Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-016-1903-9

INVITED REVIEW

Tight junctions in skin inflammation Katja Bäsler 1 & Johanna M. Brandner 1

Received: 29 September 2016 / Revised: 1 November 2016 / Accepted: 7 November 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Inflammation of the skin is found after various external stimuli, e.g., UV radiation, allergen uptake, microbial challenge, or contact with irritants, as well as due to intrinsic, not always well-defined, stimuli, e.g., in autoimmune responses. Often, it is also triggered by a combination of both. The specific processes, which mean the kind of cytokines and immune cells involved and the extent of the reaction, depend not only on the trigger but also on the predisposition of the individual. Tight junctions (TJs) in the skin have been shown to form a barrier in the granular cell layer of the epidermis. Furthermore, TJ proteins were found in several additional epidermal layers. Besides barrier function, TJ proteins have been shown to be involved in proliferation, differentiation, cell-cell adhesion, and apoptosis in keratinocytes. In inflamed skin, TJ proteins are often affected. We summarize here the impact of skin inflammation on TJs, e.g., in various forms of dermatitis including atopic dermatitis, in skin infection, and in UV-irradiated skin, and discuss the role of TJs in these inflammatory processes.

Keywords Bacteria . S. aureus . Claudin . Occludin . ZO-1 . Interleukin . Eczema

This article is published as part of the special issue on tight junctions (European Journal of Physiology). * Johanna M. Brandner [email protected] 1

Department of Dermatology and Venerology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany

Introduction Inflammation is a defense response to exogenous or endogenous (harmful) stimuli. It predominantly involves the innate immune system, but in later stages also components of the adaptive immune system contribute. After recognition of disturbances at the surface or within the cells via pattern recognition receptors, proinflammatory cytokines as well as chemokines are released in order to recruit an inflammatory infiltrate. Skin inflammation can be the result of exposure to UV radiation, ionizing radiation, allergens, pathogens or contact with chemical irritants (soaps, hair dyes, etc.) [11, 32, 79, 83, 103]. In addition, intrinsic triggers are important but often not clearly defined [44, 124]. Clinically, it is often characterized by itching rashes. Various forms of dermatitis, skin infection, rosacea, lichen planus, and psoriasis are examples of inflammatory skin diseases. Alteration of tight junction (TJ) proteins in the epidermis has been described during skin inflammation, and even a causative role of downregulation of TJ proteins for inflammation is likely in certain cases. An overview about current data is presented here.

Structure of mammalian skin The mammalian skin consists of three fundamental layers. The lowermost layer is the subcutaneous tissue (subcutis), where subcutaneous fat (stored in adipocytes) acts as an energy reservoir and padding and provides thermoregulation. The intermediate layer is the dermis. Here, extrafibrillar matrix, elastic fibers (elastin), and collagen provide stability and elasticity. The main cell population of the dermis is fibroblasts that produce and deposit collagen as well as other dermal components [4]. In addition, immune cells like mast cells, macrophages, and small amounts of lymphocytes are permanently

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within the lymph nodes. In case of an inflammatory event, transient inflammatory cells such as neutrophils, eosinophils, or lymphocytes leave the blood vessels and move through the dermis and epidermis in order to fulfill their particular functions [35, 85].

present. Furthermore, blood and lymphatic vessels as well as mechano- and thermoreceptors and the dermal compartments of hair follicles and glands are present [4, 85]. The outermost layer of the skin is the epidermis. This stratified squamous epithelium mainly consisting of keratinocytes is tightly connected to the dermis via the basement membrane. From inside to outside, it is composed of different strata: stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG), and stratum corneum (SC) [4, 85] (Fig. 1). Proliferative activity of the keratinocytes within the SB guarantees constant tissue renewing. In the SS, cells differentiate and provide tissue stability due to strong cell-cell adhesion via a large number of desmosomes. In the SG, typical TJ structures are found (for review, see [15]), and major processes for the preparation of the formation of the SC take place [95]. The SC protects the viable cell layers beneath and the whole body by serving as an air-liquid-interface barrier [71]. It is composed of corneocytes (terminally differentiated, anucleated keratinocytes) and intercorneocyte lipids [27, 95]. Even though about 90% of the cellular population of the epidermis is represented by keratinocytes, also other important cell types are residing, like melanocytes which are involved in UV protection, Merkel cells which can serve as mechanoreceptors, and Langerhans cells that belong to the skin innate immune system and can take up antigens in order to present these to lymphocytes

