Current evidence of skin barrier dysfunction in human and canine atopic dermatitis

Authors

  • Rosanna Marsella,

    1. Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
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  • Thierry Olivry,

    1. Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
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  • Didier-Noel Carlotti,

    1. Aquivet Clinique VétérinaireService de DermatologieParc d'Activités MermozAvenue de la ForêtF, Eysines, Bordeaux
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  • for the International Task Force on Canine Atopic Dermatitis


  • Sources of Funding
    This study is self-funded.

  • Conflict of Interest
    No conflicts of interest have been declared.

Rosanna Marsella, Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA. E-mail: marsella@ufl.edu

Abstract

Atopic dermatitis (AD) is a multifaceted disease resulting from a complex interaction between environmental and genetic factors. Both of these factors can shape skin barrier function and the immunological response of predisposed patients. There is increasing evidence that an impaired skin barrier plays a role in both human and canine AD. Although many primary skin barrier defects had already been documented in the past in humans, the recent identification of the filaggrin mutations and the fact that such mutations are now considered the most important risk factor for development of AD have further emphasized the relevance of epidermal dysfunction in human AD. Much less is known in veterinary medicine, but evidence is rapidly building to support a role for skin barrier dysfunction in canine AD. Canine AD shares many clinical and immunological similarities with its human counterpart. The similar distribution of clinical lesions and the importance of the epicutaneous route of allergen exposure provided the incentive to investigate the role of skin barrier impairments in canine AD. The purpose of this comparative review is to present the current evidence of barrier dysfunction in both human and canine AD.

Résumé

La dermatite atopique (DA) est une maladie à multiples facettes résultant d’interactions complexes entre facteurs génétiques et environnementaux. Ces deux facteurs peuvent modifier la fonction de barrière cutanée et la réponse immunologique des patients prédisposés. Il existe des preuves croissantes que la barrière cutanée endommagée joue un rôle dans la DA humaine et canine. Bien que des défauts de barrière cutanée aient déjàété documentés dans le passé chez l’homme, la récente identification des mutations de la filaggrine et le fait que de telles mutations sont désormais considérées comme le facteur de risque le plus important dans le développement de la DA, ont appuyés davantage la pertinence d’un dysfonctionnement épidermique dans la DA humaine. On en sait beaucoup moins en médecine vétérinaire mais des éléments se construisent rapidement autour du rôle d’un dysfonctionnement de barrière cutanée dans la DA canine. La DA canine partage de nombreuses similitudes cliniques et immunologiques avec son homologue humain. Les lésions cliniques sont distribuées de manière similaire et l’importance de la voie épicutanée d’exposition allergénique incitent à l’étude du rôle des défauts de la barrière cutanée dans la dermatite atopique canine. L’objectif de cette étude comparative est de présenter les preuves actuelles de défauts de barrière à la fois dans la DA humaine et canine.

Resumen

La dermatitis atópica (AD) es una enfermedad de múltiples facetas que resulta de una interacción compleja entre factores ambientales y genéticos. Ambos pueden influir la función de barrera de la piel y la respuesta inmunológica de los pacientes predispuestos. Existen evidencias cada vez mas claras de que la alteración de esa función de barrera juega un papel de relevancia en AD canina y humana. Aunque se han documentado muchos defectos en la función de barrera de la piel en el pasado en el ser humano, la reciente identificación de mutaciones en filagorina y el hecho de que tales mutaciones se consideran actualmente el factor de riesgo mas importante para el desarrollo de AD, solo ha hecho que enfatizar la relevancia de la disfunción epidermal en la AD humana. Se sabe mucho menos en medicina veterinaria, pero poco a poco se van acumulando evidencias que apoyan la existencia de una alteración de la función de barrera en la AD canina. La AD canina comparte muchas similitudes clínicas e inmunológicas con la enfermedad equivalente humana. La distribución similar de las lesiones clínicas y la importancia de la ruta epicutánea en la exposición a alergenos aportan un incentivo para investigar el papel de las alteraciones de la función de barrera de la piel en la AD canina. El propósito de esta revisión comparativa es presentar los últimos hallazgos de la alteración de la función de barrera tanto en la AD humana como canina.

