Somatic cell reprogramming towards a pluripotent stem cell-like state occurs in an organized way, with rapid changes in gene expression during the first days of induction. Little is known about the mechanisms that lead to DNA double strand breaks (DSB) and genome instability during reprogramming. Amongst genes upregulated during reprogramming is that encoding the transcription factor B-Myb. \(B\)-\(myb\) RNA levels increase gradually from day 2 of reprogramming, earlier than those encoding pluripotency factors such as \(Nanog\). Despite its possible function as a regulator of the pluripotent state, B-Myb has a general role in the regulation of proliferation, and genome stability throughout the animal kingdom, raising the possibility that the activity of this protein is linked to the DSBs observed during reprogramming. To explore this possibility, reprogramming studies were performed in which B-Myb expression was modified: B-\(myb\)-/- MEFs were incapable of reprogramming after transduction with OSKM lentivirus (Oct4/Sox2/Klf4/c-Myc). However, unexpectedly, co-expression of B-Myb together with OSK (OSKB lentivirus), severely impaired the efficiency of reprogramming. Interestingly, the iPSC colonies appeared more ESC-like and had less DSBs. My results show that both the level of the B-Myb expression is crucial during early stages of reprogramming, indicating that B-Myb is a gatekeeper for reprogramming.

Depression is a major disease burden worldwide and, despite its prevalence and socioeconomic costs, around 30% of patients do not respond to currently available treatments. Inflammation is increasingly associated with, not only depressive illness but also resistance to existing therapies. This highlights the need for investigation of the mechanisms of neuro-inflammation, particularly in the context of peripheral inflammatory stimuli. Specifically, the chemokine molecular family is increasingly associated with human depressive illness, and neuro-inflammation and behavioural change in rodent models, making this an attractive molecular family for study. This thesis describes research aimed at investigating the association of these molecules with human depression and analysis of their role in an animal model of peripherally stimulated neuro-inflammation, the Aldara model of psoriasis-like inflammation. Systematic review and meta-analysis of the human biomarker literature using a random effects, inverse variance model revealed that a number of chemokines (CCL2, CCL3, CCL4, CCL11, CXCL4, CXCL7, CXCL8) are significantly associated with depressive illness in a human population. However this work revealed that there are a number of limitations of the human literature primarily associated with the methodological challenges of studies in human populations and confounding factors. Alongside this work, the Aldara model, which utilises the toll-like receptor 7 (TLR7) ligand imiquimod (IMQ), was investigated as a tool for studying neuroinflammation. Initial time-course investigation revealed that significant chemokine and cytokine transcriptional alterations occur within four hours at the local site of cutaneous treatment, the peripheral tissues and the brain. In addition, protein quantification in the brain confirmed that many of these transcriptional responses are translated to protein. Interestingly, it was shown that the brain response was temporally distinct from that of the peripheral tissues, and that in general brain responses were induced slightly more slowly and persisted for a longer period of time than those in the periphery. Investigation of Iba1+ (microglia/monocytes), GFAP+ (astrocytes) and CD3+ (T-cells) cells within the brain revealed significant changes in the microglial and T-cell populations, which were consistent with microgliosis and T-cell recruitment to the brain parenchyma. Changes in astrocyte populations were more equivocal although there was evidence of astrogliosis. Mechanistic investigations into responses to the Aldara model in inflammatory chemokine receptor (iCCR) KO mice did not reveal significant alterations in chemokine and cytokine transcription or in microglial responses to cutaneous Aldara treatment in the absence of the iCCRs (CCR1, CCR2, CCR3 and CCR5), but there did appear to be evidence of reduced CD3+ T-cell recruitment. In contrast, investigations in type I interferon receptor (IFNAR) KO mice identified a clear role for type I IFN signalling through IFNAR in the induction of chemokine and cytokine gene expression in the brain, and associated changes in Iba1+ microglial and CD3+ T-cell populations in response to cutaneous Aldara treatment. Mass spectrometric analysis of IMQ, the main active ingredient of Aldara, revealed that within four hours it enters both the circulation and the brain. The finding of IMQ within the brain parenchyma suggests that, while it is not an appropriate tool for studying peripheral-central immune crosstalk, it is a useful non-invasive model of TLR7 mediated neuroinflammation. These data provide compelling evidence of a role for chemokines in human depression and in neuro-inflammation, although the precise actions of this family of molecules remain unclear. In addition, building on previous work, the Aldara model appears to be a suitable tool for the study of neuro-inflammation, particularly interferon-driven immune responses, but is less appropriate for studying peripherally driven CNS immune reactions. Further work into the specific role of chemokines and associated cellular populations will hopefully provide additional insight into how CNS immune reactions are co-ordinated.

Death Receptor 3 (DR3) is a death domain (DD) containing member of the Tumour Necrosis Factor Receptor Superfamily (TNFRSF) and has a single acknowledged TNFSF ligand called TNF-like protein 1A (TL1A). Previous research has implicated roles for DR3 in host immune defence and in various inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease (IBD), atherosclerosis and allergic lung inflammation. This thesis investigated a potential role for DR3 in co-ordinating the innate immune response, using an in vivo Staphylococcus epidermidis supernatant (SES) model of acute peritoneal inflammation. A further point of investigation looked at the effect of the absence of DR3 on thickening of the peritoneal membrane induced by repeated SES inflammation. My results showed that the DR3/TL1A pathway is not essential in maintaining the number of peritoneal or blood leukocytes during naive conditions. Stromal DR3 was found to be important in co-ordinating the innate immune response after the induction of acute SES induced inflammation, with significantly lower numbers of specific myeloid and lymphoid cell subsets accumulating in the peritoneal cavity of DR3 knockout (DR3-/-) mice. Despite this reduction in selected leukocyte numbers, the proportion of infiltrating cells exhibiting proliferation and cell death was unaffected by the absence of DR3. However reduced leukocyte numbers were associated with a significant reduction in the concentration of multiple chemoattractants in DR3-/- peritoneal supernatants compared to those from DR3+/+ mice. Quantitative RT-PCR data (qPCR) were consistent with the peritoneal membrane being a source of many of these chemoattractants. vi Results presented here identify for the first time a pro-inflammatory role for stromal DR3 in the innate immune response. However after repeatedly inducing inflammatory conditions DR3 promoted thickening of the peritoneal membrane, while an absence of DR3 prevented aberrant inflammation-induced tissue fibrosis.