Fig. 1 Schematic overview of the mammalian epidermis with tight junction (TJ) protein localization pattern and immunohistochemical staining (green) of claudin-1 (Cldn-1), occludin (Ocln), claudin-4 (Cldn-4), and zonula occludens protein-1 (ZO-1) (overlay of immunofluorescence staining and phase contrast pictures). Red dots denote functional TJ structures. JAM-A junctional adhesion molecule-A, MUPP-1 multi-PDZ domain protein 1. Bar 20 μm

Tight junctions in the skin The existence of TJs within the epidermis was assumed for a long time [42, 102]. However, undeniable confirmation of epidermal TJs in mammalian skin was just given at the turn of the century due to the availability of specific antibodies and knock-out technology [14, 33, 86, 97, 117]. Their importance for the skin barrier was impressively shown by claudin-1 (Cldn-1) knock-out and Cldn-6 overexpressing mice both of which died soon after birth due to a skin barrier defect resulting in increased water loss [33, 111, 117]. Human patients with Cldn-1 null mutations exhibit the neonatal ichthyosis sclerosing cholangitis (NISCH) syndrome. These rare patients do not die because of water loss but suffer from liver problems and show ichthyosiform skin alterations [31, 41, 63]. Epidermal TJs have been shown to form a barrier in the SG to molecules of different sizes as well as ions: from inside to

Stratum corneum Stratum granulosum

Cingulin (Cldn-5)

Cldn-1

Ocln Cldn-4 ZO-1 Cldn-7

Stratum spinosum

Ocln Cldn-1

JAM-A MUPP-1

Cldn-4

Stratum basale

ZO-1

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outside, the ion tracer lanthanum (La3+) [42, 51, 72] and tracer molecules of 557 Da [33, 65, 72, 116, 128], 1500 Da, 5 kDa, and 32 kDa [126, 128] are stopped at the TJ structures after dermal injection. Also, mathematical modeling supports the existence of an epidermal inside-out TJ barrier to ions (calcium) [1]. The barrier function of undisturbed epidermal TJs from outside to inside could not be shown up to now, as tracer molecules are already stopped at or in the SC, and when removing the SC, TJs are no longer undisturbed as the SC and TJs are interconnected (for review, see [9]). But TJs are bidirectional [5] and this most likely also holds true for epidermal TJ structures. Furthermore, in assays with cultured normal human epidermal keratinocytes (NHEKs), apically applied tracers (e.g., 4 and 40 kDa FITC dextrans) were stopped at TJs [67, 131]. Thus, if substances applied topically onto the skin were able to reach the SG, they should likely be stopped at TJs. In experiments with NHEKs, the importance of the TJ proteins Cldn-1, Cldn-4, Ocln, and ZO-1 for the TJ barrier to ions (Na+, Cl−, Ca2+) as well as to a 4-kDa tracer was shown [67, 87]. Moreover, Cldn-1 and ZO-1 also contributed to the barrier to larger molecules (40 kDa) [67]. The importance of Cldn-1 for TJ barrier function was also demonstrated in vivo as Cldn-1 knock-out mice showed a leaky barrier to a 557-Da tracer [33, 111]. In reconstructed human skin, it was demonstrated that the opening of TJs by C-terminal Clostridium perfringens enterotoxin (cCPE) which addresses Cldn-4 and additional claudins also diminished the TJ barrier to this tracer [132]. All TJ proteins colocalize at the barrier-forming TJ structures in the SG, but interestingly enough, not all TJ proteins are restricted to these barrier-forming structures or even to the SG. Occludin, for example, was additionally found in other structures in the SG [76]. Other TJ or TJassociated proteins were additionally found in the SS (e.g., Cldn-4 or zonula occludens-1 (ZO-1) and ZO-2) or in all viable cell layers (e.g., Cldn-1, Cldn-7, or junctional adhesion molecule-A (JAM-A)) [13] (Fig. 1). This suggests that TJ proteins in the epidermis also have TJ-barrierindependent tasks. For instance, involvement in proliferation and differentiation as well as apoptosis and cell-cell adhesion has been shown [29, 64, 89, 98, 111].

