The oxytocin system is downregulated in atopic skin

The OXT system is involved in the modulation of systemic and peripheral stress responses (31, 53). To adress the question whether the OXT system might be dysregulated in chronically inflamed skin, its expression levels in skin cells of atopic dermatitis (AD) patients and healthy volunteers were compared. Actually, OXT and its receptor were downregulated in dermal fibroblasts and keratinocytes derived from atopic skin. In addition, OXT release from atopic fibroblasts was also diminished. While mRNA levels of the OXTR might poorly correlate with protein levels, it is necessary to confirm these results in future experiments by determing OXTR protein concentrations in vivo. In a translational approach, the aberrant expression of the OXT system in atopic skin was confirmed. Suction-blister-derived fluids as well as epidermis derived from AD patients displayed decreased OXT protein and OXTR mRNA levels, respectively.

Regarding inflammation, OXTR gene expression has been shown to be negatively regulated by the cytokines IL1β, IL6 and interferon-γ (106, 43). This might be explained by the finding that the OXTR promoter region displays several potential interleukin response elements (10).

Furthermore, sepsis, which is associated with massive production of inflammatory mediators and neuroendocrine alterations, decreases OXT gene expression in the brain (107). As the skin of AD patients is chronically inflamed, displaying a plethora of up-regulated cytokines (108, 109, 74), elevated levels of inflammatory mediators might account for the decreased OXT and OXTR gene expression in atopic skin. As previously discussed, the expression of the OXT system might be regulated in a positive feedback mechanism in dermal fibroblasts and keratinocytes. By this mechanism, deficits in OXT signalling in atopic skin might be further aggravated.

Discussion 84

6.3.2 Oxytocin receptor knockdown increases the release of the

pro-inflammatory cytokines IL6, CCL5 and CXCL10

OXT has been shown to decrease the release of interleukins and other inflammatory mediators, on a systemic level and locally, in various tissues (87, 53, 54). Skin cells showed a cell type-specific differential response to dysbalanced OXT signalling: In contrast to dermal fibroblasts, keratinocytes released significantly elevated quantities of the cytokine IL6 as well as the chemokines CCL5 and CXCL10 after OXTR knockdown. In atopic keratinocytes, expression of the OXTR was reduced which might lead to an increased release of the pro-inflammatory cytokine IL6, aggravating skin inflammation. IL6 has been shown to negatively regulate the OXTR gene promoter (43). Thus, increased IL6 release might further downregulate OXTR expression, enhancing the deficits in OXT signalling of atopic skin cells.

Chemokines have been shown to stimulate the infiltration of immune cells into acute and chronic atopic lesions (66). The data further suggest that reduced OXT signalling might also contribute to exaggerated production of CCL5 by keratinocytes derived from AD patients (110). CCL5 mediates inflammation as it is important for the recruitment, as well as sustained survival and activation of T cells and dendritic cells (110). Hence, increased release of CCL5 further exacerbates the inflammatory state of atopic skin. Apart from this, the OXT has been previously shown to regulate CCL5 (111). In myometrial cells, OXT treatment reduces CCL5, CXCL5 and CCL20 expression. Moreover, the OXT system has also been linked to regulation of CXCL10 (112). Administration of OXT in humans reduces endotoxin-induced increases in plasma cytokines, amongst others, IL6 and CXCL10. However, CXCL10 is consistently upregulated in the epidermis of patients suffering from psoriasis but not in lesions of patients with AD (113). Regarding the dramatic release of CXCL10 by over 1000 % after OXTR knockdown, the OXT system might by also relevant for psoriasis.