Zusammenfassung

Die atopische Dermatitis (AD) ist eine facettenreiche Erkrankung, die sich aus einer komplexen Interaktion zwischen Umwelt- und genetischen Faktoren ergibt. Beide Faktoren können die Funktion der Hautbarriere und die Immunantwort prädisponierter Patienten beeinflussen. Es besteht zunehmende Evidenz, dass eine beeinträchtigte Hautbarriere sowohl bei der humanen wie auch bei der Atopie des Hundes eine Rolle spielt. Obwohl in der Vergangenheit bereits zahlreiche Primärdefekte der Hautbarriere beim Menschen beschrieben worden waren, haben die unlängst erfolgte Identifizierung von Filaggrinmutationen und die Tatsache, dass solche Mutationen mittlerweile als die wichtigsten Risikofaktoren eine AD zu entwickeln, betrachtet werden, die Bedeutung der epidermalen Dysfunktion bei der humanen AD noch unterstrichen. Obwohl man darüber in der Veterinärmedizin wesentlich weniger weiß, nimmt die Evidenz, dass eine Dysfunktion der Hautbarriere auch bei der AD des Hundes eine Rolle spielt rapide zu. Die canine AD weist viele klinische und immunologische Ähnlichkeiten mit dem humanen Gegenstück auf. Die ähnliche Verteilung der klinischen Veränderungen und die Wichtigkeit der epikutanen Route der Allergenexposition gaben den Anstoß dazu, die Rolle einer Beeinträchtigung der Hautbarrierefunktion bei der caninen AD zu untersuchen. Es ist das Ziel dieser vergleichenden Review die momentane Evidenz der Dysfunktion der Barriere sowohl bei der AD des Menschen als auch bei der des Hundes zu präsentieren.

Abstract

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Introduction

Atopic dermatitis (AD) is a multifaceted disease resulting from a complex interaction between environmental and genetic factors. Both of these factors can shape skin barrier function and the immunological response of predisposed patients. There is increasing evidence that an impaired skin barrier plays a role in both human and canine AD. Although many primary skin barrier defects had already been documented in the past in humans, the recent identification of the filaggrin mutations and the fact that such mutations are now considered the most important risk factor for development of AD have further emphasized the relevance of epidermal dysfunction in human AD.1,2 It is currently proposed that genetically inherited mutations affecting barrier function in conjunction with acquired environmental stressors lead to increased allergen penetration. This promotes a T-helper 2 (Th2) shift that further aggravates the skin barrier.3 Bacterial colonization further worsens the skin barrier (bacterial ceramidases, for example, can contribute to ceramide deficiency in atopic skin)4 creating a self-perpetuating cycle of inflammation, pruritus and additional sensitizations.3

Much less is known in veterinary medicine, but evidence is rapidly building to support a role for skin barrier dysfunction in canine AD. Canine AD shares many clinical and immunological similarities with its human counterpart.5 The similar distribution of clinical lesions6 and the importance of the epicutaneous route of allergen exposure7–9 provided the incentive to investigate the role of skin barrier impairments in canine AD. The purpose of this comparative review is to present the current evidence of barrier dysfunction in both human and canine AD.

