Progressive activation of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis

Abstract

Background: Atopic dermatitis (AD) is a common disease with an increasing prevalence. The primary pathogenesis of the disease is still elusive, resulting in the lack of specific treatments. AD is currently considered a biphasic disease, with T(H)2 predominating in acute disease and a switch to T(H)1 characterizing chronic disease. Elucidation of the molecular factors that participate in the onset of new lesions and maintenance of chronic disease is critical for the development of targeted therapeutics.

Objectives: We sought to characterize the mechanisms underlying the onset and maintenance of AD.

Methods: We investigated intrapersonal sets of transcriptomes from nonlesional skin and acute and chronic lesions of 10 patients with AD through genomic, molecular, and cellular profiling.

Results: Our study associated the onset of acute lesions with a striking increase in a subset of terminal differentiation proteins, specifically the cytokine-modulated S100A7, S100A8, and S100A9. Acute disease was also associated with significant increases in gene expression levels of major T(H)22 and T(H)2 cytokines and smaller increases in IL-17 levels. A lesser induction of T(H)1-associated genes was detected in acute disease, although some were significantly upregulated in chronic disease. Further significant intensification of major T(H)22 and T(H)2 cytokines was observed between acute and chronic lesions.

Conclusions: Our data identified increased S100A7, S100A8, and S100A9 gene expression with AD initiation and concomitant activation of T(H)2 and T(H)22 cytokines. Our findings support a model of progressive activation of T(H)2 and T(H)22 immune axes from the acute to chronic phases, expanding the prevailing view of pathogenesis with important therapeutic implications.

Conflict of interest statement

Disclosures: The authors have declared that they have no conflict of interest.

Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Figures

Figure 1. Clinical, histologic and genomic differences between non-lesional (NL), acute and chronic atopic dermatitis (AD)

A, Clinical images of NL, acute and chronic AD demonstrate bright erythema of acute and dullness and lichenification of chronic lesions. B–D, Representative immunohistochemistry stainings of B, H&E and the proliferation markers C, Keratin 16 (K16) and D, Ki67 in tissue sections of NL, acute and chronic AD skin, as well as B, quantification of epidermal thickness, C, K16 by real-time PCR (RT-PCR), represented in log2 [log2(expression/hARP)] and D, Ki67+ cell counts, showing significantly increased epidermal hyperplasia and abnormal proliferation in acute lesions, with further increases in chronic lesions. *P < .05, **P < .01, and ***P < .001, (n=10). Bar plots represent mean ± SEM. Scale bar = 100 μm. E, Venn diagrams of up- and down-regulated probe-sets in acute and chronic skin lesions, in comparison with NL skin, using criteria of fold change (FCH)>1.5 and p-value<0.1, (n=8).

Figure 2. Onset of acute lesions is associated with coordinated increases in S100A7, S100A8 and S100A9

A, Unsupervised hierarchical clustering of terminal differentiation genes across normal, non-lesional (NL), acute and chronic atopic dermatitis (AD) skin samples (red, up-regulated; blue, down-regulated). In contrast to the uniform down-regulation of well-characterized epidermal differentiation complex (EDC) genes such as filaggrin and loricrin throughout NL, acute and chronic skin lesions, there is a steep increase in expression of S100A7, S100A8 and S100A9 with the onset of acute disease, with further induction in chronic lesions, as indicated by the black highlighting. Probes with the largest fold change (FCH) were chosen when several probes represented single genes. *P < .05, **P < .01, and ***P < .001, (n=8 for AD; n=15 for normals). The significant induction of these S100 proteins has been validated by increases in both B, protein expression of S100A7 and S100A8 in representative immunohistochemistry stainings of acute and chronic lesions, compared to NL skin, and C, mRNA gene expression levels by real-time PCR (RT-PCR) represented in log2 [log2(expression/hARP)] and natural scale (expression/hARP) (~8 FCH, P<0.02). hARP, human acidic ribosomal protein. (n=10).

