Epidermal barrier function
Koji Nishifuji (Japan) thanked Sogeval for sponsoring this workshop and expressed his appreciation to all contributing speakers as well as to attendees. He then provided an overview of this workshop on the skin barrier, its structure, function and role in different diseases, particularly in canine atopic dermatitis.
Antimicrobial skin barrier in canine atopic dermatitis: how much do we really know? (D. Santoro)
Domenico Santoro (USA) remarked that only a small amount of information about antimicrobial peptides (AMPs) is available in veterinary dermatology compared to human dermatology. He therefore chose to present an overview of current knowledge about AMPs in animals. Antimicrobial peptides are small cationic proteins and, so far, 1500 AMPs have been described in different species. The most studied groups include defensins alpha, beta and theta; the greatest focus is on beta-defensins, cathelicidins and S100 proteins. AMPs are predominantly localized in the epithelia including skin, mucosae, testes and eyes, where they serve as defence mechanisms against environmental pathogens. They are also involved in toxin neutralization, function as chemokines, serve as a bridge between innate and adaptive immunity, and are involved in wound healing and skin colour.
He commented that the audience might be asking ‘what is known about beta-defensins and canine atopic dermatitis?’ Last year, using a canine atopic dermatitis model, Domenico Santoro and colleagues were able to demonstrate significant increase in mRNA expression of two different beta-defensins (cBD1 and cBD3) as well as cathelicidin in an acute lesional skin of beagles with an experimentally induced atopic dermatitis compared to healthy dogs.1 However, when the expression was analysed over time, there was decreased expression of these beta-defensins in the lesional skin of the same beagles, although this was not significant. Protein expression measured by a competitive inhibition ELISA on protein extracts from the same dogs showed reduction of measured beta-defensins (cBD3), which was similar to reports from human literature.
Only a couple of studies have addressed the relationship between AMPs and chronic atopic dermatitis. van Damme and colleagues showed decreased expression of cBD103 in skin of dogs with chronic atopic dermatitis compared to healthy control dogs, while cBD1 was increased in affected dogs compared to controls.2 During this meeting, Domenico Santoro and colleagues presented results from a study that evaluated the expression of AMPs in canine atopic skin using similar conditions as those described in van Damme’s study.3 In this study, the authors demonstrated increased cBD103 mRNA expression in the skin of chronic atopic dermatitis dogs versus healthy controls and also higher cBD103 expression in skin of atopic dogs with skin infection versus atopic dogs without an infection. The opposite findings were obtained for the cBD1-like mRNA expression. At the protein level, no significant differences were seen between atopic and healthy dogs.
For cathelicidin, mRNA expression in the skin of dogs with chronic atopic dermatitis was decreased compared to levels in the skin of healthy dogs – a contrasting finding to that reported for the skin of beagles with an experimentally induced acute atopic dermatitis.3
Finally, van Damme et al. reported a significant increase in the mRNA levels of S100A7 protein in the lesion and nonlesional skin of atopic dogs compared to healthy controls.2
Recently, Torres et al. reported the mRNA expression levels of other two AMPs, SKALP and SLPI, which appeared to be decreased in atopic dogs compared to healthy controls.4
In summary, although the results are controversial, it appears that the skin of dogs with an acute atopic dermatitis over-expresses some of the AMPs (cBD1-like, cBD3-like, cathelicidin), although data for cBD103 and cBD3 in acute atopic dermatitis are not currently available. Furthermore, older dogs have higher expression of AMPs than young dogs, a feature also reported in humans. In the skin of dogs with chronic atopic dermatitis, mRNA levels of cBD1 were shown to be increased, while cBD103 levels have been shown to be reduced2 or increased.3 The difference between these two studies could be explained by a difference in the age of the dogs, localization of the biopsy site, use of topical or systemic antimicrobials, or other factors.2,3
Several unanswered questions could be listed. For example, which AMP is critical in protection of the skin against bacteria? Is there an effect of antibiotics and immunomodulatory drugs on the production of these AMPs? Is the correct ratio of AMPs the critical factor protecting the skin from infection?