Human medicine: human atopic dermatitis

Methods to assess barrier function

Barrier function can be assessed in a variety of ways.10,11 The integrity of the skin barrier can be assessed objectively in a noninvasive way by measuring transepidermal water loss (TEWL).12,13 Skin barrier function as assessed by TEWL is intrinsically compromised in children with AD but not in children with other allergic conditions.14 The magnitude of increase in TEWL correlates with AD severity.14,15 The TEWL is higher in AD patients16–19 than in healthy control subjects, even in clinically unaffected areas.20 In humans, TEWL varies between anatomical sites,21–23 and it is affected by a variety of factors, such as the age of the patient,22,24,25 but it is not linked to particular genetic mutations.26 Measurement of TEWL, moreover, allows assessment of otherwise unnoticed damage to the epidermal water barrier in nonlesional skin. Interestingly, TEWL has been reported to be higher in patients with extrinsic AD than in patients with the intrinsic form (patients with AD in whom allergen-specific IgE is not detectable).27 Patients with the intrinsic form have been documented to retain a normal barrier function and sensory reactivity to external pruritic stimuli.27 The advantages and disadvantages of various TEWL-measuring devices (i.e. evaporimeters) have been investigated.28 In open-chamber devices, the probe consists of a cylindrical open chamber with two sensors mounted in the diffusion chamber to measure the humidity and the temperature. The open-chamber devices are based on the diffusion principle, in which the water vapour pressure gradient is indirectly measured by two pairs of combined thermistors and hygrosensors, present at two different heights inside a hollow cylinder. The water pressure measured in the chamber is used to calculate the evaporated water over a constant skin area. The probe head is placed horizontally on the skin at a constant pressure, and its small size minimizes the influence of air turbulence inside the probe. The measuring usually takes between 30 and 40 s. Criticisms of this traditional open system are related to effects of ambient and body-induced airflows near the probe, probe size, the limitation of measurement sites and application/probe angles. Other important factors to consider during TEWL measurements with an open-chamber method are air convection, room temperature and ambient humidity. Another consideration is that volatile agents, other than water, might affect the readings (e.g. when measurements are made immediately after application of moisturizers).

Closed-chamber devices use a closed, unventilated chamber system, which is not affected by ambient or body-induced airflows. Closed-chamber conditions are created upon skin contact with a surface area of 1 cm diameter, and the measuring time is between 8 and 10 s. It has been proposed that closed-chamber devices allowed measurements at any angle, had short reading times and were insensitive to external air currents. A recent study reported that both open- and closed-chamber devices are able to estimate water loss rates accurately when held in a vertical position.28 Even though closed-chamber evaporimeters might be easier to use than open-chamber devices, their tendency to become saturated in conditions of high water loss are a disadvantage when assessing TEWL dynamically.28 Open- and closed-chamber TEWL readings, furthermore, correlate well.29 The TEWL values of healthy volunteers were measured simultaneously with three closed- and open-chamber devices in the same room according to the guidelines of the standardization group of the European Society of Contact Dermatitis, and there was no statistically significant difference between the mean forearm TEWL values measured by all three instruments. The authors concluded that the TEWL values measured by all instruments were constant, with small standard deviations.30

As TEWL measurements appear to be variable, it has been suggested that continuous assessment of the epidermal water barrier by multiple TEWL measurements over longer periods of time would decrease the risk of mistakes and increase accuracy.31

Skin capacitance is another parameter used to assess the skin barrier function. Electrical skin capacitance measures the hydration of the stratum corneum and provides an estimate of cutaneous capacity to retain moisture. Capacitance has been shown to affect the balance of epidermal cell proliferation and differentiation. Higher skin hydration values indicate greater cutaneous water capacitance.32

Chemical and enzymatic abnormalities in the atopic epidermis

Lipids

The stratum corneum, composed of flattened keratinocytes and lipids, is crucial for the protective functions of the skin. Lipids in this layer are composed primarily of free fatty acids, cholesterol and ceramides. Ceramides, in particular, are the dominant lipids, constituting approximately 50% of the human stratum corneum lipids,33 and play a crucial role in skin barrier function.34,35 Ceramides are also the the most heterogeneous of the epidermal lipids and include 11 different molecules.36 Ceramides are generated from a sphingoid base and a fatty acid. Sphingoid bases include, but are not limited to, sphingosin, dihydro-sphingosin and phyto-sphingosin. Over the last few years, critical enzymes in ceramide biosynthesis, including ceramide synthases and ceramide hydroxylase/desaturase, have been identified.37 Ceramide synthesis is upregulated in the basal cell layer. Ceramides are then rapidly converted into glucosyl-ceramides and sphingomyelins that are then packed into the lamellar bodies. Once the content of these organelles is released at the interface between the stratum granulosum and stratum corneum, ceramides are regenerated by hydrolysis by β-glucocerebrosidase and sphingomyelinase Thus, ceramide levels in the stratum corneum are regulated by a balance between synthetic enzymes (e.g. β-glucocerebrosidase and sphingomyelinase) and ceramidases (which are responsible for their degradation). Ceramidases may be endogenous or exogenous (e.g. from bacteria).4

