Wong, Tsz Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 140-159). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abbreviations --- p.III / Abstract --- p.V / 摘要 --- p.VIII / Publications --- p.XI / Table of Contents --- p.XII / Chapter Chapter 1: --- General Information / Chapter 1.1 --- Characteristics and Prevalence of SLE --- p.1 / Chapter 1.2 --- Diagnosis of SLE --- p.2 / Chapter 1.3 --- Assessment of Disease Activity --- p.4 / Chapter 1.4 --- Causes of SLE --- p.4 / Chapter 1.4.1 --- Genetic Factors --- p.4 / Chapter 1.4.2 --- Hormonal Factors --- p.6 / Chapter 1.4.3 --- Environment Factors --- p.7 / Chapter 1.5 --- Drug Treatments of SLE --- p.8 / Chapter 1.6 --- Immunological Dysregulation in SLE --- p.9 / Chapter 1.6.1 --- Types and Properties of Lymphocytes --- p.9 / Chapter 1.6.2 --- Cytokines and Chemokines --- p.14 / Chapter 1.6.3 --- Toll-like Receptors --- p.17 / Chapter 1.6.4 --- Intracellular Signal Transduction Pathways --- p.21 / Chapter 1.7 --- Objectives of Our study --- p.25 / Chapter Chapter 2: --- Material and Methods / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- SLE Patients and Control Subjects --- p.27 / Chapter 2.1.2 --- Reagents for cell culture --- p.28 / Chapter 2.1.3 --- Reagents for Flow Cytometry --- p.30 / Chapter 2.1.4 --- Reagents for Phosphorylation State Analysis --- p.33 / Chapter 2.1.5 --- Reagents for Total RNA Extraction --- p.35 / Chapter 2.1.6 --- Reagents for Polymerase Chain Reaction (PCR) --- p.36 / Chapter 2.1.7 --- Reagents for Gel Electrophoresis --- p.38 / Chapter 2.1.8 --- Reagents for Real-Time Polymerase Chain Reaction --- p.39 / Chapter 2.1.9 --- Other Reagents --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Preparation of Plasma and Purification of Peripheral Blood Mononuclear Cells (PBMC) from EDTA-Blood --- p.41 / Chapter 2.2.2 --- Immunophenotyping of Cell Surface Molecules by Flow Cytometry --- p.42 / Chapter 2.2.3 --- Immunophenotyping of Intracellular Molecules by Flow Cytometry --- p.43 / Chapter 2.2.4 --- Phosphorylation State Analysis of Intracellular Signaling Molecules --- p.43 / Chapter 2.2.5 --- Cytometric Bead Array of Cytokines and Chemokines --- p.44 / Chapter 2.2.6 --- Total RNA Extraction from PBMC --- p.46 / Chapter 2.2.7 --- Reverse Transcription of the Extracted Total RNA --- p.46 / Chapter 2.2.8 --- Real-time Polymerase Chain Reaction --- p.46 / Chapter 2.2.9 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.48 / Chapter 2.2.10 --- Enzyme-Linked Immunosorbent Spot (ELISPOT) --- p.48 / Chapter 2.2.11 --- Statistical Analysis --- p.49 / Chapter Chapter 3: --- B Cell Chemokine CXCL13 in SLE / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Methods --- p.52 / Chapter 3.3 --- Results --- p.52 / Chapter 3.3.1 --- Characteristics of SLE Patients and Control Subjects --- p.52 / Chapter 3.3.2 --- Expression of Plasma CXCL13 --- p.53 / Chapter 3.3.3 --- Gene Expression of TNF-α in PBMC --- p.53 / Chapter 3.3.4 --- Expression of sTNFRl in Plasma --- p.53 / Chapter 3.4 --- Discussion --- p.57 / Chapter Chapter 4: --- T lymphocyte Co-stimulatory Molecule CD26 in SLE / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Methods --- p.64 / Chapter 4.3 --- Results --- p.66 / Chapter 4.3.1 --- Characteristic of SLE Patients and Control Subjects --- p.66 / Chapter 4.3.2 --- Expression of Human CD26 in Plasma --- p.66 / Chapter 4.3.3 --- "Cell Surface Expression of CD26 on Monocytes, Th, Tc plus Ts, B and iNKT Lymphocytes" --- p.66 / Chapter 4.3.4 --- Circulating Number of iNKT Lymphocytes --- p.67 / Chapter 4.4 --- Discussion --- p.71 / Chapter Chapter 5: --- Thl7 Lymphocytes and Expression of IL-17 in SLE / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- Methods --- p.78 / Chapter 5.3 --- Results --- p.79 / Chapter 5.3.1 --- Characteristics of SLE Patients and Control Subjects --- p.79 / Chapter 5.3.2 --- Ex vivo production of IL-17A from PBMC --- p.79 / Chapter 5.3.3 --- Circulating Number of Thl7 Lymphocytes --- p.79 / Chapter 5.4 --- Discussion --- p.82 / Chapter Chapter 6: --- Intracellular Mitogen Activated Protein Kinases in SLE / Chapter 6.1 --- Introduction --- p.87 / Chapter 6.2 --- Methods --- p.89 / Chapter 6.3 --- Results --- p.91 / Chapter 6.3.1 --- Characteristics of SLE Patients and Control Subjects --- p.91 / Chapter 6.3.2 --- Expression of Phospho-p38 MAPK in PBMC --- p.91 / Chapter 6.3.3 --- Expression of Phospho-ERK in PBMC --- p.92 / Chapter 6.3.4 --- Expression of Phospho-JNK in PBMC --- p.92 / Chapter 6.3.5 --- "Relative Percentage Increase of Phosphorylated p38 MAPK, ERK and JNK upon IL-18 Activation" --- p.93 / Chapter 6.4 --- Discussion --- p.104 / Chapter Chapter 7: --- Toll-like Receptors in SLE / Chapter 7.1 --- Introduction --- p.111 / Chapter 7.2 --- Methods --- p.113 / Chapter 7.3 --- Results --- p.115 / Chapter 7.3.1 --- Characteristics of SLE Patients and Control Subjects --- p.115 / Chapter 7.3.2 --- Expression of TLRl to TLR9 of PBMC --- p.115 / Chapter 7.3.3 --- Preliminary Results of Cytokine and Chemokine Expression Upon TLR Lignad Activation --- p.116 / Chapter 7.4 --- Discussion --- p.126 / Chapter Chapter 8: --- Conclusion and Future Perspectives --- p.133 / Appendix --- p.138 / References --- p.140

