Background Atopic dermatitis (AD) is caused by a complex interplay between immune and barrier abnormalities. Murine models of AD are essential for preclinical assessments of new treatments. Although ...many models have been used to simulate AD, their transcriptomic profiles are not fully understood, and a comparison of these models with the human AD transcriptomic fingerprint is lacking. Objective We sought to evaluate the transcriptomic profiles of 6 common murine models and determine how they relate to human AD skin. Methods Transcriptomic profiling was performed by using microarrays and quantitative RT-PCR on biopsy specimens from NC/Nga, flaky tail, Flg -mutated, ovalbumin-challenged, oxazolone-challenged, and IL-23–injected mice. Gene expression data of patients with AD, psoriasis, and contact dermatitis were obtained from previous patient cohorts. Criteria of a fold change of 2 or greater and a false discovery rate of 0.05 or less were used for gene arrays. Results IL-23–injected, NC/Nga, and oxazolone-challenged mice show the largest homology with our human meta-analysis–derived AD transcriptome (37%, 18%, 17%, respectively). Similar to human AD, robust TH 1, TH 2, and also TH 17 activation are seen in IL-23–injected and NC/Nga mice, with similar but weaker inflammation in ovalbumin-challenged mice. Oxazolone-challenged mice show a TH 1-centered reaction, and flaky tail mice demonstrate a strong TH 17 polarization. Flg -mutated mice display filaggrin downregulation without significant inflammation. Conclusion No single murine model fully captures all aspects of the AD profile; instead, each model reflects different immune or barrier disease aspects. Overall, among the 6 murine models, IL-23–injected mice best simulate human AD; still, the translational focus of the investigation should determine which model is most applicable.
Background Severe atopic dermatitis (AD) has a high unmet need for effective and safe therapeutics. In early-phase trials, dupilumab, a fully human mAb targeting IL-4 receptor α, markedly improved ...disease activity, but the effect of IL-4/IL-13 blockade on AD at the molecular level has not been characterized. Objectives We sought to evaluate dupilumab modulation of the AD molecular signature. Methods We performed transcriptomic analyses of pretreatment and posttreatment skin biopsy specimens from patients with moderate-to-severe AD treated weekly with 150 or 300 mg of dupilumab or placebo. Results Exacerbation of the AD transcriptome was observed in placebo-treated patients. Dupilumab improved the AD signature in a dose-dependent manner. Expression of genes upregulated in AD lesions decreased in patients treated with dupilumab by 26% (95% CI, 21% to 32%) and 65% (95% CI, 60% to 71%) for treatment with 150 and 300 mg, respectively. Genes downregulated in AD lesions increased by 21% (95% CI, 16% to 27%) and 32% (95% CI, 26% to 37%) with dupilumab (150 and 300 mg, respectively). The molecular changes paralleled improvements in clinical scores. A dupilumab treatment signature of 821 probes (>2-fold change, P < .05) significantly modulated in the 300-mg dupilumab group at week 4 compared with baseline was identified in this sample set. Significant ( P < .05) decreases in mRNA expression of genes related to hyperplasia ( K16 and MKI67 ), T cells, and dendritic cells ( CD1b and CD1c ) and potent inhibition of TH 2-associated chemokines ( CCL17 , CCL18 , CCL22 , and CCL26 ) were noted without significant modulation of TH 1-associated genes (IFNG). Conclusions This is the first report showing rapid improvement of the AD molecular signature with targeted anti–IL-4 receptor α therapy. These data suggest that IL-4 and IL-13 drive a complex, TH 2-centered inflammatory axis in patients with AD.
Background Allergic contact dermatitis (ACD) is the most common occupational disease. Although murine contact hypersensitivity provides a framework for understanding ACD, it carries important ...differences from its human counterpart. Unlike the contact hypersensitivity model, which is induced by potent sensitizers (ie, dinitrofluorobenzene), human ACD is induced by weak-to-moderate sensitizers (ie, nickel), which cannot induce reactions in mice. Distinct hapten-specific immune-polarizing responses to potent inducers were suggested in mice, with unclear relevance to human ACD. Objective We explored the possibility of distinct T-cell polarization responses in skin to common clinically relevant ACD allergens. Methods Gene-expression and cellular studies were performed on common allergens (ie, nickel, fragrance, and rubber) compared with petrolatum-occluded skin, using RT-PCR, gene arrays, and immunohistochemistry. Results Despite similar clinical reactions in all allergen groups, distinct immune polarizations characterized different allergens. Although the common ACD transcriptome consisted of 149 differentially expressed genes across all allergens versus petrolatum, a much larger gene set was uniquely altered by individual allergens. Nickel demonstrated the highest immune activation, with potent inductions of innate immunity, TH 1/TH 17 and a TH 22 component. Fragrance, and to a lesser extent rubber, demonstrated a strong TH 2 bias, some TH 22 polarization, and smaller TH 1/TH 17 contributions. Conclusions Our study offers new insights into the pathogenesis of ACD, expanding the understanding of T-cell activation and associated cytokines in allergen-reactive tissues. It is the first study that defines the common transcriptome of clinically relevant sensitizers in human skin and identifies unique pathways preferentially activated by different allergens, suggesting that ACD cannot be considered a single entity.
