Phytochemicals as potential inhibitors of NETosis: implications for immunothrombosis and chronic disease management

BACKGROUND: NETosis, a specialized form of neutrophil cell death, plays a dual role in immune regulation. While NET formation is essential for capturing pathogens, excessive NETosis contributes to immunothrombosis, oxidative stress, and tissue damage, affecting both acute and chronic diseases such as COVID-19, cardiovascular diseases, diabetes, cancer, and autoimmune conditions. Given the limitations of current treatments, including toxicity, high costs, and bleeding risks, phytochemicals are being explored for their therapeutic potential. METHODS: NETosis gene sets were collected through published data, and followed by Gene set enrichment analysis (GSEA) to identify potential NETosis-inhibiting natural compounds from a library of 103 phytochemicals candidates. NETosis phenotype was confirmed by assessing NET formation through immunofluorescence staining and quantification. Candidate compounds were further validated in vitro using RT-qPCR to assess the expression of NETosis-related genes, including PADI4, TREM1, S100A8/A9, and CCL7. To evaluate the procoagulant consequences of NETosis, we performed a thrombin activity assay by incubating plasma with conditioned media from treated neutrophil-like cells. RESULTS: Three phytochemicals-hesperidin, baicalin, and imperatorin-were identified as effective inhibitors of NETosis. Immunofluorescence staining confirmed NET inhibition, and RT-qPCR analysis showed significant downregulation of key genes involved in NET formation. In addition, thrombin activity was significantly reduced in plasma exposed to conditioned media from phytochemical-treated cells, indicating attenuation of NETosis-associated procoagulant activity. CONCLUSIONS: Hesperidin, baicalin, and imperatorin show promise as candidates for modulating NETosis, with implications for managing immunothrombosis and chronic diseases.