6.3.3 The oxytocin system influences the neuroendocrine stress parameters CRH and corticosterone

In mammals, physical or emotional stress leads to the induction of the hypothalamic-pituitary-adrenal axis (HPA). This includes activation of the type 1 CRH receptor (CRHR1) which regulates the secretion of ACTH from the anterior pituitary and exerts a wide spectrum of actions in the central nervous system and the periphery (114). The cutaneous CRH signalling cascade includes mediators and receptors similar to the HPA axis, thus forming an equivalent to the HPA axis to neutralize noxious stimuli and associated immune reactions (38). As OXT plays a role in modulating the HPA axis (115, 32, 116), its possible influence on cutaneous neuroendocrine stress mediators was investigated. Knockdown of the OXTR led to an upregulation of CRH and CRHR1 expression, indicating that continuous OXT signalling is needed to prevent neuroendocrine stress responses. Direct antagonizing actions between the OXT system and the CRH system have been shown in the myometrium (85). At term, CRHR signalling is inhibited by OXT via activation of protein kinase C.

The cutaneous CRH signalling pathway diverges from the systemic HPA cascade in its distal step, where CRH and ACTH stimulate production of corticosterone, instead of cortisol (117).

Corticosterone is released in response to stress and its cutaneous synthesis can be regulated, amongst others, by CRH (86). OXT-treatment of keratinocytes lowered the release of corticosterone. It remains to be elucidated in further experiments, if this decrease is due to a direct OXT-mediated inhibition of corticosterone synthesis/release or to an antagonizing action of OXT on CRH or pro-inflammatory cytokines. However, in accordance with the observation of central OXT administration reducing stress-induced corticosterone release (115), this result further supports the hypothesis that OXT moderates cutaneous neuroendocrine stress responses. In contrast to keratinocytes, no corticosterone was detected in the supernatants of fibroblasts. This finding was contrary to expectations, as Slominski et al. detected corticosterone production by fibroblasts and not by keratinocytes (117). This might be due to different experimental settings. For instance, for the corticosterone analyses, cells were used from passage 1 to 3, whereas Slominski et al. used cells from passages 4 to 6. Moreover, different culture media, harvest time points and ELISA-Kit were used.

Apart from this, the activity of the systemic/cutaneous CRH system is, amongst others, regulated by cytokine release and vice versa (118, 119, 38). Furthermore, OXTR knockdown induced cytokine release from keratinocytes. Therefore, deficits in OXT signalling might enhance the activity of the CRH system, which in turn promotes cytokine release, aggravating neuroendocrine and inflammatory stress. Taken together, the collected data suggest that OXT plays a role in counteracting not only central, but also peripheral, cutaneous, neuroendocrine stress responses.

Discussion 86

6.3.4 Oxytocin receptor knockdown increases oxidative stress

By inhibiting OXTR signalling, the presented data revealed an impact of OXT on the modulation of oxidative stress and intracellular GSH levels in dermal fibroblasts and keratinocytes. OXTR knockdown in skin cells led to increased susceptibility to oxidative stress. This might indicate a cytoprotective role of OXT with regard to oxidative stress, as the formation of ROS leads to the damage of nucleic acids, proteins and membrane lipids (120).

Noteworthy, OXT is able to exert anti-oxidative effects through both, a direct and an indirect way. On the one hand, the OXT molecule itself has direct anti-oxidative activity by scavenging radicals and thereby preventing the oxidation of e. g. lipoproteins (57). On the other hand, OXT indirectly improves the cellular anti-oxidative state via its anti-inflammatory effects. For instance, OXT ameliorates oxidative injury of colonic tissue in a colitis model in rat by a neutrophil-dependant mechanism (54). Besides, OXT has been shown to balance ROS exhibiting cardioprotective efficacy in ischaemic rat heart (121).

ROS formation is closely related to a decrease in GSH levels (122), thereby reducing the cellular anti-oxidative defense (88, 89). Consistent with higher ROS levels in OXTR knockdown dermal fibroblasts and keratinocytes, these cells also displayed decreased intracellular GSH concentrations. In accordance, a role for OXT in modulating GSH levels has been shown in sepsis-induced pelvic inflammation which exhibits increased ROS and decreased GSH levels. In this inflammatory condition, OXT administration prevents the depletion of tissue GSH levels (123). Furthermore, treatment of pregnant rats with the OXTR antagonist atosiban has been shown to elevate oxidative stress in the hearts of the newborns (124).