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with a strong and complex genetic basis. To dissect the function of the lupus susceptibility loci on New Zealand black (NZB) mouse chromosome 1, the lab had previously generated congenic mice with an introgressed homozygous NZB chromosome 1 intervals extending from ~35 or ~82 to 106 cM on the C57BL/6 background. Although both mouse strains made IgG anti-nuclear antibodies (ANAs), ANA titres and cellular activation were significantly higher in mice with the longer interval. These studies suggest the presence of two susceptibility genes. In this thesis I have sought to further characterize the cellular abnormalities and underlying genetic polymorphisms that produce them in these mice. Using mixed hematopoietic chimeric mice, with a mixture of tagged-B6 and congenic bone marrow I demonstrate that there are intrinsic B and T cell functional defects in chromosome 1 congenic mice. I further show that an intrinsic B cell defect is required for efficient recruitment of B cells into the spontaneous germinal centres and differentiation of autoantibody producing cells in these mice. To more precisely localize the susceptibility loci, I produced and characterized a number of additional subcongenic mouse strains. This revealed surprising genetic complexity with the presence of at least four lupus susceptibility loci and a suppressor locus on chromosome 1, several of which appeared to impact on T cell function. Finally, I generated bicongenic mice carrying both NZB chromosome 1 and 13 intervals, hypothesizing that since these were two of the major intervals associated with autoimmune disease in NZB mice they would fully recapitulate the autoimmune phenotypes. Although this hypothesis was incorrect, several novel phenotypes developed including marked expansion of the plasmacytoid and myeloid dendritic cell compartments and increased BAFF and IgA autoantibody production. Although this expansion was associated with TLR hyper-responsiveness, disease severity remained mild, possibly due to the lack of IFN- production, which appeared to be inhibited in these mice. Thus, lupus arises from immune defects affecting several cellular populations, which are the product of multiple genetic polymorphisms that interact in a complex fashion to produce the autoimmune phenotype.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with a strong and complex genetic basis. To dissect the function of the lupus susceptibility loci on New Zealand black (NZB) mouse chromosome 1, the lab had previously generated congenic mice with an introgressed homozygous NZB chromosome 1 intervals extending from ~35 or ~82 to 106 cM on the C57BL/6 background. Although both mouse strains made IgG anti-nuclear antibodies (ANAs), ANA titres and cellular activation were significantly higher in mice with the longer interval. These studies suggest the presence of two susceptibility genes. In this thesis I have sought to further characterize the cellular abnormalities and underlying genetic polymorphisms that produce them in these mice. Using mixed hematopoietic chimeric mice, with a mixture of tagged-B6 and congenic bone marrow I demonstrate that there are intrinsic B and T cell functional defects in chromosome 1 congenic mice. I further show that an intrinsic B cell defect is required for efficient recruitment of B cells into the spontaneous germinal centres and differentiation of autoantibody producing cells in these mice. To more precisely localize the susceptibility loci, I produced and characterized a number of additional subcongenic mouse strains. This revealed surprising genetic complexity with the presence of at least four lupus susceptibility loci and a suppressor locus on chromosome 1, several of which appeared to impact on T cell function. Finally, I generated bicongenic mice carrying both NZB chromosome 1 and 