Background Many human diseases arise from or have pathogenic contributions from a dysregulated immune response. One pathway with immunomodulatory ability is the tryptophan metabolism pathway, which ...promotes immune suppression through the enzyme indoleamine 2,3-dioxygenase (IDO) and subsequent production of kynurenine. However, in patients with chronic inflammatory skin disease, such as psoriasis and atopic dermatitis (AD), another tryptophan metabolism enzyme downstream of IDO, L-kynureninase (KYNU), is heavily upregulated. The role of KYNU has not been explored in patients with these skin diseases or in general human immunology. Objective We sought to explore the expression and potential immunologic function of the tryptophan metabolism enzyme KYNU in inflammatory skin disease and its potential contribution to general human immunology. Methods Psoriatic skin biopsy specimens, as well as normal human skin, blood, and primary cells, were used to investigate the immunologic role of KYNU and tryptophan metabolites. Results Here we show that KYNU+ cells, predominantly of myeloid origin, infiltrate psoriatic lesional skin. KYNU expression positively correlates with disease severity and inflammation and is reduced on successful treatment of psoriasis or AD. Tryptophan metabolites downstream of KYNU upregulate several cytokines, chemokines, and cell adhesions. By mining data on several human diseases, we found that in patients with cancer, IDO is preferentially upregulated compared with KYNU, whereas in patients with inflammatory diseases, such as AD, KYNU is preferentially upregulated compared with IDO. Conclusion Our results suggest that tryptophan metabolism might dichotomously modulate immune responses, with KYNU as a switch between immunosuppressive versus inflammatory outcomes. Although tryptophan metabolism is increased in many human diseases, how tryptophan metabolism is proceeding might qualitatively affect the immune response in patients with that disease.
Background Atopic dermatitis (AD) is a common inflammatory skin disease with a TH 2 and “T22” immune polarity. Despite recent data showing a genetic predisposition to epidermal barrier defects in ...some patients, a fundamental debate still exists regarding the role of barrier abnormalities versus immune responses in initiating the disease. An extensive study of nonlesional AD (ANL) skin is necessary to explore whether there is an intrinsic predisposition to barrier abnormalities, background immune activation, or both in patients with AD. Objective We sought to characterize ANL skin by determining whether epidermal differentiation and immune abnormalities that characterize lesional AD (AL) skin are also reflected in ANL skin. Methods We performed genomic and histologic profiling of both ANL and AL skin lesions (n = 12 each) compared with normal human skin (n = 10). Results We found that ANL skin is clearly distinct from normal skin with respect to terminal differentiation and some immune abnormalities and that it has a cutaneous expansion of T cells. We also showed that ANL skin has a variable immune phenotype, which is largely determined by disease extent and severity. Whereas broad terminal differentiation abnormalities were largely similar between involved and uninvolved AD skin, perhaps accounting for the “background skin phenotype,” increased expression of immune-related genes was among the most obvious differences between AL and ANL skin, potentially reflecting the “clinical disease phenotype.” Conclusion Our study implies that systemic immune activation might play a role in alteration of the normal epidermal phenotype, as suggested by the high correlation in expression of immune genes in ANL skin with the disease severity index.