Impaired skin barrier function in human AD has been linked to a variety of epidermal abnormalities, although it is unclear whether they are primary, secondary or both. The stratum corneum of atopic skin is characterized biochemically by a reduction in the amounts of ceramides, especially ceramide-1, sebum lipids and water-soluble amino acids,38 although one study reported normal levels of ceramides in uninvolved atopic skin.39 In atopic individuals, decreased epidermal sphingomyelinase activity is found in both nonlesional and, more significantly, lesional skin, correlating with reduced stratum corneum ceramide content and disturbed barrier function.40 This is associated with impaired expression of cornified envelope proteins and keratins important for skin barrier function. Another mechanism for decreased ceramides in atopic skin is the decreased biosynthesis of free glucosylceramides and ceramides.38

Some studies have proposed that ceramide deficiency is linked to a novel sphingolipid-metabolizing enzyme, termed sphingomyelin glucosylceramide deacylase,41–43 which hydrolyses sphingomyelin or glucosylceramide, decreasing their availability for synthesis of ceramides. The enzymatic characteristics of the sphingomyelin glucosylceramide deacylase are completely distinct from ceramidase as well as the other known deacylases.41,42 Sphingomyelin glucosylceramide deacylase activity is enhanced more than fivefold in lesional stratum corneum, more than threefold in uninvolved stratum corneum and approximately threefold in the involved epidermis from patients with AD compared with healthy control subjects.41,42 This increased enzymatic activity appears to be specific to AD and is not detected in patients with contact dermatitis, who have the same enzymatic activity as healthy control subjects.41 It is interesting to note that, in peripheral blood lymphocytes of AD patients, there is no increase in activity compared with healthy control subjects, indicating that the high expression of sphingomyelin deacylase is highly associated with the skin of AD patients.44 In one study, a lack of phosphatidylcholine–sphingomyelin transacylase activity was also proposed as an underlying defect in human AD, although the number of samples evaluated in that study was too small to draw final conclusions.45

In summary, lipid deficiency, especially of ceramides, in the skin of atopic patients is considered an important component of the disease. Furthermore, treatments aimed at correcting such deficiencies, by topical application of lipid preparations, have been reported to ameliorate clinical signs.46,47

Proteins

Proteins important in the epidermal differentiation/cornification process have also been reported to be decreased in patients with AD. Filaggrin is undoubtedly the most discussed protein at the moment. An early study reported decreased expression of filaggrin in the skin of atopic patients in both lesional and clinically unaffected skin.48 The more recent report of loss-of-function mutations in the gene encoding for filaggrin (FLG)49 has triggered large-scale investigation of the genetic mutations in FLG in a variety of populations.50–52 Of all the genes previously investigated as candidates for the development of AD, FLG is currently considered the most important, and FLG mutations are thought to be a major risk factor for AD.53–55 It is accepted that FLG mutations predispose to allergic sensitizations and to the development of early-onset, extrinsic disease that persists into adulthood.56–59 It is, however, important to note that decreased filaggrin expression does not necessarily imply a genetic mutation, because filaggrin can be reduced by Th2 cytokines.60 Decreased expression in the skin of some patients may therefore be a secondary phenomenon or may result from epigenetic modifications. Nevertheless, decreased filaggrin expression may still be clinically relevant in atopic individuals lacking the FLG mutations. It is estimated that only 18–48% of all eczema patients have FLG mutations,61 and that 40% of individuals with the FLG null alleles have never had signs of eczema.62 These considerations highlight the fact that that the link between FLG mutations and AD is not a simple and direct one and that AD is the result of a complex interaction between genetic and environmental factors.63

Interestingly, flaky tail (Flgft) mice, essentially deficient in filaggrin, have been used to investigate the role of filaggrin in AD. In specific pathogen-free conditions, the majority of Flgft mice develop clinical and histological eczematous skin lesions similar to human AD with outside-to-inside skin barrier dysfunction, suggesting that the Flgft mouse genotype has potential as an animal model of AD corresponding to FLG mutation in human AD.64