Tight junctions and skin inflammation Their central localization enables TJs to interact with the stratum corneum and the microbiome barrier on one hand but also with the immunological and chemical barrier on the other hand (for review, see [9]). In addition, they can react fast and dynamically on external and internal signals. As they can particularly react on changes within the immune system,

they are likely to be one of the first interaction partners for an inflammatory response. For example, after activation of various toll-like receptors (TLRs) in NHEKs, an upregulation of the TJ barrier function was observed already 3 h after activation [134]. Also, the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor alpha (TNF-α) were shown to influence TJs rapidly. Twenty-four hours after application, an increase of transepithelial resistance (TER) was observed which was accompanied by a relocalization of TJ proteins to the plasma membrane [66]. Of note, long-term treatment resulted in a decrease of TER and loss of proteins from the membranes as well as decreased protein levels. Treatment of reconstructed human epidermis with an atopic dermatitis (see below)-typical cytokine mixture of IL-4, IL-13, and IL-31 resulted in a decrease of Cldn-1 in lower epidermal layers [37]. Treatment with only IL-4 and IL-13 resulted in a downregulation of Cldn-1 in Western blot analyses in one study [88] while no alteration was shown in another study [47]. Different model systems and IL concentrations may explain these differences. IL-4 alone led to a donor-dependent up- or downregulation of TER in NHEKs [15, 24]. IL-13 induced an upregulation of Cldn-1 without clear influence on TER [24]. IL-17 resulted in a downregulation of Cldn-7 and ZO-2 messenger RNA (mRNA) in HaCaT cells [39] and a leaky TJ barrier to a 557-Da tracer in reconstructed human epidermis [133]. Also, histamine was shown to influence TJ protein and barrier function resulting in a decrease of the TJ barrier in reconstructed human skin [38]. The influence of inflammation on TJs is not restricted to the skin. Also, in intestinal epithelium, nasal epithelium, and bronchial epithelium, an influence of several inflammatory cytokines on TJs was observed [2, 46, 94, 109]. But also alteration of TJ proteins may result in an induction of inflammatory response. We observed that downregulation of ZO-1 in NHEKs resulted in an increase of IL-1β release (Fig. 2). Tokumasu et al. reported an increase of IL-1β, IL-12, IL-10, and interferon (INF)-γ in mice with a reduced level of Cldn-1 2 weeks after birth [114]. And mice overexpressing a cytoplasmic tail deletion mutant of Cldn-6 show histological signs of dermatitis, putatively dependent on mechanical injury, but only during aging [115] (inflammatory parameters were not investigated). Finally, the close collaboration between TJs and the immune system can be seen by the fact that barrier-forming TJs are formed between keratinocytes and Langerhans cells (LCs) upon their activation [72] and that LCs express several TJ proteins [66, 72, 135, 136]. Also, dendritic epidermal T cells have been described to be anchored at ZO-1-positive sites in the SG of mouse skin [18]. Thus, it is no surprise that alterations of TJs/TJ proteins are observed in skin diseases or conditions associated with inflammation.

