Role of bioactive extracts obtained from marine sources and agri-food industry by-products in ETosis modulation

ETosis is a defense mechanism implemented by the immune system of vertebrates and invertebrates, being a type of cell death that involves the release of chromatin from inflammatory cells, the formation of extracellular traps (ETs) and the death of target microorganisms. ETosis holds substantial biomedical relevance due to the implication of its down- or up- regulation in the onset of auto-immune diseases or chronic inflammation and carcinogenesis, respectively (1,2). In the present study, natural products were tested for their ability to modulate ETosis and inflammation. To this purpose, RAW 264.7 murine macrophages were immunostimulated with PMA (phorbol 12-myristate 13-acetate) and co-treated with either extracts from P. oceanica’s leaves (GLE) or rhizomes (RE), or polyphenols obtained from olive mill wastewater (OMW). The extent of ET formation was determined by spectrophotometrically assessing the amount of extracellular DNA (exDNA) under both control conditions and in the presence of the different extracts. A decrease in the release of exDNA was associated with the co-treatment with PMA and GLE. Immunofluorescence and confocal microscopy allowed the evaluation of ET generation by RAW 264.7 cells co-treated with PMA and either GLE, RE, or OMW, both with and without DNase I, and revealed a decrease in the release and formation of ETs. Dot blot analysis of the culture media was utilized to detect nuclear histones and histone H4 citrullinated at position 3, released during the process of ETosis, in the culture medium of RAW 264.7 cells co-treated with either GLE, RE or OMW and PMA. In addition, the effects of this co-treatment on the main intracellular signaling pathways were evaluated by Western blot analysis, examining the levels of both JNK, ERK, AKT proteins and their phosphorylated counterparts. Preliminary MTT assay and Griess reactions were performed using polyphenols from OMW to ascertain their dose-dependent impact and anti-inflammatory capabilities. The data obtained provide a substantial foundation for subsequent in-depth molecular inquiry into the beneficial effects of the biomolecules contained in the extracts under study, specifically regarding their influence on ETosis and inflammatory pathways, while concurrently aligning with environmentally conscious and sustainable approaches.