Nonadditive transcriptional change analysis identifies regulators for ginsenoside Rg3 to alleviate synergistic cytokine production in TLR2/3 dual-stimulated macrophages

Cytokine synergy induced by the activation of multiple Toll-like receptors (TLRs) may result in uncontrollable life-threatening inflammation, the so-called cytokine storm. Owing to the complexity of the crosstalk for individual TLR signaling pathways, analyses of transcriptional changes in addition to differentially expression genes (DEGs) are needed. In the present study, we aimed to create algorithms to obtain a profile of synergistic cytokine production and to evaluate the anti-cytokine synergistic activity of ginsenoside Rg3. RAW264.7 macrophages were activated by TLR2/3 dual ligands; Rg3 was used as an intervention. After interleukin (IL)-6 secretion was detected as a preliminary readout for cytokine synergy, RNA sequencing-based bioinformatic analysis was performed, followed by qPCR and western blotting verification. Specifically, nonadditive transcriptional responses (DIFs) were applied as a measure of synergistic genes, and an anti-synergy score was created as a measure of the antagonistic effect of Rg3. A mouse model of TLR2/3 costimulation was subsequently established to evaluate the anti-cytokine synergistic effect of Rg3 in vivo. The results show that Rg3 alleviates synergistic cytokine production both in vitro and in vivo. Nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3) are novel targets of Rg3 related to its anti-cytokine synergistic effects. Our strategy of nonadditive transcriptional change analysis will be helpful for performing high-throughput screening for drugs with anti-cytokine synergy activities.