1065 The topical AhR agonist tapinarof has broad in vivo immunosuppressive and immunomodulatory effects in human skin grafted mice

Tapinarof, a first of its kind topical AhR agonist, treats both psoriasis and atopic dermatitis and induces an average 4 month remission of psoriasis. The lack of mechanistic studies of tapinarof in living human skin is a barrier to understanding its anti-inflammatory effects. We grafted NSG mice with neonatal foreskin and infused them IV with allogeneic human PBMC, inducing a polyclonal T cell mediated inflammatory dermatitis in the human skin graft. We treated human skin grafts on NSG mice with topical tapinarof or vehicle for 3 weeks after PBMC injection and studied grafts by RNA and high throughput TCR sequencing, collagenase digestion/flow cytometry, and multiplex immunostaining. Tapinarof reduced type 1 (IFNγ, IL-12,18), type 2 (IL-4, 5, 13), type 9 (IL-9), type 17 (IL-17, 23) T cell mediated inflammatory signaling in skin, and also inhibited IL-2, 15, 21, 27, 32, TNFα and TGFβ signaling. Tapinarof down regulated JAK/STAT (1,3,5), ERK/MAPK, NF-κB, mTOR and NO signaling in skin and induced multiple immunoregulatory pathways including CTLA-4, PD-1, SOCS1/SOCS3 and IL1-RN. Pathway analysis demonstrated reduced DC and macrophage activation, reduced costimulation (CD28, CD40/CD40L) and DC/NK crosstalk, and reduced NK signaling. These changes were accompanied by reduced extracellular (TLR1,4) and intracellular nucleotide related (TLR3,7,9) DAMP sensing, reduced type I IFNs, decreased inflammasome (IL-1A,B) and CGAS/STING activation. Cytosolic nucleotide sensing (CNS), in which RNA/DNA derived from mitochondrial leakage is sensed by TLRs 3/7/9, is a critical early initiating event in inflammation. TREX1, a potent inhibitor of CNS, was the most significant upstream regulator in Tapinarof treated skin. DDX58, a measure of CNS activity, was one of the most significantly down regulated drivers. In summary, tapinarof broadly inhibits T cell mediated skin inflammation, likely by increasing mitochondrial fitness or by reducing CNS. A better understanding of this biology could lead to novel therapies for inflammatory skin disease.