13 intervals, hypothesizing that since these were two of the major intervals associated with autoimmune disease in NZB mice they would fully recapitulate the autoimmune phenotypes. Although this hypothesis was incorrect, several novel phenotypes developed including marked expansion of the plasmacytoid and myeloid dendritic cell compartments and increased BAFF and IgA autoantibody production. Although this expansion was associated with TLR hyper-responsiveness, disease severity remained mild, possibly due to the lack of IFN- production, which appeared to be inhibited in these mice. Thus, lupus arises from immune defects affecting several cellular populations, which are the product of multiple genetic polymorphisms that interact in a complex fashion to produce the autoimmune phenotype.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 228-261). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 202-219). Also available in print.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse manifestations affecting multiple organs. Current understanding of its pathogenesis remains largely inadequate despite recent progress in SLE research on the characterization of immune system dysfunction and its link with heritable and environmental factors. Dendritic cells (DCs) are immune cells that express pattern recognition receptors (PRRs) for the recognition of pathogen associated molecular patterns (PAMPs). Aberrant functions of DCs have been reported in SLE including recognition of self-nucleic acids, presentation of self-antigens and strong induction of interferon response, depending on their expression of PRRs. Dectin-1 is a non-toll like receptor PRR that is highly expressed in DCs for the recognition of pathogenic carbohydrates found mostly in fungi. Recognition of fungal carbohydrates by dectin-1 promotes DC maturation and production of pro-inflammatory cytokines that preferentially skew towards a T helper-17 (Th17) response which has been found to be engaged in the pathogenesis of SLE and other autoimmune diseases. Therefore, the current investigation aimed to study the expression of dectin-1 and the effect of dectin-1 activation in DCs from SLE patients. Dectin-1 expression on CD14+ monocytes from peripheral blood of SLE patients and healthy controls was measured by flow cytometry. SLE patients (mean+/-SD = 92.05+/-3.471%) were found to have higher dectin-1 expression on CD14+ monocytes compared to controls (mean+/-SD = 83.7+/-14.64%) (p=0.02). Monocyte derived DCs (MoDCs) were then derived from CD14+ monocytes in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. Pre-treated moDCs with curdlan, a dectin-1 specific ligand, showed increased expression of costimulatory molecules including CD83 and CD86 and enhanced production of IL-1β compared with healthy controls (all p<0.05). Curdlan treated moDCs were further co-cultured with CD4+ na?ve T cells and polarization into Th17 cells was subsequently evaluated by measuring the percentage of Th17 per moDCs-na?ve T cells and expression of intracellular IL-17. Curdlan treated moDCs from SLE patients were found to have enhanced Th17 polarization capacity (mean+/-SEM = 28.33+/-5.64%) compared with controls (mean+/-SEM = 12.77+/-2.56%) (p<0.05). To address the mechanism of Th17 polarization, expression of caspase-1 which promotes the production of IL-1β was measured. Curdlan treated moDCs in SLE patients expressed higher levels of caspase-1 detected in the cell lysate as measured using Western Blot compared with healthy controls (p<0.05). In conclusion, compared with healthy individuals, SLE moDCs were more matured and activated in response to β-glucan as shown by their higher expression of co-stimulatory molecules, enhanced production of IL-1β and stronger Th17 polarizing effect. These findings suggest functional dysregulation of dectin-1 expressing DCs in patients with SLE which may be involved in the pathogenesis of this condition. / published_or_final_version / Medicine / Master / Master of Philosophy

