DNMT1 mRNA Transfer VIA Macrophage-Derived Extracellular Vesicles Following LPS Exposure Regulates TNFα Gene...

Lipopolysaccharide (LPS) exposure results in prolonged immune dysfunction in human macrophages. Emerging evidence supports the hypothesis that epigenetic methylation of key pro-inflammatory genes contributes to this phenotype. Our data have shown that in response to LPS exposure, extracellular vesicles (EVs) contain high amounts of DNA methyltransferase (DNMT) mRNA cargo that is transferred to recipient cells with resultant methylation events and gene silencing at tumor necrosis factor alpha (TNFα). It is unknown which DNMT isoform is responsible for these methylation events.

EVs containing DNMT mRNA were generated by exposing human macrophages to LPS. Using siRNAs targeting the individual isoforms of DNMT (1, 3A, and 3B), we generated EVs with selectively packaged DNMT mRNAs for each isoform. Recipient macrophages were treated with these EVs, and methylation was assessed using bisulfate sequencing.

siRNAs targeting DNMTs 1, 3A, and 3B achieved successful knockdown. Macrophages exposed to LPS only demonstrated significant increases in DNMT1 mRNA. siRNAs directed at DNMT1 successfully knocked down mRNA expression. EVs generated from LPS exposure showed a 2-fold reduction in DNMT1 mRNA ( P < 0.001). In recipient cells exposed to these EVs, methylation of TNFα was reduced (58.3% vs. 31.3%). This reduction in methylation correlates with increased expression of TNFα mRNA after LPS exposure.

These data demonstrate that DNMT1 is the dominant mRNA isoform responsible for the methylation events and gene silencing seen in human macrophages after LPS exposure. Several pharmacologic therapies exist that selectively target DNMT1 function, which potentially can be utilized to limit the methylation events that occur at TNFα following LPS exposure.