MicroRNA regulation of macrophage activation
Hunter, Catriona Mhairi
Macrophages are mononuclear phagocytic cells that have diverse roles within the body. Tissue specific macrophages, e.g. Kupffer cells, microglia and osteoclasts, have roles in tissue homeostasis, while circulating macrophages play an important role in the innate immune system. Macrophages detect the presence of pathogen associated molecular patterns (PAMPs) via a range of receptors known collectively as pathogen recognition receptors (PRRs). Detection of pathogens causes the macrophages to become ‘activated,’ during which the macrophages undergo extreme morphological and translational changes that enable the pathogen to be neutralised and other immune system components to be recruited. Macrophage activation must be carefully regulated and promptly resolved, as chronic inflammation is damaging to the host. MicroRNAs have emerged as one mechanism by which activation is regulated. MicroRNAs are small, non-coding pieces of RNA that function as a post-transcriptional regulatory mechanism. Their action is exerted through binding with a complementary region in the 3’ untranslated region (3’UTR) of the target mRNA. This binding, facilitated by the ribonuclear protein complex RISC, prevents successful translation of the mRNA into its protein product. MicroRNAs have been shown to function across species, throughout development and during the adult life-span. In the immune system, microRNAs are known to be required for correct formation of germinal centres and normal development of B- and T-cells. MicroRNAs have also been shown to be differentially regulated during macrophage activation with different stimuli. In particular, miR-155, miR-146a and miR-21 are associated with macrophage activation by lipopolysaccharide (LPS). The objective of this work was to further understand the role of microRNAs during macrophage activation with LPS. Two approaches were adopted. Firstly, the regulation of individual microRNAs in LPS-activated bone marrow derived macrophages (BMDMs) was characterised by the use of illumina small RNA sequencing. Secondly, the requirement of the global microRNA population during macrophage biology was investigated through the use of DGCR8 and Dicer knockout systems. In keeping with the large number of changes reported in mRNA translation upon activation, expression of >400 microRNAs were found to be differentially regulated by exposure to LPS. Twelve of these microRNAs were chosen for further study (miR- 142-3p, -146a, -15b, -155, -16, -191, -21, -27b, -30b, -322-5p, -378 and -7a). Individual knock-down of these microRNAs in the RAW264.7 macrophage-like cell line mostly demonstrated subtle, rather than dramatic changes to the activation marker genes studied by RT-QPCR analysis. However, knock-down of miR-146a, -15b, - 155 and -191 were able to significantly alter the expression of the activation marker genes (Tnf-a, Cox2, Cxcl2, Il-6 and Saa3). Interestingly, knock-down of miR-142-3p, miR-146a and miR-155 appeared to show cross-regulation of these microRNAs. The cell index (CI) data suggested that miR-191 and miR-21 influence adhesion in activated macrophages. Studies with the DGCR8 and Dicer knockout systems showed that the global microRNA population was required for successful differentiation of macrophages from embryonic stem cells, and for normal expression of differentiation and activation markers in bone marrow derived macrophages. Overall, these results show that dynamic expression of microRNAs is an integral part of the macrophage response to LPS.
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