The Immune-pathogenesis of Canine Atopic Dermatitis: Skin barrier, Microbiome and Inflammation

The overall aim of the research was to contribute to further elucidation of the immune-pathogenesis of atopic dermatitis (AD) in the dog. Findings described in this PhD thesis indicated putative alterations occurring in, or inherent to the AD skin. The altered lipid composition and organization observed in both lesional and non-lesional AD skin in dogs, may support the “outside-in” hypothesis, that states that abnormalities in the skin barrier may contribute to the pathogenesis of AD. Alternatively, potentially in addition, the pathogenesis of AD is immunologically driven initially, as postulated by the “inside-out” hypothesis. The microbiome of canine skin varied between individuals and was influenced by the topical antimicrobial treatment. In general, significant differences in the microbiome composition were not observed, between AD and healthy dog skin, although the microbial diversity tended to be lower in affected compared to healthy skin. The microbiome and AD are likely to influence each other. The inflammatory state of the AD skin, partly due to infection, results in expression of both antimicrobial peptides (AMP) and further cytokines, likely to be influenced by anatomical sites and topical treatment. Distortions of the skin homeostasis may lead to significant changes in skin barrier lipid, skin microbiome, AMP and inflammatory cytokines as shown in this thesis. Study of each of these subjects not only led to a more comprehensive understanding of canine AD, but may in future contribute to design of diagnostic and therapeutic strategies

The Immune-pathogenesis of Canine Atopic Dermatitis: Skin barrier, Microbiome and Inflammation

The overall aim of the research was to contribute to further elucidation of the immune-pathogenesis of atopic dermatitis (AD) in the dog. Findings described in this PhD thesis indicated putative alterations occurring in, or inherent to the AD skin. The altered lipid composition and organization observed in both lesional and non-lesional AD skin in dogs, may support the “outside-in” hypothesis, that states that abnormalities in the skin barrier may contribute to the pathogenesis of AD. Alternatively, potentially in addition, the pathogenesis of AD is immunologically driven initially, as postulated by the “inside-out” hypothesis. The microbiome of canine skin varied between individuals and was influenced by the topical antimicrobial treatment. In general, significant differences in the microbiome composition were not observed, between AD and healthy dog skin, although the microbial diversity tended to be lower in affected compared to healthy skin. The microbiome and AD are likely to influence each other. The inflammatory state of the AD skin, partly due to infection, results in expression of both antimicrobial peptides (AMP) and further cytokines, likely to be influenced by anatomical sites and topical treatment. Distortions of the skin homeostasis may lead to significant changes in skin barrier lipid, skin microbiome, AMP and inflammatory cytokines as shown in this thesis. Study of each of these subjects not only led to a more comprehensive understanding of canine AD, but may in future contribute to design of diagnostic and therapeutic strategies

Altered lipid properties of the stratum corneum in Canine Atopic Dermatitis

Skin barrier disruption plays a role in the pathogenesis of atopic dermatitis (AD) in humans. However, little is known about skin barrier (dys-) function in Canine Atopic Dermatitis. The properties of lipids located in the outermost layer of the skin, the stratum corneum (SC) are considered to be important for the barrier. In the present study the lipid composition and lipid organization of the SC of AD dogs and control dogs were examined. The lipid composition of lesional AD skin as compared to control skin, showed a reduced free fatty acid level and a decreased ratio of ceramide[NS] C44/C34, in which C44 and C34 are the total numbers of carbon atoms of the sphingosine (S) and non-hydroxy (N) acyl chains. As a consequence of the observed changes in lipid composition in AD lesional skin the lamellar organization of lipids altered and a shift from orthorhombic to hexagonal lipid packing was monitored. Simultaneously an increased conformational disordering occurred. These changes are expected to compromise the integrity of the skin barrier. The C44/C34 chain length ratio of ceramide[NS] also showed a decreasing nonlinear relationship with the AD severity score (CADESI). Taken together, canine atopic skin showed alterations in SC lipid properties, similar to the changes observed in atopic dermatitis in humans, that correlated with a disruption of the skin barrier. Hence lipids play an important role in the pathogenesis of Canine Atopic Dermatitis

The bacterial and fungal microbiome of the skin of healthy dogs and dogs with atopic dermatitis and the impact of topical antimicrobial therapy, an exploratory study

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A canine keratinocyte cell line expresses antimicrobial peptide and cytokine genes upon stimulation with bacteria, microbial ligands and recombinant cytokines

