Medicine (RMH) - Theses
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ItemContribution of CCL17 as an inflammatory mediator in rheumatoid arthritis( 2023-10)Rheumatoid arthritis (RA) is an inflammatory, progressive, and destructive autoimmune disease. During RA, intra-articular as well as extra-articular manifestations result in morbidity and extreme cases can lead to mortality. Currently available RA therapies, such as glucocorticoids, methotrexate and TNF-inhibitors are costly and accompanied by significant adverse side effects, as well as limited effectiveness in some patients, highlighting the need for new therapies. Pro-inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF), are among the important causative mediators in the development and progression of RA. Preclinical studies identified that CCL17 is a downstream mediator of GM-CSF. GM-CSF-induced CCL17 expression in monocytes and macrophages is via the epigenetic demethylase, Jumonji domain-containing 3 protein (JMJD3), and the transcription factor, interferon regulatory factor 4 (IRF4). While GM-CSF is one of the most essential cytokines for inducing CCL17 expression, TNF has also been implicated in CCL17 expression and pain development in preclinical models of arthritis in conjunction with GM-CSF. Furthermore, GM-CSF and TNF are produced by numerous cell types in response to various stimuli. Data suggested the possibility that TNF can engage in a cytokine loop, thus potentially linking TNF biology to the GM-CSF-CCL17 pathway. However, there is little evidence about the key cells involved in this linkage. CCL17 is a potential target for treating RA, as it accumulates in the serum and synovial fluid of patients. It is speculated that CCL17 regulates T cell migration to joints via the CCR4 receptor. This receptor is common for both CCL17 and CCL22 cytokines. In contrast to CCL17, CCL22 is decreased in the RA. At the commencement of this PhD project, no specific CCL17-suppressing drugs were available in the clinics. In addition, the cells that might be responsible for CCL17-driven pathology were unknown. In this PhD thesis, FDA-approved drugs were screened in the presence of GM-CSF to identify drugs with the potential to specifically suppress CCL17 expression, but not that of CCL22. Among the 1,500 screened drugs, 5 drugs suppressed GM-CSF-induced CCL17 expression and maintained/increased GM-CSF-upregulated CCL22 in both human monocytes and mouse macrophages, with no toxicity to the cells. The identified drugs were tested in the Zymosan-induced arthritis model (ZIA) to explore their potential to ameliorate arthritic pain and disease. Among them, four drugs significantly inhibited CCL17-dependent pain with a trend towards decreasing the disease in the ZIA model. Analysis of the mechanistic pathways of these drugs in the presence of GM-CSF demonstrated that inhibition of GM-CSF-induced CCL17 expression was due to suppression of both STAT5 activity and IRF4 protein expression. Dendritic cells are thought to be a key cell type that expresses CCL17 in RA patients. There is an imbalance in Tregs/Th17 cells in RA synovial samples which suggests the possibility of a chemotactic function of the increased CCL17 on Th17 cells. However, the potential relationship(s) between CCL17 and immune cells within the RA synovium has not been explored. In this PhD, it was found that macrophage marker (CD68) was the most prevalent marker that co-expressed with CCL17 and its receptor CCR4 in synovial membranes. Significantly, elevated expression of CCL17 was observed in RA synovium compared to that from healthy controls. The thesis showed that the effect of TNF on the expression of CCL17 induced by GM-CSF is different between human and mouse macrophages. Interestingly, TNF alone had no positive effect on CCL17 expression in human monocytes/macrophages as well as in mouse macrophages. In contrast to CCL17 expression, CCL22 was significantly upregulated by TNF in monocytes and macrophages. In conclusion, this thesis indicated the promising effects of repurposed FDA-approved drugs to ameliorate arthritic pain which could be due to inhibition of the GM-CSF/CCL17 pathway. These drugs might be able to be used in future clinical trials. Furthermore, it was shown that the type of immune cells that express CCL17 in response to other cytokines, such as TNF, would be different between humans and mice. This finding once more emphasises the complex role of TNF in inflammation. Furthermore, it was demonstrated that, in addition to T cells, macrophages are among the cells that are important in the pathogenesis of RA, supported by the co-expression of both CCL17 and CCR4 in synovium tissue samples from RA patients.