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Fig. 2 Effect of knock down of ZO-1 on IL-1β release in unstimulated primary normal human keratinocytes. Mean values ± SEM. n = 3 different donors. siRNA1: HS_TJP1_6; siRNA2: HS_TJP_7 (Qiagen, Hilden, Germany). IL-1β release was measured by Ready Set Go ELISA from eBioscience (San Diego, CA, USA) 48 h after knock-down (knock-down was performed as described previously [67]; knock-down efficiency >80%)

Tight junctions and UV radiation Acute UV irradiation, especially UVB, results in an increase of proinflammatory cytokines such as IL-6, TNF-α, and IL-1β, as well as IL-8 and IL-10. In addition, release of prostaglandins and histamine is observed. Also, the number of macrophages and neutrophils rise [20]. Chronic UV irradiation results in many additional changes including hyperplasia, changes in dermal structure, and alteration of pigmentation [20, 22]. In chronically sun-exposed, healthy human skin, a decrease of Cldn-1 in the lower epidermal layers [55, 98] and a broader expression of Ocln, ZO-1, and Cldn-4 in (further) SS layers were observed [98]. In experimental models of UV irradiation, it was shown that acute UVB exposure influences TJs. Yamamoto et al. [125] showed that UVB exposure of mice resulted in a broader localization of ZO-1 after irradiation, while there were no clear changes for Ocln and Cldn-1. However, protein levels of Ocln and Cldn-1 increased. Kawada et al. showed decreased immunostaining of Cldn-1 in UVB-irradiated mice [60]. Acute UVB radiation of human skin grafted onto scid mice resulted in a decreased TJ barrier function to a 557-Da tracer 24 and 48 h after irradiation which was restored after 144 h. Changes of TJ proteins were not described [130]. In general, it is difficult to compare these studies because

different irradiation doses, different time points of evaluation, and different species were used. Also concerning the effect of UVB radiation on cultured keratinocytes, different results were described which, again, may be due to different doses of irradiation and different time points. While Yuki et al. observed unchanged levels of TJ proteins but changes in localization, i.e., a more dotty pattern of Ocln and Cldn-1 at the membranes and a dose-dependent decrease of TER [130], Borkowski et al. found an upregulation of TJ proteins [12]. The latter did not investigate the direct influence of UVon TJ functionality, but because inflammatory response to UV irradiation was shown to be partially dependent on TLR3 activation via non-coding RNA, they investigated the effect of TLR3 activation (by Poly I:C) and showed a TLR3-dependent upregulation of various TJ proteins including Ocln, Cldn-1, and Cldn-4 as well as increased TJ function (TER and sodium fluorescein) [12]. Rachow et al. [98] showed that UVB-induced apoptosis was reduced in Ocln knock-down NHEKs. In summary, UV radiation clearly influences TJs. However, the kind of effect may depend on the dose of UV and the time point of evaluation. To which extent inflammation contributes to the effect of UV on TJs still has to be clarified. Tight junctions and dermatitis Dermatitis denotes a polymorphic inflammatory reaction pattern involving the epidermis and dermis. Acute dermatitis is characterized by pruritus, erythema, and vesiculation and chronic dermatitis by pruritus, xerosis, lichenification, and hyperkeratosis [123]. Dermatitis comprises many etiologies and a wide range of clinical findings, e.g., allergic and non-allergic contact dermatitis, atopic dermatitis, and seborrheic dermatitis [123]. Sometimes, the terms Bdermatitis^ and Beczema^ are used interchangeably [123] while at times (often depending on the country) eczema is almost exclusively used for atopic dermatitis [16]. Delineation of the different forms of eczema/dermatitis is often difficult. Most knowledge has been gathered about atopic dermatitis and TJs; therefore, we will focus on this entity, but we will also briefly summarize knowledge on different forms of contact dermatitis. Information about seborrheic dermatitis can be found in BTight junctions in skin infection^ because alterations observed may be connected to fungal infection. Atopic dermatitis A common inflammatory skin disease is atopic dermatitis (AD). This chronic and non-contagious disorder is characterized by dry and scaly skin (non-lesional) which periodically exhibits severe, itchy eczema (lesional). It usually starts in infancy and affects up to 20% of school-aged children [78,