In keratinocytes, the effects of OXTR knockdown on ROS formation and GSH levels were less pronounced than in dermal fibroblasts, indicating a cell type-specific susceptibility of skin cells to oxidative stress and its modulation by OXT. Consistently with this finding, Marionnet et al. (125) reported a higher sensitivity to oxidative stress of dermal fibroblasts in comparison to keratinocytes in UV-irradiated skin models.

Among the skin’s defense system against oxidative stress exists a battery of detoxifying and repair enzymes (125, 126, 127). OXTR knockdown modulated the mRNA amount of some of those oxidative stress-related enzymes (CYP1B1, COX6B1, NQO1, TXNRD1, GCLM and GSR) and revealed a differential response between fibroblasts and keratinocytes. Compared to keratinocytes, fibroblasts exhibited a higher number of significant gene expression modulations after OXTR knockdown. Moreover, there were also differences in modulation regarding the type of gene. OXTR knockdown led to an upregulation of CYP1B1 gene expression in fibroblasts, but to a downregulation of this gene in keratinocytes. A fibroblast- and keratinocyte-specific response with respect to oxidative stress marker genes has been shown in UV-irradiated 3-D skin models, presumably reflecting differences in basal

anti-oxidant defense equipment (125, 128, 129, 130, 131). In general, OXTR knockdown-induced alterations of all screened oxidative stress marker genes was quite low on the mRNA level. It has to be elucidated in future experiments whether the effects are more pronounced studying protein levels and activities of these enzymes in non-irradiated and irradiated OXTR knockdown skin cells.

It has been reported that atopic skin comprises an elevated oxidative stress status (132, 133). Skin inflammation in AD involves intense infiltration of lymphocytes, monocytes and eosinophils which have been demonstrated to release, beside pro-inflammatory cytokines, ROS (134-137). Aside from ROS being generated during inflammation, ROS, in turn, are able to drive inflammation. For example, ROS activates the transcription factor NF-κB which regulates inflammatory mediator gene expression (51). In consideration of the collected data, deficits in OXT signalling possibly enhance both, oxidative stress and inflammation, in atopic skin resulting in a vicious circle and chronification of the inflamed skin status.

Discussion 88

6.3.5 Oxytocin inhibits proliferation

Depending on cell type, the activation of the OXTR is either associated with stimulation (25, 30) or inhibition (22, 91) of proliferation. The regulation of cell growth by OXT is complex. It depends on expression levels and coupling of G_q- and G_i-protein isoforms. Further regulatory factors are the OXT concentration, the OXTR recruitment to caveolae and the cholesterol content of cell membranes (27, 91, 138). Studies with HUVECS have demonstrated that OXT fosters proliferation through OXTR coupling to Gq-proteins leading to intracellular Ca²⁺

release (139). As activation of the OXTR induced Ca²⁺ influx in dermal fibroblasts and keratinocytes, it was expected that OXT would increase proliferation in these cells. Instead, OXT elicited a suppressive effect on proliferation in dermal fibroblasts and keratinocytes.

