This is the first report, to our knowledge, implicating a TJ defect in AD, a human skin disease that affects up to 15 million Americans. We demonstrated reduced expression of epidermal claudin-1 in AD nonlesional epidermis (). This was specific for AD and not observed in psoriasis, a Th17-driven inflammatory skin disorder (). Although previous psoriasis publications have suggested that TJ may be altered in lesional epidermis this has not been consistently observed by other groups56, 74–76
. Adapting the Ussing chamber to measure bioelectric properties of stratified squamous epithelium enabled us to characterize skin barrier properties from human subjects. Using this approach we observed a remarkable alteration in the bioelectric characteristics of AD epidermis with markedly lower electrical resistance and higher albumin permeability that was associated with the loss of ion selectivity permeability (). These defects are the signature of a TJ defect.
Our findings are highly consistent with the observations made from genetically altered mice. For example, the claudin-1 knockout mouse dies within 24 hr of birth with wrinkled skin, severe dehydration and increased epidermal permeability as measured by dye studies and TEWL27
. Importantly, these mice have no abnormalities in the expression of stratum corneum proteins (e.g., loricrin, involucrin, transglutaminase-1, or Klf4) or lipids that might explain their severe skin phenotype. Another recent study reported disruption of epidermal barrier and severe dermatitis in transgenic mice overexpressing an adhesion-deficient mutant of claudin-6 in the suprabasal compartment of the skin77
. Interestingly, a marked downregulation of claudin-1 expression was noted in these transgenic mice. It is tempting to speculate that the reduction in claudin-1 rather than the adhesion-defective claudin-6 per se was responsible for the barrier disruption observed in this mouse. Lopardo et al. also observed reduced claudin-1 expression in the epidermis of p63 mutant mice66
. These mice have a severe skin phenotype and die of dehydration within one day of birth similar to the claudin-1 KO mice. In summary, these mouse models have highlighted the critical importance of claudin-1 for a functional epidermal TJ. Recently, a human syndrome caused by an exonic mutation in the CLDN1
has been described78–80
. Patients with this syndrome called Neonatal Ichthyosis-Sclerosing Cholangitis (NISCH) have features in common with AD, namely erythema, dry flaky skin and patchy alopecia in addition to unique features such as severe liver and gallbladder abnormalities that likely arise because of the importance of claudin-1 in the barrier integrity of bile canaliculi78, 79
Our in vitro
studies with PHK monolayers demonstrating a clear association between claudin-1 levels and TEER confirmed previous work55, 57, 63, 81, 82
. We extended these findings by demonstrating that claudin-1 knockdown also enhanced TJ permeability and proliferation (). In our study, the claudin-1 siRNA was target-specific, with no changes observed in the expression and/or localization of other critical TJ proteins (occludin and ZO-1), adherens junction components (E-cadherin and nectin-1) or stratum corneum proteins (filaggrin) (Figure E2
). Additionally, we were able to reduce claudin-1 expression by ~50% - similar to the reductions we observed in claudin-1 immunoreactivity of AD epidermis and this resulted in a remarkable reduction (~50%) in TJ function (based on both electrical resistance and permeability assays) (). While immunolabeling data allow only a correlative link between claudin-1 expression and the development of epidermal barrier, our RNA interference result demonstrated a causal connection between these two events ().
Recent studies with different types of mammalian epithelia indicated that the epithelial barrier is determined by two different components, charge-selective small pores that are permeable to molecules with up to 4μ radius, and large, poorly-defined breaks of the barrier without size- or charge-selectivity (reviewed in83
). TEER reflects small-pore permeability, whereas flux of FITC with a reported Stokes’ radius of ~5.5• measures permeability of large barrier breaks. Knockdown of claudin-1 in PHK monolayers decreased TEER and increased FITC flux, which implicates claudin-1 in regulation of both paracellular pores and breaks in the epidermal barrier. These findings strongly suggest that the skin of AD subjects would also be more permissive to a number of relevant environmental allergens such as house dust mite. Purified allergens from the house dust mite, Dermatophagoides pteronyssinus
have a diameter of ~1.6• by X-ray crystallography and therefore could penetrate even small paracellular pores formed by epithelial TJs84
. Thus, defective expression and function of claudin-1 in AD provides a plausible molecular mechanism for increased sensitization to environmental antigens, allergens, irritants, or pollutants. This is in keeping with the distinction that AD is the allergic disorder with the greatest and most diverse allergen reactivity, reflected in high serum total IgE values. AD is also recognized for a reduced cutaneous irritancy threshold that could simply reflect greater epidermal penetration of irritants. Importantly, TJ disruption is a leading hypothesis to explain allergen reactivity in the airways, which manifests as asthma and allergic rhinitis or in the intestinal tract as food allergy3, 85, 86
Although we observed an inverse correlation between claudin-1 expression and markers of Th2 polarity we did not find that Th2 cytokines (IL-4 and IL-13 alone or together) reduced claudin-1 expression but in fact observed the opposite. This induction of claudin-1 was observed in conjunction with enhanced TJ function (e.g. TEER) and suggests that Th2 cytokines have a reparative effect on TJ in normal keratinocytes. Whether the actions of these Th2 cytokines would be different in AD epidermis will require further study. Interestingly Th2 cytokines have been shown to reduce the expression of several SC components important for skin barrier function72
. At this point we conclude that TJ dysfunction observed in AD epidermis is not likely caused by Th2 milieu, which is present even at nonlesional sites. Instead we would hypothesize that the connection we observed between TJ function and biomarkers of Th2 polarity suggest that AD TJ defects enable or promote Th2 responses possibly by enhancing the trafficking of non-self antigens that are responsible for triggering the Th2 response in genetically predisposed individuals. Alternatively, the upregulation of claudin-1 in response to IL-4 and IL-13 may represent a compensatory immune response to “protect” against further antigen uptake through the skin.
