The process of immune "self-nonself discrimination" is of utmost importance for the survival of all species as the biodestructive force of immune system can be directed towards the host as much as to pathogens. Thus, to shift this balance towards the latter, T cells bearing self- recognizing receptors are removed in the thymus (central tolerance) or their reactivity is harnessed through various additional mechanisms in periphery (peripheral tolerance). If the selfreactive T cells are not deleted and persist in the body, the regulation of self-tolerance can be breached, leading to the onset of autoimmunity. Presented thesis revolved around α-defensins, very effective bactericidal peptides that represent an important part of humoral innate immunity. There are two types of α-defensins: myeloid, expressed predominantly in neutrophils, and enteric, synthesized by intestinal Paneth cells. Data presented inhere are first to characterized the involvement of α-defensin- expressing cells in two types of autoimmune diseases, insulin-dependent diabetes mellitus (T1D) and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). The former relates to the identification of transcriptionally activated myeloid α-defensin- expressing eosinophils present in the thymus of diabetes prone rat. In...

O presente estudo discute o papel dos miRNAs na fisiopatologia molecular de Esclerose Múltipla Recorrente Remitente (EMRR). O estudo demonstrou que em linfócitos T CD4+ de pacientes com EMRR em surto ocorre a diminuição da expressão do miR-15a e do miR16-1 em contraposição ao aumento de seu gene alvo BCL-2, um importante gene regulador da apoptose. Esses achados sugerem a participação desses miRNAs no controle da apoptose na EM. Para explorar essa associação, foi analisado a expressão global de miRNAs nas subpopulações de linfócitos T de pacientes com EMRR no estágio de remissão. O resultado dessa análise determinou de forma inédita o aumento significativo da expressão de 9 miRNAs (miRNAs-16, miRNAs-20a, miRNAs-21, miRNAs-24, miRNAs-155, miRNAs-221, miRNAs-222, miRNAs-720 e miRNAs-1281) nos linfócitos T CD8+ de memória central. A análise in silico dos alvos desses miRNAs indicou que três vias canônicas, relacionadas à ativação da apoptose, eram enriquecidas com alvos preditos e validados experimentalmente desses miRNAs. Desse modo sugerimos a forte relação desses miRNAs no controle da apoptose nos linfócitos T dos pacientes com EMRR. A fim de aprofundar nossos estudos, selecionamos os miRNAs miR-21 e miR-24, para a realização de experimentos funcionais in vivo. Foi verificada a indução da expressão miR-21 somente nos linfócitos T CD4+ de modelo experimental da EM. Adicionalmente, experimentos in vitro demonstraram que a expressão do miR-21 e restrita as populações de células Th2 e Th17. Nesse caso, miR-21 parece ser regulado pelo fator de transcrição STAT3, sugerindo assim que o aumento da expressão do miR-21 verificada no modelo animal possa estar relacionada com a presença de linfócitos T CD4+ de perfil Th17 nesse tecido. Em resumo, o conjunto desses resultados demonstra a relevância dos miRNAs na fisiopatologia da EMRR, principalmente no controle da apoptose. / This study discusses the role of miRNA in the Molecular Pathophysiology of Relapse Remitting Multiple Sclerosis (RRMS). The study has shown that CD4+ lymphocytes from relapsed RRMS patients had lower expression of miR15a and miR-16-1 in contraposition of higher expression of the target gene BCL-2, a key regulator of apoptosis. These findings suggest the role of those on the control of apoptosis in MS. In order to explore this association, the global expression of miRNAs was analysed in T lymphocyte subpopulations from remission RRMS patients. The result of this analysis has demonstrated for the first time a significant higher expression of 9 miRNAs (miRNAs-16, miRNAs-20a, miRNAs-21, miRNAs-24, miRNAs-155, miRNAs-221, miRNAs-222, miRNAs-720 e miRNAs-1281) in central memory T CD8+ lymphocytes. In silico analysis of the miRNAs targets indicates that three canonical pathways related to the activation of apoptosis were enriched with predicted and experimental validated gene targets for those miRNAs. In this way, we suggest the strong relation of these miRNAs in the control of apoptosis in the lymphocytes from RRMS patients. In order to intensify our studies we selected miR-21 and miR-24 to perform in vivo functional experiments. It was verified miR-21 induction only in T CD4+ lymphocytes from MS animal model. Additionally, in vitro experiments have demonstrated that miR-21 expression was restricted to Th2 and Th17 cell populations. In this way, miR-21 seems to be regulated by the STAT3 transcription factor, thereby suggesting that the increase of miR-21 expression observed in vivo could be related with Th17 CD4+ present in this tissue. In summary, this set of results showed the relevance of miRNAs in the RRMS pathophysiology, mainly in the control of apoptosis.

