MicroRNAs in the regulation of alternatively activated macrophages

Macrophages play a key role in maintaining the balance and efficiency of the immune response. TH2 cytokines IL-4 & IL-13, through shared IL-4Rα signalling, trigger a state of alternative activation in macrophages and also drive their proliferation. Alternatively activated macrophages (AAMΦ) are involved in the control of helminth infections and have also been implicated in tissue repair. However, TH2 weighted imbalance can result in inflammatory disorders such as asthma and fibrosis. Hence, macrophage responses must be tightly regulated. MicroRNAs, a short (~22nt) class of non-coding RNA, are one such immunomodulatory feedback mechanism that can regulate gene expression by targeting the 3’ UTR of mRNA resulting in destabilisation of the mRNA and/or inhibition of translation. With their ability for vast gene regulation, it was hypothesised that microRNAs could play a crucial role in the regulation of AAMΦ by targeting genes and pathways critical for their induction, maintenance & proliferation. Previously generated microarrays in the lab have allowed us to identify microRNAs differentially expressed in AAMΦ. In an effort to determine which microRNAs are genuinely associated with alternative activation, the first part of this project examined the expression profiles of ten shortlisted microRNA candidates under varying conditions of alternative activation, ranging from a reductionist in vitro IL-4/13 stimulation of macrophage cell lines to a complex in vivo TH2 mouse model of filarial infection. Profiling of microRNA expression under these conditions revealed that the expression of two IL-4Rα dependent microRNAs, namely miR-199b-5p and miR-378, along with another microRNA, miR-146, was highly regulated and consistently associated with alternative activation. The subsequent chapters of this thesis investigated the contribution of these microRNAs in regulating AAMΦ responses. Interestingly, we identified miR-199b-5p as being highly expressed in AAMΦ in vivo but not in vitro. Pathway analysis identified insulin signalling and other proliferative pathways such as PI3K/AKT as being highly targeted by miR-199b-5p. Overexpression of miR-199b-5p in RAW 264.7 cells resulted in a reduction in the rate of proliferation and a change in the levels of Insulin Receptor Substrate -1 (IRS-1), suggesting that miR-199b- 5p might regulate macrophage proliferation via insulin signalling. An alteration in the expression of YM-1 and RELM-α, markers characteristic of alternative activation, was also observed. MiR-199b-5p was successfully delivered to the lung and overexpressed in alternatively activated alveolar macrophages. No effect was observed on IL-4 induced proliferation, potentially due to the lack of significant insulin receptor and IRS-1 expression in alveolar macrophages. However, secreted levels of YM-1, but not RELM-α, were significantly reduced. MiR-378 is a microRNA that has previously been shown to be associated with AAMΦ through targeting of AKT-1; however, a direct influence of this microRNA on the regulation of this phenotype is yet to be determined. In this thesis, we have provided direct evidence of the impact of miR-378 deficiency on the regulation of AAMΦ and their responses using miR-378 KO mice. The ability of macrophages isolated from WT and KO animals to alternatively activate was studied in various systems both in vitro and in vivo. The influence of miR-378 deficiency on IL-4 induced proliferation was also addressed in vivo. Although the lack of miR-378 had no significant effect on IL-4 driven macrophage proliferation, results from this chapter support a role for miR-378 in the regulation of alternative activation through regulation of YM-1 and RELM-α expression. Lastly, to determine whether this regulation by miR-378 had functional consequences, we also utilised Litomosoides sigmodontis, a murine model of filarial infection. Due to experimental limitations, a concrete role for miR-378 in the context of infection could not be established. The final chapter of this thesis focuses on examining the role of miR-146 in the regulation of AAMΦ. MiR-146a is a highly studied microRNA that has previously been linked strongly to TH1 immune responses, especially classical activation of macrophages. However, a role for this microRNA in regulating AAMΦ is yet to be determined. Expression levels of miR-146a and miR-146b, the two isoforms of miR-146, were found to be differentially regulated upon alternative activation, with a decrease in miR-146a and increase in miR-146b expression in response to IL-4 both in vitro and in vivo. Based on this difference in expression and their known functions in suppressing excessive proinflammatory responses, it was hypothesised that miR-146a/b serve to regulate proinflammatory molecules (and signals) in a fine balance to allow efficient alternative activation to occur. However, the high sequence similarity between these two isoforms proved to be a hindrance to test this hypothesis in terms of shared targets. Therefore, the latter half of this chapter was devoted to the generation and optimisation of a stable cell line for the identification of microRNA targets using CLASH (cross-linking, ligation and sequencing of hybrids). In summary, the results from this thesis provide an important foundation for further studies of the functional role of microRNAs in the regulation of AAMΦ. Firstly, it characterises the expression profiles of ten different microRNAs differentially expressed during alternative activation. Secondly, for the first time, it identifies a role for miR-199b- 5p in the regulation of macrophage proliferation and activation. Thirdly, this thesis has provided direct evidence for the effect of miR-378 deficiency on AAMΦ responses. Lastly, it identifies and demonstrates the robust differential expression of two separate isoforms of the same microRNA (miR-146) under varying conditions of alternative activation, whose functional properties as regulators of the AAMΦ phenotype await further investigation