Characterization and molecular functions of the IL-17 – IL-36 signaling axis in keratinocytes and consequences for psoriasis

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Psoriasis is a chronic inflammatory skin disease affecting about 2% of the population. Typical for the disease is epidermal hyperplasia associated with hyperproliferation and abnormal differentiation of keratinocytes. The immune system is closely associated with psoriasis, e.g. TH17 cells modulate keratinocyte function through IL-17 as well as other cytokines. Moreover, IL-36 cytokines are closely linked to psoriasis. These cytokines activate signaling pathways and control genes that encode proteins associated with keratinocyte differentiation and skin barrier formation, and factors involved in immune cell recruitment and activation. We have studied the effects of IL-17A on primary normal human epidermal keratinocyte (NHEKs) both in monolayer cultures and 3-dimensional (3D) organotypic skin models. One of the genes that is strongly activated is IL36G, which encodes IL-36, an IL-1 family cytokine. IL-36 is thought to be secreted by an unconventional protein secretion pathway and requires N-terminal processing for activation. IL-36 interferes with NHEK differentiation in 3D models and in the mouse ear. This suggests that IL-36 cytokines are effectors of IL-17. In support, IL-17A and IL-36 cooperate in controlling gene transcription and affecting mouse skin development. We propose an IL-17A – IL-36 feedforward loop, aggravating the psoriatic phenotype. Therefore, we want to understand how these two cytokines cooperate both in HaCaT keratinocytes and in NHEKs. For this we will study the signaling pathways and determine how the signals are integrated at target promoters. We will measure signaling molecules, binding of transcription factors to DNA, chromatin status, polymerase II activity, and transcription rates. Finally, post-transcriptional cooperativity will be determined by analyzing RNA stability. Together, these studies will clarify how IL-17A and IL-36 cooperate in controlling target genes. Moreover, we want to understand how the processing and secretion of IL-36 is regulated. We will use fusion proteins of IL-36 with a biotin ligase to modify interacting proteins. Biotinylated interactors will be purified using streptavidin and identified by mass spectrometry. This approach will define even short-term interacting proteins and provide us with leads to define the relevant unconventional protein secretion pathway(s). We will also stimulate cells through pattern recognition receptors to define signals that promote IL-36 secretion. The functional relevance of IL-36 will be further studied in the mouse using ear injection and imiquimod models, in which antagonistic IL-36 receptor fusion proteins as well as newly defined factors involved in IL-36 secretion and activation will be evaluated. Together, we expect from these studies to obtain molecular insight into the activation and processing of IL-36, how it cooperates with IL-17A and what its contribution is to altered keratinocyte function and differentiation in the context of psoriasis.