Tight junction defects in patients with atopic dermatitis

Abstract

Background: Atopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway.

Objective: We evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1).

Methods: Expression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illumina's BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD.

Results: We observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with T(H)2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging SNPs revealed associations with AD in 2 North American populations.

Conclusion: Collectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1.

Conflict of interest statement

Conflicts of Interest: None Declared

Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Figures

Figure 1. Intercellular junction proteins are dysregulated in AD subjects

(A) Z ratios from gene arrays performed on nonlesional epithelium (blister roofs, B-D), CLDN-1 and -23 were reduced, while the gap junction proteins, connexin-26 [GJB2] and connexin-62 [GJA10] were upregulated (A-Red). Genes indicative of de-differentiation were either unaffected or increased (A-Yellow). (E, F) Validation of the key genes in newly recruited AD/NA control samples (*P = 0.03 & **P = 0.001).

Figure 2. Claudin-1 expression is markedly reduced in AD epidermis

(A) Reduced claudin-1 immunoreactivity was noted in nonlesional AD skin (n = 11) compared to (B) NA (n = 12). Red indicates positive staining (Bar = 100 μm). Using a FITC-conjugated secondary antibody, claudin-1 had a membranous pattern in both (C) AD and (D) NA. The signal intensity was significantly reduced in (C) AD epidermis. Positive staining is indicated by green (Bar = 20 μm). The dotted line denotes the epidermal-dermal junction. (E) Semiquantitative scoring confirmed reduced epidermal expression of claudin-1 in AD (1.3 ± 0.3) compared to NA (2.9 ± 0.1; *P<0.0004).

Figure 3. AD epidermis has altered bioelectric properties compared to NA

(A) Ussing chamber measurements reveal a markedly reduced resistance (92 ± 22.0 Ohms × cm2; n = 4) in AD epithelium compared to NA (827 ± 173.3 Ohms × cm2; P = 0.01; n = 4). This was reflected in an increased permeability to FITC-conjugated albumin in AD (445 ± 24.25 O.D./cm2/h; n = 4) compared to NA (175± 68.37 O.D./cm2/h; P = 0.02; n = 4). (B) Dilution potential studies noted the preservation of membrane selectivity in NA subjects, with Na+ ions relatively more permeable than Cl− (0.77 ± 0.03 fold PCl/PNa). In contrast, the selectivity was completely lost in AD epidermis (1.1 ± 0.02 fold PCl/PNa; P = 0.001; n = 3/group) and both ions were equally permeable.

Figure 4. Claudin-1 expression from the gene arrays correlates with Th2 biomarkers

(A) The line represents the linear least square fit for log2 CLDN1 expression level vs. log10 total serum IgE (Pearson product-moment correlation coefficient n = 14; r = −0.718, P = 0.0038). (B) The plot of log2 CLDN1 expression level vs log10 total eosinophil count (n = 14; r = −0.761, P = 0.0016). The disease phenotypes (PS, AD, NA) are denoted by unique symbols.

Figure 5. Claudin-1 colocalizes with other TJ proteins at the cell membrane in differentiated keratinocytes and this coincides with maximal TJ function

(A) In Ca2+-differentiated keratinocytes (Hi Ca) claudin-1 (green) colocalizes with occludin (red) at the cell membrane. Bars = 50 μm. (B) TEER is only observed in differentiated PHK and peaks about 40h after the increase in Ca2+. (Representative of n = 5). (C) Paracellular diffusion of 0.02% sodium fluorescein is markedly reduced in differentiated PHK (3.1 ± 0.6 fold; *P = 0.046; n = 3). (D) Western Blots show enhanced expression of claudin-1 when PHK were differentiated for 48h in presence of IL-4 (50 ng/ml) and IL-13 stimulation (50 ng/ml) or both cytokines (Representative of n = 3).

Figure 6. Claudin-1 silencing reduces TEER, increases paracellular permeability and enhances cell proliferation

(A) Western blots demonstrated a dose-dependent reduction of claudin-1 with CLDN1 siRNA (48h). No change was observed for occludin. (B) Immunostaining confirmed claudin-1 expression, but not occludin, was reduced in differentiated PHK following transfection with CLDN1 siRNA (Bars = 50 μm). (C) Claudin-1 knockdown significantly reduced TEER (control: 164 ± 18.2 and CLDN1 siRNA: 80.6 ± 6.4; *P = 0.007; n = 4), (D) increased sodium fluorescein permeability (control: 27.6 ± 11.7 and CLDN1 siRNA: 52.5 ± 9.2; **P = 0.026; n = 4) and (E) enhanced cell proliferation as assessed by Click-iT™ EdU assay (**P = 0.002; n = 3).

Figure 7. Evidence for CLDN1 association with risk of atopic dermatitis (AD), early onset AD, and disease severity in two North American populations

The X-axis represents the physical position for each of 27 CLDN1 SNPS shown in relationship to the exonic structure of the CLDN1 gene on chromosome 3q.28-q29. The Y-axis denotes the association test result as -log (P-value) corresponding to representative symbols for each of the phenotypes. The standard cutoff for significance (P = 0.05) is shown as a horizontal solid line. Outcomes included risk of AD (diamond), early age of onset (<5 years; triangle) and the clinical scoring system called EASI (circle) in African American (AA; blue) and European American (EA; green) populations.

Figure 8

Skin epithelium is uniquely armed with two barrier structures, the stratum corneum (SC) and tight junctions (TJ). The SC is the outermost structure consisting of multiple layers of enucleated keratinocytes called corneocytes. The SC barrier is maintained by the complex interaction of the cornified envelope, intracytoplasmic moisturizing factor(s) and a complex lipid mixture in the extracellular space. TJ are located just below the SC at the level of the stratum granulosum (SG). The function of this structure can be accurately assessed by both resistance and permeability assays. In this paper we present evidence that this structure may be compromised in Atopic Dermatitis subjects and this may in part be due to reductions in claudin-1. These two barrier structures interact in a dynamic way to ensure that the skin maintains a formidable barrier. It is our premise that SC defects alone would not lead to the development or exacerbation of atopic dermatitis. We propose that a breach in both the SC and TJ would be required for sensitization to antigens/allergens, irritants, microbes, pollutants or possibly even nanoparticles. A recent study demonstrates that a temporary disruption in TJ results in the incorporation of Langerhans cell (LC) dendrites within a temporary TJ. This extrusion of the LC dendrite would enable antigen uptake and processing by the LC and may confer some activation signals that would determine the immunologic fate of the APC-T cell interaction that follows. A fluorescence image of healthy human epidermis (left) demonstrates the expression patterns of two key players in AD pathogenesis, namely, FLG (red) and CLDN1 (green) and pictorially highlight the orientation of the two skin barrier components.