Awuah, Dennis Kwadwo Kyeremeh
(2019)
The role of mir-511-3p in modulating human dendritic cell function.
PhD thesis, University of Nottingham.
Abstract
MicroRNAs (miRNA) are functional, non-coding RNA molecules that negatively regulate gene expression by repressing target mRNAs. Recently, microRNA-511-3p (miR-511-3p) has emerged as a key player in regulating the function of human DCs and in controlling TLR4-mediated signalling. Previously, our group showed that c-type lectin receptors on DCs such as the mannose receptor (MR) is involved in uptake of allergens and downstream events leading to Th2 allergic responses. Interestingly, miR-511-3p is embedded within the MRC1 gene that encodes MR. Therefore, in this study, it was hypothesised that miR-511-3p maybe a key player in regulating MR expression on DCs and downstream events affecting Th polarisation. Additionally, miR-511-3p is highlighted to putatively target PPARγ, a potent suppressor of immune responses; however the link between miR-511-3p and PPARγ and its influence on DC function within the context of LPS-induced inflammatory responses is unknown.
Using a selection of miR-511-3p inhibitors and mimics, this study has shown for the first time that up or down-regulation of miR-511-3p has opposing effects on mRNA and protein levels of MR and another CLR (DC-SIGN) on human DCs. In addition, knockdown of miR-511-3p induced 1) an increase in IDO enzyme activity (after treatment with mannan and LPS); 2) upregulation of the PDL-1 surface marker and 3) an increase in IL-10 production, thereby promoting an anti-inflammatory DC phenotype and generation of T cells with increased IL-4 and decreased IL-17/IFN-γ production. This was in contrast to observations with miR-511-3p mimics, which promoted a pro-inflammatory DC phenotype. Furthermore, LPS stimulation of DCs, following knockdown of miR-511-3p was also able to upregulate RelB and A20 protein levels, which are key repressors of NF-κB activation, compared to their overexpressed counterparts, further highlighting the impact of miR-511-3p expression on human DCs.
Lastly, this study has demonstrated that changes in miR-511-3p expression inversely correlate with PPARγ expression and transcriptional activity following PPARγ activation with rosiglitazone (RSG), in the presence or absence of LPS. Interestingly, inhibition of miR-511-3p was also able to promote an anti-inflammatory DC characterised by increased IL-10 production following stimulation with RSG and LPS, whereas overexpression of miR-511-3p promoted IL-6 pro-inflammatory cytokine production. This suggests miR-511-3p targets PPARγ to inhibit its suppressive role.
Taken together, these observations highlight the complexity of miR-511-3p induced regulation of human DC phenotype and function and could ultimately pave way for rational design of therapies against a range of inflammatory disorders.
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