Role of Tight Junctions and Their Protein Expression in Atopic Dermatitis

Kunwar Namrata∗ and Bing-Xue Bai

Dermatology and Venereology Department, Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001,China.

Abstract

Atopic dermatitis (AD) is a chronic inflammatory skin disease with xerosis, itchiness, as well as interconnection with immunoglobulin E (Ig E), mediated foods including airborne allergies. AD is not only related to the diminished stratum corneum barrier but also presents with an unusual expression of tight junctions (TJs) proteins. TJ barrier dysfunction leads to impairment in the stratum corneum (SC) barrier. The significant role of TJs in the epidermal barrier as indicated by Claudin-1 (Cldn-1) deficient mice that undergo high transepidermal water loss (TEWL) and skin dehydration. In atopic dermatitis, downregulation of Cldn-1 was observed due to inflammation. Still, a lack of distinct understanding exists in considering tight junction barrier impairment as a cause or outcome in atopic dermatitis. This review summarizes TJs main role in skin barrier function and TJ proteins (TJPs) expression observed in AD patients.

Keywords: atopic dermatitis, tight junctions, claudin

Introduction

Atopic dermatitis (AD) or atopic eczema is a chronic inflammatory skin disease that manifests as dry skin and pruritus, and has an association with immunoglobulin Emediated food allergies and airborne allergies.1More than 60% of children with AD have asthma or allergic rhinitis.2Skin barrier defects are an important feature of AD. The stratum corneum (SC) and tight junctions (TJs) are essential for epidermal barrier function. In the stratum granulosum, the intercellular spaces are blocked by the closeness of the TJs, which represents a fundamental characteristic of the physiological function of the skin. The significance of TJs in the epidermal barrier function is shown by the high transepidermal water loss and skin dehydration seen in claudin (CLDN)-1-deficient mice. The TJ functions as a “gate” by controlling the movement of intercellular substances like cytokines, hormones, and electrolytes, and contributes to the particular permeability of the epidermis.

We made a searching using the topic words “atopic dermatitis”, “tight junctions" and “claudin" on PubMed,Google scholar, and Wikipedia databases from September 15th, 2010 toDecember 3rd, 2019. The current data suggest that the impairment of TJs contributes to the immune dysregulation and barrier dysfunction observed in patients with AD. The present review summarizes the barrier function of TJs in AD and the regulation of TJ proteins(TJPs) in patients with AD. The components of innate immunity that contribute to improving the barrier function of TJs inAD, such as antimicrobial peptides(AMPs) and tolllike receptor 2 (TLR2) activation, are also briefly discussed.

Structure of TJs

TJs are multiprotein junctional complexes situated at the apical side of epithelial cells with function in cell adhesion and control of the paracellular transport of particles and water, including atoms.3The TJ structure provides the barrier function of the epithelium to outside substances,such as allergens, particles, and pathogens.4TJs are formed by three types of transmembrane proteins: CLDN,occludin (OCLN), and junction adhesion molecules.Moreover, the structure of the TJ is bolstered by connector proteins, such as zonula occludens (ZO) proteins.3

CLDNs are a family of proteins that encode four transmembrane domains consisting of a pair of very conservative extracellular loops. Barrier function is conducted by the extracellular loop 1, while the extracellular loop 2 is responsible for narrowing the paracellular cleft.5

OCLNs are a group of proteins with five extracellular or intracellular domains and four transmembrane domains. The C-terminus of this family is responsible for barrier function and affects cell survival by interacting with other several proteins, while the N-terminus corresponds to the TJ barrier. The penetrability of the TJ barrier is regulated by extracellular loops.6CLDN-1 contributes to epithelial barrier tightening,4while CLDN-2 impairs the epithelial barrier tightening by disturbing the barrier.

ZO proteins are also considered a foundation for TJ because of their different unbreakable areas for TJPs and actin. In addition, ZO links CLDNs or OCLNs to cytoskeletal actin.7

Barrier function of TJs in skin

TJs are found in simple and stratified mammalian epithelia, and are made up of intracellular and transmembrane proteins. TJs are present in the stratum granulosum,and TJ expression is increased after injury. In addition, TJs are implicated in the keratinization and differentiation of epidermal cells.8

The granular cell layer contains an accumulation of distinctive TJ structures that affect the barrier function.The barrier function of the TJ has been demonstrated by the failure of a 557-Da tracer applied to the dermis to permeate the granular cell layer, while a 32-kDa tracer penetrates the TJ barrier after pre-digestion of the skin with exfoliative toxin.9-10When TJs become leaky, the direction of permeation is dependent on the gradient of a specific molecule on each side of the barrier. If these molecules arrive at a TJ after damage to the SC barrier,they are likely to get stopped on their way from the outside to the inside. In the epidermis, the TJ barrier function for alternative molecules, as well as water or alternative ions has not been clarified. TJs form a barrier to water, Ca2+,Cl-, and Na+ in cultured keratinocytes showing a parallel part in the epidermis.11However, more research is required to clarify the TJ barrier function or permeability to various solutes in the epidermis.