Epidermal proteases and protease inhibitors

Skin barrier function in AD is impaired by a variety of mechanisms. Besides mutations in the genes encoding for proteins, there is also evidence of mutations in the genes encoding for proteases and protease inhibitors. This could lead to increased desquamation and an impaired skin barrier. Desquamation is determined by a cocktail of proteases (e.g. serine, cysteine and aspartic proteases) regulated by protease inhibitors.65

Human tissue kallikreins (KLKs) are a family of 15 trypsin- or chymotrypsin-like secreted serine proteases (KLK1–KLK15) important for desquamation.66 Many KLKs have been identified in normal stratum corneum and sweat, including KLK7 (also called stratum corneum chymotryptic enzyme, SCCE) and KLK5 (also called stratum corneum tryptic enzyme, SCTE). A mutation in the gene encoding for KLK7/SCCE has been described in some human patients with AD. This mutation leads to a change in activity and increased desquamation, particularly at alkaline pH.67 This genetic associationwas not confirmed in other studies, however, and there are no additive effects of mutations on phenotype.26 In the stratum corneum of AD patients, all kallikreins, except KLK11, have been reported to be significantly elevated.68 Alterations of the levels of KLKs in the stratum corneum of atopic patients are more pronounced than those in the serum.66

In a recent study investigating stratum corneum thickness and proteases, it was found that lesional atopic skin had a significantly thinner stratum corneum and that serine protease activity [stratum corneum tryptase-like enzyme (45-fold), plasmin (30-fold), urokinase (7.1-fold), trypsin-like KLKs (5.8-fold) and chymotrypsin-like KLKs (3.9-fold)] were increased in atopic skin when compared with healthy control subjects.69 The increased serine protease activities were found in acute eczematous AD, especially in deeper layers of the stratum corneum.67 These elevations in protease activities were associated with impaired barrier function, irritation and reduced skin capacitance (a measurement of the stratum corneum hydration). Elevations of some serine proteases (e.g. plasmin and urokinase) are, however, not specific for AD, but can be found in other circumstances in which the skin barrier is disrupted.70 Protease activity was also found to correlate with clinical staging of AD (i.e. clinical scores) and tended to normalize as the disease regressed.71 Whether increased proteases are the cause or simply a marker of disease activity remains unknown. Cells within the inflammatory infiltrate (e.g. mast cells) can produce proteases (e.g. chymase, a chymotrypsin-like serine proteinase),72 which can further contribute to the disruption of the skin barrier. This makes it difficult to separate cause from effect.

Proteases are regulated by protease inhibitors. A particularly important, pH-dependent protease inhibitor is lymphoepithelial Kazal-type 5 serine protease inhibitor (LEKTI), which is encoded by the serine protease inhibitor Kazal-type 5 (SPINK5) gene.73 The most convincing evidence for a role of excessive serine protease activity in AD comes from Netherton syndrome, a disease associated with loss-of-function mutations in SPINK5 and decreased LEKTI. Patients with this condition have severe AD and high IgE. Their stratum corneum is extremely thin, resulting in severe skin barrier defects. A significant association with atopy, AD and Netherton syndrome has been reported.74 The pathogenic role of serine protease inhibitor LEKTI in AD has been recently questioned owing to contradictory results from the analyses of an association between genetic polymorphisms of SPINK5 and AD. The association of AD with SPINK5 polymorphisms and AD has been reported by some studies75,76 but not confirmed by other authors.26 A recent study reported that expression of LEKTI was significantly decreased in atopic patients compared with healthy volunteers.77 Due to reduced protease inhibition, trypsin-like hydrolytic activity in AD was slightly increased, although not significantly.74 The authors of the study concluded that functional analyses in addition to genetic investigations are necessary to gain further and more detailed insights into the role of LEKTI in AD.

