1,721,307 research outputs found
Plasmacytoid dendritic cells : their functional abnormalities and regulatory mechanisms in the development of systemic lupus erythematosus
No full textSystemic 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_versionMedicineDoctoralDoctor of Philosoph
Dendritic cell and B cell interactions in systemic lupuserythematosus
No full textpublished_or_final_versionMedicineMasterMaster of Philosoph
The role of peripheral dendritic cells in systemic lupuserythematosus
No full textpublished_or_final_versionabstractMedicineDoctoralDoctor of Philosoph
The utility of patient-based disease activity score for routine practice in rheumatoid arthritis
No full textRheumatoid arthritis (RA) patients have joint pain, swelling, deformities and disability as a result of inflammation. New Disease Modifying Anti-Rheumatic
Drugs (DMARD) including biologics have made “treat-to-target” a realistic standard practice aiming at sustained remission to prevent joint damage. Remission is defined by disease activity measurements conducted by doctors and blood tests.
However, this limits the frequency of disease activity assessment. Patient-based
Disease Activity Score (PDAS) was developed in the UK to allow patients to report their symptoms and come up with a score that closely resembles the doctors’ assessment. The second formula of PDAS (PDAS2) can do it even without the need for a blood test.
This thesis explores how PDAS agrees with the doctors’ assessment, sees if PDAS2 can be further simplified, if it works as well in Chinese patients, and if home PDAS2 monitoring can give some useful predictions on the worsening of RA (flare) and the need to step up DMARD.
First, the agreement between PDAS2 and the doctors’ scales of RA activities was moderate to substantial, similar to that between two standard doctors’ scales. Few patients under-reported their symptoms and even fewer over-reported them.
Second, PDAS2 contains four components to make up a score: patient’s overall impression of RA activity, functional disability, self-rated joint swelling and stiffness in the morning. All of them were measuring different aspects of RA activity, and the total score changed as RA activity changed, particularly for flares. Although some components were heavier weighted than the others, none should be discounted.
Third, the Chinese translation of PDAS2 was shown to be valid and work equally well among Hong Kong Chinese patients to inform RA activity and flare.
Lastly, from a major hospital in Hong Kong, 92 RA patients completed home monitoring using Chinese PDAS2 questionnaire. On average, they did it every two weeks for 16 weeks between two clinic visits. PDAS2 scores of each recording were computed, and then a time-integrated cumulative PDAS2 score
(cPDAS2) was calculated to take into account of the variable time intervals between each recording. This score described the overall trends of RA activity at home and could predict doctors’ assessment at the next clinic visit. The cPDAS2 score could predict patients in sustained remission or low activity if the score remained low and without significant fluctuations. Patients who had flare would have cPDAS2 score increased to a high level or by a significant amount. More importantly, the cPDAS2 score also predicted the doctors’ intention to intensify DMARD treatment. Therefore, home PDAS2 monitoring can reassure stable RA patients, and also help to identify patients who are having a flare or worsening disease, for earlier clinic review on treatment to meet the goal of “treat-to-target”.
In summary, this thesis has shown that RA patients can use PDAS2 to monitor their own disease activity as routine care. The Chinese version of PDAS2 performed similarly to the original version. Home monitoring of disease activity using cPDAS2 may improve implementation of “treat-to-target” by identifying worsening disease prior to scheduled clinic visits.published_or_final_versionMedicineDoctoralDoctor of Philosoph
The role of AIM2 inflammasome in systemic lupus erythematosus
No full textSystemic lupus erythematosus (SLE) is a prototypic autoimmune disease with heterogeneous clinical manifestations, disease course and prognosis. Whilst lupus is originally considered to be a B and T cell-mediated disease, growing evidence highlights the critical involvement of dysregulated innate immune responses in SLE aethiopathogenesis. The inflammasomes are multiprotein complexes which oligomerise in the cytosol of monocytes/macrophages to trigger innate inflammatory responses against pathogenic ligands or endogenous stressors. An inflammasome comprises of a pattern recognition receptor (PRR), an adaptor protein named apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and a caspase-1 (CASP1) effector which cleaves and activates two potent proinflammatory cytokines, interleukin (IL)-1β and IL-18. Notably, elevated levels of IL-1β and IL-18 have been detected in the skin, kidneys and sera of SLE patients. Absent in Melanoma 2 (AIM2), which uniquely senses double-stranded DNA (dsDNA), is one of the few known PRRs that induces the assembly of inflammasomes. Although the accumulation of endogenous dsDNA (SLE autoantigen) is well documented in SLE, the role of AIM2 inflammasome remains controversial in murine SLE, and is poorly understood in human SLE. Therefore, current study aimed to characterise the functional role of AIM2 inflammasome in SLE patients.
