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Ichthyosis molecular fingerprinting shows profound Th17-skewing and a unique barrier gene expression pattern

Abstract: (250 words)

Background: Ichthyoses are a group of rare skin disorders that lack targeted effective treatments. Although DNA alterations are progressively delineated, comprehensive molecular phenotyping of ichthyotic skin could suggest much-needed pathogenesis-based therapy.

Objective: To profile the molecular fingerprint of the most common orphan ichthyosis subtypes.

Methods: Gene-arrays were performed on samples from 21 patients (congenital ichthyosiform erythroderma/CIE, n=6; lamellar ichthyosis/LI, n=7; epidermolytic ichthyosis/EI, n=5; and Netherton syndrome/NS, n=3), and age-matched healthy controls (n=14). Psoriasis (n=30), and atopic dermatitis/AD (n=16) patients, representing Th1/Th17 and Th2/Th22 polar immune responses, respectively, were included for comparison.

Results: Th17/IL-17 pathway genes (IL-17A/F, CCL20, S100As) were induced, resembling the psoriasis profile, and correlated with clinical severity (P<0.001). Up-regulation of genes considered unique classifiers of psoriasis was observed in ichthyosis (KYNU, VNN1, 3), but not in AD, and was correlated with disease severity. Contrary to AD, Th2 genes (IL-4R, IL-13, CCL26) were not up-regulated. While terminal differentiation (FLG, LOR) gene expression was preserved in ichthyosis, and even higher than psoriasis and controls (P<0.01), lipid homeostasis genes were significantly lower than controls but higher than AD. Tight junctions were differentially expressed with overall reduced expression in ichthyosis vs. controls. Gene profile differences among ichthyoses suggest the basis for phenotypic variance.

Conclusion: Similar to AD and psoriasis, where epidermal barrier impairment and cytokine dysregulation orchestrate disease phenotype, lack of barrier integrity along with psoriasis-like immune dysregulation characterized the ichthyoses. Additionally, our data support possible applicability for ichthyosis of IL-17-targeted therapeutics, which have led to great improvements in psoriasis patients.

Clinical Implications (30 words):

Capsule summary (35 words):

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Word count: 4124

Introduction

Ichthyoses are a heterogeneous group of rare inherited or acquired, debilitating inflammatory cutaneous disorders^1-4 that share defective keratinization, compromised epidermal barrier and increased transepidermal water loss/TEWL.^{2, 3, 5} Patients present with erythema, xerosis, hyperkeratosis, reduced life quality, and financial burden.^6-10

The current treatment armamentarium for ichthyosis is largely limited to topical emollients, keratolytics, and oral retinoids.^8-11 These therapeutic strategies are not specific, lack efficacy, and harbor increased toxicity.^{3, 12-15} Corrective gene therapy is evolving with continued elucidation of involved genes. However, this approach is still experimental and limited to model systems of specific ichthyosis subtypes.^{16, 17} Thus, an extensive unmet need exists for pathogenic-based targeted therapeutic approaches.

Data derived from animal or human keratinocyte models point to abnormalities in cornification, lipid homeostasis, and keratinocyte adhesion/desquamation, among the different ichthyosis subtypes, suggesting shared defects in barrier architecture.^18-25 Studies in human skin and blood, mostly limited to few patients or select ichthyosis subtypes, have noted disrupted terminal differentiation (i.e., filaggrin/FLG mutations in ichthyosis vulgaris/IV), lipid trafficking (PNPLA1, ELOVL4, ALOXE3/12B), and cornified envelope (CE) assembly (transglutaminase 1/TGM1, desmoglein 1/Dsg1).^{18, 26-35}

Several observations have linked ichthyosis to atopic dermatitis (AD), the most common inflammatory skin disease, marked by epidermal barrier defect and immune dysregulation.³⁶ Polymorphisms of genes mutated in ichthyoses (FLG, SPINK5) were associated with AD,^37-39 while Netherton syndrome (NS) was linked to eosinophilia and elevated TSLP, TARC and serum IgE.^{18, 20, 40-47} Moreover, improvement of Th2 polarized immune pathways and lipid markers in ichthyotic tissues with anti-TNFa immunomodulation imply underlying immune responses that may be amenable to therapeutic antagonism.^{42, 47, 48}