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122]. In most cases, the disease ceases during puberty, but in up to 10% of cases, it can persist into adulthood [34]. AD is often connected with other atopic diseases, often referred to as atopic march [107]. Associations are made with atopic rhinoconjunctivitis, atopic asthma as well as food allergies. AD is thought to be mainly a Th2-driven systemic disease rather than inflammation limited to the skin [121]. However, evidence is growing that barrier defects provide the initial step in the development of AD [21] (see below). Inflammation in AD is represented by increased serum IgE levels and inflammatory infiltrates (e.g., lymphocytes, macrophages, and dendritic cells) producing (pro-) inflammatory cytokines (e.g., IL-4, IL-13, and IL-31). In acute atopic eczema, the Th2 cytokine IL-4 dominates whereas in chronic eczema IFN-γ, IL-17, and IL-22 become more important [30]. AD is characterized not only by increased immune reaction but also by barrier defects. Patients show a disturbed insideout barrier as measured by a slightly increased transepidermal water loss (TEWL) in non-lesional skin, which is more pronounced in lesional skin [26, 37, 77]. Also, the outside-in barrier is disturbed: In vivo analyses of the skin of AD patients compared to that of control subjects showed increased diffusion coefficients in the SC for polyethylene glycols of various molecular sizes as well as sodium lauryl sulfate. Again, this was more pronounced in lesional compared to non-lesional skin [52, 53]. Furthermore, there is decreased microbial diversity in favor of an increased amount of Staphylococcus species in AD [69]. More than 85% of AD patients are colonized by facultative pathogenic bacteria such as Staphylococcus aureus, and the colonization of eczematous lesions with S. aureus is strongly associated with an increase in disease severity [49]. S. aureus can decrease TJ protein levels in the skin, as will be described in the next chapter in more detail. Concerning TJs, increasing evidence shows that they are involved in the pathogenesis of AD. In a northern American cohort, reduced expression of Cldn-1 and Cldn-23 has been reported in non-lesional skin and single nucleotide polymorphisms (SNPs) in the Cldn-1 gene were associated with AD in an African-American cohort [24]. In an Ethiopian cohort, a significant correlation between a Cldn-1 SNP and the onset of AD before the age of 5 years was shown [7]. Also, in a Korean study, a significant correlation between CLDN-1 SNPs and AD in a hospital-based study group was found. Nonetheless, this was not the case in a population-based Korean study group [129] and in a Danish cohort, where SNP relations were investigated in a population-based group [101]. In addition, in an Austrian cohort where immuno-intensity was investigated, no changes in Cldn-1 expression have been reported in nonlesional skin [37]. This shows that distinct populations or population subgroups may have genetic differences concerning Cldn-1. Of note, in African patients with AD, loss-offunction mutations of filaggrin are rare [122]. For Cldn-4, an

upregulation of immuno-intensity was observed in nonlesional skin of two independent European cohorts ([37] and own unpublished data). In three patients in a Japanese study, a downregulation of Cldn-4 in non-lesional skin was described based on Western blot analyses. However, there was no 100% match between the body location of patients and that of healthy controls (see influence of UV on TJs in BTight junctions and UV radiation^) and also GAPDH signals in nonlesional skin were clearly weaker than in control skin. Thus, a repetition of this study with a larger, better-controlled cohort would be of high interest. In the same study, no changes for Cldn-1 and a decrease in ZO-1 protein expression in nonlesional skin were described [133]. Also, in lesional skin, changes of tight junction proteins have been reported. In different cohorts, a clear downregulation of Cldn-1 immuno-intensity was shown [10, 37]. Additionally, reduced Cldn-1 immuno-intensity was also found in skin lesions of experimental models of AD in dogs [91]. In comparison to non-lesional skin, but not to healthy skin, a downregulation of Cldn-4 immuno-intensity was observed as well [37]. Furthermore, a broader localization of Cldn-4 was described [37, 73]. Finally, Cldn-8 alteration was found with RNAseq, when comparing lesional to nonlesional skin [110]. In mouse models for atopic dermatitis, a downregulation of Cldn-1 was described due to inflammation [37, 129]. The level of downregulation of Cldn-1 in the upper and lower layers of the epidermis correlated significantly with the density of dermal infiltrate [37]. In addition, AD typical cytokines showed an impact on TJ proteins (see above). The reasons for the development of AD are not completely elucidated yet, but an impaired epidermal barrier is thought to be a key feature of AD development [30]. It is more and more appreciated that both genetic and environmental factors, which affect skin barrier function, contribute to AD pathogenesis [8]. In industrial countries, the occurrence of AD is continually increasing, which supports a socioeconomic influence. Genetically, a very strong predisposing factor is a lossof-function mutation in the filaggrin (filament-aggregating protein) gene which was described in 2006 [92]. About one third of European patients suffering from AD show a homo- or heterozygote filaggrin mutation [92]. Consequently, filaggrin already is decreased in non-lesional AD skin [96, 106]. Filaggrin is a key protein that supports terminal differentiation and formation of the skin barrier. Filaggrin-deficient patients show a significant increase in surface pH [58], which is part of the complex barrier system of the skin, and filaggrin knockout mice show increased antigen penetration [61]. Furthermore, changes in SC lipids were described in nonlesional skin, which were linked to a decreased barrier function [50, 54, 104, 119]. In general, pathology of AD seems to be a vicious cycle of disrupted physical skin barrier and skin inflammation (Fig. 3).

