Celastrol attenuates Th1- and Th2-driven skin inflammation in 2D and 3D in vitro models

Celastrol, a bioactive compound from Tripterygium wilfordii Hook F, has been reported to exert potent anti-inflammatory effects through multiple signaling pathways. While its activity has been studied in various cell types and disease models, its effects in atopic dermatitis (AD) is limited to a few studies in mouse models and remains largely unexplored in in vitro cell models. In this study, anti-inflammatory effects of celastrol were assessed in Th1-driven 2D inflammation models of HaCaT keratinocytes and dermal fibroblasts (DF), and in a Th2-driven 3D AD skin model. Cytokine stimulation mimicked chronic inflammation. Celastrol was applied at sub-cytotoxic concentrations, and inflammatory markers were quantified on the mRNA (qPCR) and protein (ELISA) level. In 2D models, celastrol reduced interleukin (IL)-8 and IL-6 secretion in a concentration-dependent manner, with fibroblasts producing higher cytokine levels than keratinocytes. In 3D AD models, topical celastrol (10 µM) was well tolerated and markedly reduced secretion of IL-8, IL-6, IL-1α, and mRNA expression of CXCL8, IL6, IL1B, and IL23A, even under continuous Th2 stimulation. AD biomarker genes CCL26, CA2, and NELL2 were unaffected, likely due to persistent cytokine exposure. Celastrol displayed strong anti-inflammatory activity in both Th1- and Th2-driven in vitro skin inflammation models, including a physiologically relevant 3D AD model. Its multitarget action support its potential as a topical treatment candidate for chronic inflammatory skin diseases.