Submitted by admin tede (tede@pucgoias.edu.br) on 2017-04-27T14:19:15Z No. of bitstreams: 1 GLAUCIELEN GOMES DA SILVA.pdf: 1992448 bytes, checksum: 7764de752332d80cd64fa3e7a9e1b7a1 (MD5) / Made available in DSpace on 2017-04-27T14:19:16Z (GMT). No. of bitstreams: 1 GLAUCIELEN GOMES DA SILVA.pdf: 1992448 bytes, checksum: 7764de752332d80cd64fa3e7a9e1b7a1 (MD5) Previous issue date: 2017-03-08 / The search for autoantibodies in HEp-2 cells represents a relevant tool for diagnostic assistance in the investigation of autoimmune diseases, especially rheumatic diseases. This methodology, over the last years, underwent by intense process of improvement and standardization with the accomplishment of the Brazilian Consensus. The objective of this study was to evaluate the implantation of recommendations in the clinical laboratories that perform the methodology, 16 years after the accomplishment of the I Brazilian Consensus on ANA in HEp-2 cells. A research was conducted for the laboratories between February and October 2016. The laboratories were invited to answer questions that dealt with the guidelines of the Consensus, addressing technical aspects, quality control, reading of the slides, issuance of reports and educational programs. The study counted on the participation of 53 laboratories that jointly realize an estimate of 300,000 ANA by month. It has been identified that several medical specialties request the examination, and different professionals are responsible for the technical procedure and reading of the fluorescence slides. Consensus recommendations are being followed by all laboratories, in absolute by 58.5% of the laboratories. Regarding the technical procedure, 83.1% of the participants are screening at a 1:80 dilution and the title depletion, recommended by consensus, is being adopted by all participants. It was evidenced that 39.6% of the participants use more than one brand of kit and that 22.6% performs titration of the conjugate to each new kit and different lamp powers are used in laboratories with a predominance of 100 Watts. Regarding the reading of the slides, 94.3% stated that they observed the four cell compartments, 92.5% said to classify the chromosome metaphase plate negative or positive and 32.1% of the participants did not affirm to observe the cells in all phases of the cycle. In the issue of reports, 13.2% of the laboratories admitted to reporting only the name of the standard followed by the title, not presenting the descriptive report and 24.5% of the participants do not use education and quality control programs. Most laboratories were able to identify representative images of sets of patterns. The results presented here demonstrate consistent advances from the implementation of the ANA Consensus in Brazil, but also evidence the need for actions to implement continuing education programs. / A pesquisa de autoanticorpos em células HEp-2 tem auxiliado no diagnóstico na investigação de doenças autoimunes especialmente as reumáticas. Tal metodologia, ao longo dos últimos anos, passou por um intenso processo de aperfeiçoamento e padronização com a realização do Consenso Brasileiro. O presente trabalho teve como objetivo avaliar, 16 anos após a realização do I Consenso Brasileiro de FAN em Células HEp-2, a implantação das recomendações nos laboratórios clínicos que realizam a metodologia. Foi realizada uma pesquisa direcionada aos laboratórios entre fevereiro e outubro de 2016. Os laboratórios foram convidados a responder um questionário sobre as diretrizes do Consenso, abordando aspectos técnicos, controle de qualidade, leitura das lâminas, emissão de laudos e programas educativos. O estudo contou com a participação de 