Figure 3. Marked activation of Th2 and Th22 immune pathways in acute disease with progressive activation in chronic skin lesions, by real-time PCR (RT-PCR)

Mean expression estimates normalized to human acidic ribosomal protein (hARP) are represented in log2 [log2(expression/hARP)] and natural scale (expression/hARP). The largest gene-expression increases are associated with Th2- and Th22-associated products, including IL-31, IL-22 and S100A7, S100A8, S100A9. Small increases in Th1-related products are evident in both acute and chronic lesions. In contrast with the immune mediators, the mRNA expression levels of filaggrin (FLG) and corneodesmosin (CDSN) showed similarity across the disease. Bar plots represent mean ± SEM. *P < .05, **P < .01, and ***P < .001, (n=10). FOXP3, forkhead box protein 3; LCN2, lipocalin 2; MX1, myxovirus resistance 1; OX40L, OX40 ligand; PI3, peptidase inhibitor 3; TD, terminal differentiation; TSLPR, thymic stromal lymphopoietin receptor.

Figure 4. Significant increases in immune-cell infiltrates characterize acute disease onset and progression to chronic disease, as quantified by means of immunohistochemistry and cell counts

A–E, Significant increases in (CD3+) T-cells, myeloid (CD11c+) dendritic cells (DCs), mature (CD83+) DCs, Langerhans (CD1a+) cells and inflammatory dendritic epidermal cells (IDECs) [Fc receptor for IgE (Fc ε RI+)] characterize acute disease. F, Cell counts of plasmacytoid [blood dendritic cell antigen 2 (BDCA2+)] DCs, mature [dendritic cell lysosome-associated membrane glycoprotein (DC-LAMP+)] DCs, resident (CD1c+) DCs, inflammatory [TNF-related-apoptosis-inducing ligand (TRAIL+)] DCs, IDECs (CD206+) and atopic [OX40 ligand (OX40L+)] DCs, increase from non-lesional (NL) through chronic atopic dermatitis (AD) skin. *P < .05, **P < .01, and ***P < .001, (n=10). Bar plots represent mean ± SEM. Scale bar = 100 μm.

Figure 5. Disease profiles of IL-17-induced genes in acute and chronic atopic dermatitis (AD), as compared to psoriasis

A–B, A scatterplot of fold changes (FCHs) between lesional and non-lesional (NL) skin for each disease (in log2-scale) demonstrates that whereas in psoriasis, these genes are well associated with the genomic differences between NL and lesional skin, no such association was found for either acute or chronic AD. C, There is a similar distribution of the Th17-regulated genes between acute and chronic AD, both compared to NL. D, Means and 95% confidence intervals (CIs) of the log2(FCH) among IL-17 genes in psoriasis and AD transcriptomes (n=8 for AD; n=15 for psoriasis).

Figure 6. A schematic illustration of initiation of acute atopic dermatitis (AD) and progression to chronic skin lesions

Non-lesional AD skin lesions show some immune infiltrates that produce inflammatory mediators, which might contribute to a defective epidermal barrier. Barrier defects lead to penetration by epicutaneous antigens that encounter Langerhans cells in the epidermis and dermal dendritic cells in the dermis, inducing marked immune activation and recruitment of inflammatory cells in acute AD lesions. Marked activation of Th2 and Th22 axes occurs in acute disease onset. Smaller increases in Th1 and Th17 immune axes were found in acute lesions. A progressive activation of Th2 and Th22, as well as Th1 pathways is characteristic of the chronic stage of AD. The relative induction of each T-cell subset, according to disease stage, is represented pictorially by their size, relative to the other T-cell subsets. Cytokines (i.e. IL-4, IL-13) and chemokines [i.e. CCL17, CCL18, CCL19, CXCL9, CXCL10, CXCL11] produced by various T-cells and DCs induce further activation and recruitment of additional immune cells. With the onset of acute disease, Th22 cells release IL-22, which induces epidermal hyperplasia, and synergistically with the Th17 cytokine, IL-17, drives an abrupt increase in a subset of terminal differentiation genes, specifically S100A7, S100A8 and S100A9 proteins. The increases in these barrier proteins contrast with the uniformly disrupted epidermal differentiation gene products (filaggrin, loricrin, corneodesmosin, etc.) throughout non-lesional, acute and chronic AD skin. The Th2 and Th22 cytokines contribute to inhibition of the terminal differentiation proteins. IL-31 is abruptly up-regulated in acute disease, potentially reflecting its role as an itch mediator in AD.