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 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 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 Atopic dermatitis (AD) is the most common inflammatory disease. Evolving disease models link changes in epidermal growth and differentiation to TH 2/TH 22 cytokine activation. However, ...these models have not been tested by in vivo suppression of T-cell cytokines. Cyclosporine (CsA) is an immunosuppressant that is highly effective for severe disease, but its mechanism in AD skin lesions has not been studied. Objective We sought to establish the ability of a systemic immunosuppressant to modulate immune and epidermal alterations that form the pathogenic disease phenotype and to correlate changes with clinical improvement. Methods CsA's effects on AD skin pathology were evaluated by using gene expression and immunohistochemistry studies in baseline, week 2, and week 12 lesional and nonlesional biopsy specimens from 19 patients treated with 5 mg/kg/d CsA for 12 weeks. Results After 2 and 12 weeks of treatment, we observed significant reductions of 51% and 72%, respectively, in SCORAD scores. Clinical improvements were associated with significant gene expression changes in lesional but also nonlesional skin, particularly reductions in levels of TH 2-, TH 22-, and some TH 17-related molecules (ie, IL-13, IL-22, CCL17, S100As, and elafin/peptidase inhibitor 3), and modulation of epidermal hyperplasia and differentiation measures. Conclusions This is the first study that establishes a relationship between cytokine activation and molecular epidermal alterations, as well as correlations between disease biomarkers in the skin and clinical improvement. The reversal of the molecular phenotype with CsA and the associated biomarkers can serve as a reference for the successful modulation of tissue inflammation with specific immune antagonists in future studies, contributing to the understanding of the specific cytokines involved in epidermal pathology.
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.
Background Petrolatum is a common moisturizer often used in the prevention of skin infections after ambulatory surgeries and as a maintenance therapy of atopic dermatitis (AD). However, the molecular ...responses induced by petrolatum in the skin have never been assessed. Objective We sought to define the cutaneous molecular and structural effects induced by petrolatum. Methods Thirty-six healthy subjects and 13 patients with moderate AD (mean SCORAD score, 39) were studied by using RT-PCR, gene arrays, immunohistochemistry, and immunofluorescence performed on control skin, petrolatum-occluded skin, and skin occluded with a Finn chamber only. Results Significant upregulations of antimicrobial peptides (S100A8/fold change FCH, 13.04; S100A9/FCH, 11.28; CCL20/FCH, 8.36; PI3 elafin/FCH, 15.40; lipocalin 2/FCH, 6.94, human β-defensin 2 DEFB4A/FCH, 4.96; P < .001 for all) and innate immune genes ( IL6 , IL8 , and IL1B ; P < .01) were observed in petrolatum-occluded skin compared with expression in both control and occluded-only skin. Application of petrolatum also induced expression of key barrier differentiation markers (filaggrin and loricrin), increased stratum corneum thickness, and significantly reduced T-cell infiltrates in the setting of “normal-appearing” or nonlesional AD skin, which is known to harbor barrier and immune defects. Conclusions Petrolatum robustly modulates antimicrobials and epidermal differentiation barrier measures. These data shed light on the beneficial molecular responses of petrolatum in barrier-defective states, such as AD and postoperative wound care.
Background Atopic dermatitis (AD) and psoriasis pathogeneses involve skin barrier impairment and immune dysregulation; however, the contribution of B-cell imbalances to these diseases has not yet ...been determined. Objective We sought to quantify B-cell populations and antibody-secreting cells in the blood of patients with AD, patients with psoriasis, and control subjects. Methods We studied 34 adults with moderate-to-severe AD (mean SCORAD score, 65), 24 patients with psoriasis (mean Psoriasis Area and Severity Index score, 16), and 27 healthy subjects using an 11-color flow cytometric antibody panel. IgD/CD27 and CD24/CD38 core gating systems were used to determine frequencies of plasmablasts and naive, memory, transitional, and activated B cells. Results We measured increased CD19+ CD20+ B-cell counts in the skin and blood of patients with AD ( P < .01). Significantly higher frequencies of chronically activated CD27+ memory and nonswitched memory B cells were observed in patients with AD ( P < .05), with lower values of double-negative populations (4% for patients with AD vs 7% for patients with psoriasis P = .001 and 6% for control subjects P = .02). CD23 expression was highest in patients with AD and correlated with IgE levels ( P < .01) and disease severity ( r = 0.6, P = .0002). Plasmablast frequencies and IgE expression were highest in all memory subsets of patients with AD ( P < .01). Finally, CD19+ CD24++ CD38++ transitional and CD19+ CD24− CD38− new memory B-cell counts were higher in patients with AD versus those in patients with psoriasis (2.8% vs 1.4% P = .001 and 9.2% vs 5.7% P = .02, respectively). Conclusions AD is accompanied by systemic expansion of transitional and chronically activated CD27+ memory, plasmablast, and IgE-expressing memory subsets. These data create a critical basis for the future understanding of this debilitating skin disease.