The significance of TJPs like CLDN-1, CLDN-4, ZO-1,and OCLN in the effectiveness of the TJ barrier to 4-kDa tracer and ions (Na+, Cl-, Ca2+) has been shown in normal human epidermal keratinocytes.11-12Additionally,ZO-1 and CLDN-1 are essential in maintaining the barrier integrity to larger molecules (40 kDa).11In vivo experiments using 557-Da tracer in CLDN-1 knockout mice have showed the significant role that CLDN-1 plays in maintaining the TJ barrier.9,13In addition, a study performed using restored human skin showed that Cterminal Clostridium perfringens enterotoxin increases the TJ gap by inhibiting CLDN-4 and other CLDNs to reduce the function of the TJ barrier to a tracer.14

One study showed that the TJs in chronic AD leak small tracers (＜5 kDa), but not large tracers (＞30 kDa).15Furthermore, OCLN knockdown decreases sensitivity to the initiation of cell death and cell adherence.10Hence,TJPs are involved in various functions other than barrier function.

AD not only causes skin inflammation, but is also a Th2-driven systemic disease.16The proteases most commonly expressed by various Staphylococcus aureus strains are extracellular serine protease A and V8 protease.17In vivo experiments have shown that V8 protease affects the SC and damages the skin integrity of mice. Extracellular serine protease splits TJPs such as CLDN, OCLN, ZO-1, and cadherins,18ultimately leading to skin barrier dysfunction.An increasing amount of evidence suggests that TJs play a vital role in the pathogenesis of AD.

Expression of TJ proteins in AD

In the skin lesions of individuals with AD, CLDN-1 protein expression is downregulated due to inflammation. One study has shown that decreased CLDN-1 mRNA causes AD-like inflammation in fillagrin knockout mice and a NC/Nga mouse model of AD-like dermatitis.19Another study showed that reduced CLDN-1 expression in mice causes AD characteristics such as elevation of interleukin(IL)-10 and interferons, increased epidermal thickness, dry skin, and macrophage infiltration.20

A relationship between the development of AD in children younger than 5 years of age and CLDN-1 single nucleotide polymorphism (SNP) was found in an Ethiopian cohort.21A significant correlation between AD and CLDN-1 SNP was also shown in a hospital-based Korean study, but the study cohort comprised hospital patients rather than the general Korean population. Similarly, a study showed a link between AD and SNP in the Danish population.22However, an immuno-intensity investigation performed in an Austrian cohort showed no differences in the expression of CLDN-1 in non-lesional skin compared with lesional skin. Hence, CLDN-1 expression is based on genetic variations between different populations.23Increased CLDN-4 expression in non-lesional skin was shown in two individual studies performed in the European population.19RNA sequencing has shown an alteration of CLDN-8 in lesional skin compared with nonlesional skin.24Western blot analyses showed decreased expression of CLDN-4 in the non-lesional skin of three Japanese patients. Furthermore, the downregulation of ZO-1 with no changes in CLDN-1 has been observed in non-lesional skin.14

Studies have shown the association between CLDN-1 and AD. The downregulation of CLDN-1 and CLDN-23 has been shown in the non-lesional skin of patients with AD in North America, contributing to the impairment of the barrier function.25One study also reported a relationship between CLDN-1 haplotype-tagging SNPs and AD. Gene variants of CLDN-1 (related to chromosome 3q28) were shown to be associated with the risk of developing AD and disease severity in an African-American cohort.26The role of the CLDN-1 variant rs893051 in severe AD has been established in the African-American population.27

The key role of the filaggrin protein is to form a skin barrier and support terminal differentiation.28Mutations causing the loss of function of filaggrin are rare in African patients with AD.29However, an increase in skin surface pH is observed in filaggrin-deficient patients.

Immunostaining shows disorganized CLDN-1 in the skin lesions of dogs with AD exposed to house dust mites.30A recent immunohistochemistry study that investigated the TJ alteration in the epidermis in a canine model of AD showed that the expression of ZO-1 plus OCLN in TJs is decreased in dogs with AD compared with normal dogs.31Although the association between OCLN and ZO-1 and AD progression has not yet been assessed in humans, research suggests that the alteration of TJPs in individuals with AD contributes to the disease progression and development.32

There is currently no consensus onwhether the diminished TJ barrier function is the cause or the outcome of AD. Skin inflammation and the damaged physical skin barrier form a vicious cycle in the pathogenesis ofAD.Dermatitis leads toTJ leakage, and TJ barrier dysfunction leads to SC barrier impairment.13It is hypothesized that the SC barrier is impaired by dermatitis via TJ leakage and disrupted keratinocyte differentiation, making it easier for percutaneous sensitization to occur and initiating a vicious cycle between barrier deficiencies and skin inflammation.15,33This process may give rise to the chronic inflammation of AD.