Ultrastructural evaluation

It has long been known that water permeability of the stratum corneum is regulated primarily by the lamellar arrangement of lipid bilayers between the corneocytes.78 The process of postsecretory extracellular development of the lamellar body has been investigated in detail in healthy skin in electron microscopy studies.79 These investigations found that there are differences between inner and outer parts of the stratum corneum, in terms of both lipid lamellae and corneodesmosomes.77 Differences have been reported in the relative volume of lamellar bodies in the upper layers of the stratum corneum between atopic patients and healthy control subjects.80 Lamellar bodies remain undelivered within the cells of the uppermost stratum granulosum cell layer of humans with AD (26% in atopics versus 8% in control subjects, < 0.01). The authors concluded that a pathological failure to extrude lamellar bodies in AD may be at least partly responsible for the lipid abnormalities and skin barrier impairments observed in affected patients. Additionally, lipid lamellae appear misshapen and decreased in number when compared with healthy control subjects.78 No additional studies have specifically evaluated this proposed defect of extrusion.

Veterinary medicine: canine atopic dermatitis

Methods to assess the barrier function

The validity and reliability of TEWL measurements in dogs remains controversial. There are few studies that have attempted to validate either open-81 or closed-chamber82 devices to measure TEWL in healthy dogs. For example, one study evaluated the correlation between skin barrier function and TEWL in healthy dogs using a closed-chamber technique.83 This showed that increases in TEWL correlated with the frequency of tape stripping and inversely correlated with the thickness of the stratum corneum, reflecting impaired barrier function. In contrast, other reports provide evidence of very high day-to-day variation and do not recommend the use of TEWL measurements made with either open84 or closed apparatus.85 As suggested recently,85 the high variability of site-to-site, day-to-day and dog-to-dog TEWL measurements makes this technique largely unsuitable for measurement of changes associated with disease state or clinical trials testing interventions aimed at improving skin barrier function in dogs. A recent study evaluated TEWL and skin capacitance before and after anaesthesia in 14 body regions.86 The TEWL was highest for the footpad and head and lowest for the inguinal region. Following anaesthesia, TEWL and skin hydration were significantly lower on the head, upper back and footpad, and upper back and elbow, respectively, while skin pH was unaffected by this procedure. The authors concluded that while measurement of pH would seem to be valid anywhere on the body in anaesthetized dogs, regional factors should be considered when interpreting TEWL and skin hydration values and when treating regional skin diseases in veterinary practice. The variability in TEWL measurements is much larger than that reported in human medicine in recent studies comparing open- and closed-chamber devices.27,29 This may be partly attributable to the presence of hair and movement of the subject during measurements.79 A recent study indicated that clipping methods and anatomical site selection should be standardized in order to minimize the experimental variation in TEWL measurement in dogs even when closed-chamber devices are used.87 This study also reported that among the clipped sites, the upper back was the most appropriate site for TEWL measurement 48 h after clipping, whereas among the unclipped sites, the ear was the most appropriate. The TEWL values from those anatomical sites had the least fluctuation and were less affected by movement.84

Studies on TEWL in dogs with AD are limited. One study evaluating TEWL using an open-chamber method in a colony of atopic beagles found that atopic dogs had significantly higher TEWL than healthy beagles, particularly at the sites predisposed to development of AD, even if they appeared clinically unaffected.88 These findings were especially marked in young dogs. Taking into consideration the technical limitations described above, these results indicate that an impaired skin barrier might exist in experimental canine AD, even in atopic skin that appears clinically unaffected. Whether this is due to a primary defect or is secondary to subclinical inflammation is unknown at this time. A similar increase of TEWL in dogs with AD compared with healthy dogs was also found in patients with spontaneous disease using a closed-chamber technique.80 In that study, increases in TEWL were correlated with reduced proportions of ceramides in lesional and nonlesional skin.80

Chemical and enzymatic abnormalities in canine atopic epidermis

Lipids

Preliminary evidence shows that ceramides are decreased in the skin of dogs with AD, and that this defect might possibly contribute to impairment of the skin barrier. In a recent study, intercellular lipids were extracted from the stratum corneum of the inguinal area of both atopic and healthy dogs and quantified by thin-layer chromatography.80 It was found that levels of ceramides in lesional and clinically unaffected skin of atopic dogs were significantly lower than in healthy dogs, but there were no differences in cholesterol and free fatty acids.80 In another, more recent study, the stratum corneum ceramide contents of samples from clinically unaffected canine atopic skin were found to have decreased percentages of ceramides 1 and 9, and increased percentages of cholesterol compared with samples from nonatopic dogs.89 Unfortunately, the total amount of skin lipids was not determined, and whether or not there is a true deficiency of ceramides 1 and/or 9 in atopic dogs is unclear.