Pflugers Arch - Eur J Physiol Fig. 3 The vicious cycle in atopic dermatitis. Stratum corneum (SC) barrier disruption due to filaggrin deficiency, scratching, or lipid alterations leads to increased access of allergens. This results in skin inflammation, which can also be provoked by changes in the skin microbiome. Skin inflammation and changes in the skin microbiome lead to altered TJ protein expression. Disordered keratinocyte proliferation, which is provoked by skin inflammation and altered TJ protein expression, again leads to SC barrier damage. This also can be directly triggered by altered TJ protein expression

The vicious cycle in Atopic Dermatitis

Decreased skin barrier function results in an increased uptake of allergens which activates the immune system and results in inflammation. Increased number of TJ-penetrating Langerhans cells was observed in AD which may contribute to an increased uptake of allergens [127]. Inflammation in turn can reduce skin barrier function, e.g., via altered differentiation. For instance, it is known that keratinocytes differentiating in the presence of IL-4 and IL-13 exhibit reduced filaggrin levels [48]. Also, a reduction of filaggrin after treatment of epidermal models with Th2 cytokines and TNF-α was observed [23]. Altered SC lipid composition was also present in these models, further indicating decreased barrier function [23]. In addition, AD patients mostly suffer from serve pruritus which might be stimulated by IL-31 or bacterial toxins (reviewed in [28]). This leads to scratching which also disturbs the SC barrier and contributes to the vicious cycle. How do TJs fit into this vicious cycle? Cldn-1 and, at least compared to non-lesional skin, Cldn-4 are downregulated in lesional skin of AD, putatively due to inflammation, but also genetic predisposition (for Cldn-1) may play a role in some cohorts. In addition, also alterations of the chemical and the antimicrobial barrier (see BTight junctions in skin infection^) may be involved in this downregulation. The claudin downregulation supposedly results, on the one hand, in further decreasing skin barrier function by impaired TJ barrier function in cross-talk with the stratum corneum (for the interaction of TJ and SC, see [9]). On the other hand, also downregulation of Cldn-1 independent from TJ-barrierforming structures, i.e., in the lower layers of the epidermis, may play a role, because it is significantly associated with increased epidermal thickness and proliferation as well as

Altered tight junction protein expression

Genetic predisposition

altered differentiation in eczema in an AD mouse model [37] and may thus contribute to clinical AD features. This is also supported by data from Tokumasu et al. who showed that mouse models with reduced levels of Cldn-1 exhibit ADlike features like dry skin, increased epidermal thickness, infiltration of macrophages, and increased levels of IFN-γ and IL-10 [114]. In addition, it was shown that Cldn-1 downregulation can increase proliferation [24]. Whether TJs are also initiators of this vicious circle is unclear yet. In those cohorts where a decrease of Cldn-1 is already observed in non-lesional skin, this may be possible. However, in cohorts where this is not the case, it even seems that TJs try to rescue the already impaired SC barrier function by upregulation of Cldn-4 [37].