In preliminary studies we evaluated genetic associations between CLDN1
polymorphisms and AD. We undertook a haplotype-tagging SNP approach using genetic markers available in the public arena (n = 132 in dbSNP) in 414 European Americans and 328 African Americans. Interestingly, CLDN1
is localized on chromosome 3q28–q29, very close to the ATOD1
locus for AD90
. Adjusting for 10,000 permutations to reduce a Type I error due to multiple comparisons, we observed several modest associations (P
= 0.003 – 0.05) between variants throughout the CLDN1
gene and the outcomes associated with AD, especially among African American patients. In separate studies we tested for association between several of the same CLDN1
SNPs and risk of disease among a more robustly powered German dataset of AD patients and controls; however, the full set of SNPs genotyped in the ADVN study were not available for direct comparisons, and phenotyping approaches differed (data not shown). Further studies are underway for comprehensive joint analyses between ADVN and the German study. Interestingly, CLDN1
variants were also associated with asthma and its related trait, total serum IgE and FEV1
, in two independent populations of African descent (K. Barnes unpublished data). In addition, the rs893051 SNP associated with AD severity in our AA population, was also associated with asthma and disease severity in a population of African descent. As part of the ADVN study our population was also screened for the two most frequent FLG
mutations (R501X and 2282del4; ) and 9 haplotype-tagging SNPs throughtout the FLG
. There were no significant interaction effects between haplotype-tagging SNPs in FLG
SNPs. Moreover, in the AA population where we had the strongest association with CLDN1
the 2 FLG
mutations were considerable less common than in our EA population51
. Our findings provide suggestive evidence for a role of CLDN1
variants in AD and its associated phenotypes, further supporting the importance of CLDN1
in AD. In conclusion, this study provides the first evidence that epidermal TJs are defective in AD, the most common human skin disease. We observed that claudin-1 is selectively reduced in the epidermis of AD patients and that CLDN1
may be a novel AD susceptibility gene. Epidermal samples from AD subjects had remarkable defects in resistance and ion transport compared to healthy controls. Using the model of human keratinocyte monolayers, we observed enhanced claudin-1 expression and recruitment to intercellular junctions, upon cell differentiation, which coincided with the development of a paracellular barrier. Selective downregulation of claudin-1 expression markedly increased paracellular permeability, decreased resistance and enhanced proliferation indicative of a wound repair response. We hypothesize that the reduced expression of claudin-1 in AD epidermis may enhance the penetration of many relevant environmental antigens leading to greater allergen sensitization as well as greater susceptibility to irritants/pollutants and possibly even altered microbial flora. The inverse relationship between claudin-1 and serum total IgE values also suggests that this defect may promote Th2 responses. Collectively this data suggests that barrier dysfunction in subjects with AD extends beyond the stratum corneum to TJ the second barrier structure and that barrier regulation provides a novel therapeutic opportunity in AD and possibly other atopic disorders.
- Claudin-1 plays a critical role in human epidermal tight junction function and keratinocyte proliferation.
- Claudin-1 is significantly reduced in nonlesional skin of AD compared to NA and psoriasis subjects.
- Claudin-1 levels are inversely correlated with Th2 biomarkers suggesting that reductions in this key TJ barrier protein may affect the character of the immune response to environmental allergens.
- Analysis of claudin-1 (CLDN1) haplotype-tagging single nucleotide polymorphisms in two North American populations revealed associations with AD.