Studies with breast cancer cell lines have shown that OXT-induced inhibition of proliferation is mediated via coupling of the OXTR to G_i-proteins (140). It can be assumed that OXT-induced inhibition of proliferation in dermal fibroblasts and keratinocytes might be also mediated via OXTR coupling to G_i-proteins. Therefore, the apparently observed distinct responses to OXT stimulation suggest that both, Gq and Gi-protein mediated signalling cascades are activated in dermal fibroblasts and keratinocytes. Indeed, G-protein-coupled receptors, like the OXTR, are cabable of triggering different signalling pathways in the same cell type (30, 91). It depends on the localization of the OXTR in the cell membrane, which signalling cascade is activated. Localization of the OXTR outside caveolae leads to G_i -protein coupling inhibiting proliferation, whereas localization inside caveolae leads to Gq -protein coupling promoting proliferation. Therefore, it can be speculated that in skin cells, the majority of the OXTR might be located outside caveolae, whereas only a small fraction of the receptor might be located inside caveolae. This ratio would lead to domination of the Gi -pathway over the Gq-pathway, explaining the observed OXT-induced growth inhibition and the detected Ca²⁺ influx mediated by the smaller OXTR fraction, in the same cells. An example where indeed only a small fraction of the OXTR is found to be inside caveolae are HEK 293 cells (2, 141). In these cells, a fraction of solely 10-15 % of total OXTRs is localized in caveolae. However, further experiments are needed to investigate which ratio of OXTR localization inside and outside caveolae is true for skin cells.

AD is associated with hyperproliferation of keratinocytes. This results in epidermal hyperplasia, being a characteristic trait of AD. Epidermal hyperplasia has been shown to be attributed to cytokines as potent inducers of epidermal growth factors and their receptors (142-144). Another crucial factor to the hyperproliferative phenotype might be elevated oxidative stress in the inflamed skin of AD patients (145). Cell cycle progression has been shown do be regulated by ROS and to be dependant of the intracellular redox-state (146). In fact, disorders associated with free radical dysbalances are often accompanied by alterations

in cell proliferation (147). There are various pathways of triggering the mitogenic signal under conditions of oxidative stress. ROS can induce activation of NF-κB (148) and MAP-kinases (149) which are important mediators of cell growth. Thus, it is feasible to hypothesize that the OXT system might be relevant for the atopic hyperproliferative phenotype through both, direct and indirect effects. On the one hand, OXT inhibits proliferation of keratinocytes.

Therefore, a reduced OXT signalling in atopic skin might directly contribute to hyperplasia.

On the other hand, cytokine release as well as ROS levels were increased after OXTR knockdown. Thus, deficits in OXT signalling in atopic skin might indirectly promote hyperplasia through elevation of inflammatory and oxidative stress.

6.3.6 Oxytocin reduces the epidermal layer thickness in an organotypic three-dimensional skin model

Utilizing an organotypic 3-D skin culture (OTC), consisting of keratinocytes and dermal fibroblasts, revealed an impact of the OXT system on epidermal structure. Treatment of OTCs with OXT led to a reduction of the thickness of all epidermal layers. In line with this result, OTCs containing OXTR knockdown keratinocytes exhibited an increase of epidermal layer thickness. As continuous treatment with OXT reduced the proliferation rate of keratinocytes in monolayer cultures, it is reasonable to deduce that an OXT-induced decrease of proliferating keratinocytes accounts for this phenotype. This assumption is supported by a significant OXT-induced decrease of the basal/proliferating layer. To prove this hypothesis, an additional experiment needs to be conducted in which the OTCs would be stained with the proliferation marker Ki67 six days after culturing (150). Furthermore, OXT-mediated modulation of differentiation processes in keratinocytes could be another explanation for the observed altered epidermal structure. Terminal differentiation is associated with the induction of loricrin expression as well as of other proteins like involucrin, filaggrin, cytokeratin 1 and 10 (151, 152, 153). OXT-treatment increased the expression of loricrin in the OTC-derived epidermis. In accordance, loricrin expression was reduced in the epidermis of OXTR antagonist-treated OTCs as well as of OTCs containing OXTR knockdown fibroblasts and keratinocytes. Therefore, OXTR signalling might favor early transition from proliferating, undifferentiated to non-proliferating, differentiated keratinocytes accounting for the reduction in epidermal layer thickness. Nevertheless, the contribution of this effect to the observed epidermal thinning might only be low, as no influence of OXT on the terminal differentiation markers involucrin and filaggrin was detected. It is also possible that both OXT-mediated effects, inhibition of proliferation and induction of loricrin expression, might act synergistically leading to a thinner epidermis.

