.
      • Sansome A.
      • Hazleman B.L.
      • Ostör A.J.
      Harlequin ichthyosis and juvenile idiopathic arthritis: a rare combination.
      ,
      • Rajpopat S.
      • Moss C.
      • Mellerio J.
      • Vahlquist A.
      • Gånemo A.
      • Hellstrom-Pigg M.
      • et al.
      Harlequin ichthyosis: a review of clinical and molecular findings in 45 cases.
      ).

      What Is the Significance of Th17 Skewing In Ichthyosis?

      The significance of Th17 skewing in ichthyosis and its specificity as a marker is unclear. It is possible that the shared Th17 activation is merely the response to the impaired barrier and a compensatory attempt to protect against organisms. Although innate immune response markers are known to be increased after barrier impairment by tape stripping (
      • Dickel H.
      • Gambichler T.
      • Kamphowe J.
      • Altmeyer P.
      • Skrygan M.
      Standardized tape stripping prior to patch testing induces upregulation of Hsp90, Hsp70, IL-33, TNF-α and IL-8/CXCL8 mRNA: new insights into the involvement of ‘alarmins’.
      ), no studies have looked at the Th17 pathway response. Similarly, few studies have broadly assessed the T-cell immune molecular phenotype for inflammatory disorders. AD, characterized by barrier impairment, features skewing toward Th2/Th22 immune responses, with Th17 response to a much lesser extent and only in select populations (Asians, young children) (
      • Esaki H.
      • Brunner P.M.
      • Renert-Yuval Y.
      • Czarnowicki T.
      • Huynh T.
      • Tran G.
      • et al.
      Early-onset pediatric atopic dermatitis is TH2 but also TH17 polarized in skin.
      ,
      • Noda S.
      • Suárez-Fariñas M.
      • Ungar B.
      • Kim S.J.
      • de Guzman Strong C.
      • Xu H.
      • et al.
      The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.
      ). The psoriasiform dermatitis induced by TNF inhibitors, particularly in patients with Crohn disease, has now been shown to have a biomarker pattern distinct from either AD or psoriasis, with a strong IFN-γ activation signature (
      • Stoffel E.
      • Maier H.
      • Riedl E.
      • Brüggen M.C.
      • Reininger B.
      • Schaschinger M.
      • et al.
      Analysis of anti-tumour necrosis factor-induced skin lesions reveals strong T helper 1 activation with some distinct immunological characteristics.
      ). Molecular immunophenotyping of skin samples has shown allergic contact dermatitis to be a heterogenous disorder in which some allergens trigger a Th1/Th17 response (e.g., nickel) and others a Th2 response (e.g., fragrance) (
      • Dhingra N.
      • Shemer A.
      • Correa da Rosa J.
      • Rozenblit M.
      • Fuentes-Duculan J.
      • Gittler J.K.
      • et al.
      Molecular profiling of contact dermatitis skin identifies allergen-dependent differences in immune response.
      ). In a recent gene array analysis of chronic venous wound skin (vs. healthy skin), 2-fold increases were noted for almost 3,500 genes, among them some encoding innate immune responses (IL-6; IL-8; β-defensin 2; S100As) and a few IL-17 and TNF-α co-regulated genes (IL36G, KYNU), but not CCL20, CXCL1, or IL17A (
      • Stone R.C.
      • Stojadinovic O.
      • Rosa A.M.
      • Ramirez H.A.
      • Badiavas E.
      • Blumenberg M.
      • et al.
      A bioengineered living cell construct activates an acute wound healing response in venous leg ulcers.
      ). In a form of epidermolysis bullosa simplex, a chronic blistering disorder with associated inflammation, five immune response genes were differentially up-regulated compared with normal skin, based on gene arrays; these included CCL22 (Th1) and CCL5/RANTES (Th2), but none from the Th17 pathway (
      • Bchetnia M.
      • Farez T.
      • Lacroix J.
      • Leclerc G.
      • Powell J.
      • McCuaig C.
      • et al.
      Gene expression analysis of epidermolysis bullosa simplex with mottled pigmentation.
      ). Response to medication has recently been suggested as an additional marker for correlating suspected immune phenotype with clinical morphology (
      • Eyerich K.
      • Eyerich S.
      Immune response patterns in non-communicable inflammatory skin diseases.
      ). In addition to the Th2 and Th17/Th22 responses of AD and psoriasis, respectively, Th1 lymphocyte subsets were proposed to drive lichenoid responses (including in lupus erythematosus) (biomarkers such as IFN-γ and CXCL10) and regulatory T cells (biomarkers IL-10 and TGF-β) fibrogenic responses. Taken together, these data suggest that the strong Th17 skewing across the ichthyoses as a group is disease-specific and not a common phenotype for chronic inflammatory disease, even with barrier abnormalities.
      The other consideration is that the Th17 skewing is a response to the barrier defects that leads to inflammation and scaling. Th17 and TNF co-regulated genes are thought to cause the inflammation and scaling of psoriasis, and administration of Th17-targeting medications dramatically improves disease severity. Recently, two patients with syndromic ichthyosis from mutations in DSP, encoding desmoplakin, experienced dramatic improvement in ichthyosis severity (both erythema and scaling) and TEWL from use of ustekinumab, which inhibits IL-12/23p40 (
      • Paller A.S.
      • Czarnowicki T.
      • Renert-Yuval Y.
      • Holland K.
      • Huynh T.
      • Sadlier M.
      • et al.
      The spectrum of manifestations in DSP (desmoplakin) SR6 domain mutations: immunophenotyping and response to ustekinumab.
      ). In one of those patients, immunophenotyping of skin and blood was performed. In skin, expression of IL-17A, but also IL-22/IL-23p40 and IL-22, was increased as shown by RT-PCR analysis, and the frequency of IL-17– and IL-22–producing T cells was shown to be elevated by single-cell flow cytometry, which led to the decision to treat with ustekinumab.
      The shared Th17 signal among the orphan ichthyoses and the ameliorative result of Th17/IL-23 inhibition in a patient with a form of syndromic ichthyosis suggest that the Th17 skewing plays a pathogenic role in ichthyosis. To further test this possibility, a 16-week double-blind, randomized, placebo-controlled trial of the Th17 inhibitor secukinumab has been initiated (NCT03041038). This trial will help unravel the complex relationship between IL-17 and clinical features/barrier issues related to ichthyosis.
      In conclusion, there is a significant unmet need for new treatments for patients with genetic skin disorders. Looking beyond the underlying genetic basis toward the molecular changes that may lead to phenotypic characteristics may provide insight, not only on understanding these genetic disorders but also on new therapeutic directions. The rapidly expanding repertoire of systemic therapies that target specific immune-polarized T-cell subsets provides the opportunity for repurposing commercially available medications toward a variety of inflammatory skin disorders, including monogenic disease.

      Conflict of Interest

      ASP is an investigator for Novartis Eli Lilly and Janssen, makers of anti-Th17/IL-23 monoclonal antibodies, and has been a consultant with honoraria for Novartis and Eli Lilly.

      Acknowledgments

      The Montagna Symposium is an National Institutes of Health -supported conference (grant no. R13-AR009431-52 ; principal investigator, Kulesz-Martin); Thanks to Emma Guttman-Yassky, James Krueger, Tali Czarnowicki, Patrick Brunner, and many others for their collaboration in discovering the biomarkers in skin and blood associated with ichthyosis.

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