53 laboratórios que, em conjunto, realizam uma estimativa de 300.000 FAN/mês. Foi identificada uma heterogeneidade em especialidades médicas solicitantes do teste e profissionais responsáveis pelo procedimento técnico e leitura das lâminas de fluorescência. As recomendações do Consenso estão sendo seguidas em sua totalidade por 58,5% dos laboratórios. Em relação ao procedimento técnico, 83,1% dos participantes fazem triagem com diluição 1:80 e o esgotamento de título, recomendado pelo Consenso, está sendo adotado por todos os participantes. Evidenciou-se que 39,6% dos participantes utilizam mais de uma marca de kit e que 22,6% realiza a titulação do conjugado a cada nova kit, e diferentes potências de lâmpadas são utilizadas nos laboratórios com predomínio das de 100 Watts. Em relação à leitura das lâminas, 94,3% afirmam observar os quatro compartimentos celulares, 92,5% afirmam classificar a placa metafásica cromossômica em negativa ou positiva e 32,1% dos participantes não afirmaram observar as células em todas as fases do ciclo celular. Na emissão de laudos, 13,2% dos laboratórios admitiram relatar somente o nome do padrão seguido pelo título, não apresentando o laudo descritivo e 24,5% dos participantes não utilizam programas de educação e controle de qualidade. A maioria dos laboratórios foi capaz de identificar imagens representativas de grupos de padrões. Os resultados aqui apresentados demonstram consistentes avanços a partir da implantação do Consenso de FAN no Brasil, porém, evidenciam também a necessidade de ações para implementação dos programas de educação continuada.

Encefalite auto-imune experimental (EAE) é o modelo animal da doença humana esclerose múltipla. Foi demonstrado que as estatinas podem ter efeitos benéficos na EAE. Diversos mecanismos de ação foram descritos para explicar esses efeitos, entre eles: estímulo a expressão de HO-1, inibição da expressão de Toll-like receptors (TLR) e inibição da produção de citocinas de padrão Th1. Estudamos o efeito de uma estatina, a sinvastatina, na evolução clínica da EAE e seus mecanismos de ação. Também avaliamos o papel do TLR4 na EAE. O tratamento com sinvastatina melhorou a evolução clínica da EAE nas doses de 1 e 5 mg/kg/dia. A análise do infiltrado celular no SNC mostrou mudança do padrão de diferenciação dos linfócitos com menor porcentagem de células produtoras de IL-17 nos grupos tratados em relação aos controles. Os animais TLR-4 -/- apresentaram melhor evolução da doença. Concluímos que o tratamento com sinvastatina melhora a evolução clínica da EAE através da inibição da produção de IL-17 e que animais TLR-4 -/- têm melhor desfecho clínico na EAE que os controles. / Experimental autoimmune encephalomyelitis (EAE) is considered the experimental model for the human multiple sclerosis disease. It has been demonstrated that statins, used as lipid-lowering agents, can have beneficial effects on EAE. Several actions have been described to explain these effects: enhancement of HO-1 expression, inhibition of Toll-like receptors expression and inhibition of Th1 cytokines. We have investigated the effect of a statin, simvastatin, on EAE clinical development and the mechanisms behind those effects. Simvastatin treatment ameliorated EAE clinical outcome at doses 1 and 5 mg/kg/day. SNC cellular infiltrates analysis showed altered pattern of differentiation with decreased percentage of IL-17-producing T lymphocytes in treated group comparing with controls. TLR4 -/- mice showed better clinical development. We concluded that simvastatin treatment ameliorates EAE clinical outcome through inhibition of IL-17 production. Mice TLR4 -/- have better EAE clinical outcome than WT controls.