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ItemContribution of CCL17 as an inflammatory mediator in rheumatoid arthritis( 2023-10)Rheumatoid arthritis (RA) is an inflammatory, progressive, and destructive autoimmune disease. During RA, intra-articular as well as extra-articular manifestations result in morbidity and extreme cases can lead to mortality. Currently available RA therapies, such as glucocorticoids, methotrexate and TNF-inhibitors are costly and accompanied by significant adverse side effects, as well as limited effectiveness in some patients, highlighting the need for new therapies. Pro-inflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF), are among the important causative mediators in the development and progression of RA. Preclinical studies identified that CCL17 is a downstream mediator of GM-CSF. GM-CSF-induced CCL17 expression in monocytes and macrophages is via the epigenetic demethylase, Jumonji domain-containing 3 protein (JMJD3), and the transcription factor, interferon regulatory factor 4 (IRF4). While GM-CSF is one of the most essential cytokines for inducing CCL17 expression, TNF has also been implicated in CCL17 expression and pain development in preclinical models of arthritis in conjunction with GM-CSF. Furthermore, GM-CSF and TNF are produced by numerous cell types in response to various stimuli. Data suggested the possibility that TNF can engage in a cytokine loop, thus potentially linking TNF biology to the GM-CSF-CCL17 pathway. However, there is little evidence about the key cells involved in this linkage. CCL17 is a potential target for treating RA, as it accumulates in the serum and synovial fluid of patients. It is speculated that CCL17 regulates T cell migration to joints via the CCR4 receptor. This receptor is common for both CCL17 and CCL22 cytokines. In contrast to CCL17, CCL22 is decreased in the RA. At the commencement of this PhD project, no specific CCL17-suppressing drugs were available in the clinics. In addition, the cells that might be responsible for CCL17-driven pathology were unknown. In this PhD thesis, FDA-approved drugs were screened in the presence of GM-CSF to identify drugs with the potential to specifically suppress CCL17 expression, but not that of CCL22. Among the 1,500 screened drugs, 5 drugs suppressed GM-CSF-induced CCL17 expression and maintained/increased GM-CSF-upregulated CCL22 in both human monocytes and mouse macrophages, with no toxicity to the cells. The identified drugs were tested in the Zymosan-induced arthritis model (ZIA) to explore their potential to ameliorate arthritic pain and disease. Among them, four drugs significantly inhibited CCL17-dependent pain with a trend towards decreasing the disease in the ZIA model. Analysis of the mechanistic pathways of these drugs in the presence of GM-CSF demonstrated that inhibition of GM-CSF-induced CCL17 expression was due to suppression of both STAT5 activity and IRF4 protein expression. Dendritic cells are thought to be a key cell type that expresses CCL17 in RA patients. There is an imbalance in Tregs/Th17 cells in RA synovial samples which suggests the possibility of a chemotactic function of the increased CCL17 on Th17 cells. However, the potential relationship(s) between CCL17 and immune cells within the RA synovium has not been explored. In this PhD, it was found that macrophage marker (CD68) was the most prevalent marker that co-expressed with CCL17 and its receptor CCR4 in synovial membranes. Significantly, elevated expression of CCL17 was observed in RA synovium compared to that from healthy controls. The thesis showed that the effect of TNF on the expression of CCL17 induced by GM-CSF is different between human and mouse macrophages. Interestingly, TNF alone had no positive effect on CCL17 expression in human monocytes/macrophages as well as in mouse macrophages. In contrast to CCL17 expression, CCL22 was significantly upregulated by TNF in monocytes and macrophages. In conclusion, this thesis indicated the promising effects of repurposed FDA-approved drugs to ameliorate arthritic pain which could be due to inhibition of the GM-CSF/CCL17 pathway. These drugs might be able to be used in future clinical trials. Furthermore, it was shown that the type of immune cells that express CCL17 in response to other cytokines, such as TNF, would be different between humans and mice. This finding once more emphasises the complex role of TNF in inflammation. Furthermore, it was demonstrated that, in addition to T cells, macrophages are among the cells that are important in the pathogenesis of RA, supported by the co-expression of both CCL17 and CCR4 in synovium tissue samples from RA patients.