Proteins

Limited information exists on the expression of proteins, such as filaggrin, in atopic and healthy canine skin. A pilot study investigated expression of filaggrin in the skin of various animal species, including the dog.90 Immunostaining of healthy canine skin with a polyclonal antihuman filaggrin antiserum established that filaggrin was expressed in keratohyalin granules in the cytoplasm of the stratum granulosum, as in other species.90 A preliminary study in an experimental model of atopic beagles using another polyclonal antibody directed against human filaggrin showed an abnormal pattern and decreased immunostaining for skin filaggrin in atopic dogs when compared with healthy beagles.91 This antiserum recognized intracellular granular antigenic epitopes in both the stratum granulosum and the lower epidermal layers, suggesting that proteins other than filaggrins (perhaps keratins) were also identified. A more recent study evaluated the same colony of atopic beagles and age- and breed-matched healthy control dogs using a polyclonal antibody specific for canine filaggrin.92 Skin biopsies were taken before and after allergen challenge, and filaggrin mRNA was quantified using quantitative real-time PCR. Indirect immunofluorescence was also used to localize filaggrin protein expression. It was found that filaggrin fluorescence was distributed homogeneously in the stratum granulosum and the lower layers of the stratum corneum of healthy dogs, while in atopic dogs a patchy pattern was visible after house dust mite allergen challenge, suggesting that the changes might be secondary to atopic inflammation. In another recent study, immunofluorescence microscopy was performed in healthy and atopic dogs with naturally occurring disease using an antibody raised against the canine filaggrin C-terminus and a commercial antibody directed against the N-terminal segment of human filaggrin.93 Four distinctive filaggrin expression patterns were identified in nonlesional skin. It was found that 10 of 18 dogs with AD exhibited an identical pattern for both antibodies with comparable (category I, three of 18) or reduced expression (category II, seven of 18) compared with that of control dogs. In contrast, four of 18 dogs displayed aberrant large vesicles revealed by the C-terminal but not the N-terminal antibody (category III), while four of 18 showed a control-like N-terminal expression but lacked the C-terminal protein (category IV). The missing C-terminal filaggrin in category IV strongly points towards mutations resulting in a truncated filaggrin in four of 18 (22%) of all atopic dogs analysed. The authors concluded that, in dogs with filaggrin-deficient AD, the disease is a suitable model to study the barrier dysfunction typical of the human disease. Finally, a recent study evaluating possible filaggrin mutations in West Highland white terriers excluded a major causative role for the Flg orthologue in AD in this breed.94 The authors did not, however, exclude the possibility of Flg mutations with small effect sizes. A lack of linkage of AD in West Highland white terriers with the FLG has also been excluded in West Highland white terriers of US lines (C. Salzman, T. Olivry, D. Nielsen and N. Olby, unpublished observations).

Proteases and protease inhibitors

Very limited information is currently available on the role of proteases and their inhibitors in the canine disease. A recent study investigating gene transcription in biopsies from dogs with AD reported that significant differences existed in mRNA expression between normal and atopic dogs for SPINK5, mast cell protease I, dipeptidyl-peptidase-4, phosphatidylinositol-3,4,5-trisphosphate-5-phosphatase-2 and sphingosine-1-phosphate lyase-1.95 Whether these differences reflect a primary defect or changes secondary to skin inflammation is not known. A recent study has demonstrated that it is highly unlikely that KLK7 is a candidate gene for AD development in boxer and West Highland white terriers.96