Firstly, gene expression of AIM2, ASC and CASP1 were found significantly higher in SLE monocytes than healthy control (HC) monocytes. Secondly, SLE monocytes were found to exhibit augmented production of IL-1β and IL-18 by AIM2 inflammasome. Together, current study revealed an anomalous state of the AIM2 inflammasome in SLE patients. Additionally, dysregulated expression of the AIM2 inflammasome components were associated with lower platelet count, haemoglobin level and the use of hydroxychloroquine in SLE patients. Next, I showed that SLE serum could upregulate both mRNA and protein levels of AIM2 in healthy monocytes, and correspondingly, mediate higher AIM2 inflammasome activity. Type I IFNs was later delineated to be the pathogenic factor which could potentiate AIM2 inflammasome response in human monocytes through upregulating AIM2 expression. Transcriptional profiling further revealed elevated expression of signal transducer and activator of transcription (STAT) 1 and STAT2, in SLE monocytes, which might in turn regulate AIM2 expression. Using chromatin immunoprecipitation assay, I confirmed that type I IFNs could increase the binding of these two transcription factors to an ISRE (interferon-stimulated regulatory element)-like site in AIM2 promoter to mediate higher gene expression.
This study demonstrates that the dysregulation of AIM2 inflammasome in SLE patients is mediated by elevated levels of type I IFNs. Additionally, these findings further support the pathogenic role of type I IFNs in SLE development through a novel mechanism by which AIM2 inflammasome response is amplified via the STAT1/STAT2/AIM2 axis in monocytes. Hitherto, there is no cure for lupus and patients are mainly managed using corticosteroids and immunosuppressants. Hence, novel inhibitors targeting AIM2 and its upstream TFs (STAT1/2) or type I IFNs might constitute an alternate strategy to help dampen chronic inflammation and damage in SLE patients, especially those with high levels of IL-1β and IL-18.published_or_final_versionMedicineDoctoralDoctor of Philosoph
Effects of Ganoderma lucidum on rheumatoid synovial fibroblasts
No full textpublished_or_final_versionabstracttocPharmacologyMasterMaster of Philosoph
The role of dectin-1 expressing dendritic cells in the pathogenesis ofsystemic lupus erythematosus
No full textSystemic 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_versionMedicineMasterMaster of Philosoph
Cellular and molecular dysregulations of neutrophils in systemic lupus erythematosus
No full textSystemic lupus erythematosus (SLE) is a female-biased chronic autoimmune disorder presenting a broad spectrum of clinical manifestations in multiple organ systems. As the most abundant immune cells in innate immunity, neutrophils are actively involved in the regulation of inflammation and immune responses in SLE. However, the molecular and cellular dysfunctions of neutrophils in lupus remain ambiguous. The aim of this study was to investigate the expression and functional significance of two molecular targets, namely interleukin-18 receptor accessory protein (IL18RAP) and peptidylarginine deiminase 4 (PADI4), in neutrophils of SLE patients.
IL18RAP is an indispensable subunit for the IL-18 receptor (IL-18R) complex’s ability to mediate high-affinity IL-18 binding and signaling transduction. Research in IL-18 in SLE has been mostly focused on its role as a type 1 T helper cell-driving cytokine. The functional significance of IL18RAP in mediating the IL-18-driven response in myeloid cells in SLE remains largely unexplored. Elevated IL18RAP expression in association with neutrophil activation network was initially identified in a pilot transcriptome profile analysis of SLE leukocytes. In a larger patient cohort, an increased expression of IL18RAP was observed in neutrophils from SLE patients, particularly those with a history of nephritis. IL18RAP expression was positively correlated with SLE disease activity. The increased IL18RAP expression in SLE neutrophils could be attributed to the elevated type I interferon level in sera. Functionally, neutrophils from SLE patients showed higher IL-18-mediated enhancement in reactive oxygen species (ROS) generation, which showed positive correlation with IL18RAP expression and could be neutralized by anti-IL18RAP blocking antibodies. The findings suggest that IL-18 could contribute to SLE pathogenesis through mediation of neutrophil dysfunction via the upregulation of IL18RAP expression.