Subsets of systemic lupus erythematosus (SLE) patients with distinct patterns of disease manifestations and autoantibody production have been reported, but seldom have these two phenomena been analysed together. Cluster analysis was performed on 1928 Chinese SLE patients based on autoantibody profile and the frequencies of various clinical manifestations were compared between each cluster. Separate association analyses between individual autoantibodies and clinical manifestations, as well as between clinical manifestations, were also performed. This study identifies three separate autoantibody clusters each with different clinical manifestations, and proposes that the phenomena of autoantibody clustering and clinical subsets may be inter-related. Patient clusters could also be stratified into a bipolar spectrum. On one end are patients with over-representation of anti-dsDNA and renal disorder; whilst on the other end are two distinct autoantibody clusters (anti-Sm/anti-RNP/aPL and aPL/anti-Ro/anti-La) with overlapping of other non-renal manifestations. Patient stratification could aid disease prediction and subsequent management. These findings may also elucidate disease pathogenesis and guide future study on potential common pathological processes within autoantibody clusters. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that brings physical and psychological turmoil to patients. Neuropsychiatric (NP) involvement in SLE also complicates disease management compared with non-NP SLE. The joint pain caused by the dysfunctional inflammatory response, as well as the negative mood induced by the disease have been found to predict the patients’ quality of life. The primary goal of this study is to examine the mediation effect of negative mood on the relationship between pain and quality of life in patients with SLE based on a biopsychosocial approach. Results revealed that negative mood partially mediated the relationship between pain and the physical aspect of quality of life. On the other hand, negative mood completely mediated the relationship between pain and psychological health. The mediation relationship lends support to the biopsychosocial perspective that physical distress, psychological state, and one’s adaptive functioning are closely related. The secondary goal of this study is to explore the effect of NP involvement in patients with SLE on self-report variables, including perceived pain intensity, negative mood, fatigue, sleep quality, perceived cognitive difficulties, and quality of life in comparison with patients with non-NP SLE as well as with healthy controls. / published_or_final_version / Clinical Psychology / Master / Master of Social Sciences

Systemic lupus erythematosus (SLE) is a chronic multi-organ autoimmune disease that is characterised by diverse clinical manifestations. Immunologically, SLE features a prominent “interferon (IFN) signature” which is marked by an elevated expression of type I IFN-regulated genes in blood and tissue cells of patients with this condition. Plasmacytoid dendritic cells (pDCs), also known as the most potent type I IFN-producing cells, are therefore considered the major culprit in SLE pathogenesis. Previous studies from our group have demonstrated abnormalities in circulating and bone marrow (BM)-derived pDCs from SLE patients. In the light of this, the present study was undertaken to further evaluate the role of pDCs in SLE development and to seek for key mediator(s) that might lead to functional aberrations of pDCs in this condition. Recently, a growing attention has been drawn to microRNAs (miRNAs) for their critical role in regulating immune cell function and strong association with autoimmune diseases. Therefore, the current study hypothesised that microRNAs played an important role in modulating pDC response(s) to toll-like receptor (TLR) stimulation, and that dysregulated microRNA expression induction was responsible for pDC abnormalities in SLE pathogenesis. The spontaneous lupus mouse model, F1 hybrid of New Zealand Black and White strains (NZB/W F1), was used in this study. The disease profile of NZB/W F1 was characterised based on the development of serum antinuclear antibodies and proteinuria. Specifically, the development of lupus in these mice (symptomatic mice) was illustrated by high titres of serum antinuclear antibodies, persistent proteinuria, glomerular immune complex deposition and elevated expression of pro-inflammatory cytokine genes in the kidney. Young NZB/W F1 (pre-symptomatic) as well as age- and sex-matched non-lupus maternal NZW mice were used as controls. While the development of pDCs appeared to be unaffected by lupus, elevated upregulation of MHC class II and co-stimulatory molecules, and induction of IFN-stimulated gene Ifitm3 in TLR7-stimulated lupus pDCs suggested phenotypic and functional hypersensitivity of these cells. Furthermore, analysis of the expression profile of miRNAs in pDCs upon TLR7 activation identified six differentially regulated targets. Among these, miR-155 was the most highly induced and its induction was