Antibody combining sites are constructed from limited set of germ-line gene segments, yet are capable of both recognizing a broad range of common epitopes and eliciting an adaptive response to newly encountered pathogens. Carbohydrate antigens generally do not draw T cell help and concomitant affinity maturation in the humoral response. Therefore, anti-carbohydrate responses must rely more heavily on the primary germ-line gene repertoire. Antibodies are usually thought of as highly specific. It has been suggested that polyspecificity and cross-reactivity in germ-line antibodies is necessary to provide the protective mechanisms required to broaden the potential number of antigens recognized; however, the molecular mechanism underlying polyspecificity is poorly understood. To investigate the phenomena of specificity, cross-reactivity and polyspecificity in germ-line antibodies my thesis focuses first on the unique LPS inner core oligosaccharide of Chlamydiaceae, which contains variations within the conserved inner core trisaccharide Kdo(2→8)Kdo(2→4)Kdo (3-deoxy-D-manno-oct-2-ulosonic acid). Antibodies raised against this family-specific trisaccharide showed strong V-region restriction with two sets of heavy and light chain V genes accounting for almost all clones isolated. These groups were named after their prototypic clones as the ‘S25-2 type’ and the ‘S25-23 type’. In contrast to the cross-reactive S25-2 and related antibodies, the S25-23 family of antibodies were shown to be specific for the Chlamydiaceae-specific trisaccharide antigen with no cross-reactivity to Kdo mono or disaccharides or to the Kdo(2→4)Kdo(2→4)Kdo trisaccharide antigen. Interest in S25-23 was sparked by its rare high μM affinity and strict specificity for the family-specific trisaccharide antigen. The structures of the antigen binding fragments of four S25-23-type mAbs have been determined to high resolution in complex with the Chlamydiaceae-specific epitope, revealing the molecular basis for their binding behaviour. The germ-line-encoded paratopes of these antibodies differ significantly from previously characterized S25-2-type mAbs. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the length of the trisaccharide antigen. Further S25-23-type antibodies are glycosylated on the variable heavy chain. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of a Gal(α1→3)Gal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic responses in patients receiving therapeutic antibodies. The second focus of my thesis research is the lipid A moiety of LPS, which is involved in septic shock. Though the lipid A epitope appears to be cryptic during infection with Gram-negative bacteria, there have been several reported instances of lipid A specific antibodies isolated from human sera. While these antibodies are strictly selective for lipid A, there are reports of polyspecificity of some anti-lipid A antibodies for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity has been reported to result in the unfortunate consequences of autoimmune disease. This thesis reports the first crystal structures of antibodies in complex with lipid A and single stranded nucleic acids, elucidating their mechanism for polyspecificity. Perhaps more importantly, the structures may yield clues to the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases. / Graduate / 2020-04-18 / 0487 / 0982

Wun Hau Ling. / "August 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 177-208). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Atualmente, ainda é incerto se a hidrólise de L-arginil-β-naftilamida (ArgNA) e do leucotrieno (LT) A4 pela LT-A4-hidrolase (LT-A4-H) (EC 3.3.2.6) e pela aminopeptidase básica (APB) (EC 3.4.11.6) tem influência no desenvolvimento da artrite induzida por colágeno (CIA). O objetivo deste estudo foi investigar a inter-relação entre LT-A4-H, APB e LT-B4 em ratos submetidos à CIA. Cromatografia líquida de alta eficiência (HPLC), ensaio imunoenzimático (EIA), espectrofluorimetria e reação quantitativa em tempo real em cadeia da polimerase (qPCR) foram usados como metodologias. A existência dos genes para as proteínas EC 3.3.2.6 e EC 3.4.11.6 foi confirmada no tecido sinovial (TS) de ratos controles sadios. A hidrólise