microRNA Expression Profile and Response of Alveolar and Peritoneal Derived Macrophages to Mycobacterial Infection

Tuberculosis (TB) is one of the devastating infectious diseases worldwide, with over 9 million new cases reported in 2013. Mycobacterium tuberculosis is the causative agent and it primarily infects lungs. Upon infection, lung macrophages are activated to contain the bacilli. The interaction between host macrophages and the bacilli determines the outcome of the disease. In 1993, the discovery of miRNAs has opened a new door to a deeper understanding on cell biology and the host inflammatory response against various diseases. Recent studies on miRNA have also revealed its importance during M. tuberculosis infection. This study investigated how mycobacterial infection modulates the miRNA expression profile in murine alveolar (AMJ2.C11) and peritoneal (IC-21) macrophage cell lines. The effect on bacterial virulence on miRNA expression was also examined by comparing infection with virulent M. tuberculosis and the vaccine strain M. bovis BCG. The result showed that different strain of Mycobacterium induced different miRNA expressions on both types of murine macrophage cell lines. Moreover, the pattern of bacterial killing was different on both types of cells. When a particular miRNA was knockdown, miR-146a chose in this study, AMJ2.C11 cells showed a reduction of bacterial load compared to IC-21 cells. This finding has opened a new path to further examine the significance of miRNAs including miR-146a in regulating host immunity to tuberculosis. Although further complex investigations need to be established, determining how miRNAs regulate immunity to M. tuberculosis may identify new targets for future therapeutic intervention

ROLE OF MICRORNA IN THE REGULATION OF TLR SIGNALING PATHWAY

Toll-like receptors (TLRs) play key roles in detecting pathogens and initiating inflammatory responses via the activation of specific signaling pathways. The TLRs activity must be tightly regulated to avoid excessive inflammation and consequent immunopathology, ranging from autoimmunity to cancer. MicroRNAs (miRNAs) are a new class of negative regulators involved in setting the balance of the immune response to inflammatory triggers. In this study, we identified miR-125a~99b~let-7e cluster and miR-146b as miRNAs that, after LPS engagement on human monocytes, are induced by the anti-inflammatory IL-10 and TGF\u3b2, but are inhibited by the pro-inflammatory IFN\u3b3. Bioinformatic analysis predicted and experimental evidence demonstrated that miR-125a-5p, let-7e-5p and miR-146b directly target the TLR pathway at multiple levels, including receptors (TLR4, CD14), signaling molecules (IRAK1, MyD88, TRAF6), and effectors (TNF\u3b1, IL-6, CCL3, CCL7, CXCL8). We showed that over-expression or inhibition of miR-125a, let-7e and miR-146b expression with lentiviral vector in human monocytes had a significant impact on the production of pro-inflammatory cytokines in response to LPS. In particular, we identified a role for miR-125a-5p and miR-146b in mediating the LPS hyporensponsiveness observed after IL-10 or TGF\u3b2 priming or during the endotoxin tolerance, the phenomenon of reduced sensitivity to subsequent challenge of LPS. The up-regulation of miR-125a-5p and miR-146b into THP-1 cells mimicked the LPS, IL-10 or TGF\u3b2 priming, whereas the inhibition of them by lentiviral vector or a pre-treatment with IFN\u3b3 reverted, partially, the tolerant phenotype. In an in vivo model of acute inflammatory response, we obtained that miR-125a-5p, miR-99b-5p and miR-146b were induced in macrophages recruited at the site of inflammation during the resolution process, and this was impaired in macrophages of IL-10 KO mice. Our studies indicated that miRNA cluster and miR-146b represent a new negative feedback mechanism of the TLR signaling pathway

Current perspectives on the role of interleukin-1 signalling in the pathogenesis of asthma and COPD

Asthma and chronic obstructive pulmonary disease (COPD) cause significant morbidity and mortality worldwide. In the context of disease pathogenesis, both asthma and COPD involve chronic inflammation of the lung and are characterised by the abnormal release of inflammatory cytokines, dysregulated immune cell activity and remodelling of the airways. To date, current treatments still only manage symptoms and do not reverse the primary disease processes. In recent work, interleukin (IL)-1α and IL-1β have been suggested to play important roles in both asthma and COPD. In this review, we summarise overwhelming pre-clinical evidence for dysregulated signalling of IL-1α and IL-1β contributing to disease pathogenesis and discuss the paradox of IL-1 therapeutic studies in asthma and COPD. This is particularly important given recent completed and ongoing clinical trials with IL-1 biologics that have had varying degrees of failure and success as therapeutics for disease modification in asthma and COPD

MicroRNA‐146 represses endothelial activation by inhibiting pro‐inflammatory pathways

