| Summary: | Introduction: Liver X receptors (LXRα and LXRβ) are members of the nuclear receptor superfamily of ligand-activated transcription factors that regulate many biological and physiological processes. LXRs are important regulators of cholesterol and lipid metabolism and this is mediated by regulating a wide range of genes such as ABCA1 and ABCG1 that are involved in lipogenesis, cholesterol efflux and absorption, and bile acid synthesis. Since there is a relationship between chronic inflammatory diseases and lipid metabolic dysfunction, the role of LXRs has been investigated in different inflammatory diseases and disease models. Generally, LXRs have an anti-inflammatory effector function, however occasionally pro-inflammatory effects have also been reported. MicroRNAs (MiRNAs) are small, evolutionary conserved, single-stranded, non-coding RNA molecules with 20- 22 nucleotide base pairs. They regulate mRNA translation by fine tuning the production of proteins involved in the initiation or maintenance of inflammation. MiR-155 is one of the most studied members of miRNAs, and it has a regulatory role in certain inflammatory diseases such as collagen induced arthritis, lung fibrosis, and cardiovascular diseases. Idiopathic pulmonary fibrosis (IPF) is a devastating inflammatory disease of unknown aetiopathogenesis characterised by progressive breathlessness. IPF is characterised by approximately 50% survival of around 3 years after diagnosis, and there is no effective treatment. The main imperative for pulmonary fibrosis research is to identify potential causal inflammatory and remodelling pathways that contribute to IPF initiation and progression in order to determine possible candidate pathways for therapeutic intervention. Hypothesis: LXRs play an important role in lipid metabolism and cholesterol homeostasis and because there is a strong relationship between metabolic disease and chronic inflammatory and fibrotic diseases, e.g. LXR agonists may be beneficial for the treatment of RA. We proposed the following hypothesis ‘’ Liver X Receptors can modulate bleomycin-induced pulmonary fibrosis and therapeutic intervention with LXR agonists may be beneficial for the treatment of pulmonary fibrosis’. Methods & Results: Administration of the LXR agonist GW3965 to LXRα-/-β-/- or LXRαβ wild type mice given bleomycin to induce pulmonary fibrosis significantly exacerbated the severity of the disease only in LXRαβ wild type mice. The worsening of disease was seen as enhanced loss of body weight, increased inflammatory and fibrotic pathomorphological changes in the lung, increased inflammatory cells in the bronchoalveolar lavage, increased concentrations of several pro-inflammatory and pro-fibrotic mediators, and increased expression of genes that regulate inflammation and fibrosis, such as collagen and TGFβ, increased lung collagen content, and finally up-regulation of the expression of the alternative activated macrophages (M2)markers arginase 2 and IL-13 receptor. The effect of the LXR agonist was mediated specifically by LXRs because the severity of disease did not change in LXRα-/-β-/- mice given bleomycin and treated with GW3965, nor on similarly treated single LXRα or LXRβ gene-deleted mice. Furthermore, similar activation of LXRs in primary human or murine fibroblasts demonstrated up-regulation of the expression of collagen. The function of LXR agonist was directly on collagen gene expression and did not require de novo protein synthesis as demonstrated by the addition of cycloheximide as a translation inhibitor to murine primary fibroblasts activated with LXR agonist. This suggested that the LXR may have acted directly on the promoter region of the collagen gene. Also I investigated if the collagen genes have response elements for LXR in their promoter regions using a cell reporter system. I demonstrated that the collagen genes have response elements for LXR in their promoter regions. In a parallel set of experiments, administration of bleomycin or PBS to miR-155-/- and miR-155 wild type mice demonstrated a significantly stronger inflammatory and fibrotic process in the miR-155 gene-deleted mice. This worsening of disease was seen as an enhanced loss of body weight, increased inflammatory and fibrotic pathomorphological changes in the lung, increased inflammatory cells in the bronchoalveolar lavage, increased concentration of several pro-inflammatory and pro-fibrotic mediators, and increased expression of genes that regulate inflammation and fibrosis; such as collagen and TGFb, increased lung collagen content, and finally up-regulation of the expression of the alternative activated macrophages (M2)markers arginase 2 and IL-13 receptor. The effect was mediated specifically by miR-155 because the severity of disease was increased in miR-155-/- mice compared with miR-155 wild type mice given bleomycin. Also no differences were observed in miR-155-/- and miR-155 wild type mice given PBS. Conclusion: My results demonstrate that both the LXR and miR-155 have an important impact on the progression and extent of murine pulmonary fibrosis. Since completing the work for this thesis some of my additional pilot work has shown that LXR is a target for miR-155 and therefore both may have an important role in lung homeostasis. My results suggest the therapeutic approaches for IPF might include targeting the LXRs or LXR-regulated pathways, including potential fine tuning levels of LXRα with miR-155 antagonists. The aim would be to prevent excessive remodelling. Furthermore, any such therapeutic intervention would need to be done in a very careful way because LXRs are involved in many other physiological processes. Therefore, more targeted therapy perhaps controlling miR-155 may be of clinical relevance for therapeutic strategies.
|
|---|