de ArgNA aumentou na fração solúvel (FS) dos animais submetidos à CIA que desenvolveram a doença (artríticos-CIA) em comparação com aqueles que não desenvolveram a doença (resistentes-CIA) e com os controles sadios. No líquido sinovial (SY) e no plasma sanguíneo houve menor hidrólise de ArgNA em resistentes em comparação aos artríticos e controles. Nas células mononucleares do sangue periférico (PBMCs), os níveis de hidrólise de ArgNA aumentaram na FS de controles e na fração de membrana (FM) dos resistentes em comparação aos artríticos. Em comparação com controles sadios, a hidrólise de LT-A4 aumentou no SY e na FS de PBMCs de artríticos e resistentes. A hidrólise de LT-A4 também aumentou na FM do TS de resistentes e diminuiu na FM de PBMCs em artríticos e resistentes. Em todos estes compartimentos a hidrólise de ArgNA permaneceu inalterada ou relacionou-se inversamente com a hidrólise de LT-A4, comparativamente aos controles sadios. A hidrólise de ArgNA diferiu entre os artríticos e resistentes em FM-TS, FS-TS, FM-PBMCs, SY e no plasma sanguíneo. Uma relação no mesmo sentido foi encontrada entre alterações na hidrólise de LT-A4 e nos níveis de LT-B4 apenas em SY e FM-PBMCs dos artríticos e resistentes e em FM-TS dos resistentes, comparativamente aos controles sadios. Em conclusão, a atividade APB é um novo marcador que distingue ratos artríticos e resistentes no modelo CIA. Os níveis de LT-B4 em ratos não são controlados somente pela LT-A4-H. Alterações na atividade LT-A4-H e nos níveis de LT-B4 são indistinguíveis entre artríticos e resistentes, mas tais alterações marcadamente distinguem essas duas condições da condição saudável. LT-A4-H e APB estão relacionadas de uma forma compartimento-dependente, atuando como enzimas independentes, com modulação diferencial das suas especificidades, eficiências e/ou afinidades catalíticas sobre os substratos epóxi e peptídico, ou como enzimas bifuncionais, cujas atividades são inversamente relacionadas devido à inibição concorrente de uma destas atividades / Whether L-arginyl-β-naphthylamide (ArgNA) and leukotriene (LT)-A4 hydrolyses by LT-A4 hydrolase (LT-A4-H) (EC 3.3.2.6) and basic aminopeptidase (APB) (EC 3.4.11.6) influence the development of collagen-induced arthritis (CIA) is presently uncertain. The objective of this study was to investigate the interrelationship among LT-A4-H, APB and LT-B4 in CIA rats. High-performance liquid chromatography (HPLC), enzyme immunoassay (EIA), spectrofluorometry and quantitative real time polymerase chain reaction (qPCR) were used as methodologies. The existence of genes for EC 3.3.2.6 and EC 3.4.11.6 proteins were confirmed in the synovial tissue (TS) of healthy control rats. ArgNA hydrolysis was higher in soluble fraction (FS) in rats submitted to CIA that developed the disease (CIA-arthritic) than in those that did not develop the disease (CIA-resistant) or healthy control. Synovial fluid (SY) and blood plasma had lower ArgNA hydrolysis in CIA-resistant than in CIA-arthritic or control. In the peripheral blood mononuclear cells (PBMCs) the levels of ArgNA hydrolysis increased in FS of control and in membrane-bound fraction (FM) of CIA-resistant in comparison with CIA-arthritic. Compared with healthy control, LT-A4 hydrolysis increased in SY and in FS from PBMCs of CIA-arthritic and CIA-resistant. LT-A4 hydrolysis also increased in FM from TS of CIA-resistant and decreased in PBMCs-FM of CIA-arthritic and CIA-resistant. In all these locations hydrolysis of ArgNA remained unchanged or it was inversely related with LT-A4 hydrolysis, comparatively to healthy control. ArgNA hydrolysis differed between CIA-arthritic and CIA-resistant in TS-FM, TS-FS, PBMCs-FM, SY and blood plasma. A same-sense relationship was found between changes on LT-A4 hydrolysis and LT-B4 levels only in SY and PBMCs-FM of CIA-arthritic and CIA-resistant and in TS-FM of CIA-resistant, comparatively to healthy control. In conclusion, the APB activity is a novel distinctive marker of CIA-arthritic and CIA-resistant statuses. The levels of LT-B4 in rats are not controlled only by LT-A4-H. Changes on LT-A4-H activity and LT-B4 levels are indistinguishable between CIA-resistant and CIA-arthritic, but such variations markedly distinguish these two statuses from healthy status. LT-A4-H and APB are related in a compartment-dependent manner acting as independent enzymes with differential modulation of their specificity, efficiency and/or catalytic affinity on the aminoacyl and epoxy substrates, or as bifunctional enzymes which activities are inversely related due to the concurrent inhibition of one of these

