MCs SEEM TO PLAY A ROLE BETWEEN TNF-DEFICIENCY AND TSLP

TNF deficiency through an indirect micromilieiu mechanism as TNF can directly induce TSLP as described earlier²⁰⁹. The increase of the number of mast cells in lesional skin of TNF^-/- mice and its significant correlation with the severity clearly indicate that MCs are a hot candidate to explain the relationship between TNF-deficiency and TSLP. Based on previous literature we assumed that MCs are upstream of TSLP and downstream of TNF in this scenario. MCs have been linked with TSLP in different contexts such as they are known to produce TSLP themselves^103,210, are responsive to TSLP¹²², and can enhance TSLP production by epithelial cells¹³³. Based on this literature, we aimed to clarify whether an experimental manipulation of MC density or abrogation of MC function is facilitating amelioration of AD.

Intradermal administration of cKit neutralizing antibodies to TNF^-/- mice, resulted in an improvement of the symptom score of the TNF^-/- mice, compared to their isotype control. Similar results were obtained with measurements by TEWL although these data did not reach statistical significance due to a high variation within the groups.

The decrease in the symptom score of AD as well as in TEWL observed in mice treated with cKit antibodies indicate that mast cells are involved in the process of AD aggravation under TNF deficiency. Recently, Tomoaki Ando et al (2014) also have demonstrated that mast cells but not the B or T cells are crucial for the onset of spontaneous dermatitis in Plcb3^-/- mice²¹¹.

Our next question was whether mast cells might contribute to TSLP production.

MCs are known to produce TSLP themselves103,210,212. Additional data from Yong-Jun Liu (2006) suggest that mast cells which were activated by IgE receptor cross-linking express high levels of TSLP¹²¹. In accordance to the above findings, we activated BMcMcs with IgE cross linking in combination with other cytokines.

BMcMcs from TNF^-/- mouse as well as wt mice failed to produce TSLP, indicating that under TNF^-/- deficiency MCs are not likely to be the major source of an 81

increased TSLP production in an AD environment. Thus, we speculated that as MCs are not able to produce TSLP, but rather may act as inducers of TSLP production since they are also known to regulate epithelial cells regarding TSLP expression in allergic rhinitis¹³³.

Next our aim was to investigate the role of mast cells as triggers of TSLP production by instructing keratinocytes as they are the best known TSLP producers¹²⁷. For this purpose mast cell supernatants and mast cell mediators were studied regarding the onset of TSLP production. Different experimental settings with skin biopsies and MCs were used to pin down the molecular cascade which promotes TSLP production and consecutively support AD aggravation in a TNF deficient environment.

Stimulation with anti-IgE led to crosslinking of the FcεRI and hence MCs degranulate which progressively leads to the release of MC mediators like histamine and proteases^213,214. To investigate the role of MCs mediators that may enhance TSLP expression by acting on keratinocytes in the skin, skin biopsies were stimulated with the supernatants of either IgE cross linked stimulated or resting MCs ex vivo. Stimulated mast cells were not able to enhance TSLP levels. This implicates that high concentrations of MC mediators in the supernatants may even inhibit TSLP production compared to their non-stimulated counterparts. This was also observed by Okayama et al., 2009. These authors have shown that TSLP can be degraded by MC-derived proteases²¹⁰. Moreover, MCs release a plethora of other mediators upon stimulation^215-218 which can all have an impact on TSLP production in the skin. Based on that, it is clear that mast cell supernatants contain highly concentrated MC-derived proteases which can further promote TSLP degradation.

Nevertheless, the supernatants from resting MCs contain only mediators released spontaneously (e.g. through so called piecemeal degranulation²¹⁹ (Dvorak and Kissell, 1991)), granula-associated mediators are at lower concentrations in these supernatants, and they are also virtually devoid of activation-induced mediators like LTC4 and PGD2²²⁰. Interestingly, the supernatants from resting mast cells

82

significantly increased TSLP levels in skin biopsies. These data allow to presume that some of the MCs mediators instruct KCs to produce enhanced TSLP under TNF deficiency.

In order to identify which factor of the mast cell supernatant may be responsible for the increased TSLP expression by keratinocytes, the two most abundant mast cell mediators, histamine and tryptase, were evaluated for their ability to enhance TSLP expression.

Histamine failed to increase TSLP expression at protein levels. A dose response of histamine was performed, but no single concentration elicited significant TSLP expression. Histamine is believed to be a major player in the crosstalk between mast cells and keratinocytes^221,222. For example, human keratinocytes and organotypic skin models revealed that histamine is able to down regulate the expression of differentiation-associated proteins like filaggrin, keratin and loricrin, as well as tight junction and desmosomal junction proteins²²³. These data suggest that mast cell activation and histamine release contribute to skin barrier defects which have been implicated in the initiation of AD²²³. In addition, histamine is one of the major pruritogenic factors. Itch is a hallmark of AD, whereas histamine 1 receptor (H1R) antagonist cannot ameliorate the itch in lesional skin of AD patients²²⁴. In contrast it has been shown in a model of chronic allergic dermatitis in NC/Nga mice, that the combined treatment of a H1R and histamine 4 receptor (H4R) antagonist displayed anti-pruritogenic and anti-inflammatory effects²²⁵. Based on this evidence, it is clear that, one of the most abundant mast cell mediators does not appear to play a role for increased TSLP levels in AD.

The next major factor is the protease β-tryptase which is expressed in mast cells²²⁶. Mast cell proteases constitute between 30-50% of the total mast cell protein content²¹⁸. Therefore this mast cell mediator was analyzed next as a possible trigger of TSLP. The mouse analogue of human β-tryptase is mMCP6, which was indeed resulting in an increased TSLP production.

83

Based on our data it is difficult to answer the question whether tryptase is the main component from the MC supernatant that triggers TSLP in skin. The application of anti mMCP6 neutralizing antibody needs to be used to neutralize the induction of TSLP by either resting mast cell supernatants or by mMCP6. Mast cells from mMCP6^-/- mice would be also required to confirm these data. But in support, Thakurdas et al. (2007), showed that mMCP6 is not essential for migration, retention and overall maturation of MC-committed progenitors in connective tissues in mMCP6^-/- mice²²⁷. These mice however had a reduced ability to combat K.

pneumonia infections, suggesting a critical immune protective role of mMCP6 in bacterial infections²²⁷. Interestingly, it was reported that tryptase-like enzymes in the stratum corneum were highly unregulated in lesional AD skin²²⁸. In addition, trends of elevated tryptase levels in MCs from AD patients were observed²²⁹. Although, mast cell tryptase serum levels were investigated for their suitability as a serum marker for AD, two studies have shown no correlation between mast cell tryptase serum levels and the severity of AD^230,231. However, tryptase has been more and more implicated with AD-mediated itch²³².

Taken together, TNF^-/- mice have increased mast cell numbers in lesional skin which correlate with clinical severity and TSLP mRNA levels. Anti- cKit improved the development of AD and reduced TEWL in TNF^-/- mice compared to the controls.

BMcMcs data indicate that MCs are not producing TSLP as this is rather produced by keratinocytes. In search of MCs mediators which enhance TSLP levels in TNF -/-mice, we identified that supernatants from resting MCs increased TSLP production in skin biopsies. Histamine was not able to modulate TSLP production whereas mMCP6 was significantly able to induce TSLP production by keratinocytes in skin biopsies, indicating that tryptase might be the relevant factor involved in instructing KCs to produce TSLP under TNF deficiency.