研究背景 / 系統性紅斑狼瘡（SLE）是一種較常見的累及多系統多器官的自身免疫性疾病，由於細胞和體液免疫功能障礙，產生多種自身抗體，確切病因不明。研究一般認為是在遺傳、環境等諸多因素的共同作用下導致了機體固有免疫及獲得性免疫系統功能紊亂，從而發病。作為機體抵抗病原體入侵的第一道防線，固有免疫系統通過模式識別受體（PRRs），不僅可以識別結合外源性的病原體相關模式分子（PAMPs），也可識別機體自身細胞所釋放的內源性危險信號又稱破壞相關模式分子（DAMPs），從而啟動信號傳導途徑，激活天然免疫細胞，從而導致一系列免疫應答的發生。本文將初步探討三類PRRs在SLE發病機制及病毒感染中的作用：（i）胞質中識別細胞壁肽聚糖的NOD樣受體（NLR），（ii）識別危險信號分子（DAMP）的膜結合型晚期糖基化終末產物受體（RAGE）及（iii）識別核酸的胞內Toll樣受體（TLR）。 / NLR是一種新發現的PAMP識別受體，在配體識別及信號傳導方面有別於膜型PRRs，在固有免疫應答中發揮重要作用。目前報道中NLR至少有23個成員，其中最有代表性的是NOD1和NOD2，通過特異性識別細菌胞壁肽聚糖產物從而參與固有免疫應答並誘導炎症反應和細胞凋亡。前期研究多集中於NODs與SLE基因易感性的探討，而SLE患者體內免疫細胞是否功能性表達NOD2及其下遊 的效應如何，仍有待進一步探討。 / RAGE是一種多配體受體，廣泛分布於上皮細胞、血管及炎症細胞表面，低表達於正常組織細胞，但與其配體結合後可啟動激活細胞內部各種信號傳導機制從而產生相應的生物學效應。HMGB1作為RAGE的重要配體在細胞和組織中分佈十分廣泛，近年研究證實，胞外高表達的HMGB1為一種重要的內源性危險信號分子，通過RAGE受體通路，可促進趨化作用，並通過激活NF-κB途徑誘導炎症反應。越來越多的證據表明HMGB1在自身免疫性疾病中起積極作用， RAGE-HMGB1軸在SLE的炎症反應及組織損傷中的重要作用值得研究。 / RAGE基因可因RNA的選擇性剪接而分為：全長RAGE（flRAGE）、截去N端的RAGE及截去C端的可溶性RAGE（sRAGE）。sRAGE有兩種來源，其中由細胞分泌而來的又稱為內分泌性RAGE（esRAGE）。sRAGE通過與flRAGE競爭性與結合配體從可抑制RAGE誘導的細胞信號傳導途徑，故又稱為作為“誘餌受體，作為潛在的治療靶點，對疾病的進展有保護作用。因此，評估sRAGE/esRAGE與flRAGE及配體HMGB1在SLE患者體內的動態平衡具有顯著臨床意義。 / 人類乳頭狀病毒（HPV）感染是子宮頸癌的致病因素，高危型HPV感染的持續存在是子宮頸癌的重要風險因素之一。早期病例對照研究已提示伴高炎症狀態及長期多重高危型HPV感染的SLE患者其子宮頸巴氏塗片異常和宮頸癌的發病率顯著高於對照人群，但在前瞻性隊列研究中其致高危致病因素及預測因子並未得到證實。TLR家族在早期固有免疫中對入侵病原微生物的識別發揮重要作用，胞內TLR3、TLR7、TLR8、TLR9通過識別病毒核酸成分通過介導下遊信號轉導誘導免疫反應。是否固有免疫系統異常參與SLE患者體內HPV持續及高危感染，仍有待進一步探討。 / 研究目的 / 1.研究NOD2受體通路及RAGE-HMGB1軸在SLE發病機制中的作用； / 2.闡明sRAGE/esRAGE作為“保護因子與flRAGE及其配體HMGB1在SLE患者體內的動態平衡及評估與疾病活動相關性； / 3.探討TLR受體通路在宿主SLE抗 HPV感染中的作用。 / 研究方法 / 本文通過三項臨床病例對照研究分別探討NLR、RAGE、TLR在SLE發病機制及病毒感染中的作用。 / 研究結果 / 1.SLE外周高表達於單核細胞內的NOD2可通過特異性識別配體誘導外周血單個核細胞的異常活化及促炎細胞因子的產生；而免疫抑制治療可下調CD8+ T細胞及抗原體提呈細胞內NOD2表達及抗炎細胞因子的產生； / 2.FlRAG高表達於SLE患者外周血單核細胞；血漿sRAGE作為獨立風險因素，與SLE疾病活動指數呈負相關；HMGB1單獨或與TLR9配體協同作用可刺激單核細胞分泌促炎細胞因子並激活信號轉導通路； / 3.TLR拮抗劑（羥氯喹）及強的松治療作為獨立風險因素可下調SLE患者子宮頸上皮細胞中TLR7和TLR9的表達；腫瘤相關的高危型HPV細胞株內核酸識別受體TLR及幹擾素刺激基因（ISGs）表達明顯下調伴功能異常。 / 研究結論 / 一方面，異常活化的PRRs通過識別結合外源及內源性病原體相關分子從而啟動固有免疫應答，激活一系列信號轉導通路，參與SLE的自身免疫反應： / 1.胞內受體NOD2可能通過特異性識別細菌胞壁酰二肽及誘導炎症反應，參與固有免疫應答反應抵禦外源性病原體侵襲，為感染因素導致SLE發病的假說尋找進一步理論依據； / 2.膜表面受體RAGE與配體HMGB1結合可激活細胞內多信號轉導機制，參與固有免疫應答反應抵禦內源性病原體侵襲，為胞內危險信號分子释放導致SLE無菌性炎症的假說提供初步理論依據； / 3.可溶性“誘餌受體RAGE作為潛在治療靶點可抑制SLE體內高炎症反應。 / 另一方面，多重因素交叉作用可引起SLE患者體內PRR轉錄及表達下調，從而抑制固有免疫應答，導致病毒逃避宿主免疫系統的監視及清除而長期潛伏： / 1.TLR拮抗劑（羥氯喹）和強的松治療可能引起SLE患者體內TLR7和TLR9表達下調，從而抑制固有免疫系統對外源侵入性病原體HPV的識別； / 2.腫瘤相關的高危型HPV細胞株亦可通過抑制TLR7和TLR9轉錄、下調受體表達及功能致 HPV逃避宿主免疫防禦而長期潛伏。 / Introduction / The pathogenesis of systemic lupus erythematosus (SLE) is a complicated process caused by genetic and environmental factors resulting in abnormalities of both the innate and the adaptive immune system. Sensing the presence of a pathogen is the first step for the immune system to mount an effective response to eliminate invading microorganisms and establish protective immunity. The innate immune system constitutes an important defense system to respond rapidly to both endogenous and exogenous molecules, in which the pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs) can interact with the pattern recognition receptors (PRRs) and then activate the antigen presenting cells (APCs), T, B cells. / Effective sensing of endogenous and exogenous molecules promotes autoreactivity via immune activation and antigen presentation. In lupus, these molecules may have a special role in the pathogenesis since they can serve as targets of autoreactivity as well as inducers. In this series of experiments, we focused on the roles of various PRRs including nucleic acid sensing toll-like receptors (TLRs), bacterial peptidoglycans sensing NOD-like receptors (NLRs) and dangerous signals sensing receptor for advanced glycation end products (RAGE), which are involved in the recognition of PAMPs and DAMPs sharing between microbes and the host in the pathogenesis of SLE. / In contrast to the well elucidated membrane-bound TLRs, cytoplasmic NLRs are a new family of PRRs for the recognition of extracellular PAMPs. NLRs can participate in the signaling events triggered by host recognition of specific motifs of