ABSTRACT We have demonstrated that mast cells entering the skin change phenotype triggered by contact with dermal fibroblasts (DF). In the skin, Mast Cells (MCs) down regulate their TLR2 and increase the expression of genes that downregulate the NF?B, a known inflammatory pathway. This contributes to maintaining skin homeostasis and makes mast cell tolerant to commensal bacteria. We found that DKK2, a specific protein secreted by DFs, modulates the huMC NF??B pathway, by increasing gene expression of TNFAIP3 (A20) and NFKBIA in huMCs. This modulation results in a decreased inflammatory response to commensal bacteria. In this proposal, we will clarify the mechanisms of DF - MC signaling, which induces a MC skin tolerant phenotype. This is an important mechanism for skin inflammation and atopic dermatitis (AD) in particular. We will present preliminary data that show that in AD, DFs fail to maintain human MC (huMC) tolerance to commensal bacteria which allows huMC to release proinflammatory cytokines. The idea that inflammatory diseases develop because MC tolerance is broken represents a shift in science paradigm. This work will identify the mechanisms underlying the roles of huMCs in building and breaking tolerance to the skin?s rich environment. We propose the followings: 1. To determine whether TNFAIP3 (A20) and NFKBIA in human MCs are required for preventing proinflammatory interleukin responses (tolerance) to commensal supernatant. In Aim 1 of this proposal, we will use a variety of structural, biophysical, biochemical, and functional assays to determine how dermal MCs collaborate with DFs. More specifically, we will analyze the MC expression of the NF??B related genes, TNFAIP3 (A20) and NFKBIA, as well as receptors related to the innate immune system in dermal huMCs. 2. To confirm that MC tolerance can be induced by DKK2 from DFs In Aim 2, we will determine whether DF DKK2 is required for inducing huMC tolerance to commensal supernatant and whether different DF sub-populations vary in their ability to induce tolerance in huMCs. 3. To confirm that DF-induced MC tolerance to commensal bacteria is critical for suppressing human skin inflammation. In Aim 3 of this proposal we will look at the consequences of the failure of huMCs to maintain tolerance and their role in skin inflammation, specifically in Atopic Dermatitis skin.