Allergic contact dermatitis In allergic contact dermatitis (ACD), a specific immunological sensitization and subsequent elicitation due to contact allergens triggers eczema formation. In the sensitization phase, IL6, IL-12, TNF-α, IL-1β, and IL-18 play a role [79, 80]; in the elicitation phase, infiltrating T cells and IL-1β, TNF-α, IL-18, IFN-γ, IL-4, and IL-17 are involved [59, 103] (for review, see [70]). Data about TJs and ACD are sparse. In humans, up to now, only a genetic correlation of allergic contact sensitization with Cldn-1 SNPs [101] was described. In a mouse model of allergic dermatitis, an increase of TJ permeability could be shown for molecules from 557 to 5000 Da. This was accompanied by a decrease of Cldn-1. Of note, permeability for molecules of approximately 30 kDa was unchanged [126].

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Irritant contact dermatitis Single or cumulative exposure to physical or chemical irritants leads to irritant (non-allergic) contact dermatitis (ICD). ICD is characterized by an increase of IL-1α, IL-1β, IL-6, TNF-α, IL-8, CCL20, CCL27, IL-10, IL-12, and IL-18 [45, 103]. Concerning TJs, it was shown that cumulative application of sodium lauryl sulfate (SLS) and nonanoic acid resulted in downregulation of claudin-23 gene expression in human volunteers [19]. Data concerning TJ protein localization and TJ functionality are, to our knowledge, absent. Tight junctions in skin infection Regarding skin infection, one has to distinguish between colonization and infection. A classical definition of infection is the passive or active invasion of an organism’s tissues by disease-causing pathogens (e.g., bacteria, fungi, or viruses), their residence, and multiplication. The development of skin infections strongly depends on the pathogenic characteristics of the microorganism, on the status of the overall skin barrier, and on the immune response [57]. Examples of infectious microorganisms in the skin are Trichophyton spp. or Microsporum spp. (tinea) as dermatophytes and Candida albicans (candidiasis) as yeast in the context of fungal infections as well as herpes simplex virus or human papilloma virus in the context of viral infections. The most prominent example of bacterial skin infection is impetigo contagiosa caused by infection with S. aureus or Streptococcus pyogenes [99]. For skin infection, one has to have in mind that not only skin inflammation which is provoked by the infection may alter TJs but also the microbes themselves may interact with this structure and TJ proteins. Tight junctions in bacterial skin infection A hallmark of, e.g., S. aureus infection is neutrophil abscess formation. This is mediated by proinflammatory cytokines (IL-1β, TNF-α, or IL-6) and especially by chemokines like CXCL2 or IL-8 [83]. In in vivo skin diagnosed with impetigo contagiosa, TJ proteins Ocln, ZO-1, and Cldn-1 are downregulated at infected sites, but there is an upregulation as well as a broader localization in areas only colonized by the bacteria [90]. This clearly shows that pathogenic bacteria can downregulate TJ proteins, but it also hints of an upregulation of the TJ barrier and thus a mechanism to reduce bacterial invasion at sites with bacterial colonization. Under controlled conditions in a porcine skin infection model, it was shown that S. aureus as well as Staphylococcus epidermidis induced an upregulation of TJ proteins at early time points of inoculation (reflecting colonization), followed by a reduction of protein

