SKIN IRRITATION LEADS TO RAPID INDUCTION OF TSLP, INDEPENDENT FROM

A disrupted barrier makes the skin more susceptible to the environment as shown by Mogbekeloluwa O Danso et al. (2014)¹⁶³. This allows allergens or irritants to enter through the skin and to induce a Th2 response which in turn activates keratinocytes to produce TSLP to activate dDCs thereby promoting the inflammatory process. Many genetically modified mouse models have been used to 71

show an essential role for TSLP in allergic diseases^60,129. As our understanding about functional aspects of TSLP in different pathophysiological conditions is slowly increasing, the regulatory role of endogenous TSLP has been hardly well-defined.

Moreover, there are a lot of discrepancies between in vitro and in vivo studies¹⁶⁴. Various genetic studies with transgenic mice show that gene manipulation of different genes in the skin was followed by an increase of the TSLP expression51,125,146,165-167. The manipulation of different unrelated genes such as Notch and lymphoepithelial Kazal-type-related inhibitor (LEKTI) display similar outcomes. Therefore we hypothesized that skin perturbation caused by either genetic manipulation or by environmental factors can initiate a specific cascade that leads to TSLP production, even outside of an allergic scenario.

To further investigate our hypothesis and to understand whether barrier disruption would be sufficient for the initiation of TSLP production in mouse skin, a range of irritants were used. The data show that TSLP induction was an elicited as a common consequence in inflamed skin when skin homeostasis was deviated. This observation was made and confirmed by the variety of TSLP inducers, ranging from physical trauma i.e. taking punch biopsy of the skin to mild physical irritation (wet shaving, tape stripping) and chemical insults (croton oil, SDS). These data was supported by a previous finding with the detergent SDS. SDS is used in different models as irritants and can alter the stratum corneum due to its action on surface tension. Therefore it has been used in different patch tests models and animal assays as it is able to enhance the penetration of other substances¹⁶⁸. Moreover SDS can cause to a large extend alterations of the skin barrier function¹⁶⁹. On the other hand, croton oil has been used to induce ear edema in a mouse model^170,171. One important component of croton oil is the phenol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Furstenberger et al. (1994) have shown that topical application of croton oil can initiate local inflammation accompanied by edema formation, polymorphonuclear leukocyte infiltration and epidermal hyperproliferation, as a consequence of the production of inflammatory mediators, such as prostaglandin E2, leukotrienes, histamine, serotonin and IL-1¹⁷². The

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specific skin inflammation elicited by SDS and croton oil resemble early phase events during AD development in certain mouse models^173,174.

In accordance with the literature, our data show a link between TSLP expression in skin even in the absence of any particular allergic scenario. 2010, Angelova-Fischer et al. show that injury of the stratum corneum by tape stripping and 2% SLS leads to an increase of TSLP in human epidermis¹¹⁷. 2007, Allakhverdi et al. observed that a trauma driven by a punch biopsy was sufficient to induce TSLP expression in human skin¹²². These findings are in line with our data, derived from the acute mouse skin model. Using this model we show that the increase of TSLP mRNA in the skin but also protein in the skin and the serum was more pronounced after physical irritation although the highest increase of TSLP expression was determined after croton oil application. These data were further confirmed in our ex vivo cell culture settings. Additional, evidence for the susceptibility of the TSLP gene to various insults are known from physical irritation, UV irradiation, malignancy, xerosis, and even in the absence of intestinal microbiota117,175-180. In support to previous observations, our acute skin mouse model not only revealed that the different types of stressors can lead to TSLP induction, but we could also for the first time that TSLP induced after mild skin irritation can be measured in the serum within in a short period of time i.e only after 4 hrs and increases over the time (18 hrs).

In search of a mechanism by which the gentle irritation and other distress can induce TSLP in murine skin, we first looked for factors which can induce TSLP in murine skin. As mentioned above several factors have been described to induce TSLP in human keratinocytes. These include e.g. TNF-α, IL-1, IL-4, IL-25, IL-33 and TLR3-ligand123,148,149,151, although their exact effects in murine keratinocytes are not well known in detail yet. Our in vitro and ex vivo findings indicate that primary keratinocytes and skin explants are capable to produce TSLP upon treatment with different stimuli in a similar manner as in humans in spite of some unexpected results. These findings are in accordance with a report from Takai et al. (2012)¹⁶⁴. These authors demonstrate that the gene expression of TSLP was induced in bronchial epithelial cells (NHBE) after the exposure with different proinflammatory 73

cytokines such as IL-1β, TNF-α and TLR2, TLR8, and TLR9 ligands. They also indicated that the effects of these cytokines in both species are derived from the NFκB pathway. In contrast, our data from the skin epithelial cells show that there is considerable species dependence. TNF-α for example was able to induce TSLP to a greater extend in mouse keratinocytes compared to human keratinocytes. By contrast, IL-1β was more active in human keratinocytes compared to mouse. In addition, TLR3 ligand was significantly stronger effective in human than in mouse keratinocytes as described previously^149,153.

