Langerhans and inflammatory dendritic epidermal cells in atopic dermatitis tolerate Toll-like receptor 2-specific activation

Abstract

Atopic dermatitis (AD) is a chronic multifactorial inflammatory skin disease. The skin of AD patients shows a significant dysbalance of the microbiome with high colonization of S. aureus, which positively correlates with the disease severity. Langerhans cells (LC) are immune system sentinels and reside in the epidermis, where they sense invading pathogens by pattern recognition receptors, such as Toll-like receptors (TLR). LC bridge the innate and the adaptive immune systems, orchestrate primary immune responses to pathogens, allergens, and maintain tolerance. Inflammatory dendritic epidermal cells (IDEC) migrate in the epidermis of inflamed AD skin and contribute to the pro-inflammatory microenvironment of the AD skin. <br /> The aim of this thesis was to investigate the TLR2-specific functional differences between LC from healthy and AD individuals in an ex vivo human skin model that is very close to the in situ situation of the skin. Therefore, an ex vivo skin model was established from which the epidermal cells were isolated and analysed. The synthetic protein Pam3CSK4 (P3C) mimics S. aureus structures and P3C was used to activate the TLR2 on immature DC. Furthermore, the migration activity and the ability to activate naïve T cells by TLR2-stimulated DC was analysed. <br /> The results of this thesis show that the maturation status of freshly isolated LC from AD patients was similar to LC from healthy controls, as witnessed by equal expression levels of CD83, CD86, MHC class I and II. The main finding of this thesis is that LC and IDEC from AD skin showed tolerance towards TLR2 ligation. The LC did not further increase their maturation status or alter their cytokine profile in response to TLR2 ligand P3C, as witnessed by no alteration of CD83, CD40, CD80, CD86, MHC class II, TLR2, FcεRIα , IL-1β , hBD2, TSLP, and IL-11 when compared to healthy controls. Interestingly, steroid treatment seemed to slightly correct the tolerant behaviour and change the phenotype towards a healthy-like status, as witnessed by P3C induced up-regulation of CD83, CD80, a down-regulation of TLR2, and a slightly higher MHC class II expression. The co-culture of epidermal cells with naïve T cells showed that P3C-stimulated cells from healthy skin increased the T cell proliferation and the IL-17 level in the supernatant, whereas this was not the case for AD skin, which supported the tolerant behaviour of epidermal cells from AD skin. The analysis of receptors that allow the LC to remain in the epidermis showed that Integrin α3 (CD49c) and CCR6 were down-regulated in LC from AD patients, which indicated, that the LC migrated spontaneously from the tissue to the lymph nodes. Moreover, CCR7 that regulates the specific migration was slightly up-regulated in LC from AD donors. IL-18 and TNF-α induce migration out of the epidermis and both were elevated in the supernatant of cultured ex vivo skin from AD, which underlined the strong spontaneous migration. However, P3C ligation did not increase the specific migration rate of LC from AD patients, while it was up-regulated in healthy controls. Taken together, the TLR2-induced maturation of LC, the capability of LC to induce T cell activation, and the migration rate was different in AD skin when compared to healthy controls. The results of this thesis show that LC from AD skin were tolerant towards TLR2-mediated activation, which may be due to prolonged exposure of S. aureus, which can influence the TLR2 pathway and lead to a desensitisation of LC from AD patients. The TLR2 tolerance may contribute to the immune deviation in AD and the lack of S. aureus clearance, where S. aureus changes the local cytokine environment and directly or indirectly inhibits the TLR2 signalling.