Background Genomic profiling of lesional and nonlesional skin of patients with atopic dermatitis (AD) using microarrays has led to increased understanding of AD and identification of novel ...therapeutic targets. However, the limitations of microarrays might decrease detection of AD genes. These limitations might be lessened with next-generation RNA sequencing (RNA-seq). Objective We sought to define the lesional AD transcriptome using RNA-seq and compare it using microarrays performed on the same cohort. Methods RNA-seq and microarrays were performed to identify differentially expressed genes (criteria: fold change, ≥2.0; false discovery rate ≤0.05) in lesional versus nonlesional skin from 18 patients with moderate-to-severe AD, with real-time PCR (RT-PCR) and immunohistochemistry used for validation. Results Both platforms showed robust disease transcriptomes and correlated well with RT-PCR. The common AD transcriptome identified by using both techniques contained 217 genes, including inflammatory ( S100A8/A9/A12, CXCL1 , and 2′-5′-oligoadenylate synthetase-like OASL ) and barrier ( MKi67 , keratin 16 K16 , and claudin 8 CLDN8 ) AD-related genes. Although fold change estimates determined by using RNA-seq showed somewhat better agreement with RT-PCR (intraclass correlation coefficient, 0.57 and 0.70 for microarrays and RNA-seq vs RT-PCR, respectively), bias was not eliminated. Among genes uniquely identified by using RNA-seq were triggering receptor expressed on myeloid cells 1 (TREM-1) signaling (eg, CCL2 , CCL3 , and single immunoglobulin domain IL1R1 related SIGIRR ) and IL-36 isoform genes. TREM-1 is a surface receptor implicated in innate and adaptive immunity that amplifies infection-related inflammation. Conclusions This is the first report of a lesional AD phenotype using RNA-seq and the first direct comparison between platforms in this disease. Both platforms robustly characterize the AD transcriptome. Through RNA-seq, we unraveled novel disease pathology, including increased expression of the novel TREM-1 pathway and the IL-36 cytokine in patients with AD.
Background The molecular signature of atopic dermatitis (AD) lesions is associated with TH 2 and TH 22 activation and epidermal alterations. However, the epidermal and dermal AD transcriptomes and ...their respective contributions to abnormalities in respective immune and barrier phenotypes are unknown. Objective We sought to establish the genomic profile of the epidermal and dermal compartments of lesional and nonlesional AD skin compared with normal skin. Methods Laser capture microdissection was performed to separate the epidermis and dermis of lesional and nonlesional skin from patients with AD and normal skin from healthy volunteers, followed by gene expression (microarrays and real-time PCR) and immunostaining studies. Results Our study identified novel immune and barrier genes, including the IL-34 cytokine and claudins 4 and 8, and showed increased detection of key AD genes usually undetectable on arrays (ie, IL22 , thymic stromal lymphopoietin TSLP , CCL22 , and CCL26 ). Overall, the combined epidermal and dermal transcriptomes enlarged the AD transcriptome, adding 674 upregulated and 405 downregulated differentially expressed genes between lesional and nonlesional skin to the AD transcriptome. We were also able to localize individual transcripts as primarily epidermal (defensin, beta 4A DEFB4A ) or dermal ( IL22 , cytotoxic T-lymphocyte antigen 4 CTLA4 , and CCR7 ) and link their expressions to possible cellular sources. Conclusions This is the first report that establishes robust epidermal and dermal genomic signatures of lesional and nonlesional AD skin and normal skin compared with whole tissues. These data establish the utility of laser capture microdissection to separate different compartments and cellular subsets in patients with AD, allowing localization of key barrier or immune molecules and enabling detection of gene products usually not detected on arrays.
Background Tofacitinib is an oral Janus kinase inhibitor being investigated for psoriasis. Objective We sought to elucidate the molecular mechanisms underlying the clinical efficacy of tofacitinib in ...patients with psoriasis. Methods Twelve patients with plaque psoriasis were randomized (3:1) to receive 10 mg of tofacitinib or placebo twice daily for 12 weeks. Biopsy specimens were taken from nonlesional (baseline) and lesional (baseline, days 1 and 3, and weeks 1, 2, 4, and 12) skin. Biopsy specimens were examined for psoriatic epidermal features (thickness, Ki67+ keratinocytes and keratin 16 KRT16 mRNA expression, and phosphorylated signal transducer and activator of transcription pSTAT+ nuclei) and T-cell and dendritic cell (DC) subsets by using immunohistochemistry, and mRNA transcripts were quantified by using a microarray. Results In lesional skin keratinocyte pSTAT1 and pSTAT3 staining was increased at baseline but reduced after 1 day of tofacitinib (baseline, median of 1290 pSTAT1+ cells/μm2 ; day 1, median of 332 pSTAT1+ cells/μm2 ; and nonlesional, median of 155 pSTAT1+ cells/μm2 ). Epidermal thickness and KRT16 mRNA expression were significantly and progressively reduced after days 1 and 3 of tofacitinib administration, respectively (eg, KRT16 decreased 2.74-fold, day 3 vs baseline, P = .016). Decreases in DC and T-cell numbers were observed after weeks 1 and 2, respectively. At week 4, significant decreases in IL-23/TH 17 pathways were observed that persisted through week 12. Improvements in clinical and histologic features were strongly associated with changes in expression of psoriasis-related genes and reduction in IL-17 gene expression. Conclusions Tofacitinib has a multitiered response in patients with psoriasis: (1) rapid attenuation of keratinocyte Janus kinase/STAT signaling; (2) removal of keratinocyte-induced cytokine signaling, leading to reductions in pathologic DC and T-cell numbers to nonlesional levels; and (3) inhibition of the IL-23/TH 17 pathway.