Activation of inflammatory pathways in the endothelium contributes to vascular diseases, including sepsis and atherosclerosis. We demonstrate that miR-146a and miR-146b are induced in endothelial cells upon exposure to pro-inflammatory cytokines. Despite the rapid transcriptional induction of the miR-146a/b loci, which is in part mediated by EGR-3, miR-146a/b induction is delayed and sustained compared to the expression of leukocyte adhesion molecules, and in fact coincides with the down-regulation of inflammatory gene expression. We demonstrate that miR-146 negatively regulates inflammation. Over-expression of miR-146a blunts endothelial activation, while knock-down of miR-146a/b in vitro or deletion of miR-146a in mice has the opposite effect. MiR-146 represses the pro-inflammatory NF-κB pathway as well as the MAP kinase pathway and downstream EGR transcription factors. Finally, we demonstrate that HuR, an RNA binding protein that promotes endothelial activation by suppressing expression of endothelial nitric oxide synthase (eNOS), is a novel miR-146 target. Thus, we uncover an important negative feedback regulatory loop that controls pro-inflammatory signalling in endothelial cells that may impact vascular inflammatory diseases

A review of macrophage microRNAs' role in human asthma

There is an imbalance in asthma between classically activated macrophages (M1 cells) and alternatively activated macrophages (M2 cells) in favor of the latter. MicroRNAs (miRNAs) play a critical role in regulating macrophage proliferation and differentiation and control the balance of M1 and M2 macrophage polarization, thereby controlling immune responses. Here we review the current published data concerning miRNAs with known correlation to a specific human macrophage phenotype and polarization, and their association with adult asthma. MiRNA-targeted therapy is still in the initial stages, but clinical trials are under recruitment or currently running for some miRNAs in other diseases. Regulating miRNA expression via their upregulation or downregulation could show potential as a novel therapy for improving treatment efficacy in asthma

MicroRNAs in skin immunity and psoriasis

The skin protects the organism from the environment and shields it from the constant danger of infections by microorganisms. Keratinocytes are epithelial cells in the skin that constitute a physical barrier towards the environment. More than that, they are essential players in innate immunity: Keratinocytes can recognize invading pathogens by a variety of receptors, among them Toll-like receptors (TLRs). Activation of keratinocytes by pathogenic triggers leads to the induction of an inflammatory reaction in the skin, finally leading to the destruction and elimination of the pathogens. After clearance of the infection, homeostasis needs to be restored in order to avoid pathophysiological chronic inflammation. Psoriasis is a common chronic inflammatory skin disease characterized by local and systemic activation of both the innate and the adaptive immune system. In psoriasis skin lesions, hyperproliferation and activation of keratinocytes is combined with a massive infiltration of immune cells into the skin. MicroRNAs are endogenous short RNA molecules that regulate gene expression at the posttranscriptional level. They have been shown to be involved in the regulation of all basic biological processes. The aim of this thesis was to study the role of microRNAs in skin immunity, with a focus on their regulation and function in keratinocytes under homeostatic and inflammatory conditions. We have characterized systematically the microRNA expression profile of keratinocytes treated with ligands for TLR2, TLR5 and TLR3, showing that a distinct subset of microRNAs is regulated by different TLR ligands (Paper I). MiR-146a was strongly induced by all studied TLR ligands, while other microRNAs were regulated in a TLR- or time pointspecific manner. A detailed analysis of the regulation of miR-146a in keratinocytes revealed its long-lasting induction upon TLR2 stimulation, leading to a global repression of the inflammatory response (Paper II). Functionally, miR-146a acts as a negative feedback to counteract TLR2-induced inflammation and to restore tissue homeostasis by suppressing the production of inflammatory mediators and the chemotactic attraction of immune cells. Moreover, endogenous miR-146a was essential to prevent unstimulated keratinocytes from producing inflammatory mediators, thus protecting from unwanted inflammation in the absence of a trigger. In the chronically inflamed skin of psoriasis patients, miR-146a was overexpressed and keratinocytes were partially responsible for this phenotype (Paper III). Pro-inflammatory cytokines of the IL-1 family were shown to be strong inducers of miR- 146a, plausibly responsible for the miR-146a overexpression in psoriasis keratinocytes. Taken together, these results propose that miR-146a regulates skin immune responses after infection or skin injury and may set the threshold of activation in keratinocytes. We have identified miR-31 as another microRNA overexpressed in psoriasis keratinocytes and regulating the keratinocyte immune responses (Paper IV). MiR-31 could be induced by TGF-β1 in vitro and in vivo. Inhibition of endogenous miR-31 decreased the inflammatory activity of keratinocytes, suggesting that miR-31 acts as a pro-inflammatory microRNA and contributes to the chronic inflammation in psoriasis lesions. In conclusion, the data presented in this thesis underline the crucial importance of microRNAs in the innate immune response of keratinocytes. The modulation of the local inflammatory environment by microRNAs may explain more of the unknown underlying factors regulating susceptibility to autoimmune diseases such as psoriasis

The Yin and Yang of microRNAs: leukemia and immunity

Yin and Yang are two complementary forces that together describe the nature of real-world elements. Yin is the dark side; Yang is the light side. We describe microRNAs having both Yin and Yang characteristics because they can contribute to normal function (Yang) but also to autoimmunity, myeloproliferation, and cancer (Yin). We have been working on a number of microRNAs that have these dual characteristics and here we focus on two, miR-125b and miR-146a. We have concentrated on these two RNAs because we have very extensive knowledge of them, much of it from our laboratory, and also because they provide a strong contrast: the effects of overexpression of miR-125b are rapid, suggesting that it acts directly, whereas the effects of miR-146a are slow to develop, suggesting that they arise from chronic alterations in cellular behavior

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