Neutrophil extracellular traps promote macrophage activation through Toll-like receptor engagement and PKA and NF-kB signaling pathways.

Neutrophils play key roles in innate immune responses and can release extracellular traps (NETs), characterized by chromatin exteriorization associated with cytoplasmic and granule proteins, such as neutrophil elastase, myeloperoxidase, HMGB1, and S100 family. These traps are released upon neutrophil activation by several factors, including inflammatory mediators and infectious agents. Since NETs interact with macrophages in various tissues in physiological contexts or pathological conditions, we aimed to elucidate the molecular mechanisms underlying cellular activation upon this interaction. Human monocyte-derived macrophages from healthy donors were in vitro exposed to NETs induced by the inactivated viruses HIV-1 (inHIV) or SARS-CoV-2 (inSARS), and ELISA was used to analyze the production of inflammatory mediators. The involvement of Toll-like receptors (TLRs) and the engaged signaling pathway was elucidated using pharmacological inhibitors. RNA sequencing was applied to analyze the transcriptional profile of macrophages exposed to IL-8-induced NETs. NETs increased macrophage production of reactive oxygen species and promoted NF-κB activation. Furthermore, NETs induced the release of the β-chemokines MIP-1α, MIP-1β, and RANTES, as well as the cytokines IL-6, IL-8, IL-10, and TNF-α. The inhibition of TLR2, TLR4, and NF-κB signaling abrogated macrophage production of inflammatory mediators induced by NETs. NET-mediated macrophage activation was also reduced upon inhibition of protein kinase A and blockade of actin polymerization, suggesting that both pathways are required for NET effects. RNA-seq revealed that 406 genes were differentially expressed, such as chemokines, transcription factors and metabolism-related genes. Gene Ontology analysis showed ten biological processes, six cellular components and seven molecular functions enriched in NET-treated macrophages, including chemoattractant activity, regulation of protein kinase activity, protein phosphorylation, and response to oxygen levels. Our findings show that NETs modulate the macrophage transcriptional profile and function through TLR2 and TLR4 engagement, and PKA and NF-kB recruitment.