bacterial peptidoglycans and, upon activation, induce the production of proinflammatory mediators. Apart from the putative link between genetic mutations of NOD2 and SLE, little is known regarding the expression and function of NOD2 in SLE. / RAGE is a transmembrane cell-surface receptor on a variety of immune effector cells, which is expressed at low levels in normal tissues and vasculature, but is upregulated wherever the accumulation of its proinflammatory ligands, especially the key ligand, high mobility group box protein 1 (HMGB1). Both endogenous secretory RAGE (esRAGE) as well as soluble RAGE (sRAGE) can be detected in blood serum and are able to bind the circulating ligands, neutralizing their actions. In those conditions characterized by high concentrations of the circulating ligands, the decoy receptors are reduced drastically, revealing the system function. Therefore, the relationship between the upregulation of full-length (fl) RAGE/RAGE ligands and the levels of “protective“ esRAGE/sRAGE in SLE is of obvious clinical interest. / Apart from inducing and perpatating autoreactivity, abnormal innate response may also be responsible for the increased risk of infection in patients with lupus. The prevalence of abnormal Papanicolaou (Pap) smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk and multiple human papillomavirous (HPV) infections. However, none of the clinical, lifestyle, gynecological and treatment parameters was predictive of persistent HPV infection. Innate immune recognition of viral infection triggers antiviral immune responses. Whether the abnormal host innate immune response in lupus patients may play a role in enhancing HPV persistence remained unknown. / Hypothesis / 1.Aberrant activation of NLR and RAGE pathways by endogenous or exogenous ligands lead to the initation and/or perpetuation of autoimmune responses in SLE; / 2.HPV infection suppresses the host immune response by deregulating the TLRs transcript, leading to increased viral persistence in SLE. / Aims / 1.To evaluate the role of NOD2 pathway in the pathogenesis of SLE; / 2.To elucidate the relationship and regulatory mechanisms among members of the RAGE axis in the pathogenesis of SLE; / 3.To investigate the role of TLR in the defense against HPV infection in SLE. / Methods / The present thesis comprised of three cross-sectional studies in Chinese patients with SLE and controls in Hong Kong. Clinical assessments and review of medical records were performed to obtain information regarding disease status. / Results / 1.Over-expression of NOD2 in monocytes was observed in immunosuppressant naive SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8⁺ T, monocytes and dendritic cells (DCs). Ex vivo basal productions of cytokines [Interleukin (IL)-6, IL-8 and IL-10] were significantly increased in immunosuppressant naive patients and patients with active disease despite immunosuppressants compared with healthy controls. Upon muramyl dipeptide (MDP) stimulaiton, relative induction (%) of cytokines (IL-1β) from peripheral blood mononuclear cells (PBMCs) was significantly increased in immunosuppressant naive patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with healthy controls. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naive patients and healthy controls. / 2.Plasma sRAGE level was negatively correlated with SLE disease activities. The reduction in sRAGE levels in SLE patients with flare indicates that sRAGE may play a regulatory role on disease activity. HMGB1 alone could only mildly induce IL-6 production, which resulted in a transient phosporylation of intracellular p38 mitogen activated protein kinase (MAPK), c-Jun NH2- terminal protein kinase (JNK) and nuclear factor (NF)-κB. On the other hand, CpG-oligodeoxynucleotides (ODN) (TLR9 ligand) together with HMGB1 not only had a additive effect on IL-6 and IL-12p70 secretions compared with each agent alone, but also activated the phosphorylation of p38 MAPK and NF-κB. / 3.TLR