TNF -/- MICE DEVELOP AGGRAVATED AD AND DISPLAY INCREASED TSLP

SEVERITY

AD is a chronic inflammatory skin disease with a complex pathogenesis. It is caused by a combination of an epidermal barrier dysfunction and an immune dysregulation¹⁶³. A variety of factors contribute to the pathogenesis of AD, including environmental or genetic factors. Immunological factors as well as pharmacological abnormalities play a role in disease progression¹⁹².

TNF-α is known for its pleiotropic functions in host defense but also for its role on the elicitation of inflammatory diseases during immune dysregulation^79,80. Anti-TNF therapies are approved and effective for autoimmune disorders such as rheumatoid arthritis, inflammatory bowel disease and psoriasis with relatively few side-effects⁷⁷. The two monoclonal antibodies adalimumab and infliximab and the soluble receptor etanercept are the most commonly prescribed therapeutics in autoimmune diseases interfering with the TNF function^193,194. A direct role of TNF-α in AD initiation and development is not well understood. A detailed analysis of the literature revealed more evidence for negative than a positive association. 1992, Takahashi et al, has 76

shown that the PBMCs from AD patients had decreased levels of TNF-α compared to healthy controls⁸⁶. Nomura et al. (2003) also observed lower levels of TNF-α, IL-1β and IFN-γ in the skin from AD patients when compared to skin samples derived from psoriasis⁸⁹. Similarly dermal inflammatory DCs which produce TNF-α were found to be reduced in the skin of AD vs psoriasis patients¹⁹⁵. The most striking evidence came from epidemiological studies from different clinics including our institution, which reported the onset of AD like symptoms as a side effect of anti TNF therapy90,91,196-198. These findings point towards a rather protective role of TNF in the context of AD, but the experimental evidence was missing. To support previous findings and clinical observations we used a murine model. To delineate the underlying mechanisms, we applied an OVA-allergen AD mouse model in TNF -/-mice to mimic the human AD scenario. Indeed, TNF-α protected against AD development as reported in literature as worsening of the severity of AD-like lesions occurred in TNF^-/- mice. The measurement of the severity in the AD model was adopted from the SCORAD score a tool to assess clinical severity in human AD¹⁹⁹. The majority of AD in human is characterized by increased levels of IgE (extrinsic AD) and the presence of mostly Th2 cells in lesional skin³². Woodward, A.L et al (2001) have shown that T cells play a critical role in skin inflammation²⁰⁰. The key role of effector T cells and their major relevance in AD pathology was further supported by the fact that under T cell immunodeficiency, elevated IgE levels with eczematous skin lesions were observed³². Other studies have shown that epidermal CD8+ cells are involved in the pathogenesis of AD²⁰¹. In search of mechanism behind AD their accumulation under TNF absence and also to understand the clinical observations, various factors were analyzed. The analysis of the humoral immune response from sera of mice from the AD model revealed a decrease of the specific IgE and IgG1 concentrations in the TNF^-/- mice whereas no change was observed in infiltrating CD4+ and CD8+ T cells.

AD patients have shown to have higher frequencies of Th1, Th2, Treg and Th22 subsets as reported by Turner et al. 2012²⁰². In addition these cells show a higher expression of 5, 13, 1β 4, IFN-γ, 12, Gm-CSF, 10, TGF-β and IL-6⁸⁸. The analysis of different proinflammatory cytokines and Th subsets; Th1, Th2,

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Treg, Th17 (e.g. IL-4, IL-10, IL-17, IFN-γ IL-1β, IL-1α, IL-13, IL-33 and β-defensins) revealed no impact or slight increase on their expression under TNF deficiency indicating that T cells were most likely not responsible for AD aggravation under TNF deficiency.

Barrier dysfunctioning is well linked to AD pathogenesis. Aioi, A. et al. (2001) have shown a crucial role of skin barrier function in AD manifestation using NC/Nga mice, a spontaneous mouse AD model displaying skin barrier abnormalities like increased TEWL and an abnormal skin conductivity under conventional conditions, but not under specific pathogen-free conditions²⁰³. Similarly, Gupta et al. (2007) have shown that the pathogenesis of AD is at least in part related to barrier dysfunction²⁰⁴. In line with these findings, we investigated the expression of several skin barrier genes in these mice. The expression level of transglutamase, involucrin, loricrin and filaggrin were either comparable or slightly increased in TNF^-/- mice and did not correlate with AD symptom score (data not shown).

TSLP is a well-known key initiator of allergic diseases including AD and asthma¹²⁶. TSLP has been shown to be increased in lesional skin from AD patients but not in nickel induced contact dermatitis¹⁰³. TSLP is an IL-7 type of cytokine belonging to the IL-2 family, which can act on DCs and can promote Th2 cell differentiation and recruitment. TSLP can also directly act on naïve CD4+ cells to promote proliferation in response to antigen¹¹⁴. Ziegler et al. (2013) demonstrated that epithelial cell-derived TSLP can activate T cells, DCs and mast cells²⁰⁵. The overexpression of TSLP in murine skin leads to the development of a spontaneous dermatitis^49,103. In agreement with the literature, TSLP was significantly overexpressed in AD skin and also correlated with disease severity. TNF-α is known to regulate TSLP expression.

2011, Brandt and Sivaprasad provided evidence that human skin explants produce higher levels of TSLP, when treated with a combination of pro-inflammatory cytokines i.e. TNF-α, IL-1α and Th2 cytokines i.e IL-4 and IL-13, but not alone⁴⁴. Similar results were obtained from keratinocyte cultures^149,206.

To understand why TSLP was enhanced in TNF^-/- mice although TNF-α is a well-recognized inducer of TSLP¹⁶⁴ different factors and cell types were evaluated in

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TNF^-/- mice. Another presumably important component in AD pathogenesis is the mast cell. Various studies have shown that mast cells are commonly increased in human but also the AD mouse model¹⁶². Therefore we analyzed the mast cell numbers in lesional skin from the TNF^-/- mice.

Mast cells, but not T cells as mentioned earlier were increased in AD lesions of TNF^-/- mice. Its number not only correlated with disease severity but also with mRNA levels of lesional TSLP in the skin. In agreement with this finding Yong-Jun Liu et al. (2006) also described that mast cells activated by IgE receptor cross-linking express high levels of TSLP, which may support allergic inflammation¹²¹. Interestingly, Na-Ra Han et al. in 2014 have shown as well that numbers of mast cells in different organs of wildtype mice were significantly higher compared with TSLP-deficient mice as well as TSLP was capable of inducing the proliferation and differentiation of mast cells²⁰⁷.