To the Editor: Alopecia areata (AA) is a prevalent (approximately 1.7% lifetime risk) disease.1 Although it has a large effect on patients' quality of life and poses a large economic burden, ...treatment options for patients with AA are limited.2,3 For more extensive alopecia forms, such as total scalp (totalis) or body (universalis) AA, for which spontaneous regrowth is rare,1 immunosuppressants (systemic corticosteroids, cyclosporine A, and Janus kinase inhibitors) have shown some efficacy but are associated with side effects that preclude long-term use.2,3 Furthermore, hair loss recurs shortly after cessation of treatment.3 Cytokines driving hair loss are not well understood,1 hindering the targeted therapeutic development seen with other skin diseases.4 Our recent study in a well-characterized group of 27 patients with AA associated the AA signature with robust TH2 and IL-23p19 and IL-23/IL-12p40 activation in addition to the TH1 skewing that has been the previous focus.5,6 This provides a rationale for exploring cytokine-targeted therapeutics in patients with AA, which are approved or tested for psoriasis or atopic dermatitis.4 Our data show a large and significant increase in IL-12/IL-23p40 cytokine levels in scalp from patients with lesional AA versus normal scalp.5 Ustekinumab, an IL-12/IL-23p40 blocker that is US Food and Drug Administration approved for psoriasis, induces high-grade improvement in 70% to 80% of patients with psoriasis.4 Here we show that ustekinumab has impressive ability to improve hair growth in patients with extensive AA. Substantial TH2 downregulation was observed in the 2 patients with dramatic clinical improvement, whereas all patients had reduction of PDE gene expression (Fig 2, C). Because PDE genes were significantly upregulated at baseline and downregulated with treatment, a PDE score might also provide insights into disease resolution with treatment.
Background B cells undergo maturation and class-switching in response to antigen exposure and T-cell help. Early B-cell differentiation has not been defined in patients with early-onset atopic ...dermatitis (AD). Objective We sought to define the frequency of B-cell subsets associated with progressive B-cell maturation and IgE class-switching. Methods We studied 27 children and 34 adults with moderate-to-severe AD (mean SCORAD score, 55 and 65, respectively) and age-matched control subjects (15 children and 27 adults). IgD/CD27 and CD24/CD38 core gating systems and an 11-color flow cytometric panel were used to determine the frequencies of circulating B-cell subsets. Serum total and allergen-specific IgE (sIgE) levels were measured by using ImmunoCAP. Results Compared with adults, children showed T-cell predominance in the skin. Circulating CD19+ CD20+ B-cell counts were lower in patients with pediatric AD than in control subjects (24% vs 33%, P = .04), whereas CD3+ T-cell counts were higher (62% vs 52%, P = .05). A decreased B-cell/T-cell lymphocyte ratio with age was observed only in pediatric control subjects ( r = −0.48, P = .07). In pediatric patients with AD, a positive correlation was observed between B-cell/T-cell ratio and nonswitched memory B-cell counts ( r = 0.42, P = .03). Higher frequencies of positive sIgE levels were seen in pediatric patients with AD ( P < .0001). Diverse sIgE levels correlated with SCORAD scores and age of pediatric patients with AD ( P < .01). Positive correlations were observed between activated B-cell and memory T-cell counts ( P < .02). In patients with AD, IgE sensitization to most allergens clustered with age, TH 1, TH 2, total IgE levels, and B-cell memory subsets. Conclusions Peripheral B and T cells are altered in pediatric patients with early AD, but T cells predominate in skin lesions.
Background Topical glucocorticosteroids are considered an efficient treatment option for atopic dermatitis (AD), but a global assessment of glucocorticosteroid responses on key disease circuits upon ...weeks to months of treatment is currently lacking. Objective We sought to assess short (4 weeks) and long-term (16 weeks) application of topical glucocorticosteroids on AD skin and define response biomarkers. Methods The effects of triamcinolone acetonide cream 0.025% were assessed based on gene expression and immunohistochemistry studies at baseline, 4 weeks, and 16 weeks in biopsy specimens from 15 patients with moderate-to-severe AD. Results At 16 weeks, only 3 patients were clinical responders (by using SCORAD50 criteria), but 6 patients qualified as responders based on histologic criteria. Baseline characteristics indicated more severe disease in nonresponders. While 3 of 15 patients experienced only transient benefit after 4 weeks, others showed progressive improvements toward 16 weeks. Topical glucocorticosteroid use in patients with AD resulted in improvements of the AD genomic signature of 25.6% at 4 weeks and 71.8% at 16 weeks, respectively, and even 123.9% in the histologic responder group. Cytokines (IL-12p40, IL-13, IL-22, CCL17, CCL18, peptidase inhibitor 3 PI3/elafin, and S100As) showed consistent decreases from baseline toward 16 weeks with corresponding improvements in epidermal disease hallmarks (keratin 16 and loricrin) in lesional skin from responders ( P < .05). Nonresponders largely showed lesser/nonsignificant reductions in key inflammatory and barrier markers (keratin 16, IL-13, IL-22, CCL17, CCL18, PI3/elafin, S100As, and loricrin). The combination of IL-21 and IFN-γ baseline expression closely predicted individual clinical glucocorticosteroid responses at 16 weeks of treatment. Conclusion Our study indicates that even low-potency glucocorticosteroids can broadly affect immune and barrier responses in patients with moderate-to-severe AD, associating higher baseline severity with increased steroid resistance in patients with AD.
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