inhibitor (hydroxychloroquine) and prednisolone may down-regulate protein levels of TLRs 7 and 9 in cervical epithelial cells of lupus patients. In the cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral interferon-stimulated genes (ISG) 15 and myxovirus resistance (Mx) 1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands [R837, single stranded (ss) RNA and CpG-ODN] was greatly impaired in CaSki and HeLa than C33A cells. / Conclusions / Aberrant activation of PRR pathways by endogenous or exogenous molecules triggers the initation and/or perpetuation of autoimmune responses as follows: / 1.NOD2 may participate in the pathogenesis of lupus via the recognition of MDP and induction of proinflammatory effects, implicating the innate immune response for endogenous pathogens in the immunopathological mechanisms in SLE; / 2.Over-expression of RAGE may amplify the pro-inflammatory effects of DAMP such as HMGB1, while soluble RAGE may serve as a decoy receptor to suppress inflammation in patients with lupus nephritis. / 3.Upregulated HMGB1 may act alone or in combine with TLR9 ligand through the phosphorylation of p38 MAPK and NF-κB to promote inflammation in lupus. / On the other hand, the immune evasion strategy via avoidance of stimulation and downregulation of PRRs may promote establishment of persistent infection as listed below: / 1.TLR inhibitor (hydroxychloroquine) and prednisolone may down-regulate protein levels of TLRs 7 and 9 in lupus patients, thereby decreasing the innate immune response against HPV infection. / 2.Upon infection, HPV further down-regulate TLRs 7 and 9 levels for viral persistence. / 3.Reduction of TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs on a biologically relevant antiviral response. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yu, Shuilian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 114-141). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.v / ACKNOWLEDGEMENTS --- p.viii / LIST OF PUBLICATIONS --- p.ix / LIST OF AWARDS AND GRANDT RECORD --- p.x / CONTENTS --- p.viii / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xv / LIST OF APPENDIXS --- p.xvi / ABBREVIATIONS --- p.xvii / Chapter CHAPTER 1 --- REVIEW OF THE LITERATURE --- p.1 / Chapter 1.1 --- What is systemic lupus erythematosus (SLE)? --- p.1 / Chapter 1.2 --- Epidemiology of SLE --- p.1 / Chapter 1.3 --- Etiology and pathogenesis of SLE --- p.3 / Chapter 1.3.1 --- Genetic factors --- p.5 / Chapter 1.3.2 --- Environmental triggers --- p.5 / Chapter 1.3.3 --- Cellular abnormalities --- p.6 / Chapter 1.3.3.1 --- APCs and cell debris clearance --- p.6 / Chapter 1.3.3.2 --- B cell and the production of autoantibodies --- p.8 / Chapter 1.3.3.3 --- T cell and the regulation of the immune responses --- p.9 / Chapter 1.3.4 --- Disturbance of the innate immune response --- p.10 / Chapter 1.3.4.1 --- PAMPs and DAPMs: all we need to know about danger in SLE --- p.10 / Chapter 1.3.4.2 --- Sensors of innate immunity --- p.12 / Chapter 1.3.4.2.1 --- TLRs sensing nucleic acids --- p.14 / Chapter 1.3.4.2.2 --- NLRs sensing bacterial peptidoglycans --- p.16 / Chapter 1.3.4.2.3 --- RAGE sensing dangerous signals --- p.16 / Chapter 1.3.5 --- Dysregulation of cytokine networks --- p.19 / Chapter 1.3.5.1 --- Anti-inflammatory cytokines --- p.20 / Chapter 1.3.5.2 --- Proinflammatory cytokines --- p.20 / Chapter 1.3.6 --- Abnormal signaling transduction --- p.22 / Chapter 1.4 --- Clinical features of SLE --- p.22 / Chapter 1.5 --- Laboratory features of SLE --- p.26 / Chapter 1.6 --- Assessing disease activity and damage of SLE --- p.28 / Chapter 1.7 --- Treatment of SLE --- p.28 / Chapter 1.7.1 --- Current immunosuppressive therapy --- p.28 / Chapter 1.7.2 --- Novel biologic therapies --- p.30 / Chapter 1.8 --- Human papillomavirus (HPV) infection in SLE --- p.32 / Chapter 1.8.1 --- Are women with lupus at higher risk of HPV infection? --- p.32 / Chapter 1.8.2 --- Abnormalities of TLR-IFN axis potentially increases HPV risk --- p.32 / Chapter 1.8.3 --- TLR suppressing mediciation potentially increases HPV risk --- p.34 / Chapter CHAPTER 2 --- HYPOTHESISS AND AIMS --- p.35 / Chapter CHAPTER 3 --- METHODOLOGIES --- p.36 / Chapter 3.1 --- Materials --- p.36 / Chapter 3.1.1 --- Selection of patients and controls --- p.36 / Chapter 3.1.2 --- Blood and cervical samples --- p.36 / Chapter 3.1.3 --- Cervical epithelial cell lines --- p.37 / Chapter 3.1.4 --- Culture medium and serum supplement --- p.37 / Chapter 3.1.5 --- Culture ligands --- p.37 / Chapter 3.1.6 --- Reagents for flow cytometric analysis (FCM) --- p.37 / Chapter 3.1.7 --- Antibodies for FCM --- p.38 / Chapter 3.1.8 --- Quantative assay kits --- p.39 / Chapter 3.1.9 --- Membrane array of phosphorylated intracellular kinases --- p.39 / Chapter 3.1.10 --- Primers for qPCR --- p.40 / Chapter 3.2 --- Methods --- p.40 / Chapter 3.2.1 --- Study design and patient assessment --- p.40 / Chapter 3.2.2 --- Isolation of PBMCs --- p.41 / Chapter 3.2.3 --- Isolation of monocytes --- p.42 / Chapter 3.2.4 --- Cell culture --- p.42 / Chapter 3.2.5 --- Sampling procedure of cervical epithelial cells --- p.42 / Chapter 3.2.6 --- HPV identification --- p.43 / Chapter 3.2.7 --- Flow cytometry gating of target cells --- p.43 / Chapter 3.2.8 --- FCM of target molecules and phosphorylated signaling molecules --- p.45 / Chapter 3.2.9 --- Membrane array of phosphorylated intracellular kinases --- p.46 / Chapter 3.2.10 --- Cytokine cytometric bead array --- p.46 / Chapter 3.2.11 --- Enzyme-linked immunosorbent assay --- p.46 / Chapter 3.2.12 --- Real-time qPCR --- p.46 / Chapter 3.2.13 --- Statistical analysis --- p.47 / Chapter CHAPTER 4 --- DOWN-REGULATED NOD2 BY IMMUNOSUPPRESSANTS IN PERIPHERAL BLOOD CELLS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS REDUCES THE MURAMYL DIPEPTIDE-INDUCED IL-10 PRODUCTION --- p.48 / Chapter 4.1 --- INTRODUCTION --- p.48 / Chapter 4.2 --- METHODS --- p.49 / Chapter 4.2.1 --- Patient selection and assessment --- p.49 / Chapter 4.2.2 --- FCM of NOD2 expression in T, B cells, monocytes and DCs --- p.49 / Chapter 4.2.3 --- Cell culture --- p.49 / Chapter 4.2.4 --- Quantitative assay --- p.49 / Chapter 4.2.5 --- Statistical analyses --- p.49 / Chapter 4.3 --- RESULTS --- p.50 / Chapter 4.3.1 --- Characteristics of lupus patients and control subjects --- p.50 / Chapter 4.3.2 --- Identification of DCs, T, B lymphocytes and monocytes --- p.50 / Chapter 4.3.3 --- Protein level of NOD2 in DCs, T, B lymphocytes and monocytes --- p.53 / Chapter 4.3.4 --- Potential explanatory variables associated with NOD2 levels in lupus patients --- p.55 / Chapter 4.3.5 --- Induction of inflammatory cytokines by NOD2 ligand --- p.61 / Chapter 4.4 --- DISCUSSION --- p.63 / Chapter 4. --- 5 CONCLUSIONS --- p.67 / Chapter CHAPTER 5 --- MEMBERS OF THE RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS AXIS AS POTENTIAL THERAPEUTIC TARGETS IN SYSTEMIC LUPUS ERYTHEMATOSUS --- p.68 / Chapter 5.1 --- INTRODUCTION --- p.68 / Chapter 5.2 --- METHODS --- p.68 / Chapter 5.2.1 --- Patients selection and assessment --- p.68 / Chapter 5.2.2 --- FCM of monocyte-surface flRAGE --- p.69 / Chapter 5.2.3 --- Cell culture --- p.69 / Chapter 5.2.4 --- Quantitative assay --- p.69 / Chapter 5.2.5 --- Membrane array of phosphorylated of intracellular kinases --- p.69 / Chapter 5.2.6 --- FCM of activated intracellular signaling molecules --- p.69 / Chapter 5.2.7 --- Statistical analysis --- p.69 / Chapter 5.3 --- RESULTS --- p.69 / Chapter 5.3.1 --- Characteristics of SLE patients --- p.69 / Chapter 5.3.3 --- Relationships between RAGE isoforms and HMGB1 --- p.75 / Chapter 5.3.4 --- Potential explanatory variables associated with levels of RAGE isoforms and HMGB1 in LN patients --- p.77 / Chapter 5.3.5 --- Activity of HMGB1 alone or in combine with TLR9 ligand --- p.81 / Chapter 5.4 --- DISCUSSION --- p.84 / Chapter 5.5 --- CONCLUSIONS --- p.88 / Chapter CHAPTER 6 --- ANTAGONIST-MEDIATED DOWN-REGULATION OF TOLL-LIKE RECEPTOR INCREASES