Medicine (RMH) - Theses
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ItemHigh mobility group box protein 1 in paediatric traumatic brain injury( 2018)Traumatic brain injury (TBI) is a leading cause of mortality and morbidity for children and young adults. Accumulating research suggests that children may be more vulnerable to poor long-term outcomes after TBI compared to adults, including a higher risk of developing post traumatic epilepsy (PTE). The neuroinflammatory response, known to contribute to neuropathology after TBI and the development of PTE, has been reported to differ depending upon age at the time of injury, though the time course and key factors in this response have not been well-characterized. One of the major initiators of the inflammatory cascade, high mobility group box protein 1 (HMGB1), shows age-dependent expression under basal conditions, and its elevation after TBI has been associated with worsened outcomes in young patients. This thesis therefore aims to characterize the acute time course of key neuroinflammatory cells and HMGB1 after paediatric and adult TBI, using an experimental mouse model. It further aims to evaluate the effect of acute inhibition of HMGB1 on the acute and long-term outcomes after paediatric TBI. To investigate this, we used a controlled cortical impact model in C57Bl/6 mice with a 3-week age-of-injury to represent a paediatric age and an 8- to 10-week age-of-injury to represent early adult age. Western Blot (WB) revealed a temporal profile of Iba1, GFAP and HMGB1 across the time course post-injury, which showed earlier and more robust expression in the paediatric than the adult cohort. . HMGB1 extracellular release was confirmed at 3 d post-injury by immunofluorescence. An increase in peripheral HMGB1 was found in serum from paediatric TBI mice, which was not evident in adult serum. Acute inhibition of HMGB1 with Glycyrrhizin (Gly) showed reduced oedema with a pre-treatment paradigm but a reduction of gliosis only with a post-treatment paradigm, suggesting that Gly actions were dependent upon the timing of treatment initiation after paediatric TBI. In a more chronic study, acute inhibition of HMGB1 improved some long-term cognitive deficits and reduced persistent microglia activation in the hippocampus but did not improve PTE outcomes or lesion size. Together, these findings demonstrate that the post-injury inflammatory cascade is influenced by age at the time of insult, which may be an important consideration for treatment strategies aiming to ameliorate this response after TBI. They also demonstrate that pharmacological inhibition of HMGB1 is both time-dependent and age-dependent after TBI. Therefore, HMGB1 is involved in secondary damage after paediatric TBI and may represent an important therapeutic target.
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ItemRegulation of a cytokine pathway in inflammation and arthritis( 2015)Rheumatoid arthritis (RA) is an idiopathic autoimmune/inflammatory joint disease. The involvement of granulocyte macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of RA is being indicated in clinical trials, but the underlying mechanism of action is still unclear. GM-CSF exhibits macrophage (MΦ)-polarising properties, where it can generate a more pro-inflammatory MΦ population. This thesis illustrates, for the first time, an important signalling cascade involving GM-CSF that is important for the development of murine inflammatory arthritis models.
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ItemCytokines, pain and regulation of inflammation( 2015)A mechanism termed the “inflammatory reflex” has been proposed as the basis for autonomic regulation of immune function. The efferent arm of this reflex – the neural-to-immune link – is thought to be the “cholinergic anti-inflammatory pathway”. According to this concept, an immune challenge is relayed by afferent nerves and/or by humoral signals to the brain, whereupon the brain drives efferent nerve fibres in the vagus that act ultimately to prevent the damaging consequences of excessive inflammation. The cholinergic anti-inflammatory pathway has been extensively studied in vivo and in vitro in terms of its protective effects against a wide range of inflammation-related diseases and immunomodulating functions. However, whether the in vivo anti-inflammatory effect of the cholinergic system can be mirrored and modelled by in vitro culture systems remains controversial and needs to be readdressed. The reported inhibitory effect of nicotine and the selective cholinergic agonist, AR-R17779, on the disease severity in murine collagen-induced arthritis could not be confirmed. Moreover, in a systematic in vitro study with LPS-stimulated murine macrophage populations and human monocyte/macrophage populations, it was found that nicotine did not reduce tumour necrosis factor (TNF) release even though in vivo it inhibited such expression in splenic macrophages. This suggests caution should be taken in the use of these particular in vitro systems as surrogate assays to study how the cholinergic pathway suppresses systemic inflammation. Inflammatory