Rheumatoid Arthritis (RA) is a common chronic autoimmune disease that is characterized by synovial tissue inflammation eventually leading to joint destruction with severe functional deterioration and increased mortality – the underlying pathogenesis of RA remains unsolved. The principle of new therapeutic development is to define and characterize a molecular pathway both in terms of its basic biology and also its context-dependent effects in the synovial compartment. A hallmark pathological feature of RA is a rapid influx and accumulation of immune cells such as monocytes/macrophages into the synovium. Monocytes/ macrophages are major effector cells in RA synovitis, principally acting by releasing TNF-α, IL-6 and other inflammatory cytokines and chemokines. The recruitment of effectors cells is an important step in RA progression and is mediated by chemokines and their receptors. Two pathways will be studied in this project, both with potential relevance to the accumulation and activation of inflammatory leukocytes to the synovium, namely microRNAs and sphingolipid enzymes. MicroRNAs are a recently discovered class of post-transcriptional regulators that induce mRNA target degradation or translation inhibition. They have been shown to be involved in the regulation of the immune response and the development of autoimmunity. Of particular interest in the context of RA is miR-155, which is upregulated in RA synovial macrophages where it regulates cytokine expression such as TNF-α. Until now little was known about the role of miR-155 in regulating monocyte migration. Therefore, we sought to focus on the functional contribution of miR-155 in monocyte migration by the modulation of the expression of chemokines and their reciprocal chemokine receptors. Firstly the absolute copy numbers of miR- 155 transcripts in peripheral blood (PB) and synovial fluid (SF) monocytes of RA and healthy controls were assessed. To examine the role of miR-155 in monocyte migration and retention in the joint space, I overexpressed miR-155 in PB CD14+ monocytes of healthy controls and RA patients and examined the expression of chemokines and chemokine receptors mRNA levels by taqman low-density array (TLDA) and quantified the production of these chemokines in culture supernatant by multiplex assay. The role of miR-155 was investigated further using bone marrow monocytes (BMMO) from miR-155−/− and wild type (WT) mice. RA PB and SF CD14+ monocytes expressed higher copy numbers of miR-155 compared with healthy controls. RA SF monocytes exhibited the highest expression levels of miR- 155. The copy number of miR-155 expression was significantly increased in anti- citrullinated protein antibody (ACPA) positive RA compared with ACPA negative RA. The RA PB monocyte miR-155 copy number correlated positively and significantly with DAS28. Overexpression of miR-155 in PB monocytes led to an increased production of chemokines (CCL3/MIP-1α, CCL4/MIP-1β, CCL5/ RANTES, and CCL8/MCP2) and a reduction in expression of inflammatory chemokine receptor CCR2 while homeostatic CCR7 was up regulated. Commensurate with this, these receptors were expressed in an opposite direction inBMMO from miR-155 deficient cells; CCR7 was significantly down regulated and CCR2 expression level was increased. These observations suggest that CCR2 and CCR7 were under the tight control of miR-155 and that this regulation is preserved across the species; together suggesting that miR-155 can act as an important regulator of these receptors. We conclude that deregulation of miR- 155 in RA monocytes contributes to monocyte retention at sites of inflammation due to induction of chemokine production and down-regulation of inflammatory chemokine receptors. Furthermore, these data imply that miR-155 levels may reflect RA disease activity and could be a potential diagnostic or clinical disease activity biomarker for RA. Sphingosine kinases (SPHKs), SPHK1 and SPHK2, are isozymes that phosphorylate sphingosine into sphingosine-1- phosphate (S1P). S1P, a pleiotropic lipid mediator of inflammation, subsequently binds with any of the five G-protein coupled protein S1P receptors (S1PR1-5) and stimulates an array of cellular responses. Defects in S1P/S1PRs signalling have been shown to be associated with various pathologies. Until now however, no comprehensive analysis of expression of its components in RA has been performed. My data show that S1P concentrations were significantly elevated in the serum of RA patients with active disease compared to RA patients in remission and in healthy controls. Moreover, S1P1, S1P3, S1P5 and SPHK1 were differentially regulated in RA immune cell subsets, such as neutrophils, monocytes (CD14+), lymphocytes (CD4+ and CD8+) compared to healthy controls. In addition, compared with osteoarthritis (OA) pathological control, RA synovial tissues were strongly positive for the SPHK1, S1P1, and S1P3. Interestingly, RA patients treated with biological DMARDs had attenuated levels of SPHK1, S1P3 and S1P5, but not S1P1, when compared with patients treated with conventional DMARDs. Therefore, my study warrants further investigation of the clinical significance of S1P as a biomarker for disease activity and to explore the utility of novel therapeutic tools available to modulate the SPHK/S1PR/S1P axis in RA with a view to defining new therapeutic possibilities.