THE PREVALENCE OF HUMAN PAPILLOMAVIRUS INFECTION IN SYSTEMIC LUPUS ERYTHEMATOSUS --- p.89 / Chapter 6.1 --- INTRODUCTION --- p.89 / Chapter 6.2 --- METHODS --- p.90 / Chapter 6.2.1 --- Patient selection and assessment --- p.90 / Chapter 6.2.2 --- HPV sampling procedure and identification --- p.90 / Chapter 6.2.3 --- FCM of TLRs 3, 7, 8 and 9 in cervical epithelial cells --- p.90 / Chapter 6.2.4 --- Cell culture --- p.90 / Chapter 6.2.5 --- Quantitative assay --- p.90 / Chapter 6.2.6 --- Real-time qPCR of Interferon-stimulated genes (ISGs) --- p.90 / Chapter 6.2.7 --- Statistical analysis --- p.91 / Chapter 6.3 --- RESULTS --- p.91 / Chapter 6.3.1 --- Pap smear findings, socio-demographic and clinical characteristics --- p.91 / Chapter 6.3.2 --- Identification of cervical epithelial cells --- p.95 / Chapter 6.3.3 --- Protein level of TLRs 3, 7, 8 and 9 in cervical epithelial cells --- p.96 / Chapter 6.3.4 --- Potential explanatory variables associated with TLR levels in lupus patients --- p.98 / Chapter 6.3.5 --- TLRs and ISGs expressions are inhibited by oncogenic HPVs --- p.102 / Chapter 6.3.6 --- Induction of inflammatory cytokines by TLR agonists was impaired in oncogenic HPVs --- p.103 / Chapter 6.4 --- DISCUSSION --- p.105 / Chapter 6.5 --- CONCLUSIONS --- p.107 / Chapter CHAPTER 7 --- CONCLUSIONS OF THE THESIS --- p.108 / Chapter 7.1 --- Answers to the hypotheses --- p.108 / Chapter 7.2 --- Conclusions and implications --- p.109 / Chapter 7.3 --- Liminations and future plan --- p.110 / Chapter 7.3.1 --- Liminations of study design --- p.110 / Chapter 7.3.2 --- Liminations of methodology --- p.111 / Chapter 7.3.3 --- Liminations of CHAPTER 4 and future plan --- p.111 / Chapter 7.3.4 --- Liminations of CHAPTER 5 and future plan --- p.112 / Chapter 7.3.5 --- Liminations of CHAPTER 6 and future plan --- p.112 / Chapter CHAPTER 8 --- REFERENCES --- p.114 / Chapter CHAPTER 9 --- APPENDIX --- p.142

A flagelina é reconhecida pelo receptor TLR5, e pelos receptores NLRs, NLRC4/Naip5. Estes últimos induzem ativação de caspase-1 e liberação de IL-1b e IL-18, além da morte do macrófago por piroptose. Para investigar os mecanismos moleculares envolvidos nesses processos, MOs peritoneais foram estimulados com flagelina de B. subtilis e S. typhimurium em sua forma livre (capaz de ativar TLR5), ou inserida em vesículas lipídicas (capaz de ativar os NLRs). Demonstramos que ambas as flagelinas citosólicas induzem produção de IL-1b e morte celular, embora a flagelina de S. typhimurium tenha mostrado maior potencial em induzir produção de IL-1b e IL-6 pelos MOs. Verificamos que a produção de IL-1b e lise celular de MOs se mostraram eventos subseqüentes à formação de poros na membrana celular. Embora a liberação de IL-1b após reconhecimento de ambas as flagelinas ser um fenômeno dependente do eixo NLRC4/caspase-1, a morte celular induzida pelas flagelinas citosólicas ocorre na ausência destas moléculas, ao contrário do que prevê a literatura atual. / Flagellin is recognized by TLR5, and NLRs NLRC4/Naip5. The latter induce activation of caspase-1 and release of IL-1b and IL-18, besides the death of macrophages by pyroptosis. To investigate the molecular mechanisms involved in these processes, peritoneal MOs were stimulated with flagellin from B. subtilis and S. typhimurium in its free form (activating TLR5), or inserted into lipid vesicles (able to activate NLRs). We demonstrated that both cytosolic flagellins induce the production of IL-1b and cell death, while the flagellin of S. typhimurium has shown greater potential to induce production of IL-1b and IL-6 by MOs. We found that the production of IL-1b and cell lysis by MOs are subsequent events proven by the formation of pores in cell membranes. Although the release of IL-1b after recognition of both flagellins is a phenomenon dependent on the axis NLRC4/caspase-1, cell death induced by cytosolic flagellin occurs in the absence of these molecules, unlike that provided by the current literature.

背根神經節（DRG）上的初級感覺神經元通常負責感覺從環境中有害的刺激，但新出現的證據表明，它亦負責對危險的感覺。Toll-樣受體-4（TLR4）通常見於小膠質細胞，它是負責識別病原體相關分子模式（PAMPs）或損傷相關分子模式（DAMPs）並誘發炎症。奇怪的是，儘管TLR4在中樞神經系統通常見於神經膠質細胞，在DRG發現的TLR4僅見於初級感覺神經元，但從未見於周邊的衛星膠質細胞（SGC）。而重要的是，在感覺神經節中激活TLR4是會導致神經病理性疼痛的，但我們仍然未知道初級感覺神經元上的TLR4是否導致疼痛的唯一來源。本研究旨在探討在DRG細胞的TLR4信號傳導的分子和細胞機理，並探討在DRG的神經元和膠質細胞上TLR4活動的差異，在生物學上有甚麼意義。 / 為了研究在DRG神經元和膠質細胞的相互作用，我們首先在一個既定的混合DRG細胞培養模型上研究了谷氨酰胺合成酶（ GS ）的表達模式。GS是一種只會在SGC上表達的特異性酶，並於神經元和神經膠質細胞之間的谷氨酰胺 - 谷氨酸循環產生相互作用。在典型的DRG細胞培養，神經元通過擴散因子促進了GS在神經膠質細胞上的表達，然而，GS的表達亦受到TLR4激動劑，即脂多醣（LPS），的抑制。這表明DRG神經元和神經膠質細胞的關係受到TLR4介導的炎症之影響。在混合DRG細胞中，我們對TLR4-免疫反應（IR）進行了鑑定，發現TLR4最主要的是表達在神經元細胞的表面。另外，LPS（ 1微克/毫升，2小時）會刺激混合DRG細胞，通過在DRG細胞中MyD88依賴性信令，誘導環加氧酶-2（COX -2），白細胞介素-1β（ IL-1β）和腫瘤壞死因子-α（TNFα）的轉錄。此外，在DRG細胞， LPS（ 1微克/毫升， 24小時）亦會觸發依賴COX-2 的前列腺素E2（PGE₂）和的前列環素（PGI₂）的產生。但在LPS刺激後，我們發現DRG神經元和神經膠質細胞都對 COX-2-IR呈陽性反應。這證明DRG神經膠質細胞對TLR4誘發的神經炎症也擔任一定的角色。 / 為了純粹研究神經膠質細胞有沒有任何TLR4活性，我們把神經元從混合DRG細胞中除去，從而把神經膠質細胞純化。出乎意料的是，在純化後，大約80的神經膠質細胞對TLR4 -IR呈陽性反應。而且，時間和濃度依賴性的研究表明，純化後的神經膠質細胞對LPS刺激的COX-2表達反應在有效性和效率上比混合DRG細胞的顯著更高。明顯地，神經元對神經膠質細胞的TLR4活性有抑制作用。我們並且發現，神經元的抑制作用是透過由細胞與細胞之間的接觸介導，而不是由擴散因子介導。 / 重要的是， LPS也能誘導純化後的神經膠質細胞去產生依賴COX-2活性的前列腺素E2（PGE₂）。反過來， PGE₂能區別地調節依賴TLR4的炎症基因轉錄，說明在DRG 由TLR4介導的神經炎症是受多重複雜的機理控制。然而有趣的是，從受熱休克性損害的感覺神經元所收集的培養基可以激活純化膠質細胞，並通過對TLR4局部依賴性的方式，誘導COX-2的轉錄。此外，我們利用斑馬魚作為疼痛行為反應的模型，發現COXs的活性與瞬時受體電位通道亞家族V1（ TRPV1）有密切關係。斑馬魚幼蟲的疼痛行為反應是一個適合於篩選新型鎮痛化合物的體內模型。 / 總括來說，透過細胞與細胞之間的接觸和擴散因子，感覺神經元可以控制神經膠質細胞的表型。我們的研究確定感覺神經元是在DRG中表達TLR4的主要細胞類型，但當神經元施加的抑制被削弱，SGC可以成為完全勝任TLR4信息傳遞的細胞。因此我們推測TLR4的活性在DRG中被嚴格調控，以防止不必要的神經炎症發生。至於未來，我們認為在DRG中的TLR4/COX-2/PGE₂信號通路可以成為研究方的新型鎮痛化合物的方向。而轉基因斑馬魚則可用作篩選新型鎮痛化合物的工具。 / Primary sensory neurons of the dorsal root ganglia (DRG) are classically responsible for the detection of physiological stimuli from the environment, but emerging evidences suggests that they are also involved in the sensation of danger. Toll-like receptor 4 (TLR4) is commonly found on microglia for the recognition of pathogen- or damage- associated molecular patterns (PAMPs or DAMPs) and to the activation of TLR4 leads to inflammation. Curiously, while commonly found in glial cells in central nervous system, TLR4 expression was only found in primary sensory neurons but not the satellite glial cells (SGCs) in the DRG. Importantly, activation of TLR4 in sensory ganglia mediates neuropathic pain, but it remains unknown whether neurons are the only source of TLR4 activity. The present study aims to study the cellular and molecular mechanism(s) of TLR4 signalings and explore the biological significance of differential cellular TLR4 activity in the DRG. / To investigate neuron-glia interactions in the DRG, the expression of glutamine synthetase (GS), a SGC-specific enzyme in the glutamine-glutamate shuttle between neuron and glia, was studied in an established model of mixed DRG cells culture. In typical mixed DRG cell cultures, neurons promoted the GS expression in glial cells through diffusible factors. However, GS expression was negatively regulated by theTLR4 agonist, lipopolysaccharide (LPS), indicative of a change in neuron-glia relationships by TLR4 mediated inflammation. In mixed DRG cells, cell surface TLR4-immunoreactivity (-ir) was predominantly identified on the neurons. LPS (1 μg/mL, 2 h) stimulation induced cyclooxygenases-2 (COX-2), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) transcription through MyD88-dependent signalings in DRG cells. Furthermore, LPS (1 μg/mL, 24 h) triggered COX-2-dependent production of prostaglandin E₂ (PGE₂) and prostacyclin (PGI₂) in mixed DRG cells. / To study the TLR4 activity of glial cells, glial cell cultures were purified by removing neurons from mixed DRG cell culture. Unexpectedly, approximately 80% of purified glial cells become TLR4-ir positive. Moreover, a time- and concentration-dependent study showed that the efficacy and efficiency of purified glial cells to express COX-2 in response to LPS was significantly higher than that of mixed DRG cells. We found that neuron inhibited glial cells through cell-cell contact, but not by diffusible factors. Importantly, LPS also induced COX-2 dependent PGE₂ production in purified glial cells. In turn, PGE₂ can differentially modulate TLR4-dependent gene transcription, suggestive of a complex regulation of TLR4-mediated inflammation in the DRG. Intriguingly, conditioned media from heat-shocked damaged sensory neurons activated purified glial cells to induce COX-2-transcription through a partially TLR4-dependent mechanism. Using zebrafish as a model of nocifensive behavior, we found that the activity of COXs was closely associated with the transient receptor potential channel subfamily V1 (TRPV1), and the nocifensive behavior of zebrafish larvae is suitable for in vivo screening of novel analgesic compounds. / To conclude, sensory neurons regulate the phenotypes of DRG glial cells through cell-cell contact and diffusible factors. Here, sensory neurons are confirmed to be the predominant cell type expressing TLR4 in the DRG, but SGCs become fully competent for TLR4 signalings when the neuronal inhibitions are diminished. We therefore hypothesize that TLR4 activity is tightly regulated in the DRG to prevent unwanted neuroinflammation. Future studies with genetically modified zebrafish can be used for the screening of novel analgesic compound targeting the TLR4/COX-2/PGE₂ signaling pathway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tse, Kai Hei. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 190-222). / Abstracts also in Chinese.

An in vitro model of the follicle-associated epithelia that overlie the Peyer's patches of the small intestine was developed and validated to examine the mechanisms of mucosal antigen sampling. This model displays many phenotypic and physiological characteristics of M cells including apical expression of [alpha]5[beta]l integrin and enhanced energy dependent participate transport. CD4+ T-cells were shown to be an important influence on the development of Mlike cells. The model was used to examine the M cell mediated uptake of several putative whole-cell killed bacterial vaccines. Greater numbers of non-typeable Haemophilus influenzae NTHi 289, NTHi 2019, Escherichia coli 075 HMN and Streptococcus pneumoniae were transported by model M cells compared to control Caco-2 enterocyte-like cells. Studies in isolated murine intestine segments confirmed the selective uptake of NTHi 289 and Escherichia coli demonstrating that intestinal mucosal sampling of these antigens is performed by M cells. Pseudomonas aeruginosa was not absorbed as whole cell bacteria but as soluble antigen, as indicated by the presence of bacterial DNA in the cytoplasm of epithelial cells. These results suggest that bacteria such as NTHi and E. coli are sampled by the mucosal immune system in a different manner to that of bacteria such as Pseudomonas. A number of potential cell surface receptors were investigated to identify which molecules are responsible for intestinal uptake whole-cell killed bacteria. Immunofluorescence studies detected the presence of toll-like receptor-2, toll-like receptor-4, PAF-R and [alpha]5[beta]l integrin on in vitro M-like cell cultures. Examinations of murine intestine confirmed the presence of TLR-4 and PAF-R. TLR-4 was found in small quantities and on M cells. In contrast to the M cell model, TLR-2 expression in the murine intestine was sparse. Receptor inhibition experiments provided evidence for the involvement of TLR-4, PAF-R and [alpha]5[beta]l integrin in M cell uptake of killed bacteria both in vitro and in vivo. This thesis has contributed valuable information regarding the mechanisms of uptake of whole-cell killed bacteria by the intestinal mucosal immune system. For the first time, M cell sampling of whole-cell killed bacteria has been demonstrated. Furthermore, the receptors involved in these processes have been identified. This information will be of great use in the development and optimisation of new oral vaccines.

Bibliography: leaves 129-145.

Biased immunoglobulin heavy variable (IGHV) gene usage and distinctive B-cell receptor (BCR) features have been reported in chronic lymphocytic leukaemia (CLL), which may reflect clonal selection by antigens during disease development. Furthermore, the IGHV gene mutation status distinguishes two clinical entities of CLL, where patients with unmutated IGHV genes have an inferior prognosis compared to those with mutated IGHV genes. Recently, one subgroup of CLL patients expressing the IGHV3-21 gene was found to display highly similar immunoglobulin (IG) gene features, even within the heavy chain complementarity-determining region 3 (HCDR3). Patients in this subgroup typically had a poor prognosis. In paper I, we aimed to identify further subgroups with restricted BCR features among 346 CLL cases. Six subsets were defined which carried virtually identical BCRs in terms of rearranged heavy and light chain (LC) IG genes and CDR3 length and composition. In paper II, we investigated 90 IGHV3-21 cases from diverse geographical locations. We confirmed the highly restricted HCDR3 characteristics in 56% of patients and a biased usage of the IGLV3-21 gene in 72% of cases. Survival analysis also confirmed the poor outcome of this group, irrespective of IGHV gene mutation status and geographical origin. Papers III and IV involved a large-scale analysis of IGH and IG kappa and lambda (IGK/L) gene rearrangements, to define subsets with ‘stereotyped’ BCRs and also to systematically examine the somatic hypermutation (SHM) features of the IG genes in CLL. We studied a cohort of 1967 IGH and 891 IGK/L gene sequences from 1939 patients from 6 European institutions. Over 5300 IGH and ~4700 IGK/L sequences from non-CLL B cells were used as a control data set. In total, 110 CLL stereotyped subsets were defined according to HCDR3 homology. Striking IGK/L gene biases were also evident within subsets, along with distinctive K/LCDR3 features, such as length and amino acid composition. At cohort level, the patterns of mutation appeared to be consistent with that of a canonical SHM mechanism. However, at a subgroup level, certain stereotyped subsets, e.g. IGHV3-21/IGLV3-21 and IGHV4-34/IGKV2-30 CLL, deviated from this pattern. Furthermore, recurrent ‘stereotyped’ mutations occurred in cases belonging to subsets with restricted HCDR3s, in both IGHV and IGK/LV genes, which were subset- and CLL-biased when compared to non-CLL B cells. In conclusion, our findings implicate antigen selection as a significant factor in the pathogenesis of CLL, particularly in cases carrying stereotyped BCRs. The presence of stereotyped mutations throughout the VH and VL domain also indicates involvement of IG regions other than the CDR3 in antigen recognition. Finally, biased IGK/L gene usage and specific K/LCDR3 features are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains.

Cellular adhesion research has recently focused on the small scale at the level of individual receptor-ligand bonds. This trend in research is primarily due to experimental advances which allow such individual bond force measurements. Here, one of these techniques, micromanipulation, has been extended to not only determine the bond force of individual receptor-ligand pairs, but also the intrinsic kinetic rates of the interaction. Using transmembrane (TM ) Fc gamma receptor III (CD16a-TM) and human IgG (hIgG), the dependence of adhesion probability on receptor-ligand expression densities, contract duration and contact area was quantitated. A probabilistic based theoretical formulation was developed and validated that relates the intrinsic molecular kinetic rates of the receptorVligand interaction to the experimentally determined adhesion probability. This theoretical formulation describing individual receptor-ligand kinetics has also allowed direct evaluation of existing biophysical bond strength/kinetics paradigms at the extreme condition of single bonds. A force-displacement model was also developed to quantitate the force exerted on the RBC membrane transducer during the micropipette retraction process and found to be in agreement with previous work. In addition to CD16a-TM, the kinetic rates of CD16a anchored via a glycosyl phosphatidylinositol (GPI) moiety (CD16a-GPI) and the two alleles of CD16b (NA1 and NA2) were determined for human, rabbit, and mouse IgG species. The binding affinity of these CD16 interactions to soluble IgG was also measured by traditional bulk chemistry approaches and compared to those measured via the micromanipulation protocol in which the IgG ligand is membrane bound in the solid phase. These data suggest that the membrane anchor itself can alter CD16 binding properties. This represents the first reported effect of the anchor on an intrinsic receptor property, its kinetic rates and binding affinity. This thesis presents two specific aims or goals. These goals were achieved and reported in this thesis. During the course of this research, I also explored other directions and gathered initial data. These directions were further explored by other researchers but the initial data is also presented here.

The purpose of this study is to determine whether or not adipocytes harvested directly from fat pads or induced from bone marrow in lean and obese mice exhibit a sex-dependent rapid response to vitamin D metabolite 1á,25(OH)2D3 and if so to elucidate if it is via an ERp60 receptor mediated signaling pathway. The role of 1á,25(OH)2D3 and specifically the membrane effect will be examined in two genetically distinct mice to see if their cells have a differing sensitivity. The results indicate that there are differing responses in adipocytes that are induced from bone marrow versus differentiated fat pad adipocytes, and the function of 1á,25(OH)2D3 is sex-specific in some cases. Additionally, all the adipocytes tested demonstrated a rapid response to 1á,25(OH)2D3; mRNA for nVDR and ERp60 were found in all cells however the only functional protein found in the plasma membrane was ERp60 indicating that it may be necessary for the rapid response whereas nVDR is not required.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 228-272). Also available on the Internet.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Committee Chair: Zhu, Cheng; Committee Member: Babensee, Julia; Committee Member: Dustin, Michael; Committee Member: Garcia, Andres; Committee Member: Jo, Hanjoong; Committee Member: van der Merwe, Anton. Part of the SMARTech Electronic Thesis and Dissertation Collection. Non-Latin script record