Recently, our group found ichthyoses to harbor enhanced Th17/IL-23 immune responses with minimal Th2 inflammation and preserved terminal differentiation.⁴⁹ These data, obtained by a limited panel of genes by RT-PCR, associated ichthyoses to psoriasis, a common inflammatory skin disease driven by pathogenic Th17/IL-23 activation. Prior ichthyosis studies noted variable increases of diverse inflammatory markers (e.g., TNFα, IL-1β, IL-4) and chemokines (e.g., TSLP, PAR2, TARC).^50-52 The majority of these data was derived from animal ^53-55 and keratinocyte cultures^{21, 25} or limited to specific disease subtypes in humans,⁵² focusing only on few markers and necessitating a global, comprehensive molecular phenotyping across ichthyoses.

The present study is the first detailed genomic profiling of the most common orphan forms of ichthyosis (NS, LI, congenital ichthyosiform erythroderma/CIE, and epidermolytic ichthyosis/EI) compared to controls. psoriasis, and AD. In light of prior data linking ichthyosis with either Th2 AD-like or Th17/IL-23 psoriasis-like immune features, psoriasis and AD patients were used as controls. Our fingerprinting showed ichthyotic skin to harbor robust, severity-related Th17/IL-17 skewing, with activation of genes considered molecular classifiers of psoriasis [KYNU, vanin (VNN) 1/3]. Unlike AD, Ichthyoses failed to induce Th2-markers and did not suppress terminal differentiation or many lipid products greatly (FLG, LOR, FABP4), but did suppress claudins that were even more decreased in AD (CLDN1,8,23). Increased Th17 signal in ichthyosis blood, infers systemic activation of this immune axis, supporting the therapeutic potential of IL-17-antagonism in ichthyosis.

Methods

Patients’ characteristics and samples

21 patients (aged 10.8-56.9 years, 12 female, 9 male), with ichthyosis and known mutations were enrolled, as previously reported (Table 1).⁴⁹ Written institutional review board-approved consent was provided by subjects (≥12 years) and parents (<18 years), and patient demographics, medical history, physical examination, clinical severity scores (Ichthyosis Area and Severity Index/IASI, including IASI-erythema/IASI-E, and IASI-scaling/IASI-S), pruritus (5-D itch and Numeric Rating Scale) were obtained. Four-millimeter skin biopsies were obtained.  Age-matched healthy controls (n=14), and lesions from psoriasis (n=30),⁵⁷ as well as from moderate-to-severe AD patients (n=16)⁵⁶ were included for comparative analyses. Notably, ichthyosis patients were significantly younger than control, AD, and psoriasis cohorts, thus age was considered a covariate in all analyses. Among the ichthyoses, there were no significant differences in IASI or IASI-E scores, racial distribution, gender, or age.  See Table 1 for demographics.

All LI patients had known TGM1/transglutaminase mutations, EI patients had KRT10 mutations, NS patients had SPINK5 mutations, and patients with CIE had primarily hepoxilin-pathway encoding genes mutated.⁴⁹ 4 AD patients were previously genotyped for FLG mutations, and all were negative. Microarray data are deposited in Gene Expression Omnibus repository (accession no. XXX).

Gene-expression analyses

RNA was extracted for RT-PCR with EZ-PCR Core Reagents (Life Technologies, Grand Island, NY), as described.^{59, 74} Expression values were normalized to hARP. Primers are in Table E5. HGU133Plus2.0 microarrays (Affymetrix, Santa Clara, CA) were used for gene-arrays, as described.⁵⁶

Serum immunoassays

Electrochemiluminescence immunoassay was used in a Singulex format to quantify concentrations of IL-17A in each ichthyosis sample, as described.⁷⁵

Immunohistochemistry/IHC and Nile Red immunofluorescence/IF

IHC was performed on frozen sections usine antibodies listed in Table E6, as described.⁶⁹ IF was performed on fresh cryostat sections that were stained with Nile red solution (500μg/mL) in acetone, and images were acquired by using appropriate filters of Zeiss Axioplan 2 (LSM 510) (Carl Zeiss Microimaging, Thornwood, NY) as described.⁶¹ See Supplementary methods.