TJ barrier repair in AD

TLR2 activation

Compared with non-atopic cohorts, patients with AD are frequently infected by S. aureus, indicating that the alteration of TLR2 promotes susceptibility to such infection. A study performed in a human wound model and TLR2-deficient mouse model suggested that TLR2 plays a role in maintaining the TJ integrity as a response to barrier insults, and that the barrier repair mechanism may be deficient in patients with AD.34

Role of p63

An experimental study has shown wound healing and reduced keratinocyte differentiation in N-terminal truncated transcription factor p63 (ΔNp63) knockout mice,which demonstrates the role of ΔNp63 in the integrity of the epidermis.35Increased CLDN-4 plays an important role in the undefined restoration of impaired keratinocytes and provides supportive responses to the diminished barrier in AD.36In the epidermis of patients with AD,higher CLDN-4 expression with reduced ΔNp63 expression is observed in the cell membranes of keratinocytes treated with TLR3 ligand.37

AMPs function

AMPs play a significant role in the innate immunity of the skin and act as endogenous antibiotics. Among the various AMPs, human β-defensin (HBD) and cathelicidin (LL-37)are considered important.38In individuals with AD,decreased expression of AMPs might cause greater vulnerability to skin infections caused by bacteria, fungi,or viruses,34thus explaining the recurrent bacterial infections in patients with AD. Western blot analysis has shown that LL-37 increases the expression of TJPs,including CLDN-1, CLDN-4, CLDN-3, CLDN-9, CLDN-7, CLDN-14, and OCLN, while HBD-3 upgrades the regulation of CLDN-1, CLDN-3, CLDN-4, CLDN-14,and CLDN-23, including their membrane distribution.Furthermore, HBD-3 promotes the TJ barrier function by decreasing paracellular flux in the keratinocyte layers and elevating the transepithelial electrical resistance.39

A recent study established the role of IL-1β in inducing keratinocyte protection against the S. aureus proteases serine protease A/V8, and indicated the role of IL-1β in the upregulation of CLDN-1 expression in immortalized human epidermal keratinocyte (HaCat) cells. The degradation of CLDN-1 is not prohibited in primary cells, and this keratinocyte protection is independent of the CLDN-1 level. This process of protection against V8 protease is induced by HBD-2.40

IL-17A improves the function of TJs

A recent study performed using a human skin model and primary human keratinocytes showed that IL-17A increases the progression of the TJ barrier function of the epidermis by elevating the transepithelial electrical resistance and reducing paracellular flux proteins; this protective effect of IL-17A is inhibited after treatment with IL-4. This adds further evidence for the improvement of the TJ barrier function by IL-17A, and the reduction of the TJ barrier function by IL-4. Re-establishment of the appropriate proportions of IL-17 and IL-4 improves barrier function in the skin of patients with AD.41

Clinical indications in the treatment of AD

AD treatment includes the application of moisturizers such as lipid mixtures, petrolatum, and ceramide-controlling triple-physiologic lipid (ceramide:cholesterol:free fatty acid molar ratio of 3:1:1).42-43Petrolatum enhances the barrier functions of the skin by upregulating AMPs such as HBD-2, LL-37, S100 proteins, and elafin.44The use of coagulase-negative Staphylococcus strains reduces colonization by S. aureus in the skin of patients with AD.44

An experiment showed that the Lactobacillus strain CJLP55 (isolated from kimchi) causes decreased eosinophils, mast cell infiltration, and Th2 cytokine production in AD-induced mouse skin.45A recent study demonstrated the role of prebiotics and probiotics in decreasing the symptoms and improving the disease severity and quality of life in patients with AD.46Furthermore, the genes involved in the barrier function of the skin are upregulated by the administration of dupilumab, which is an anti-IL-4 Rα monoclonal antibody.47

Conclusions

In summary, the TJ barrier is an essential part of the barrier system and is important in maintaining the skin barrier integrity in skin inflammatory diseases such as AD.However, the studies have following limitations: 1) The importance of TJ barrier function hasn’t to be investigated in other skin diseases/ conditions; 2) TJ barrier independent roles of TJ proteins haven’t be investigated in more details.

Alterations of TJPs can lead to the imbalance of the skin barrier system. Inflammation in AD leads to epidermal barrier leakage of TJs and disturbs the barrier formation of the SC. Upregulation of TJPs enhances epidermal TJ barrier integrity and improves the TJ barrier function in AD, thus contributing to the development of new strategies to better control various inflammatory skin diseases.Further investigation is needed to determine the function of the TJ barrier and its clinical relevance in inflammatory skin diseases.