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Nanosecond pulsed electric fields induce extracellular release of chromosomal DNA and histone citrullination in neutrophil-differentiated HL-60 cells.

ABSTRACT: Nanosecond pulsed electric fields (nsPEFs) have gained attention as a novel physical stimulus for life sciences. Although cancer therapy is currently their promising application, nsPEFs have further potential owing to their ability to elicit various cellular responses. This study aimed to explore stimulatory actions of nsPEFs, and we used HL-60 cells that were differentiated into neutrophils under cultured conditions. Exposure of neutrophil-differentiated HL-60 cells to nsPEFs led to the extracellular release of chromosomal DNA, which appears to be equivalent to neutrophil extracellular traps (NETs) that serve as a host defense mechanism against pathogens. Fluorometric measurement of extracellular DNA showed that DNA extrusion was rapidly induced after nsPEF exposure and increased over time. Western blot analysis demonstrated that nsPEFs induced histone citrullination that is the hydrolytic conversion of arginine to citrulline on histones and facilitates chromatin decondensation. DNA extrusion and histone citrullination by nsPEFs were cell type-specific and Ca²⁺-dependent events. Taken together, these observations suggest that nsPEFs drive the mechanism for neutrophil-specific immune response without infection, highlighting a novel aspect of nsPEFs as a physical stimulus.

SUBMITTER: Koga T

PROVIDER: S-EPMC6559984 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Toll-like receptor 2 (TLR2) is an important sensor for innate immune cells, including neutrophils, for the recognition of pathogen infection. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is a TLR2 ligand. LTA-induced TLR2 signaling pathways are well established in neutrophils. However, experimental studies regarding transcriptional regulation and the molecular mechanisms in primary human neutrophils are limited due to their short lifespan. The promyelocytic leukemia cell line, HL-60, can differentiate into a neutrophil-like phenotype following treatment with dimethyl sulfoxide. The aim of the present study was to investigate whether differentiated HL-60 (dHL-60) cells induced a similar gene expression profile upon LTA treatment as that previously determined for primary human neutrophils. After 4 or 24 h of Staphylococcus aureus LTA treatment, undifferentiated HL-60 (uHL-60) and dHL-60 cells were collected for RNA sequencing. The results demonstrated that hundreds of identical differentially expressed genes (DEGs) were observed in 1 and 10 µg/ml LTA-treated dHL-60 cells following 4 and 24 h of incubation, while almost no DEGs between LTA-treated HL-60 and dHL-60 cells were observed. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG), it was noted that the pathways of shared DEGs between the 1 and 10 µg/ml LTA-treated dHL-60 cells at both time points were significantly enriched in immune and inflammatory response-related pathways, such as cellular response to tumor necrosis factor, interleukin-1, interferon γ, neutrophil chemotaxis, the NF-κB signaling pathway and the Toll-like receptor signaling pathway. In addition, when comparing the effect of 1 and 10 µg/ml LTA treatment on dHL60 cells, it was found that all enriched GO and KEGG pathways were associated with the TLR signaling pathways of neutrophils. The results of the present study provided important information for the implementation of mRNA profiling in LTA-treated dHL-60 cells and may indicate the feasibility of using dHL-60 cells as a research model for TLR2 signaling in human neutrophils.

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Nanosecond pulsed electric fields induce extracellular release of chromosomal DNA and histone citrullination in neutrophil-differentiated HL-60 cells.

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