Statistical analyses

Statistical analysis was performed using R-language (www.R-project.org) and packages available through bioconductor (www.bioconductor.org).

For gene-arrays, quality control was performed as described.⁵⁶  Expression values were obtained using the GCRMA algorithm. Batch effects were adjusted using ComBat.⁷⁶  Probe-sets with1sample with expression values of>3 were kept for analyses.  Data was log2-transformed prior to analysis with the linear mixed-effect model, with disease (AD, psoriasis, ichthyosis) and tissue (lesional and control) as fixed factors and a random effect for each patient. FCHs between comparisons of interest were estimated and hypothesis testing was conducted using contrasts under general framework for linear models in R’s limma package. P-values from the moderated (paired) t-test were adjusted for multiple hypotheses using the Benjamini–Hochberg procedure. Genes with FCH>2 and FDR<0.05 were considered differentially expressed. Hierarchical clustering of samples/conditions used a McQuitty agglomeration algorithm. GSVA was performed using unsupervised sample-wise enrichment.⁷⁷ Gene-set over-representation analysis was performed using XGR software⁷⁸ and CREEDs disease signatures.⁷⁰ RT-PCR data were analyzed using the linear mixed effect model with the fixed factors as above. See supplementary methods.

 

Results

Patients’ demographics and clinical characteristics

To characterize the ichthyosis molecular phenotype, we performed gene-arrays on 21 patients age 10yrs or older, including 6 with autosomal recessive congenital ichthyosis/ARCI-CIE, 7 ARCI-LI, 5 EI, and 3 NS. Age-matched healthy controls (n=14), and previously reported data on lesions from moderate-to-severe AD (n=16)⁵⁶ and psoriasis (n=30)⁵⁷ were included as comparators.

All LI patients had known TGM1/transglutaminase mutations, EI patients had KRT10 mutations, NS patients had SPINK5 mutations, and patients with CIE had mainly hepoxilin-pathway encoding genes mutated. Clinically, there were no significant differences in Ichthyosis area and severity index/IASI among subtypes. See methods and Table 1 for details.

Global genomic profiling of ichthyotic skin shows great similarity to psoriasis

We evaluated gene expression by microarrays, using criteria of fold change/FCH >2 and false-discovery rate/FDR<0.05, to define differentially expressed genes (DEGs) in ichthyotic skin. Ichthyosis induced 699 DEGs (375 upregulated and 324 downregulated; Fig. 1A; Table E2). 242 DEGs (34.6% of total ichthyosis DEGs) induced by ichthyosis were shared with AD and psoriasis. These included upregulations of the innate immunity marker NOD2 and barrier gene LCE3D, and downregulation of lipid (FAR2, FABP7, GAL) as well as tight junction (TJ) (CLDN8/23) genes. Of all ichthyosis DEGs, only 28 (4%) were shared exclusively with AD, including down-regulation of the lipid gene APOE. A much larger proportion of DEGs [223 (31.9%)] were common to ichthyosis and psoriasis, including upregulation of IL-17 associated markers VNN1/3, KYNU, IL-36A, LCN2, the barrier genes KLK10/13, and downregulation of IL-31RA and periostin/POSTN.

A heatmap representing top dysregulated DEGs in ichthyosis as compared to healthy, psoriatic, and AD skin, illustrates ichthyosis to show overall greater similarity to psoriasis and less to AD (red: upregulated genes; blue: downregulated genes; Fig. 1B; Fig. E2; Table E2). Key Th2-markers increased in AD (CCL4/11/13/17/18/22/26, IL4R) were minimally induced (or even reduced) in ichthyosis.⁵⁸ Similarly, Th1-associated markers (CXCL9, CXCL10, CXCL11) showed slight activation in ichthyotic lesions (Fig. E3).

