The Th2-promoting cytokine, thymic stromal lymphopoietin (TSLP), has been implicated in the pathogenesis of the chronic inflammatory skin condition, atopic dermatitis (AD). Understanding immune mechanisms that increase responsiveness to TSLP in AD could aid in developing new treatments that inhibit the inflammatory cascade underlying this disease. Responsiveness to TSLP is enhanced in AD patients through a process that depends upon the capacity for dendritic cells (DCs) to upregulate TSLP receptor (TSLPR) following ligation of Fc receptors by allergen. The objective is to elucidate the immunogenetic mechanisms involved in modulating expression of TSLPR in AD and to determine the functional relevance of this receptor to Th2-driven inflammatory responses in the skin. Complementary approaches in humans and mice will incorporate in vitro studies of cells from AD patients (Aims 1 and 2) with an in vivo mouse model of AD (Aim 3).
In Aim 1, an array of molecules that bind surface receptors on DCs (allergens and toll-like receptor ligands) will be tested for their capacity to upregulate TSLPR and the relevant signaling components elucidated using flow cytometry and gene silencing techniques. The requirement for TSLPR upregulation in amplification of TSLP-mediated Th2 responses will be verified in DC/T cell co-cultures. As an adjunct, cytokine-mediated suppression of TSLPR will be explored in order to determine whether this process is dysregulated in AD. To assess in vivo relevance, TSLPR expression will be analyzed on discrete DC types in lesional skin and at atopy patch test sites by flow cytometry and confocal microscopy techniques.
In Aim 2, the influence of genetic variants of the TSLPR gene and epigenetic changes at this locus on TSLPR upregulation will be examined. Sequencing of the TSLPR gene will be carried out using samples from a large cohort of patients in order to confirm the 5' upstream region and to identify single nucleotide polymorphisms. Bioinformatics and luciferase reporter assays will then be used to identify and confirm promoter elements and haplotypes that enhance TSLPR gene activity. Chromatin remodeling at the TSLPR locus will be assessed in DCs by chromatin immunoprecipitation and DNase hypersensitivity assays. Loss-of-function mutations in the gene encoding filaggrin, a protein essential to skin barrier function, will also be genotyped to probe the relationship between an inherited defect in the skin barrier and factors predisposing to TSLPR upregulation.
In Aim 3, the role of epidermal and dermal DCs in TSLP-driven inflammation will be examined in mice that lack Langerhans cells or CD11c+ DCs. The requirement for Langerhans cells to respond directly to TSLP will be tested in bone marrow reconstitution studies. To identify the principal cell type(s) responsible for TSLP-driven Th2-type inflammation in the skin, mixed chimeric mice will be used to limit TSLP responsiveness to specific cell types. Mice will also be generated with a conditional null allele of the TSLPR gene, allowing TSLP non-responsiveness to be limited to specific lineages.
The cytokine, TSLP, has been implicated in driving Th2 responses that underlie the chronic inflammatory skin condition, atopic dermatitis. Nothing is known about how TSLP receptor may modulate responses to TSLP. The proposed studies, which investigate TSLP receptor expression and function in mice and man could yield new insight into why some individuals are susceptible to the effects of TSLP while others are not. Such studies could identify new molecular targets for treatment of this debilitating disease.
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