Discussion 90 Defects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like AD (154). The impaired skin barrier in AD facilitates increased allergen and pathogen penetration initiating immunological reactions and inflammation. Loricrin is the major protein of the epidermal cornified envelope, contributing approximately 70 % by mass (155) and important for skin barrier formation and integrity. A decreased loricrin expression has been found in peri-lesional and lesional skin of AD and psoriatic subjects (156, 157).

Downregulation of loricrin by over 70 % in OTCs containing OXTR-depleted fibroblasts and keratinocytes suggests that OXT-signalling could be important for epidermal barrier integrity with relevance for AD.

7 C ONCLUSION AND PERSPECTIVE

In the present thesis, the OXT system was identified and characterized as a novel neuroendocrine mediator relevant for human skin physiology and morphology. OXT turns out to be a new candidate of brain- and skin-derived neuropeptides, supposed to be involved in cutaneous neuroendocrine networks. OXTR expression and its functionality in dermal fibroblasts and keratinocytes were demonstrated, enabling either skin cell-derived or plasma-derived OXT to signal in an autocrine, as well as in a paracrine way in the skin. This would link into the hypothesis of a bidirectional brain-skin connection (37, 158, 159). The proposed communication between the brain and the skin involves local neuroimmunoendocrine circuitries. These underlie the pathogenesis of allergic and inflammatory skin diseases such as AD, which can be triggered or aggravated by psychological stress. Considering that OXT has been shown to antagonize the HPA-axis and to influence mental state (32, 160, 161), lower levels of systemic or skin-derived OXT might aggravate stress-triggered skin diseases both, locally and systemically. Actually, downregulation of the OXT system was detected in atopic skin, indicating its relevance to AD and possibly other chronic inflammatory skin diseases. Indeed, the data revealed an impact of OXT on the modulation of processes dysregulated in atopic skin like proliferation, epidermal structure, ROS formation, GSH content and cytokine release in dermal fibroblasts and keratinocytes, pointing to a role of OXT in cutaneous inflammation. Moreover, the analyses suggest that continuous OXT signalling is needed to balance neuroendocrine, oxidative and inflammatory stress in the skin: OXTR knockdown was sufficient to upregulate stress parameters like CRH, CRHR1, ROS, IL6, CCL5 and CXCL10 without applying any further stress stimulus to the cells. Figure 34. displays a model, based on the presented data in this thesis, of the hypothesized effects occurring in human skin after downregulation of the OXT system.

Although the presented data reflect the first attempts to identify and elucidate OXT-mediated functions in human skin, the arising knowledge opens the field for new therapeutical approaches. OXT has been shown to reduce corticosterone levels systemically (115) and own data show that OXT reduces corticosterone release from keratinocytes. Since decades, it is well known that corticosteroid-treatment of inflamed skin bears the risk of skin atrophy/degeneration leading to deterioriation of epidermal barrier function (162, 163).

Considering the variety of diseases, which are associated with inflamed skin such as AD, psoriasis and diabetes, new therapeutical approaches might permit the reduction of corticosteroid treatment through modulating the OXT system. Certainly, one has to keep in mind that OXT might even share some corticosteroid-mediated actions such as anti-inflammatory and anti-mitotic effects. Therefore, to evaluate its potential as a therapeutical

Conclusion and perspective 92 target, detailed investigation of OXT-mediated signalling cascades and effects on human skin physiology and morphology need to be performed in future studies.

Figure 34. Downregulation of the oxytocin system alters stress parameters and influences skin structure, exacerbating inflammatory skin conditions such as in AD.

Based on the presented data in this thesis, this figure displays a model of hyphothesized effects occurring in human skin after chronic and dramatic downregulation of the OXT system. Poor epidermal barrier functionality, due to abberant hyperproliferation and reduction of the structural protein loricrin, in combination with increased inflammatory parameters might further downregulate the cutaneous oxytocin system resulting in a viscious circle.