Multiple epidermal barrier markers, including terminal differentiation and lipid genes, that are downregulated in AD,^{58, 59} were not dysregulated in ichthyotic skin (FLG, CDSN, LOR, PPL, PSORS1C2, ELOVL3, FABP7; Fig. E3B). TJ genes known to be downregulated in AD (CLDN8/23)⁵⁹ showed diminished expression in ichthyosis, albeit to a lesser extent. Striking fingerprint similarity was found between ichthyosis and psoriasis, but not AD skin (Fig E4). This included but was not limited to the epidermal growth factor receptor (EGFR) ligand EREG,⁵⁶ and barrier genes known to be increased in psoriasis (SPRR2 class, KLK13).^{60, 61} A strong Th17 signature, highly pathogenic to psoriasis,⁶² was shared in ichthyosis skin, including increases in IL-36A/B, IL-36RN, KYNU, VNN1/3, LCN2, CCL20, PI3, S100A12, and the IL-17A/IFNγ induced antimicrobial RNASE7 (Fig. E4; Table S2).

IL-17 related markers are significantly increased in ichthyosis and correlate with disease severity

To validate induction of psoriasis hallmark genes in ichthyosis skin,^63-65 we further performed RT-PCR in ichthyosis, psoriasis, AD, and healthy skin. The IL-17 associated markers IL-17F, IL-36α, IL-36β, IL-36γ, KYNU, EREG, and VNN1/3 showed highly significant mRNA levels in both psoriasis and ichthyosis lesions vs. healthy skin (P<0.001; Fig. 2A). While some markers (IL-36α, IL-36γ, KYNU, VNN1/3) were significantly elevated in AD vs. healthy skin, highest expressions were seen in ichthyosis and psoriasis.  Some markers were insignificantly induced (EREG, IL-17F, KLK13; P>0.1) or even reduced (IL-36β) in AD vs. healthy skin, but highly increased in ichthyosis, analogous to psoriasis (Fig. 2B).

Spearman correlations were performed to determine how clinical severity (by IASI) and barrier disruption (by TEWL) are linked to individual cellular and molecular markers. Markers showing the highest correlations with IASI-E included IL-17F (r= 0.71,P= 9.54×10^-6), IL-36γ (r=0.62, P=0.0002), KYNU (r=0.57, P=0.001), and KLK13 (r=0.57, P=0.001) (Fig. 2C; Fig. E5; Table E3). Highly significant correlations were also noted between select markers, IASI score (IL-17F, KLK13; P<0.05) and TEWL (IL-17F, EREG, IL-36α, IL-36β, IL-36γ, KLK13, VNN3; all p<0.01). The 5-D itch scale showed few non-significant correlations (data not shown).

 

Increased systemic Th17 activation, lipid gene down-regulation and non-altered Th2 axis characterize ichthyosis

Few reports have noted ichthyosis blood to harbor inflammatory changes.^{42, 46, 47, 51, 52} To evaluate for systemic activation, we measured IL-17A levels in blood. We found highly significant (P<0.001) increases of circulating IL-17A levels in ichthyosis vs. control (Fig. 3A), that correlated with the erythema (inflammatory) component of ichthyosis severity (r=0.42, P=0.07; Fig 3B). To examine differences across ichthyoses, we performed a subgroup analysis, which showed most ichthyoses to harbor significant increases of circulating IL-17A vs. control (P<0.01), with NS showing highest levels (Fig 3C).

Aiming to evaluate for distinctions between different ichthyosis phenotypes, we next compared between ichthyoses subtypes, AD, and psoriasis. In our prior RT-PCR profiling that was limited to a small set of inflammatory markers, we observed IL-17 related factors, including those displaying synergistic/additive effect with TNF- α, to show upregulation in ichthyosis patients to a similar extent as in those with psoriasis. We further explored genes showing Th17/TNFa synergism (Fig. 3D).