A artrite induzida por pristane (PIA) em camundongos HIII (resistentes) e LIII (suscetíveis) foi usada para estudar mecanismos inflamatórios e imunes atuantes na fase pré-clínica da doença, os quais são pouco conhecidos. Estudos anteriores mostraram diferenças significativas na produção de citocinas nos animais HIII e LIII na fase pré-clínica da PIA, sugerindo forte influênica no fenótipo de PIA. A PIA foi induzida apenas por via intraperitoneal nos animais LIII, com intensa infiltração de neutrófilos, linfócitos, monócitos e macrófagos, altos níveis de IL-12p40 e maior expressão de genes de citocinas inflamatórias após a injeção de pristane. Por outro lado, na linhagem HIII houve aumento de eosinófilos e neutrófilos, mas redução de monócitos e linfócitos. Não observamos diferenças nos níveis de TNF-<font face=\"Symbol\">&#945;, IFN-<font face=\"Symbol\">g, IL-12p70, IL-17, IL-10. Concluímos que a intensidade e o tipo de resposta inflamatória na fase inicial da PIA podem ser mecanismos envolvidos na diferença de resistência/ susceptibilidade entre as linhagens HIII e LIII. / Pristane-induced arthritis (PIA) in HIII (resistant) and LIII (susceptible) mice was used in this work to characterize the cellular and molecular alterations of the pre-clinical phase of the disease, of which little is known. Previous reports showed significant differences in cytokine production of HIII and LIII mice in the pre-clinical phase of PIA, suggesting a strong influence on PIA phenotype. PIA was induced only by the intraperitoneal route in LIII animals, which showed intense infiltration of neutrophils, lymphocytes, monocytes and macrophages, with high levels of IL-12p40 after pristane injection. Inflammatory cytokine genes were also upregulated in LIII mice. On the other hand, HIII strain had increased eosinophils and neutrophils, but reduced monocytes and lymphocytes. No significant differences were found in TNF-<font face=\"Symbol\">&#945;, IFN-<font face=\"Symbol\">g, IL-12p70, IL-17, IL-10 levels. We conclude that the intensity and type of inflammatory response in the initial phase of PIA may be different mechanisms involved in resistance / susceptibility between HIII and LIII mice.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease where failure to remove apoptotic cells, due to a defect in phagocytic cells, or deficient opsonisation, leads to secondary necrosis and the release of DNA and chromatin. The nuclear constituents from apoptotic cells are targeted by autoantibodies, which form immune complexes. Immune complex-mediated TLR9 activation of plasmacytoid dendritic cells (pDCs) and subsequent secretion of interferon (IFN-α) is thought to drive inflammation in SLE. It is currently believed that pDCs do not normally respond to apoptotic cells, as self-DNA is hidden from TLR9. However, DNA and chromatin expressed on membrane bound apoptotic bodies is essential for inducing IL-10 secreting regulatory B cells through TLR9 stimulation. The overall objective of this thesis was to understand how apoptotic cells influence immune responses in health and in patients with SLE. Splenic mouse pDCs were activated with the synthetic TLR7 agonist R848 and TLR9 agonists CpGB and CpGA and were co-cultured with apoptotic cells, or with freeze-thawed necrotic cells. PDCs co-cultured with apoptotic cells down-regulated the expression of CD40 and CD86. When pDCs were activated by R848 or CpGB, IL-10, IFN-γ and IL-6 secretion was significantly induced in the presence of apoptotic cells. PDCs so cultured induced T cells to secrete immune-regulatory IL- 10. In contrast, co-culturing apoptotic cells with pDCs activated by CpGA, augmented IFN-α secretion. These cytokine responses by pDCs were only stimulated by DNA on whole apoptotic cells; not by free nucleic acids derived from necrotic cells. This data demonstrates that the inflammatory context