pain is multifaceted. Granulocyte macrophage-colony stimulating factor (GM-CSF) and TNF are potential drug targets for inflammatory and pain-related disorders based on the successful translation of animal data into clinical outcomes. In this thesis, TNF and GM-CSF were found to induce pain in an inflammatory footpad model in mice. It was found that neutrophil infiltration was required for TNF- and GM-CSF-induced pain. Colony stimulating factor-1 receptor signaling in macrophages was also required for inflammatory pain. In addition, TNF- and GM-CSF-induced pain was found to be abrogated in B6-KitW-sh/W-sh mice suggesting mast cell involvement. Ion channels, such as TRPV1, TRPA1, NaV1.7, and the neuropeptides, substance P and calcitonin gene-related peptide, were also found to be required for TNF- and GM-CSF-induced inflammatory pain development. The requirement for similar cell types and ion channels suggests that the pathways leading to TNF- and GM-CSF-driven pain might be similar and that TNF and GM-CSF may, in fact, be linked in this system. Utilizing GM-CSF-/- mice and an anti-GM-CSF mAb-based strategy, it was found that GM-CSF is required for TNF-induced pain. Moreover, treatment with anti-TNF mAb abolished GM-CSF-induced pain, indicating that TNF and GM-CSF are interdependent likely causing an amplification of the inflammatory response. Mice that lacked CCL17 were protected from inflammatory pain evoked by TNF and GM-CSF; CCL17 itself was able to induce inflammatory pain which remained unaffected in TNF-/- and GM-CSF-/- mice. These data indicate that CCL17 is a novel key downstream mediator of TNF and GM-CSF in these cytokine-driven inflammatory pain models. The pathway(s) linking these cytokines which ultimately leads to pain induction was found to be COX-2 dependent.
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ItemRetinal vascular calibre in rheumatoid arthritis: an evaluation of the relationship between systemic inflammation, immunosuppression and retinal vascular calibre( 2013)Background Retinal vascular calibre (RVC) measurement is currently a non-invasive research tool for assessing the human microcirculation for early vascular changes associated with systemic diseases. Widened retinal venular calibre is associated with systemic inflammation, increased cardiovascular (CV) risk and rheumatic diseases, particularly rheumatoid arthritis (RA) and high disease activity. To date there have been no longitudinal studies evaluating the impact of suppressing systemic inflammation on RVC in RA. Aims The primary aim of this thesis was to investigate the effects of suppressing systemic inflammation on serial RVC measurements in patients with RA with moderate to high disease activity. A secondary aim was to examine the stability of RVC measurements in patients with RA with well controlled, low disease activity receiving stable maintenance immunosuppression, to serve as a comparison for our primary analysis. Methods Two groups of patients with RA were recruited and studied concurrently. Group A included patients with moderate-to-high disease activity (DAS28-CRP>3.2) who required treatment escalation as standard of care. Group B had stable, low disease activity (DAS28-CRP≤3.2), did not require alteration of medical therapy and were the control for Group A. Patients in Group A underwent retinal photography at baseline, week six and week twenty-four to assess the respective early and late changes in RVC associated with escalation of systemic immunosuppression. Patients in Group B underwent retinal photography at baseline and at week twelve. Images were analysed by purpose designed software and a trained assessor masked to subject identity and timing of retinal photography. Linear regression and paired t-tests were used to compare serial RVC measurements in Groups A and B respectively, with a p value <0.05 considered significant. Results Group A included 26 patients (69% female) with a mean (SD) age of 50.7 (3.5) years and a mean (SD) disease duration of 7.1 (8.0) years. Disease activity significantly improved between baseline and follow-up at week six (mean reduction in DAS28-CRP score of -1.8 units; 95% CI -2.3 to -1.3 units, p<0.001) and week twenty-four (mean reduction in DAS28-CRP of -2.4 units; 95% CI -3.0 to -1.8 units, p<0.001). This improvement was accompanied by a significant reduction in retinal venular calibre (mean difference (MD) -10.6µm (95% CI -16.4 to -4.8µm, p=0.001) at week six, and MD -8.1μm (95% CI -14.1 to -2.1μm, p=0.009) at week twenty-four), which persisted after adjusting for companion retinal arteriolar calibre. Retinal arteriolar calibre significantly decreased at week six (MD -3.8μm; 95% CI -7.7 to -0.01μm, p=0.05) but not week twenty-four (MD -1.8μm; 95% CI -5.8 to 2.1μm, p=0.36). No significant change in retinal arteriolar calibre was found after adjusting for companion retinal venular calibre. Group B included 27 patients (81% female) with a mean (SD) age of 54.6 (1.8) years and a mean (SD) disease duration of 14.5 (10.9) years. As expected, disease activity and therapy remained unchanged between baseline and week twelve (DAS28-CRP MD -0.1, 95% CI -0.36 to 0.15, p=0.40). There was no significant change in retinal venular calibre (MD 1.37µm; 95% CI -2.83 to 5.58µm, p=0.51) or retinal arteriolar calibre (MD 0.39µm; 95% CI -3.05 to 3.83µm, p=0.82) in Group B during this period. Conclusion This is the first study to demonstrate that suppressing inflammatory disease activity in RA reduces retinal venular widening. This change was evident as early as six-weeks after treatment escalation and was maintained during twenty-four weeks follow-up. In contrast, retinal venular calibre measurements were unchanged in patients with RA with stable, low-level systemic inflammation during short-term follow-up. Taken together, these findings suggest that RVC measurement may be a biomarker of CV risk, and possibly endothelial dysfunction, in patients with RA and supports the concept that immunosuppressive treatment may have vascular benefits in RA.
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ItemThe role of myeloid cells in breast cancer metastasis( 2012)Metastasis is the leading cause of death in breast cancer patients. The current methods for predicting likelihood of metastasis are insufficient and treatment options are limited once distant metastasis occurs. Emerging data implicate myeloid cells as active participants in breast cancer metastasis. Myeloid cells in cancer are often studied as Gr-1+CD11b+ myeloid derived suppressor cells. However, this group of cells is comprised of many different cell types, including macrophages and neutrophils. Tumour-associated macrophages (TAMs) can play key roles in metastatic events but little is known about the roles of monocytes, i.e. macrophage precursors, in metastasis, and it has been suggested that monocyte/macrophage subpopulations may have different functions in breast tumour metastasis. Recent data link tumour-associated neutrophils (TANs) to the progression of malignancy in other cancers, but the link between TANs and breast cancer metastasis has not been well studied. This thesis is the first body of work to include a comprehensive analysis of monocytes/macrophages/TAMs and neutrophils/TANs during various stages of mammary tumour metastasis. Utilising three isogenic murine mammary tumour variants, namely 4T1.2 (highly metastatic), 66cl4 (weakly metastatic) and 67NR (non-metastatic), as well as the myeloid cell markers Ly6C (a marker for monocytes and neutrophils) and Ly6G (a marker for neutrophils), it was found that increases in both primary tumour Ly6Clow (non-classical, mature) TAM and TAN numbers, as well as that of circulating Ly6Chigh (classical, immature) monocytes and neutrophils were associated with metastasis. Furthermore, increases in these cells occurred early during tumour progression. Pro-MMP-9 was increased in the serum around this time; hence a model is proposed where increased production of MMP-9, either from the tumour or from the surrounding stroma, enhances mobilisation of monocytes and neutrophils leading to increased TAM and TAN numbers that promote metastasis. Furthermore, preliminary data indicate that co-injection of either TAMs and TANs isolated from highly metastatic tumours may promote the growth of less metastatic tumours. Colony stimulating factor-1 (CSF-1/M-CSF), a key macrophage growth factor, has been implicated in advanced breast cancer. Since inhibition of CSF-1 receptor (CSF-1R) signalling depletes TAMs and reduces metastasis in some mammary tumour models, CSF-1 and CSF-1R are considered targets for the treatment of metastatic disease. It is shown in this thesis, for the first time, that treatment of 4T1.2 tumour-bearing mice with neutralising anti-CSF-1R antibody can increase metastasis to lung and bone, without altering primary tumour growth. Furthermore, CSF-1R blockade leads to increased neutrophils in the primary tumour, metastasis-associated lung and peripheral blood, as well as to increased ‘classical’ Ly6Chigh blood monocytes. These data indicate that neutrophils can also play a critical role in 4T1.2 tumour metastasis. In support of this, it was found that increased expression of a key neutrophil CSF, G-CSF, in weakly metastatic primary tumours enhances circulating neutrophil and Ly6Chigh monocytes and is associated with increased metastasis. Thus, cellular changes in neutrophils and Ly6Chigh monocytes may be linked to metastasis and their subsequent increase upon anti-CSF-1R treatment may be related to the enhanced metastasis observed. The above data indicate that both macrophages and neutrophils may be involved during the early stages of breast tumour metastasis. Further analysis of monocyte/macrophage/TAM subpopulations, as well as neutrophils/TANs, may lead to improved treatment of aggressive breast tumours. Targeting both macrophages and neutrophils may be required for successful treatment of certain breast tumour subtypes. It may also be important to target monocytes and neutrophils both in the circulation and at metastatic sites and, if possible, during early stages of metastasis. Stratification of patients based on primary tumour TAM or TAN numbers may better predict the likelihood of metastasis and also guide treatment strategies.