Koji Nishifuji asked for clarification of whether the expression of cBD1 and cBD3 is increased in acute atopic dermatitis skin lesions while cBD2 expression is not.
Domenico Santoro confirmed that cBD2 expression was not increased.
Koji Nishifuji asked whether there is a difference in the expression of these molecules in relation to the different body areas.
Domenico Santoro replied there probably is, although he was not aware of any published study in animals. In human dermatology, however, it has been demonstrated that there are large differences in the expression of AMPs in different body areas. Higher expression has been shown in areas exposed to the environment (e.g. forehead, palms, feet). Therefore, it is possible that his data could have been different if he had collected samples from other areas such as the interdigital skin.
Koji Nishifuji asked if there is any difference in the function of cBD1, cBD2 and cBD3.
Domenico Santoro answered that this has not yet been determined, although his research group is currently working on experiments that test immunological and antimicrobial functions of different AMPs. The problem is that some AMPs such as cBD1-like, cBD2-like and cBD3-like molecules have very similar, often overlapping amino acid sequences as exemplified by the cBD2/122 group. This makes it difficult to design a unique peptide to test an individual AMP. He is currently working with cBD103 and some other members of the cBD2 group.
Christoph Klinger (Germany) asked if Domenico Santoro had any knowledge about different drugs affecting the expression of AMPs, such as the speculation about ciclosporin’s positive effect on beta-defensins and maybe cathelicidins.
Domenico Santoro replied that drugs do affect AMPs and that in humans, tacrolimus and ciclosporin have been shown to increase the expression of AMPs, while steroids appear to inhibit the expression of these proteins. Also, stress seems to inhibit the production of AMPs. On the other hand, vitamin D3 has recently been discussed as a strong stimulator of cathelicidin and human beta-defensin 2 (hBD2) expression. One must be careful, however, as most of the beta-defensins studied in dogs are not true orthologs of those described in humans. The only one is cBD103, which is the ortholog of hBD3. There are studies in progress that look at different drugs and cytokines and their effect on keratinocyte expression of AMPs.
Changes in epidermal ceramides in canine atopic dermatitis (J.S. Yoon)
Ji Seon Yoon (South Korea) reviewed the functions of ceramides in the stratum corneum in maintaining the epidermal barrier function. The molecular structure of ceramides consists of sphingosine linked to fatty acids. In the stratum corneum, ceramides are present in extracellular spaces with other lipids. These extracellular lamellar lipids are important contributors to barrier function and maintain skin hydration. If ceramide content is decreased in the stratum corneum, transepidermal water loss as well as penetration of allergens or microbes are increased.
Recently her group reported that the stratum corneum of dogs contains 11 free ceramide classes, a finding similar to that reported in humans. The classes depend on the ceramide composition in which the following molecules play a critical role: sphingosine, dihydrosphingosine, phytosphingosine, 6-hydroxyl-sphingosine, and non-hydroxylated-, alpha-hydroxylated and omega-hydroxylated fatty acids.5
Abnormalities of ceramides in the skin of people with atopic dermatitis have been intensely studied. Studies have shown that quantities (μg/mg of protein) of the ceramides EOS (combination of omega-hydroxy fatty acids and sphingosines), EOP (combination of omega-hydroxy fatty acids and phytosphingosines), NP (combination of non-hydroxy fatty acids and phytosphingosines) and NS (combination of non-hydroxy fatty acids and sphingosines) are decreased in humans with atopic dermatitis.
In veterinary medicine, the proportions of ceramides, cholesterol and free fatty acids have been analysed in healthy dogs and dogs with atopic dermatitis.6 This study demonstrated that the proportion of ceramides in lesional and nonlesional skin of dogs with atopic dermatitis is significantly lower than in healthy controls. Furthermore, Reiter et al. reported that the proportion of free extractable ceramides 1 and 9 was lower in nonlesional skin of dogs with atopic dermatitis than in that from breed- and age-matched controls.6