Initiation and maintenance of Th17 inflammation in EKC syndrome - Epidermal differentiation disorders (EDD, a.k.a. the ichthyoses) are caused by pathogenic variants in over 50 genes and feature generalized skin scaling, thickening, and redness often associated with itch, a clinical marker of inflammation. Prior work provides evidence of T helper (Th) 17 inflammation across EDD with an immune signature resembling the common inflammatory skin disease psoriasis. Despite this, clinical response to Th17 inhibition has generally been poor or incomplete. Our lab discovered a novel genetic EDD, the erythrokeratodermia cardiomyopathy (EKC) syndrome which results from heterozygous genetic variants affecting the SR6 domain of desmoplakin. EKC features early childhood onset of inflammatory skin lesions and progressive dilated cardiomyopathy. In contrast to the majority of individuals with EDD due to other genetic causes, individuals with EKC have shown consistent and profound improvement in skin redness, inflammation and, sometimes, cardiac function, when treated with therapies inhibiting the Th17 pathway, suggesting that inflammation contributes to disease pathogenesis. We propose that EKC can serve as a model for Th17 inflammation due to genetic defects in skin barrier dysfunction, and our preliminary data shows increased innate immune activity in EKC skin and keratinocytes expressing DSP variants. We have developed unique resources that make us well-posed to study this phenomenon: skin tissue and primary cultured keratinocytes from individuals with EKC, keratinocytes engineered to stably express EKC-associated DSP variants, and a transgenic knock-in mouse model which allows us to express EKC-associated DSP variants across the entire organism or specifically in skin. We will interrogate effects on desmosomes by assessing desmoplakin molecular turnover at the cell membrane and structure and function of desmosomes using electron microscopy and dispase-based mechanical dissociation assays. In addition, we will use unbiased transcriptomic profiling to uncover cell-intrinsic innate immune signatures in cultured keratinocytes and molecular cross-talk between keratinocytes, immune cells, and stromal cells in murine skin using single cell RNA sequencing. We generated a transgenic knock-in mouse model of EKC with skin-specific expression of mutant Dsp driven by Keratin14-Cre and found that K14-DspL622P/WT mice are viable with intact barrier at birth, mirroring EKC patient presentation. Over time, K14-DspL622P/WT mice develop progressive inflammatory lesions and elevated markers of Th17 immunity, establishes the utility of this model to study Th17 inflammation due to skin barrier dysfunction. We will use flow cytometric analysis of skin tissue and plasma cytokine profiling to assess inflammatory markers in the mouse model and test how modulating the cutaneous microbiome (through both depletion and diversification of skin microbes) affects markers of Th17 inflammation. Studying the cellular determinants of innate immune activation and specific cellular interactions which drive initiation and maintenance of Th17 inflammation in EKC may elucidate mechanism underlying the pattern of Th17 polarization that is shared across EDD.