PADI4 is a calcium dependent enzyme that catalyzes the conversion of positively charged arginine into neutrally charged citrulline. PADI4 is mainly expressed in neutrophils, is responsible for histones citrullination and was shown to correlate with chromatin decondensation during neutrophil extracellular traps (NETs) formation. However, the effects of PADI4 on lupus progression were contradictory in different lupus mouse models. In this study, elevated expressions of PADI4 were observed in neutrophils of SLE patients. Functionally, SLE neutrophils were prone to form NETs. Significantly decreased NETs formation was exhibited in PADI4 specific inhibitor GSK484-treated neutrophils and PADI4-knockdown neutrophil-like HL-60 cells (dHL-60). Neutrophils from SLE patients produced more ROS upon stimulation and expressed higher levels of the NADPH oxidase complex subunits. Furthermore, PADI4-deficient dHL-60 cells showed reduced chromatin accessibility in gene regions of NADPH subunits, which could account for their suppressed expression and led to reduced ROS induction. Hence, dysregulated PADI4 expression in SLE neutrophils could mediate enhanced NETs formation and ROS induction, thereby contributing to SLE pathogenesis through providing excessive autoantigens and facilitating oxidative damage.
Taken together, these findings reveal the overactivity of IL18RAP and PADI4 in SLE neutrophils and provide expression and functional bases for their use as potential therapeutic targets or biomarkers for lupus.published_or_final_versionMedicineDoctoralDoctor of Philosoph
Dissecting the physiological role of the novel lupus-associated C-type lectin-like protein CLEC16A
No full textThe CLEC16A locus has been identified as a susceptibility gene for multiple autoimmune diseases, including multiple sclerosis, type-I diabetes and systemic lupus erythematosus (SLE), in genome-wide association studies. CLEC16A encodes a novel C-type lectin-like protein, by virtue of a predicted C-type lectinlike domain (CTLD), with unclear function. Studies on the disease-associated SNPs have suggested that CLEC16A polymorphisms affect the expression of neighboring genes, while the effect on its own expression is unclear. Several functional studies have interrogated the physiological role(s) of CLEC16A in disparate directions. The Drosophila ortholog of CLEC16A, Ema, has been reported to regulate endosomal protein trafficking and the autophagic process, while CLEC16A has been found to participate in LPS-induced inflammatory cytokine response in rat astrocytes. Since there is not a consenting role ascribed to CLEC16A, this study was undertaken to investigate the functional involvement(s) of CLEC16A in mammalian cells and the expression of CLEC16A in lupus patients, with the attempt to comprehend the association between CLEC16A and SLE.
By overexpressing in non-immune epithelial cells, CLEC16A was revealed to be an intracellular protein of ~130 kDa in size. CLEC16A displayed a punctated expression pattern, which did not co-localize with endosomes, lysosomes, autophagosomes or endoplasmic reticulum in steady state. When treated with rapamycin or serum-starved, CLEC16A-overexpressing cells exhibited a reduced autophagic response, suggesting that CLEC16A may have an inhibitory role in autophagy. Besides the predicted CTLD, motif prediction has also implicated an immunomodulatory role for CLEC16A. Due to the observed inhibition on autophagy, coupled with recent findings linking autophagy and inflammasome activation, the involvement of CLEC16A in NLRP3 inflammasome was investigated. By knocking down CLEC16A in the human macrophage-like THP-1 cells, CLEC16A was found to potentially regulate NLRP3 inflammasome activation via inhibiting the LPS-induced pro-IL-1aasynthesis. Finally, the expressions of the long and short isoforms, CLEC16A_V1 and CLEC16A_V2 of CLEC16A in PBMCs were compared between healthy controls and SLE patients. A higher CLEC16A_V1 expression was observed in SLE patients, whereas the reverse was found for CLEC16A_V2. The expressions of the isoforms, however, were not correlated with the disease severity and clinical manifestations.