AD showed the highest inflammatory/immune activation markers (e.g., MMP12, ICOS, CCR7; Fig 3D). Among the ichthyoses, NS demonstrated the greatest upregulation of these markers (Fig. Exx; Table E4). Th1 responses (CXCL9, CXCL10, CXCL11) were relatively attenuated among the ichthyoses (except EI), and similarly, the Th2 axis pathogenic to AD,^{66, 67} showed minimal or no activation across the ichthyoses (IL-4R, IL-5, IL-6, IL-13, CCL11, CCL13, CCL17, CCL18, CCL26), with NS harboring greatest Th2 inductions vs. other subtypes. A strong Th17 signal (PI3, S100A9/12, CCL20) was demonstrated across ichthyosis subtypes, with NS showing highest elevations, and LI characterized by significant LCN2, CCL20, and MAPK8 expressions. Upregulations of KYNU, VNN3, SERPINB4, as well as the Keratoconus associated gene DOCK9⁶⁸ were seen across all ichthyoses, similar to psoriasis skin (Fig. 3D).

To comprehensively assess barrier features we next looked at expression levels of epidermal differentiation complex (EDC),cornified envelope (CE) and lipid-associated genes. EDC and CE genes downregulated in AD⁵⁸ were not decreased among the ichthyoses (LOR, FLG, PPL, EVPL, CDSN, LCE1B/E, ANXA9; Fig. 4 yellow box), while lipid metabolism genes (FAR2, FADS1, ELOVL3, GAL, HAO2, FABP7; Fig. 4, green box) and claudins (CLDN1,8,23) reduced in AD, were also downregulated in majority of ichthyoses. EI did not demonstrated lipid abnormalities (Fig 4). Notably, psoriasis lesions also exhibited some decreases in terminal differentiation (LOR) and lipid markers (ELOVL3, FABP7) vs. healthy skin.

IL-17 and psoriasis related markers are increased among all ichthyosis subtypes and correlate with disease severity

To validate Th17-related marker expressions among ichthyosis subtypes, we performed RT-PCR analyses. All ichthyoses showed significantly increased IL-36α, IL-36β, IL-36γ, KYNU, EREG, and VNN1/3 mRNA vs. healthy skin (P<0.05 for all; Fig 5A)  with majority of these also significantly increased in ichthyoses vs. AD (P<0.05 for all; Fig 5A). NS was typified by superior IL-17F, IL-36α, EREG, and VNN3 induction compared to other ichthyosis subtypes (P<0.05 for all; Fig 5A). Notably, EREG demonstrated larger and more significant expression in all ichthyoses than even psoriasis (P<0.05; Fig 5A).

Comparing the magnitude of expression of lesional vs. normal tissues revealed almost all IL-17-related markers (except IL-36α and VNN1) to be increased in ichthyoses, even higher than in psoriasis (Fig. 5B), with NS and CIE showing highest expressions vs. other ichthyoses. All subtypes showed greatest and most significant correlations of several markers with IASI-E, including CIE/KLK13 (r=0.85, P=0.003), EI/KYNU (r=0.86, P=0.0102), LI/VNN1 (r=0.81, P=0.0149), and LI/IL-36α (r=0.71, P=0.0465) (Fig.5C). Highly significant correlations were also noted with total IASI scores (KLK13, KYNU; P<0.05) and TEWL (VNN1, VNN3; P<0.05) for all subtypes (Fig. E6). Correlations within NS could not be performed due to limited sample size.

Pathway analysis of the global ichthyosis molecular profile shows Th17 polarization

We next evaluated the extent to which individual ichthyosis subtypes activated major inflammatory and regulatory pathways represented in gene arrays (compared to psoriasis and AD), by gene set variance analysis (GSVA) using previously defined gene lists for these axes.⁶⁹ The bar-plots in Fig. 6A-B depict IL-17 and Th17/TNF synergistically induced genes to be highly expressed in all ichthyoses, compared to AD and healthy skin (p<0.05 for all), aligning closer to psoriasis. CE genes (Fig. 6C) were significantly downregulated only in AD vs. healthy skin, but were relatively unchanged or even increased among the ichthyoses. Lipid genes (Fig. 6D) showed significant downregulation in AD (P<0.001) compared to healthy skin, but with relatively less suppression among the ichthyoses, albeit significant reductions were noted vs. control skin. Of note, EI was an exception and showed no respective lipid deficiencies vs. healthy skin.

