TRANSCRIPTOMIC PROFILING OF HIGH-SALT STIMULATED HUMAN NEUTROPHILS

RIVELLI, FEDERICO; MAZZITELLI, IGNACIO; BLEICHMAR, LUCÍA; BOGADO, MELISA; ERRA DIAZ, FERNANDO; GEFFNER, JORGE

Congreso; Reunion Anual Sociedad Argentina Inmunologia 2023; 2023

Neutrophils are the first cells recruited into sites of inflammation or infection, where they play a pivotal role in eliminating pathogens through a wide array of microbicidal mechanisms. Once recruited, neutrophil function can be modulated by physicochemical changes in the inflammatory milieu, including the accumulation of sodium. In fact, high salt (NaCl) concentrations are found in a number of tissues under physiological and pathological conditions. We have previously reported that human neutrophils exposed for a short period of time to high salt concentrations display a decreased ability to produce IL-8 and oxygen reactive intermediates (ROS in response to conventional agonists. By contrast, exposure to high salt concentrations for longer periods of time resulted in a strong activation signal, leading to the production of high levels of IL-8, an increased respiratory burst, and a marked synergistic effect on TNF-α production induced by LPS. To gain insight into the mechanisms by which high salt modulates neutrophil phenotype and function, we performed transcriptional profiling by RNAseq. Neutrophils were isolated from peripheral blood of healthy donors by conventional methods and were cultured for 4 hours in culture medium supplemented, or not, with NaCl (50 mM) and/or LPS (100 ng/ml) (n=4). We found that only 75 genes were differentially expressed between untreated (controls) vs LPS-stimulated neutrophils, being NFkB- signaling associated genes the top enriched pathway among upregulated genes (padj4). In contrast, 4812 genes were differentially expressed between untreated vs. high salt-treated neutrophils (padj4), revealing a broader and distinct transcriptional profile compared to the changes induced by LPS. Retinoic acid signaling and cell-cycle associated genes, as well as paraspeckle components (subnuclear bodies involved in the regulation of cell function), were among the top enriched pathways upregulated by exposure to high salt concentrations. Interestingly, the genes that were markedly up-regulated (>200X) in neutrophils by high salt, but not by LPS, included genes whose expression and function in neutrophils have not been previously analyzed such as ICAM-4, CENPU (Centromere Protein U), OSM (Oncostatin M), CYB5D1 (Cytochrome b5 domain containing 1), DDIT3 (DNA damage-inducible transcript 3), among others. Further studies are required to determine the role of these genes in neutrophil function.