br Results br Discussion Tapinarof GSK is a naturally
Discussion Tapinarof (GSK2894512) is a naturally derived investigational topical medicine with demonstrated clinical efficacy for patients with AD and psoriasis (Bissonnette et al., 2010, Bissonnette et al., 2012a). The drug substance is a polyphenol produced by bacteria that is structurally and functionally distinct from plant-derived polyphenols such as resveratrol. We show that tapinarof is a direct binding partner and agonist ligand of AhR. Furthermore, IMQ-induced inflammation can be countered with 1% tapinarof cream in AhR-sufficient, but not AhR-deficient mice. Together these data identify AhR as the primary molecular target for tapinarof. In addition to AhR agonist activity, tapinarof has antioxidant properties inherent to its chemical structure that may also derive from Nrf2 pathway activation. It has been proposed that AhR/Nrf2 dual activation drives the efficacy of coal tar, a traditional topical treatment for psoriasis and AD that contains complex mixtures of polyaromatic hydrocarbons (van den Bogaard et al., 2013). Antioxidant activity is not present after AhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Rather, 2,3,7,8-tetrachlorodibenzo-p-dioxin is a potent inducer of reactive oxygen species (Kennedy et al., 2013). Thus, dual AhR/Nrf2 activation may be key to the anti-inflammatory activity of AhR agonists such as tapinarof and coal tar extracts. However, in alk5 to coal tar, tapinarof is a single, chemically defined natural product. Many genes are transcriptionally regulated by AhR. The epidermal differentiation complex contains more than 50 structurally, functionally, and evolutionarily related genes involved in the terminal differentiation and cornification of keratinocytes. Several of these genes, including filaggrin, hornerin, and involucrin, play important roles in barrier function and can be modulated by AhR agonists, including coal tar and soybean tar (Sutter et al., 2011, Takei et al., 2015, van den Bogaard et al., 2013). Our results showing that tapinarof has no effect on KRT1 mRNA expression are in concert with that of coal tar. Coal tar had no effect on KRT10, KRT5, and KRT14, which do not exist in the epidermal differentiation complex and do not appear to be regulated by AhR. Together, these data show that tapinarof increases the expression of barrier genes that are regulated by xenobiotic response elements in their promoter region. An impairment of skin barrier function has been well described in specific inflammatory skin disease, including AD. Barrier genes are reduced in keratinocytes in response to proinflammatory cytokines, and these proinflammatory mechanisms are exacerbated in AhR-deficient keratinocytes (Di Meglio et al., 2014), which also suggests that AhR agonism by tapinarof would impact skin barrier function in wild-type mice. As such, we acknowledge one drawback of our study design, which utilizes pretreatment with tapinarof cream, could be that improved barrier function would reduce the overimpact of IMQ treatment, thereby leading to the reduced clinical score. However, that tapinarof acts as an anti-inflammatory agent, rather than a preventative one, is evidenced by (i) therapeutic improvement in patients with chronic disease, (ii) that not all cytokines are reduced with tapinarof treatment, suggesting a more selective anti-inflammatory mechanism, and (iii) FICZ also led to reduced inflammation in the IMQ mouse following a different treatment schedule (Di Meglio et al., 2014). AhR controls the expression of cytokines, such as IL-10, IL-21, and IL-22, and plays a role in the differentiation of regulatory T and Th17 cells (Quintana and Sherr, 2013) implicating Th17 cells as target cells for tapinarof. We show that IL-17A expression levels are reduced in human peripheral blood T cells and ex vivo sRICA cultures after tapinarof treatment. Likewise, topical treatment of tapinarof suppresses IMQ-induced Il17a and Il17f gene expression in AhR-sufficient, but not AhR KO mice, similar to topical FICZ treatment. Treatment with tapinarof also led to modestly increased Il10 in IMQ mice, consistent with the role of AhR in IL-10 production from type 1 regulatory T cells (Apetoh et al., 2010). IL-22 exists at high levels in human psoriatic skin, is correlated with disease severity, and increases proliferation of keratinocytes (Wolk et al., 2006). AhR is required for production of IL-22 in Th17 cells and IL-17+ γδ T cells, which do not produce IL-22 in the absence of AhR (Martin et al., 2009). We observe a significant increase in IL-22 levels in vitro. That this was not recapitulated in mice likely reflects the complex interactions of multiple cell types leading to an overall reduction in the inflammatory response by tapinarof. IL-17A is implicated as a key player in psoriasis and has been clinically validated via the efficacy of neutralizing antibodies on plaque psoriasis (Griffiths et al., 2015). Here we report that tapinarof significantly reduces IL-17A in vitro, ex vivo, and in IMQ mice. Yet, tapinarof also demonstrates clinical efficacy in AD, which is largely driven by Th2 cytokines (Bissonnette et al., 2012b). AhR agonists may similarly reduce these pathogenic cytokines. GATA3 contains a high frequency of potential xenobiotic response element binding sites (Hanieh, 2014) and agonism of AhR in naive T cells results in reduced polarization to the Th2 lineage and favored Th1 T-cell development (Negishi et al., 2005). Thus, AhR agonism, through modulation of cytokines and improved barrier function, may be driving tapinarof’s efficacy in both patients with psoriasis and AD. Identifying which cellular subset is key to this response will be critical for the full mechanistic understanding of tapinarof’s action.