To relate the molecular profile of ichthyosis to other inflammatory diseases, we performed enrichment analyses with signatures of inflammatory conditions marked by polar immune activation.⁷⁰ Ichthyosis showed high overlap with the psoriasis fingerprint, as well as other Th17-mediated dermatoses (acne), but also somewhat shared its transcriptome with Th2 conditions (eczema, ulcerative colitis)⁷¹ (Fig. 6B).⁷²

 

Cutaneous Th17 activation and lipid abnormalities extend to the protein and ultrastructural level across Ichthyoses

Ultimately, to corroborate our gene profile findings, we examined Th17-related activation at the protein level, utilizing immunohistochemistry studies to evaluate expression of IL-36R, that is known to form feedback loops driving Th17-related cytokine expression in psoriasis.^{72, 73} Overall, wide epidermal expression of IL-36R was noted among all ichthyosis subtypes, similar to psoriasis, and strikingly different from AD tissues, in which IL-36R was minimally expressed (Fig. 7A-G).

We further examined lipid distribution at a closer level. To enhance visualization of stratum corneum intercellular lipids, we performed Nile red immunofluorescence staining in ichthyoses as well as AD, psoriasis, and healthy skin (Fig. 7H-N). While healthy skin was characterized by well-packed extracellular lipids between compacted corneocytes (Fig. 7N), the ichthyoses showed noticeable reductions in extracellular lipid layers with diminished corneocyte compaction, with NS demonstrating more prominent extracellular lipid layers and EI showing xxx. Psoriasis (Fig. 7L), also harbored decreases in corneocyte compaction, and illustrated preservation of extracellular lipid layers, whereas AD (Fig. 7M) revealed minimal corneocyte compaction and almost non-existent lipid layers, congruent with its greatest suppression of lipid-related genes.

DISCUSSION

FLG mutations, increased TEWL, dysregulated lipid homeostasis, and frequent concomitancy with allergic phenotypes, have linked ichthyoses to AD.³⁶ (more refs, RTPCR-101) However, we recently profiled ichthyotic skin using a limited RT-PCR based approach and found enhanced Th17/IL-23 inflammatory responses, minimal Th2-skewing, and no defects in terminal differentiation, aligning it closer to the psoriasis profile.⁴⁹ The present report is the first comprehensive genomic characterization of the most common orphan ichthyosis subtypes in humans, with comparisons not only to healthy controls, but also to AD and psoriasis, representing Th2/Th22 and Th17/IL-23 polar immune activation, respectively.

Our data shows ichthyoses to express a predominant Th17/IL-23 signature, in line with our prior report (ref) and a recent skin profiling of an Ichthyosis Curth-Macklin patient (Refs Relcade 2017). We show striking induction of Th17/TNF synergistic genes and molecular hallmarks of psoriasis (IL-17F, KYNU, VNN1,3, IL-36 family, EREG), across ichthyoses but most remarkably in NS, that strongly correlate with the inflammatory component of disease severity. KYNU, a psoriasis classifier, is involved in tryptophan metabolism, and enhanced by IL-17/TNF synergism. VNN3, a mediator in Th17/Th1 driven skin disease, has likewise been shown to be uniquely upregulated in psoriasis (Jansen 2009, kamsteeg 2010). Moreover, VNN1 and VNN3 were reportedly induced by Th17/Th1, but not Th2 cytokines (Jansen 2009). Hence, their increased expression across ichthyoses, shown here, is possibly related to increased Th17 and non-altered Th2 levels, highlighting resemblance between ichthyosis and psoriasis. IL-36γ, a proinflammatory cytokine overexpressed in psoriasis skin 28043870Johnston 2016, was implicated in epidermal thickening and hyperkeratosis. IL-36γ is induced in keratinocytes by IL-17 and TNFα, 27259168Dietrich 2016is known to amplify feedback loops driving Th17/TNF-related cytokine expressions, and precisely discriminates psoriasis from AD lesions.^{72, 73}(refs Derme 2015) Psoriasis-level elevations of the IL-36 and VNN family, as well as KYNU, correlated with ichthyosis inflammation, seen across subtypes, underscoring the strong overlap of ichthyosis with psoriasis. These IL-36γ elevations also mirror our prior RT-PCR data that showed hyperkeratosis and epidermal thickening, which correlated with ichthyosis severity (ref).