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ItemDissecting autoimmune disease susceptibility in Lyn-deficient mice( 2011)This thesis dissects the nature of autoimmune disease development in mice deficient in the tyrosine kinase Lyn. In particular, the role of cellular defects, genetics, inflammation and its consequences that influence the pathogenesis of autoimmune disease are studied. Lyn-deficient mice develop antibody-mediated autoimmune disease resembling systemic lupus erythematosus (SLE). In this disease hyperactive B cells are major contributors to pathology. It is believed that the primary defects in B cell development, including an expansion of antibody-producing plasma cells, are responsible for disease, as interference with molecules essential for B cell function and development renders the mice disease-free. This thesis dissects the role of other immune cell types and molecules that as well as B cells make important contributions to disease. In addition to B cell developmental defects, several immune cell populations are perturbed in diseased Lyn-/- mice, including the expansion of immunosuppressive regulatory T cells, activated pathogenic T cells, dendritic cells, myeloid cells and erythroid cells. Despite expansion of functional Tregs, Lyn-/- mice remain autoimmune-disease prone, suggesting that the inflammatory environment in these mice may alter the suppressive capacity of these cells. In Lyn-/- mice, B cells produce interleukin-6 (IL-6), which facilitates activation of B and T cells, enhanced myelopoiesis, splenomegaly, and ultimately, generation of pathogenic autoreactive antibodies. These pathogenic autoantibodies deposit in the kidneys causing kidney pathology. Genetic deletion of IL-6 on a Lyn-/- background ablates disease in these mice without altering B cell development or plasma cell accumulation, partitioning cellular changes that are intrinsic to loss of Lyn from induction of autoimmunity. Lyn is an important regulator of autoimmune susceptibility as genetic deletion of Lyn on multiple genetic backgrounds results in autoimmune disease. Lyn is also a haploinsufficient gene for autoimmune disease as Lyn+/- mice develop autoimmune disease of varying severity, albeit milder and delayed. Genetic interaction between heterozygote mutations of Lyn and the inhibitory phosphatase SHP-1 result in amplification of cellular activation and autoantibody levels, ultimately resulting in exacerbated kidney pathology. This synergistic interaction between loss of key inhibitory molecules is highly relevant to human autoimmune disease, which is extensively polygenic and relies on multiple genetic anomalies. This thesis also examines the role of lymphangiogenesis in the development and exacerbation of autoimmune disease. Lymphangiogenesis occurs during development and de novo, where lymphatic vessels form in response to a stimulus. The lymphatic vasculature in Lyn-/- and Mev mutants are severely perturbed. These perturbations occur in a setting of autoimmune disease, characterized by expansion and activation of several immune cell subsets. The role of immune cells in producing lymphatic growth factors in response to an inflammatory stimulus was examined, with stimulated immune cells showing rapid and robust expression of vascular endothelial growth factors. These findings highlight the putative contribution of immune cell-derived growth factors to the expansion of lymphatic vasculature involved in the establishment of pathology. Collectively, the studies presented in this thesis provide strong support for the study of autoimmune disease in Lyn-/- mice and together highlight the complexity of autoimmune disease and pathogenic mechanisms present in this model.