Background In subjects with psoriasis, inflammation and epidermal hyperplasia are thought to be controlled by T cell–derived cytokines. Evidence suggests that the TH 17 cell cytokine IL-17A (IL-17) ...might play a role in disease pathogenesis. Objective We sought to understand the effect that neutralization of IL-17 has on the clinical features of psoriasis and to understand the role that IL-17 has in inflammatory pathways underlying psoriasis in human subjects. Methods We examined skin lesions obtained from 40 subjects participating in a phase I, randomized, double-blind, placebo-controlled trial of the anti–IL-17 mAb ixekizumab (previously LY2439821) in which subjects received 5, 15, 50, or 150 mg of subcutaneous ixekizumab or placebo at weeks 0, 2, and 4. Results There were significant dose-dependent reductions from baseline in keratinocyte proliferation, hyperplasia, epidermal thickness, infiltration into the dermis and epidermis by T cells and dendritic cells, and keratinocyte expression of innate defense peptides at 2 weeks. By week 6, the skin appeared normal. Quantitative RT-PCR and microarrays revealed an ablation of the disease-defining mRNA expression profile by 2 weeks after the first dose of study drug. The effect of IL-17 blockade on expression of genes synergistically regulated by IL-17 and TNF-α was of higher magnitude at 2 weeks than in prior studies with TNF-α antagonism. Conclusion Our data suggest that IL-17 is a key “driver” cytokine that activates pathogenic inflammation in subjects with psoriasis. Neutralizing IL-17 with ixekizumab might be a successful therapeutic strategy in psoriasis.
Background The ichthyoses are rare genetic disorders associated with generalized scaling, erythema, and epidermal barrier impairment. Pathogenesis-based therapy is largely lacking because the ...underlying molecular basis is poorly understood. Objective We sought to characterize molecularly cutaneous inflammation and its correlation with clinical and barrier characteristics. Methods We analyzed biopsy specimens from 21 genotyped patients with ichthyosis (congenital ichthyosiform erythroderma, n = 6; lamellar ichthyosis, n = 7; epidermolytic ichthyosis, n = 5; and Netherton syndrome, n = 3) using immunohistochemistry and RT-PCR and compared them with specimens from healthy control subjects, patients with atopic dermatitis (AD), and patients with psoriasis. Clinical measures included the Ichthyosis Area Severity Index (IASI), which integrates erythema (IASI-E) and scaling (IASI-S); transepidermal water loss; and pruritus. Results Ichthyosis samples showed increased epidermal hyperplasia (increased thickness and keratin 16 expression) and T-cell and dendritic cell infiltrates. Increases of general inflammatory (IL-2), innate (IL-1β), and some TH 1/interferon (IFN-γ) markers in patients with ichthyosis were comparable with those in patients with psoriasis or AD. TNF-α levels in patients with ichthyosis were increased only in those with Netherton syndrome but were much lower than in patients with psoriasis and those with AD. Expression of TH 2 cytokines (IL-13 and IL-31) was similar to that seen in control subjects. The striking induction of IL-17–related genes or markers synergistically induced by IL-17 and TNF-α (IL-17A/C, IL-19, CXCL1, PI3, CCL20, and IL36G; P < .05) in patients with ichthyosis was similar to that seen in patients with psoriasis. IASI and IASI-E scores strongly correlated with IL-17A ( r = 0.74, P < .001) and IL-17/TNF–synergistic/additive gene expression. These markers also significantly correlated with transepidermal water loss, suggesting a link between the barrier defect and inflammation in patients with ichthyosis. Conclusion Our data associate a shared TH 17/IL-23 immune fingerprint with the major orphan forms of ichthyosis and raise the possibility of IL-17–targeting strategies.
PDF
