Identification and characterization of the keratinocyte kinome and secretome in pemphigoid disease

Pemphigoid diseases (PD) are chronic inflammatory diseases of the skin and/or mucous membranes caused by autoantibodies targeting specific autoantigens in the skin. Despite manifold insights into PD pathogenesis, PD treatment still relies on unspecific and mostly systemic immunosuppressive treatment. Current drug developments almost exclusively focus on the modulation of B cells and autoantibody concentrations. However, as shown in PD patients treated with the anti-CD20 antibody rituximab, time to remission requires several weeks. In addition, remission rates in rituximab treated PD patients are relatively low. Hence, treatments targeting the pathogenic effects induced after autoantibody binding to their target cells (keratinocytes) may offer a window of therapeutic intervention that is currently not in the focus of drug development in PD. To fill this gap, we have determined the kinome and secretome of keratinocytes incubated with different anti-PD autoantibodies. Specifically, we incubated the immortalized keratinocyte HaCaT cell line with affinity-purified anti-type VII collagen IgG or anti-BP180-NC16A IgG from epidermolysis bullosa or bullous pemphigoid patients respectively. Kinase activity of a total of 176 kinases was evaluated using the PamGene technology, whereas the concentration of approximately 500 proteins was determined using the Olink platform. Based on these findings, we here will (1) identify the molecular mechanisms how autoantibody binding to keratinocytes activates kinases and induces mediator release, validate the autoantibody binding-induced (kinome) and (3) secretome of keratinocytes, (4) establish causal relationships between the kinome and secretome in autoantibody-stimulated keratinocytes, and ultimately (5) functionally evaluate the autoantibody-induced kerationocyte secretome on immune cells. In perspective, to translate our findings into clinical application, we aim to validate our findings in mouse models of pemphigoid disease. We also plan to investigate the kinome in PD patients in at least 2 compartments (skin and leukocytes). Collectively, we here will provide detailed insights on keratinocyte biology in the context of PD, as well as potentially identify druggable pathways that may be amendable for topical treatment of PD.