The finding that CLEC16A may inhibit autophagy is in contrast with the reported function of Ema in supporting autophagy, and such discrepancy could be because of the different cell systems used. The finding that CLEC16A may downregulate NLRP3 inflammasome activation has not been previously reported, and the mechanism(s) of such regulation warrant(s) future studies. The molecular basis of how CLEC16A regulates autophagy and inflammasome waits to be delineated. Such knowledge, together with information of where endogenous CLEC16A is expressed, shall incite better understanding of the contribution of CLEC16A to SLE development.published_or_final_versionMedicineDoctoralDoctor of Philosoph
Characterization of expression and function of dectin-1 on monocyte-derived dendritic cells in patients with systemic lupus erythematosus
No full textSystemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with diverse clinical manifestations on multiple organ systems. It is characterized by dysregulated innate and adaptive immunity. The exact pathogenesis of SLE is still unknown but current studies suggest the immunological abnormalities of lupus patients are triggered by environmental factors on genetically susceptible individuals. Dectin-1 is an innate immune receptor expressed on antigen presenting cells such as monocytes and dendritic cells (DCs). It is responsible for a wide range of cellular responses including the detection of pathogens, reactive oxygen species (ROS) production and induction of the potent inflammatory T helper 17 (Th17) cells. Excessive production of ROS and Th17 are associated with SLE pathogenesis. Interleukin-1β (IL-1β) is a crucial cytokine for the differentiation of Th17 and its production requires a two-step process: pro-IL-1β synthesis and IL-1β maturation by inflammasome. Both processes can be regulated by dectin-1 signaling mediated through spleen tyrosine kinase (Syk) phosphorylation. Recently, dectin-1 has been found to induce autoimmune arthritis in genetically susceptible mice and its ligand exacerbates disease development in a SLE mouse model. Therefore, we hypothesized that dectin-1 plays an important role in the immunopathogenesis of SLE involving the activation of inflammasome and production of pro-inflammatory cytokines.
In the present study, the expressions and functions of dectin-1 were characterized on monocytes and monocyte-derived DCs (MDDC) from SLE patients. A diminished frequency of dectin-1^+ cells was found among total monocytes from lupus patients compared to normal controls. It was inversely correlated with disease activity and anti-dsDNA antibody levels suggesting factors related to disease activity may affect dectin-1 expression. However, soluble factors in sera from patients with active disease and high levels of anti-dsDNA antibodies failed to down-regulate dectin-1 expression on monocytes from normal controls. Despite the decreased expression of dectin-1 on SLE monocytes, the higher levels of ROS produced upon activation suggested functional hypersensitivity of dectin-1 in SLE patients. In contrary to monocytes, the expression of dectin-1 was comparable between patients and normal controls after differentiation into DC. While the production of cytokines was not different upon dectin-1 stimulation alone, SLE MDDC exhibited augmented dectin-1 signaling indicated by higher levels of Syk phosphorylation and pro-IL-1β synthesis. Furthermore, dectin-1 has been reported to interact with other immune receptors especially toll-like receptor 2 (TLR2). This study showed that co-activation of the two receptors led to significantly higher production of IL-1β and IL-6 on SLE MDDC compared to controls. In addition, elevated Syk phosphorylation in SLE MDDC was also observed suggesting abnormal cross-talk between dectin-1 and TLR2 signaling pathways in lupus patients. However, caspase-1 inflammasome activation was not different between patients and normal controls despite the higher levels of Syk phosphorylation. Therefore, the involvement of the non-canonical caspase-8 inflammasome requires to be further explored.
In conclusion, the results from this study showed that dectin-1 is reduced in expression and functionally abnormal on SLE monocytes and MDDC. These findings provide a novel understanding on a role of dectin-1 in the complex pathogenesis of SLE.published_or_final_versionMedicineDoctoralDoctor of Philosoph
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