Ultrastructural evaluation

Preliminary studies of the epidermis suggest that ultrastructural differences exist between atopic and healthy dogs. The first report came from a pilot study in dogs with spontaneous AD, which described abnormal lipid lamellae in atopic dogs.97 This study documented significantly reduced thickness and continuity of epidermal lipid lamellae in atopic compared with healthy dogs.97 Similar observations were made recently in a small number of dogs with naturally occurring AD.98 A further evaluation in an experimental model of atopic beagles reported irregular and highly disorganized lipid lamellae in the atopic beagles when compared compared with healthy control dogs.99 In atopic specimens, lamellar bodies were detected inside corneocytes, similar to what is observed in human patients. These findings were also reported in clinically unaffected atopic skin. Exposure to allergen and development of clinically evident lesions was ultrastructurally associated with copious release of irregularly organized amorphous lipid material in the intercellular spaces of the lower stratum corneum and widening of the intercellular spaces in the stratum corneum.99

Where do we go from here? Implications for research and practice

Although investigations of alterations to the skin barrier in atopic dogs are still in their infancy, there is increasing, albeit preliminary, evidence to suggest that they play a role, be it primary or secondary to underlying inflammation. More research is clearly warranted. Investigation of differences in lipid composition with particular emphasis on the evaluation of ceramides is also warranted, as preliminary studies indicated that major differences in lipids can exist between breeds and at different ages (e.g. cholesterol content appears to be variable with age, and wax diesters increase with age).100

It is tempting to speculate that some of the cutaneous abnormalities in atopic dogs could be corrected by oral administration or topical application of lipid preparations, as in human medicine.45,46,101,102 The role of nutrition in skin health has long been recognized,103,104 and it is known that oral supplementation with essential fatty acids increases skin essential fatty acids, reduces TEWL and improves skin barrier function.105 The recent study by Piekutowska et al.98 showed that topical application of a lipid preparation in atopic dogs may be beneficial. Repeated topical applications to clinically unaffected skin of dogs with AD led to a significant increase in lipid lamellae in the deepest part of the stratum corneum and to the release of lipid material at the junction between the stratum granulosum and stratum corneum; however, the clinical benefit, if any, was not reported. A recent study in Japan using a topical preparation containing ceramides showed that some atopic dogs responded very well to this form of therapy, while others did not respond or worsened during treatment.106 Clinical benefit was evident around 4–6 weeks, but did not reach a maximum until 8–12 weeks in some cases.106 It is unclear whether a change in lipid ultrastructure, and any potential clinical benefit, can occur at sites distant from the point of application.

At present, studies are still needed to evaluate whether correction of ultrastructural abnormalities results in appreciable clinical benefit, as it is unknown whether clinical signs in canine AD correlate with the severity of the skin impairment, whether assessed by TEWL or electron microscopy.

This area of research is important and potentially promising. Therapy to improve the skin barrier could combine both topical and systemic therapy (e.g. oral essential fatty acids). Thorough studies are necessary to avoid using inappropriately formulated products that could further aggravate an already impaired barrier. It is important that clinical trials document both the degree of clinical improvement and improvements to skin barrier function. While the documentation of chemical or ultrastructural changes in epidermal lipids is important to validate a proof of concept for the relevance of barrier-targeting interventions, it is ultimately the demonstration of meaningful clinical improvement in well-conducted trials that will confirm whether or not the approach of correcting a barrier dysfunction, if present, is valid. In order to help sort out whether skin barrier impairment is primary or secondary to even subclinical inflammation, it could also be interesting to test barrier function in atopic dogs before and after treatments such as cyclosporine. Logic would suggest that if the impairment is primary, a complete improvement should not be seen after treatment, because the defect would be permanent and of genetic origin. A partial improvement might be expected, however, as atopic inflammation might have aggravated a primary defect.

If future studies confirm that impaired skin barrier function in canine AD leads to increased allergen penetration and increased risk for allergic sensitization, it is tempting to speculate that proactive restoration of the skin barrier could alter the course of the disease and minimize the development of an allergic response. This ‘preventative’ approach could change the way we manage dogs predisposed to AD.

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