TNFα has been implicated in Th17/IL-23 responses in psoriasis (chiricozzi 2011), and TNFα blockade has been shown to reduce IL-17/TNF synergistically induced genes better than TNFα modulated genes alone. (zaba 2009) Across ichthyoses, NS showed the highest elevation of psoriasis classifiers, with differences most prominent in the blood, supporting an accentuated systemic inflammatory phenotype in NS versus other ichthyoses. Tissues in NS patients have been documented to harbor Th17 elevations⁵³(refs furio 2015), that were reduced with TNF-antagonism (refs fontao 2011).  While data in ichthyotic blood is scarce, NS serum showed increases in TNFα, interferon induced responses (IL-18), and Th17/Th22 (IL-12) (renner 2009, hosomi 2008, with only a single case describing IL-17A expansion successfully attenuated with omalizumab (REFS yalcin 2016). Severity-associated Th17 dominance in the blood shown here across all ichthyoses, and strikingly in NS, supports the need for systemic therapeutic interventions in the severe forms of ichthyoses.

While Th2 signaling is key to AD pathogenesis, (refs) it was minimally induced (CCL4/11/13/22/26, IL-4R, IL-13) in ichthyoses, besides NS, which has been classically linked with atopic diathesis (REFS). Lack of Th2 induction might be secondary to several factors. KYNU metabolites were implicated in Th2 downregulation, particularly IL-4 expression (REFS).  Another putative explanation lies in the recognized reciprocal regulatory capacity of Th17 and Th2 axes (Choy 2015, Newcomb 2011, Newcomb 2012).  Plausibly, Th17 upregulation in ichthyosis may be the outcome of complex regulatory interactions between different arms of adaptive immunity that extend to the systemic level, as evidenced by our blood data.

The ichthyoses are known to harbor barrier impairment, reflected by increased TEWL (refs).To dissect relative contributions of barrier components to functional barrier impairment underlying ichthyosis, we comprehensively analyzed different aspects of the epidermal barrier, including terminal differentiation and TJ genes, as well as lipid markers. Wide gene sets demonstrated a preserved terminal differentiation profile across ichthyoses, mirroring findings from prior studies 24534191, Kawashima 2005), and potentially reflecting lack of Th2 mediated epidermal differentiation suppression (20147633,16546102, 18166499).  Claudins-1, 8, and 23, that are crucial for TJ formation, are suppressed in AD lesions (refs laser capture, de bennedeto 2011), and inversely correlate with high Th2 signals observed in AD (de bennedeto 2010). Intriguingly, in ichthyosis, various TJ genes were differentially expressed, with some showing downregulation (CLDN1,8,23), similar to AD, whereas other were unchanged (CLDN5,7,10). This dichotomy may be attributable to both cytokine dysregulations seen in ichthyosis and/or the diverse regulation of different TJ genes (Gruber 2015). The influence of the Th17 axis to enhance some claudins, harboring a TJ-protective effect (de benne 2011, 2013), but also to suppress other TJ and adhesion molecules (Yuki 2016, Owsiak), may account for diverse TJ expression levels seen in ichthyosis. Relative Th2 increases in NS may contribute to TJ downregulation, further compromising barrier integrity in these patients.

The ichthyoses are characterized by defects in lamellar body (LB) trafficking and lipid homeostasis (refs). Our gene profiling, corroborated by a Nile-red lipid staining, demonstrates unique abnormalities in SC lipids and related genes, across subtypes. While overall lipid measures (FA2H, FABP7, ELOVL3) were suppressed in ichthyosis compared to controls, this downregulation was much less dominant than in AD. While lipid gene suppression was similar across ichthyosis subtypes, EI, that is associated with KRT1 and 10 mutations, showed minimal lipid defects. Abnormal KRT1 and 10 were previously linked to disrupted LB exocytosis (PMC3607361 and 11676820), but were also shown to induce pro-inflammatory, Th17-skewed networks in murine keratinocytes (refs). Additionally, IL-17 is a regulator of adipogenesis, inhibiting adipocyte differentiation (Lee 2017), and potentially curbing epidermal lipid synthesis (ref). EI had relatively low Th17 induction, raising possibility for the lack of lipid abnormalities observed in this subtype. Decreased lipids have been shown to upregulate psoriasis-related gene expression (IL-17, IL-22, IL-23, S100As, and beta-defensins) Nakajima 2013), potentially explaining upregulation of these genes in ichthyosis. Alternatively, increased VNN3 levels, implicated in lipid homeostasis, may account for higher lipid levels seen in ichthyosis compared to AD (refs Jansen 2009).

