De Palma, Giacomo (2024) Evaluation of vitamin D effects in psoriatic lesion development through in vitro and in vivo models. [Tesi di dottorato]

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Abstract

Background and aim Psoriasis is a chronic, immune-mediated inflammatory skin disease characterized by keratinocyte hyperproliferation and abnormal differentiation. Vitamin D3 and its derivatives are used therapeutically for psoriasis although the mechanism behind their anti-inflammatory effects is unclear. Experimental studies show that vitamin D3 immunomodulatory activity may be linked to its interaction with purinergic signalling, particularly through the upregulation of ecto-5'-nucleotidase CD73 on human CD4+ Foxp3- T cells. On the other hand, vitamin D3 antiproliferative effect on human cutaneous melanoma cell lines may be dependent upon the inhibition of CD73 activity. Thus, the aim of this research project is to investigate on the possible role of CD73/adenosine signalling in the effects of vitamin D3 in normal and diseased skin, by using in vitro and in vivo approaches. Experimental approach In vitro studies were conducted on HaCaT cell line in order to assess CD73 role in keratinocytes proliferation and inflammation. Then, impact of vitamin D3 on keratinocytes proliferation was investigated. Furthermore, CD73 expression and activity were assessed following treatment with vitamin D3. For in vivo studies, the mouse model of imiquimod induced psoriasis-like lesion was conducted. The first aim was to investigate the involvement of CD73 in psoriasis-like lesion development and CD73 functional role in psoriasis inflammation with a double approach consisting of pharmaceutical blockade with CD73 inhibitor APCP and by using CD73-/- mice. The second aim was to evaluate the anti-inflammatory effect and the possible modulation of CD73 expression by calcipotriol, a vitamin D derivative. Results HaCaT cells express CD73 and pharmacological blockade of the enzyme with APCP resulted in increased proliferation and increased keratin-14 expression. CD73 expression was found to be increased following incubation with a mix of proinflammatory cytokines involved in psoriasis pathogenesis (TNF, IL-17A, IL-6 and IL-1α all at the concentration of 10 ng/ml). Vitamin D3 treatment on HaCaT cells resulted in increased proliferative rate and increased expression of KRT-14, which were paralleled by a reduction in CD73 expression and increased CD73 activity following 48h of treatment. In vivo experiments provided further evidence of CD73 involvement in skin inflammation. CD73 expression was found to be increased in imiquimod treated mice, while topical treatment with clobetasol prevented this increase. In CD73-/- mice, imiquimod-induced lesion was exacerbated compared to the development of lesion in WT mice, as shown by ear thickness, erythema and scaling. Pharmaceutical blockade of CD73 with APCP seemed to have contrasting effects depending upon the type of treatment either from the beginning or after the spread of the psoriasis-like lesion. Topical treatment with calcipotriol, as curative intervention, reduced both clinical signs of psoriasis–like lesion and splenomegaly (p<0.05, N=4); this reduction was paralleled by decreased tissue levels of IL-17, and decreased CD73 expression. Conclusion In vitro experiments on keratinocytes show a modulation of CD73 by vitamin D. In vivo studies provide evidence for CD73 involvement in psoriasis-like inflammation; calcipotriol anti-inflammatory treatment results in a tissue reduction of CD73. Further investigation is needed to clarify whether CD73/adenosine signalling is involved in the anti-inflammatory effect of vitamin D.

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