Wedelolactone, a Novel TLR2 Agonist, Promotes Neutrophil Differentiation and Ameliorates Neutropenia: A Multi‐Omics Approach to Unravel the Mechanism

Neutropenia, a common complication in cancer patients undergoing radiotherapy, heightens the risk of infection and mortality, with limited treatment options. This study investigates wedelolactone (WED), a natural coumarin, as a potential therapeutic agent. WED is found to promote neutrophil differentiation and enhance bactericidal function in vitro. Its in vivo efficacy is validated in radiation-induced neutropenic mouse and zebrafish models, where it facilitates rapid recovery of leukocyte and neutrophil levels. An integrated approach using the GEO database, RNA sequencing, molecular docking, and Drug Affinity Responsive Target Stability (DARTS) assays identifies TLR2 and its downstream MAPK signaling pathway as essential for WED's anti-neutropenic effects. DARTS confirms significant binding of WED to TLR2. Knockdown of TLR2 with siRNA or inhibition of TLR2 with C29 reduces WED-induced neutrophil differentiation, MEK1/2 and ERK1/2 phosphorylation, and expression of transcription factors (PU.1, CEBPβ). Similarly, ERK1/2 inhibition by SCH772984 impairs WED-induced neutrophil differentiation and bactericidal activity, decreasing PU.1 and CEBPβ expression without affecting TLR2 levels. These findings position TLR2 as a key therapeutic target for neutropenia, with WED effectively promoting neutrophil differentiation via TLR2 and MEK/ERK pathway activation. This study highlights the therapeutic potential of targeting TLR2 to alleviate neutropenia and underscores the utility of a multi-omics approach in uncovering drug mechanisms.