Similar to psoriasis,ref ichthyoses profiling revealed elevations of several IL-17/TNF-induced antimicrobial peptides (AMPs) (RNASE7, EREG, LCN2, CCL20, and LL37), with NS showing most robust increases (refs Haneda 2016, Li 2014). LL37 was shown to stimulate production of various proinflammatory cytokines and to recruit Th17 cells through IL36g induction. 25305315 Li 2014This LL37-IL36g link is further accentuated by our results that show parallel increases of these two markers alongside a prominent Th17 signature. AMP production accelerates in response to barrier disruption and pathogen challenges (chan 2015), but also increases in diverse inflammatory dermatoses (chan 2015-36). With these data, it is important to delineate whether IL-17 induction is a reactive attempt to curtail infection risk, a primarily pathogenic immune response, or if it is secondary to intrinsic abnormalities of the ichthyotic epidermis.

We recently reported two cases of syndromic ichthyoses (Severe dermatitis, multiple allergies, and metabolic wasting/SAM, and erythrokeratodermia-cardiomyopathy/EKC syndromes) treated successfully with IL12-23p40 antagonism (ustekinumab) (REFS). Clinical improvement was striking with decreased erythroderma and scaling, and reductions in IASI, IASI-E, and TEWL, providing compelling support that Th17/IL-23 activation plays a major role in ichthyosis, and that immune dysregulation is beyond a response of the impaired barrier to generate antimicrobials (REFS). Taken together, we propose that IL-17 could potentially be a pathogenic cytokine in ichthyosis, partially driving epidermal pathologic features. These data additionally form the basis for an ongoing clinical trial (NCT03041038) to test therapeutic efficacy of IL-17A antagonism in patients with ichthyosis, which may elucidate the functional role of IL-17 in disease pathogenesis and more closely dissect its complex relationship with underlying barrier defect.

Our study has few limitations. Ichthyosis rarity limited our sample size, nevertheless, the present cohort is the largest assessed in a comparative molecular approach. Second, due to limited serum, we only performed IL-17A measurements in ichthyosis blood. Third, as there are currently no validated ichthyosis severity scores known, we utilized IASI, which is ongoing validation. IASI better defines clinical severity but omits the non-common alopecia measurement. Finally, skin biomarker correlations with TEWL and IASI could not be performed within NS due to small sample size.

In conclusion, our immunophenotyping at the genomic, mRNA and protein level demonstrates IL-17/TNFα synergism underlying cutaneous inflammatory responses in ichthyoses, and extending to the blood compartment in a severity-related manner. These changes parallel preserved EDC and CE genes and downregulation of lipid genes. Functional and targeted therapeutic studies are needed to clarify the pathogenic role of IL-17 in ichthyosis and determine whether its elevations are primary or a compensatory response to a defective barrier. Similar to AD and psoriasis pathogenesis, where crosstalk between immune dysregulation and barrier impairment drives the disease phenotype (refs), we offer novel insights into Th17 driven immune circuits in diseases of defective skin barrier, linking communication between these two components in ichthyosis. While future studies will need to dissect the relationship of epidermal abnormalities with immune-driven cutaneous inflammation in ichthyoses, our data advocates for further development and testing of cytokine-specific targeting modalities in ichthyosis that can potentially change the therapeutic standard for these devastating diseases.

Acknowledgements: We thank Drs. Adam Berry, Jayla Gray, Isabel Haugh, Lisa Shen, Anjali Shroff, and Robalee Wanderman for helping with patient enrollment. We are grateful to the Foundation for Ichthyosis and Related Skin Types for allowing this research

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