consistently higher in pDCs from symptomatic NZB/W F1 mice. Nevertheless, transfection of miR-155 mimics into pre-symptomatic pDCs resulted in a reduced expression of Ifitm3, suggesting that miR-155 has a negative regulatory role in IFN production in pDCs. The finding of upregulated induction of miR-155 in lupus pDCs reported in this thesis is in line with previous studies, which showed increased expression of miR-155 in splenic lymphocytes of lupus NZB/W F1 mice. Results obtained from the transfection experiments are also in accordance with other previous studies, which showed miR-155 functioned as a negative feedback regulator of IFN production in pDCs. However, the mechanism of the association between miR-155 expression and increased IFN response in SLE requires further investigations. It is hoped that findings from this study contribute to a better understanding of SLE pathogenesis and ignite future interests in evaluating the molecular layer of regulation in autoimmunity. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is characterized by auto-reactive T and B lymphocytes and abundant auto-antibodies against nuclear components that form immune-complexes and lead to inflammation, organ dysfunction and failure. The current treatment for SLE includes corticosteroid and immunosuppressant agents which are associated with side effects. Immunotherapy such as tolerogenic dendritic cells (DCs) and regulatory T cells have potential therapeutic implications in autoimmune diseases. DCs are professional antigen presenting cells with important role in promoting immune response and maintaining peripheral tolerance. Alternatively activated DC (aaDC) derived from treating monocyte-derived DCs with vitamin D3 and dexamethasone has demonstrated tolerogenicity and suppressed activation and proliferation of allogeneic T cells in in vitro human studies. As circulating DCs in SLE patients were reported to be hyperactive with increased expression of co-stimulatory molecules and hyper-responsiveness to immunostimulatory stimuli. Therefore, this study aims to examine if aaDCs derived from SLE patients possess tolerogenic properties, to delineate the underlying mechanisms and to examine for therapeutic effect by adoptive transfer of tolerogenic DCs in lupus mouse model. We found that lupus aaDCs derived in vitro displayed semi-mature phenotype with lower expression of co-stimulatory molecules compared with mature DCs. The tolerogenic phenotype remained stable despite challenge by CD40L, CpG-DNA and SLE serum. Lupus aaDCs showed comparable tolerogenic properties as aaDCs from healthy subjects with suppressive effect on allogeneic T cell activation and proliferation. In addition, lupus and normal aaDCs were shown to polarize normal and lupus naïve T cells into IL-10+ suppressive T cells that showed antigen-nonspecific suppressive effect on allogeneic third-party T cells. On the other hand, lupus and normal aaDCs skewed memory T cells to less inflammatory phenotype with reduced expression of IFN-ɤ and IL-17. Although aaDCs displayed a cytokine profile of IL-12loIL-10hi, addition of neutralizing anti-IL-10 and exogenous IL-12 did not reverse the suppressive effect of aaDCs on allogeneic T cells, suggesting their tolerogenicity was not related to cytokine imbalance between IL-12 and IL-10. Furthermore, aaDCs were found to express reduced level of RelB, a transcription factor regulating DC differentiation and maturation. As RelB can be a potential target to induce stable tolerogenic DCs, we constructed RelB shRNA to silence RelB in bone marrow derived DCs (BMDCs) from MRL/MPJ mice. The RelB shRNA transduced BMDCs showed lower level of RelB compared with scramble control shRNA, and displayed tolerogenic phenotype with decreased co-stimulatory molecules, but had no effect on the expression of chemokine receptors. When co-cultured with allogenic CD4+ T cells, RelB shRNA modified BMDCs showed suppressive function on T cell activation and proliferation and increased the production of IL-10 by T cells. However, in vivo study based on 5 mice per treatment group did not show significant effect of RelB shRNA modified BMDCs on disease progress of lupus mice compared to control mice. In conclusion, lupus aaDCs demonstrated tolerogenic properties with induction of IL-10 producing T cells with regulatory functions. RelB shRNA modified BMDCs showed tolerogenic properties in vitro but their in vivo effect on alleviation of murine lupus disease needs further study. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy