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

Introdução: O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune multissistêmica de etiologia complexa que envolve fatores ambientais, genéticos e hormonais. É caracterizada pela produção de autoanticorpos e mediadores inflamatórios, ativação e proliferação de células T autorreativas e perda da autotolerância imunológica. Em pacientes com LES, a expressão do receptor primário de ativação CD69 é aumentada e a de células T supressoras/reguladoras (Treg) CD4+CD25+FoxP3+ é reduzida. O CD69 é essencial para ativação de células T CD4 autorreativas enquanto que as células Treg são importantes na manutenção da autotolerância. Desta forma, células T tem um papel central na patogênese do LES, mas os mecanismos implicados na falência da autotolerância ainda não são elucidados, destacando a importância de estudos em modelos experimentais da doença, como o de LES-induzido por pristane. Objetivo: Quantificar células T CD4+CD69+ ativadas e Treg CD4+CD25+FoxP3+ no sangue, baço e LP de camundongos Balb/c LESinduzido por pristane no sentido de avaliar a falência de autotolerância neste modelo. Métodos: Analisamos 84 camundongos Balb/c fêmeas: 52 receberam por via intraperitoneal uma dose única de 0,5 ml de pristane e 32 a mesma dose de salina. Amostras de sangue, baço e LP dos camundongos eutanasiados foram coletadas 90, 120, 180 e 300 (T90, T120, T180 e T300) dias após a inoculação de pristane ou salina. Células mononucleares do sangue periférico (CMSP), do LP (CMLP) e esplenócitos foram obtidos por lise das hemácias seguida de lavagens com RPMI medium 1640 e centrifugação, e posteriormente criopreservadas até a avaliação por citometria de fluxo usando o aparelho Guava EasyCyteTM HT (Millipore). Para esta etapa, as células foram descongeladas, lavadas com RPMI medium 1640 e incubadas com anticorpos monoclonais dirigidos contra CD3, CD4, CD25, CD28, CD69, CTLA-4, FoxP3, CD14 e Ly6C (BD PharmingenTM). Os resultados foram expressos como média ± DP e teste de Mann-Whitney foi utilizado para análises estatísticas, sendo p<0,05 considerado significante. Resultados: Comparados aos animais controles, animais com LES-induzido por pristane apresentaram aumento de células T CD4+CD69+ no sangue nos T90, T120 e T180 (p < 0,022, p=0,008 e p=0,010, respectivamente) e no baço no T120 (p=0,049), enquanto que, no LP, houve redução destas células nos T120, T180 e T300 (p=0,001, p=0,001 e p < 0,001, respectivamente). A porcentagem de células Treg CD4+CD25+FoxP3+ foi menor no sangue nos T90, T120 e T180 (p=0,018, p=0,012, p < 0,046, respectivamente), no baço, nos T120 e T180 (p=0,018 e p=0,013), e no LP nos T90 e T300 (p=0,008 e p=0,005). Conclusão: Aumento da expressão de células T CD4+CD69+ e redução da expressão de Treg CD4+CD25+FoxP3+ sugerem células T CD4 ativadas e perda da autotolerância periférica em camundongos com LES-induzido por pristane. Estas alterações são semelhantes às observadas no lúpus humano, de modo que demonstramos que este modelo também pode ser útil na avaliação de mecanismos de ativação celular, tolerância periférica desequilíbrio imune homeostático envolvidos no LES / Introduction: Systemic Lupus Erythematosus (SLE) is multisystemic autoimmune disease with complex etiology that involves environmental, genetic and hormonal factors. Is characterized by auto-antibodies and inflammatory mediators production, autoreactive T cells activation and proliferation and loss of immunogenic autotolerance. In patients with SLE, expression of CD69 activation primary receptor is increased and the CD4+CD25+FoxP3+ suppressor/regulatory T cell (Treg) is reduced. CD69 is essential for activation of autoreactive CD4 T cells while Treg cells are important in autotolerance maintenance. In this way, T cells have a central role in the pathogenesis of SLE however, the mechanisms implied in the autotolerance failure are still not elucidated, highlighting the importance of studies in this disease\'s experimental models, such as pristane-induced SLE. Objective: Quantify activated CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg in blood, spleen and peritoneal lavage (PL) of Balb/c mice with pristane-induced SLE in order to evaluate autotolerance failure in this model. Methods: 84 female Balb/c mice were analyzed: 52 received a single intraperitoneal 0,5 ml dose of pristane and 32 the same dose of saline. Euthanized mice samples of blood, spleen and peritoneal lavage were collected 90, 120, 180 and 300 (T90, T120, T180 and T300) days after inoculation of pristane or saline. Mononuclear cells from peripheral blood (PBMC), PL (PLMC) and splenocytes were obtained by lysis of erythrocytes followed by washings with RPMI medium 1640 and centrifugation, subsequently criopreserved until evaluation by flow cytometry using the appliance GuavaEasyCyteTM HT (Millipore). For this step, cells were unfrozen, washed with RPMI medium 1640 and incubated with monoclonal antibodies against CD3, CD4, CD25, CD28, CD69, CTLA-4, FoxP3, CD14 and Ly6C (BD PharmingenTM). The results were expressed as mean ± SD and Mann-Whitney 11 test was used for statistical analysis, being considered significant p < 0,05. Results: Compared to control animals, SLE pristane-induced animals presented increase of CD4+CD69+ T cells in blood on T90, T120 and T180 (p=0.022, p=0.008 and p=0.010, respectively) and in spleen on T120 (p=0.049), while, in PL, there was reduction of these cells on T120, T180 and T300 (p=0.001, p=0.001 and p < 0.001, respectively). The porcentage of Treg CD4+CD25+FoxP3+ was smaller in blood on T90, T120 and T180 (p=0.018, p=0.012 and p < 0.046, respectively), in spleen on T120 and T180 (p=0.018 and p=0.013), and in PL on T90 and T300 (p=0.008 and p=0.005). Conclusion: Increase of CD4+CD69+ T cell and reduction of CD4+CD25+FoxP3+ Treg expression suggests activated T CD4 cells and loss of peripheral autotolerance in pristane-induced SLE mice. These alterations are similar to observed in human lupus, in order we showed that this model can also be useful in evaluating the mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in the LES

Introdução: O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune multissistêmica de etiologia complexa que envolve fatores ambientais, genéticos e hormonais. É caracterizada pela produção de autoanticorpos e mediadores inflamatórios, ativação e proliferação de células T autorreativas e perda da autotolerância imunológica. Em pacientes com LES, a expressão do receptor primário de ativação CD69 é aumentada e a de células T supressoras/reguladoras (Treg) CD4+CD25+FoxP3+ é reduzida. O CD69 é essencial para ativação de células T CD4 autorreativas enquanto que as células Treg são importantes na manutenção da autotolerância. Desta forma, células T tem um papel central na patogênese do LES, mas os mecanismos implicados na falência da autotolerância ainda não são elucidados, destacando a importância de estudos em modelos experimentais da doença, como o de LES-induzido por pristane. Objetivo: Quantificar células T CD4+CD69+ ativadas e Treg CD4+CD25+FoxP3+ no sangue, baço e LP de camundongos Balb/c LESinduzido por pristane no sentido de avaliar a falência de autotolerância neste modelo. Métodos: Analisamos 84 camundongos Balb/c fêmeas: 52 receberam por via intraperitoneal uma dose única de 0,5 ml de pristane e 32 a mesma dose de salina. Amostras de sangue, baço e LP dos camundongos eutanasiados foram coletadas 90, 120, 180 e 300 (T90, T120, T180 e T300) dias após a inoculação de pristane ou salina. Células mononucleares do sangue periférico (CMSP), do LP (CMLP) e esplenócitos foram obtidos por lise das hemácias seguida de lavagens com RPMI medium 1640 e centrifugação, e posteriormente criopreservadas até a avaliação por citometria de fluxo usando o aparelho Guava EasyCyteTM HT (Millipore). Para esta etapa, as células foram descongeladas, lavadas com RPMI medium 1640 e incubadas com anticorpos monoclonais dirigidos contra CD3, CD4, CD25, CD28, CD69, CTLA-4, FoxP3, CD14 e Ly6C (BD PharmingenTM). Os resultados foram expressos como média ± DP e teste de Mann-Whitney foi utilizado para análises estatísticas, sendo p<0,05 considerado significante. Resultados: Comparados aos animais controles, animais com LES-induzido por pristane apresentaram aumento de células T CD4+CD69+ no sangue nos T90, T120 e T180 (p < 0,022, p=0,008 e p=0,010, respectivamente) e no baço no T120 (p=0,049), enquanto que, no LP, houve redução destas células nos T120, T180 e T300 (p=0,001, p=0,001 e p < 0,001, respectivamente). A porcentagem de células Treg CD4+CD25+FoxP3+ foi menor no sangue nos T90, T120 e T180 (p=0,018, p=0,012, p < 0,046, respectivamente), no baço, nos T120 e T180 (p=0,018 e p=0,013), e no LP nos T90 e T300 (p=0,008 e p=0,005). Conclusão: Aumento da expressão de células T CD4+CD69+ e redução da expressão de Treg CD4+CD25+FoxP3+ sugerem células T CD4 ativadas e perda da autotolerância periférica em camundongos com LES-induzido por pristane. Estas alterações são semelhantes às observadas no lúpus humano, de modo que demonstramos que este modelo também pode ser útil na avaliação de mecanismos de ativação celular, tolerância periférica desequilíbrio imune homeostático envolvidos no LES / Introduction: Systemic Lupus Erythematosus (SLE) is multisystemic autoimmune disease with complex etiology that involves environmental, genetic and hormonal factors. Is characterized by auto-antibodies and inflammatory mediators production, autoreactive T cells activation and proliferation and loss of immunogenic autotolerance. In patients with SLE, expression of CD69 activation primary receptor is increased and the CD4+CD25+FoxP3+ suppressor/regulatory T cell (Treg) is reduced. CD69 is essential for activation of autoreactive CD4 T cells while Treg cells are important in autotolerance maintenance. In this way, T cells have a central role in the pathogenesis of SLE however, the mechanisms implied in the autotolerance failure are still not elucidated, highlighting the importance of studies in this disease\'s experimental models, such as pristane-induced SLE. Objective: Quantify activated CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg in blood, spleen and peritoneal lavage (PL) of Balb/c mice with pristane-induced SLE in order to evaluate autotolerance failure in this model. Methods: 84 female Balb/c mice were analyzed: 52 received a single intraperitoneal 0,5 ml dose of pristane and 32 the same dose of saline. Euthanized mice samples of blood, spleen and peritoneal lavage were collected 90, 120, 180 and 300 (T90, T120, T180 and T300) days after inoculation of pristane or saline. Mononuclear cells from peripheral blood (PBMC), PL (PLMC) and splenocytes were obtained by lysis of erythrocytes followed by washings with RPMI medium 1640 and centrifugation, subsequently criopreserved until evaluation by flow cytometry using the appliance GuavaEasyCyteTM HT (Millipore). For this step, cells were unfrozen, washed with RPMI medium 1640 and incubated with monoclonal antibodies against CD3, CD4, CD25, CD28, CD69, CTLA-4, FoxP3, CD14 and Ly6C (BD PharmingenTM). The results were expressed as mean ± SD and Mann-Whitney 11 test was used for statistical analysis, being considered significant p < 0,05. Results: Compared to control animals, SLE pristane-induced animals presented increase of CD4+CD69+ T cells in blood on T90, T120 and T180 (p=0.022, p=0.008 and p=0.010, respectively) and in spleen on T120 (p=0.049), while, in PL, there was reduction of these cells on T120, T180 and T300 (p=0.001, p=0.001 and p < 0.001, respectively). The porcentage of Treg CD4+CD25+FoxP3+ was smaller in blood on T90, T120 and T180 (p=0.018, p=0.012 and p < 0.046, respectively), in spleen on T120 and T180 (p=0.018 and p=0.013), and in PL on T90 and T300 (p=0.008 and p=0.005). Conclusion: Increase of CD4+CD69+ T cell and reduction of CD4+CD25+FoxP3+ Treg expression suggests activated T CD4 cells and loss of peripheral autotolerance in pristane-induced SLE mice. These alterations are similar to observed in human lupus, in order we showed that this model can also be useful in evaluating the mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in the LES