Keratinocytes (KC) are the main cellular components of the stratum corneum that constitutes a solid physical skin barrier representing the first line of defense against pathogens. Moreover, KC are potent producers of inflammatory mediators and antimicrobial peptides (AMP) when activated through their pattern recognition receptors. In atopic dermatitis (AD) the protective skin barrier may be compromised due to barrier disruption, secondary infection and accelerated secretion of inflammatory cytokines which may also affect AMP expression in the skin. In the present study, we addressed the responses of a canine KC cell line upon exposure to Staphylococcus pseudintermedius, typically found on canine atopic skin during secondary infections, and stimulation by individual AD-associated ligands and cytokines. All stimuli induced a significant increase in expression of the pro-inflammatory cytokine genes tumor necrosis factor (TNF)-α and interleukin (IL)-8, but with different kinetics. Limited effects were observed on AMP gene expression except for K9CATH which was significantly upregulated upon bacterial infection but with none of the individual AD-associated ligands. Interestingly, K9CATH possessed antimicrobial activity towards Staphylococcus pseudintermedius, indicating that K9CATH expression is a specific defense reaction towards bacterial infection and not part of a general pro-inflammatory profile of KC

Altered lipid properties of the stratum corneum in Canine Atopic Dermatitis

Skin barrier disruption plays a role in the pathogenesis of atopic dermatitis (AD) in humans. However, little is known about skin barrier (dys-) function in Canine Atopic Dermatitis. The properties of lipids located in the outermost layer of the skin, the stratum corneum (SC) are considered to be important for the barrier. In the present study the lipid composition and lipid organization of the SC of AD dogs and control dogs were examined. The lipid composition of lesional AD skin as compared to control skin, showed a reduced free fatty acid level and a decreased ratio of ceramide[NS] C44/C34, in which C44 and C34 are the total numbers of carbon atoms of the sphingosine (S) and non-hydroxy (N) acyl chains. As a consequence of the observed changes in lipid composition in AD lesional skin the lamellar organization of lipids altered and a shift from orthorhombic to hexagonal lipid packing was monitored. Simultaneously an increased conformational disordering occurred. These changes are expected to compromise the integrity of the skin barrier. The C44/C34 chain length ratio of ceramide[NS] also showed a decreasing nonlinear relationship with the AD severity score (CADESI). Taken together, canine atopic skin showed alterations in SC lipid properties, similar to the changes observed in atopic dermatitis in humans, that correlated with a disruption of the skin barrier. Hence lipids play an important role in the pathogenesis of Canine Atopic Dermatitis

The bacterial and fungal microbiome of the skin of healthy dogs and dogs with atopic dermatitis and the impact of topical antimicrobial therapy, an exploratory study

Canine atopic dermatitis is a genetically predisposed inflammatory and pruritic allergic skin disease that is often complicated by (secondary) bacterial and fungal (yeast) infections. High-throughput DNA sequencing was used to characterize the composition of the microbiome (bacteria and fungi) inhabiting specific sites of skin in healthy dogs and dogs with atopic dermatitis (AD) before and after topical antimicrobial treatment. Skin microbiome samples were collected from six healthy control dogs and three dogs spontaneously affected by AD by swabbing at (non-) predilection sites before, during and after treatment. Bacteria and fungi were profiled by Illumina sequencing of the 16S ribosomal RNA gene of bacteria (16S) and the internally transcribed spacer of the ribosomal gene cassette in fungi (ITS). The total cohort of dogs showed a high diversity of microbes on skin with a strong individual variability of both 16S and ITS profiles. The genera of Staphylococcus and Porphyromonas were dominantly present both on atopic and healthy skin and across all skin sites studied. In addition, bacterial and fungal alpha diversity were similar at the different skin sites. The topical antimicrobial treatment increased the diversity of bacterial and fungal compositions in course of time on both AD and healthy skin

A canine keratinocyte cell line expresses antimicrobial peptide and cytokine genes upon stimulation with bacteria, microbial ligands and recombinant cytokines

Keratinocytes (KC) are the main cellular components of the stratum corneum that constitutes a solid physical skin barrier representing the first line of defense against pathogens. Moreover, KC are potent producers of inflammatory mediators and antimicrobial peptides (AMP) when activated through their pattern recognition receptors. In atopic dermatitis (AD) the protective skin barrier may be compromised due to barrier disruption, secondary infection and accelerated secretion of inflammatory cytokines which may also affect AMP expression in the skin. In the present study, we addressed the responses of a canine KC cell line upon exposure to Staphylococcus pseudintermedius, typically found on canine atopic skin during secondary infections, and stimulation by individual AD-associated ligands and cytokines. All stimuli induced a significant increase in expression of the pro-inflammatory cytokine genes tumor necrosis factor (TNF)-α and interleukin (IL)-8, but with different kinetics. Limited effects were observed on AMP gene expression except for K9CATH which was significantly upregulated upon bacterial infection but with none of the individual AD-associated ligands. Interestingly, K9CATH possessed antimicrobial activity towards Staphylococcus pseudintermedius, indicating that K9CATH expression is a specific defense reaction towards bacterial infection and not part of a general pro-inflammatory profile of KC