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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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Canine atopic dermatitis (AD) is a chronic inflammatory and pruritic skin disease which shares several characteristics with its human counterpart. In chronic patch test lesions of human with AD mainly a Th1-type cellular response is found. Besides, non-lesional AD skin is already skewed for inflammation and therefore different from healthy skin. The goal of this study was to characterize local immune responsiveness in chronic canine AD lesions as compared to that in non-lesional AD skin by defining T cell subset relevant cytokine- and transcription factor expression profiles. The gene expression of the Th1 cytokines IL-12p35, IL-12p40 and IFN-γ and their related transcription factors STAT4, SOCS5 and T-bet, the Th2 cytokines IL-4 and IL-13 and transcription factors STAT6, SOCS3 and GATA-3 and the regulatory cytokines IL-10 and TGF-β and the transcription factor FOXP3 was evaluated in healthy control and atopic dogs. In non-lesional (NLS) and chronic lesional skin (LS) of atopic dogs and control skin (CS) from healthy dogs mRNA expression of cytokines and transcription factors were measured by quantitative real-time PCR. Significantly different values were found for the following factors: IL-12p40 mRNA was lower in LS when compared to NLS. Expression of STAT4 was higher in LS compared to CS and NLS. More IL-13 and SOCS3 were found in LS and NLS when compared to CS and also in LS compared to NLS. GATA-3 was lower in LS compared to NLS. IL-10 expression was higher in both LS and NLS compared to CS and more IL-10 was present in LS compared to NLS. These findings indicate that both Th1- and Th2-type as well as T regulatory cells are present in NLS and LS in canine atopic skin., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Quantitative real time PCR (Q-PCR) is the method of choice to study mRNA expression levels. Since Q-PCR is very sensitive, normalization of the data with stably expressed reference genes if of utmost importance. The stability of reference genes depends on the tissue and the species of interest. Therefore, evaluation of the stability of reference genes must be performed for each new tissue and species under study. The stability of B2M, GAPDH, HPRT, SRPR, hnRNPH, GUSB, RPL8, RPS5, and RPS19 was analyzed with the GeNorm software in snap frozen canine skin biopsies. Healthy dogs (n=7) and dogs with confirmed atopic dermatitis (n=28) were included. Lesional and non-lesional skin was analyzed. The study indicated that the most appropriate reference genes in canine skin are the ribosomal gene products RPL8, RPS5 and RPS19 besides GUSB and HPRT. As little as three reference genes will reveal highly reliable Q-PCR calculations.