Discovery of immune-activating molecules targeting TLR7/8 receptors from natural products.

This study aimed to identify natural compounds activating Toll-like receptors 7 and 8 (TLR7/8) via high-throughput screening, offering candidate immunomodulators targeting these pathways. Using NF-κB/SEAP reporter assay, three compounds (3-Butylidenephthalide, BC-44b and Hydroxytyrosol) were selected from a library of 109 natural products as putative dual TLR7/8 agonists. These agonists induced TLR7/8-dependent signaling, enhanced maturation and activation of macrophages and dendritic cells, upregulated the expression of co-stimulatory molecules (CD80, CD86, CD40) and major histocompatibility complex molecules (MHC-I, MHC-II), and stimulated the secretion of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and chemokines (MCP-1, MIP-1α). Genetic silencing with siRNA confirmed the essential roles of TLR7 and TLR8 in mediating these effects. Western blot analysis further demonstrated activation of the NF-κB and mitogen-activated protein kinases (MAPKs) signalings downstream of TLR7/8. In vivo, these compounds exhibited potent immunostimulatory activity, promoting the proliferation of T and B lymphocytes, increasing the frequencies of CD3+, CD4+ and CD8+ T-cell subsets, and eliciting a mixed Th1/Th2 immune response characterized by elevated levels of IgG2a, IgG2b, IgG3 (Th1) and IgG1 (Th2) antibodies. In summary, 3-Butylidenephthalide, BC-44b and Hydroxytyrosol activate antigen-presenting cells and enhance humoral immunity through the TLR7/8-MAPK-NF-κB axis, highlighting their potential as novel vaccine adjuvants.