expression at later time points of inoculation (reflecting infection). Of note, the downregulation was not due to cell death [90]. Similar results could also be obtained in S. aureus-treated reconstructed human epidermis. This decrease of TJ proteins was associated with impaired TJ barrier function (Bäsler et al., in preparation). In HaCat cells, S. aureus induced a rapid (within 3 to 5 h) decrease in TER values which was associated with reduced cell membrane localization of key TJ components (e.g., Cldn1, Cldn-4, and ZO-1) [90]. This does not exactly fit into the in vivo, ex vivo, and 3D-in vitro results for early time points showing an upregulation of TJ proteins. However, we observed a significant increase in TER at early time points followed by a decrease at later time points after inoculation of NHEKs with different S. aureus strains (Bäsler et al., in preparation). This discrepancy could be explained by different expression patterns of pattern recognition receptors (PRRs) in NHEKs and HaCat cells [68]. Additionally, HaCaT cells are spontaneously transformed aneuploidy immortal keratinocytes and it is still unclear if they fully differentiate or form stable TJs, as TER values are lower compared to NHEKs [6, 36]. Also, the treatment of primary keratinocytes with lysates of probiotic bacteria (Lactobacillus spp. and Bifidobacterium longum) enhanced TJ barrier function in a dose-dependent manner as measured by TER and increased protein levels of Cldn-1, Ocln, and ZO-1 [112]. The alterations of TJ proteins in infected skin might be a result of the inflammatory response of the skin as described above. However, also a direct regulation of the TJ barrier via activation of PRRs like TLRs probably plays a role. It was shown that activation of various TLRs strengthens the TJ barrier [12, 74, 134]. The role of PRRs for the overall skin barrier has been reviewed in [75]. In addition, the TJ barrier could also be regulated indirectly via the chemical barrier because S. aureus influences AMPs and AMPs, in turn, influence TJs. For example, treatment with heat-inactivated S. aureus enhanced the production of AMPs like hBD-2 and (slightly) hBD-3 [82], while S. aureus proteinases were able to degrade cathelicidin in a time- and concentration-dependent manner [108]. hBD-3 and cathelicidin were shown to increase TJ barrier function, i.e., they result in increased TER and reduced tracer permeability [3, 43, 62]. TJ proteins are also affected by bacteria in other tissues. Depending on the tissue and on the bacteria used, TJs were either opened (e.g., enteropathogenic Escherichia coli in CaCo2 cells [17, 40, 113]) or strengthened (e.g., probiotic treatment of MDCK cells [56, 118]). Tight junctions in viral and fungal infections On the one hand, viruses can influence TJ proteins. Foreskin tissue samples of herpes simplex virus type 2

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(HSV-2)-seropositive Kenyan men showed lower Cldn-1 mRNA levels than those of seronegative men [100]. Furthermore, infection of HaCaT cells with West Nile virus resulted in a degradation of Cldn-1 [81]. On the other hand, it is probable that the integrity of the TJ barrier prevents viral infection. Downregulation of Cldn-1 in cultured human keratinocytes resulted in increased HSV-1 infectivity [25]. This may be due to increased contact of the viruses with their cellular receptors which is normally limited by an intact TJ barrier. For fungi, it was shown that the mycotoxin patulin which is produced by molds reduced Cldn-1 expression in cultured human keratinocytes and in an AD mouse model [129]. Seborrheic dermatitis, an inflammatory disease of the scalp, which is thought to be correlated with Malassezia yeasts and which is characterized by an increase of various cytokines, e.g., IL-1α, IL-β, IL-4, IL-6, IL-8, and TNF-α, as well as the infiltration by neutrophils and impaired skin barrier [84, 105], showed a downregulation on mRNA levels for Cldn-8 (personal communication with Kevin Mills, based on [84]).

References 1.

2.

3.

4.

5. 6.

7.

Tight junctions and further inflammatory skin diseases 8.

In lichen planus, a broader expression of occludin and ZO1 was described [97]. The same is true for psoriasis, where in addition a broader expression of Cldn-4 and a more or less pronounced downregulation of Cldn-1 were described [66, 93, 120]. Of note, the TJ barrier is still present in psoriatic plaques, at least in lesions with residual Cldn-1 [65]. Detailed data for psoriasis and TJs have recently been summarized in [9].

Conclusion Skin inflammation can have various causes and result in different clinical entities. TJ proteins are altered in these entities as far as they have been investigated up to now. This emphasizes the important role of TJ proteins in the skin. However, in most of the diseases/skin conditions, TJ barrier function has not been investigated yet. This is an important task for the future. In addition, TJ barrierindependent roles of TJ proteins will have to be investigated in more detail.

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