Transient up-regulation of miR-155-3p by lipopolysaccharide in primary human monocyte-derived macrophages results in RISC incorporation but does not alter TNF expression

Abstract

Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating that microRNAs contribute to this, although few studies have examined the early events that constitute the “primary” response. Methods: LPS-dependent changes to miRNA expression were studied in primary human monocyte-derived macrophages (1°MDMs). An unbiased screen by microarray was validated by qPCR and a method for the absolute quantitation of miRNAs was also developed, utilising 5’ phosphorylated RNA oligonucleotide templates. RNA immunoprecipitation was performed to explore incorporation of miRNAs into the RNA-induced silencing complex (RISC). The effect of miRNA functional inhibition on TNF expression (mRNA and secretion) was investigated. Results: Of the 197 miRNAs expressed in 1°MDMs, only five were induced >1.5-fold. The most strongly induced was miR-155-3p, the partner strand to miR-155-5p, which are both derived from the MIR155HG/BIC gene (pri-miR-155). The abundance of miR-155-3p was induced transiently ~250-fold at 2-4hrs and then returned towards baseline, mirroring pri-miR-155. Other PAMPs, IL-1β, and TNF caused similar responses. IL-10, NF-κB, and JNK inhibition reduced these responses, unlike cytokine-suppressing mycolactone. Absolute quantitation revealed that miRNA abundance varies widely from donor-to-donor, and showed that miR-155-3p abundance is substantially less than miR-155-5p in unstimulated cells. However, at its peak there were 446-1,113 copies/cell, and miR-155-3p was incorporated into the RISC with an efficiency similar to miR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNF secretion after 2hrs in 1°MDMs, but technical challenges here are noted. Conclusions: Dynamic regulation of miRNAs during the primary response is rare, with the exception of miR-155-3p. Further work is required to establish whether its low abundance, even at the transient peak, is sufficient for biological activity and to determine whether there are specific mechanisms determining its biogenesis from miR-155 precursors