In vitro studies with human primary keratinocytes show that cytokines like TNF-α in combination with Th2 cytokines such as IL-4 and IL-13 can synergistically increase the poly I:C (TLR-3 ligand) induced TSLP¹⁴⁹. 2007, Bogiatzi et al. also found similar results from human skin explants stimulated with different combinations of pro-inflammatory cytokines such as TNF-α or IL-1α, in combination with Th2 cytokines such as IL-4 and IL-13¹²³. These results were further confirmed by many other groups using different cell types. For example, Allakhverdi et al. (2007) show in human airway epithelial cells, that a combination of TNF-α and IL-1α augmented TSLP expression¹²². In accordance with these findings, we were able to confirm this data in our in vitro and ex vivo settings. TSLP expression was increased in mouse keratinocytes and in skin explants upon treatment with TNF-α and IL-1β in combination with IL-4.

Finally, we addressed the mechanisms by which the acute skin irritation and other physical stress can elicit TSLP in murine skin. TNF-α was the primary candidate to be investigated as this cytokine is well-known to be induced upon skin irritation^82,156 and to increase TSLP in the skin¹²³. This was also observed in our in vitro and ex vivo results making TNF-α to a probable intermediary in the cascade to mount TSLP production. However, surprisingly we found that endogenous TNF-α was expendable for TSLP production under all different settings i.e in vitro, in vivo and ex vivo.

Taken together, it is obvious that exogenous TNF-α can induce TSLP in murine skin under different settings, but endogenously it is not able to reach threshold levels which may be required for its effect during skin damage. Although the impact of 74

TNF-α regarding TSLP production in human compared to mouse skin was lower in vitro, we investigated the role of endogenous TNF-α upon skin perturbation in human epidermal sheets. As expected, endogenous TNF-α induced lesser TSLP protein in the human in comparison to the mouse system emphasizing on its dispensability in both the murine and the human skin.

Since TNF-α was found to be not required for TSLP production in the skin, an alternative mechanism was needed to be identified. Upon trauma cells may die and defined defense and repair processes in the respective host are activated. Such processes or pathways include in part the activation of the ancient IL-1 family^181,182. Previous studies have shown that cytokines from the IL-1 family are involved in SDS-mediated skin irritation¹⁵⁶. Physical disruption of the skin can induce the release of IL-1α. The dispensability of TNF-α and based on the literature, IL-1 seemed to be a reasonable candidate to explain the present findings. By using IL-1Ra and IL-1α neutralizing antibodies, IL-1 was certainly shown to participate in TSLP production in the skin when triggered by SDS. Around 20% of TSLP production was reduced when treated with IL-1Ra or by the according neutralizing antibodies. This indicates that IL-1 is involved in SDS mediated TSLP production in damaged skin. As the reduction of TSLP reached only 20% we believe that other factors may be also be involved and are likely to play an additional role in the TSLP production upon SDS treatment. However, the other IL-1 family member IL-33¹⁸³ is not likely to be a suitable candidate as it was not able to induce TSLP in skin constituents. Other new members of the IL-1 family whose specific contribution to inflammatory skin conditions has only begun to be cleared¹⁸². So it is still not clear whether these mediators such as high mobility group box chromosomal Protein 1 (HMGB1)¹⁸⁴ might serve as a driving force for TSLP production in skin.

Nevertheless, IL-1 had a better impact on the irritation-induced TSLP production in human skin, which was in the range of around 50%. The higher responsiveness of human KCs towards IL-1 compared to murine KCs suggest that IL-1 is more important in human skin compared to mouse skin. Therefore TSLP regulation in different species will not be fully the same¹⁸⁵. The apparent species variation in the actions of TSLP provides another example of significant differences between the human and murine immune system¹⁸⁵. This is further supported by our observation

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that murine KCs were basically resistant to TLR3-mediated TSLP induction compared to human KCs, although murine skin was susceptible to TLR3-ligands in other settings¹⁸⁶.

In summary it can be proposed based on the findings from this thesis that TSLP acts as an executer of the innate alarm system aiming to protect the host defense and restoring barrier function. That TSLP is critical to healing and barrier restoration in mucosal tissues^187-189, providing anti-tumor and antimicrobial effects in the skin has already been demonstrated175,176,190 and that is moreover able to induce extramedullary hematopoiesis¹⁹¹. Therefore it seems that the role of TSLP outside of the allergic scenario will require a thorough investigation in the future.

4.2 TNF^-/- MICE DEVELOP AGGRAVATED AD AND DISPLAY INCREASED