in which pDCs sense whole apoptotic cells is crucial to determining the threshold of tolerance to apoptotic self. It questions the perception that pDCs see all apoptotic cells and their necrotic cellular debris as dangerous and suggests that there may be something intrinsically different about SLE apoptotic cells, which causes inflammation. SNPs near ATG5, a protein of the cell survival pathway autophagy, have been linked to SLE susceptibility, but the role of autophagy in SLE pathogenesis is unclear. We hypothesised that dysfunctional autophagy is linked to abnormal apoptosis of SLE lymphocytes. Western blotting revealed that ATG5-ATG12 protein complex expression was significantly reduced in SLE lymphocytes and they failed to convert LC3-I to LC3- II, the hallmark of a functioning autophagy pathway, which caused accelerated secondary necrosis. Apoptotic SLE lymphocytes had an impaired ability to stimulate IL-10 secreting regulatory B cells and they induced pro-inflammatory cytokine secretion by monocyte-derived macrophages. Phagocytosis of apoptotic SLE lymphocytes by healthy macrophages was also impaired; however this was independent of ATG5 protein expression. The novel findings of this thesis suggest SLE apoptotic lymphocytes are intrinsically pro-inflammatory, which may be caused by diminished autophagy leading to an inability of lymphocytes to correctly execute apoptosis. Furthermore, inefficient clearance of SLE apoptotic cells results from a defect in the apoptotic cell, rather than the phagocytic cell.

BAFF, «facteur d'activation des lymphocytes B (LB) » contribue à l'expansion des LB autoréactifs de faible affinité lors de la mise en place de la tolérance. Cependant, les mécanismes menant à la surexpression de BAFF dans les maladies auto-immunes ne sont pas compris. Nous avons découvert un nouveau variant de BAFF, 4BAFF (dans lequel l'exon 4 est épissé), qui agit comme un facteur de transcription de son propre gène et participe à sa régulation. Ainsi, 4BAFF est préférentiellement observé dans les cellules isolées de patients atteints de maladies auto-immunes. De plus, 4BAFF régule un grand nombre de gènes associés à la réponse immunitaire innée et à la régulation de l’apoptose. Une autre constatation importante est que 4BAFF est un élément clé pour comprendre l’activité des LB régulateurs. Notre travail présente un concept entièrement nouveau suggérant qu'une cytokine peut être régulée par l'activité de l'un de ses variants d'épissage. Par ailleurs, nous avons observé que les cellules épithéliales expriment le récepteur de BAFF : BR3. Le blocage de BR3 se traduit par la translocation nucléaire de PKC et l'apoptose des cellules épithéliales. Par un effet autocrine, nous démontrons que seules certaines formes de BAFF participent à la survie des cellules épithéliales. Enfin , nous avons étudié les conséquences de l'expression du TLR9 à la surface des LB et démontrons que ce TLR9 membranaire ne fixe pas le CpG et agit comme un co-récepteur négatif du BCR. En effet, l'activation des LB par le CpG capté au niveau endosomal, est inhibée par l’action d’un anticorps anti-TLR9 se fixant au niveau membranaire. Tous ces résultats contribuent à une meilleure compréhension des mécanismes impliqués dans l'immunopathologie des maladies autoimmunes avec des applications potentielles en thérapeutique. / Elevated expression of ‘B cell activating factor’ (BAFF), a potent B cell survival factor contributes to the expansion of low-affinity self-reactive B cells during the establishment of tolerance. However, mechanisms leading to BAFF over-expression in autoimmune diseases are not understood. We reported the discovery of a new variant for BAFF, 4BAFF in humans (in which exon 4 is excised) or 5BAFF in mice (in which exon 5 is excised), which acts as a transcription factor of the full-length form of BAFF, and which is preferentially found in cells isolated from patients with autoimmune diseases. When transfected in human B cells, D4BAFF upregulates a large number of genes associated with immune response and especially innate immunity and regulation of apoptotis. Furthermore D4BAFF acts, in association with p50 from the NF- B pathway, as a transcription factor for its own parent gene. Another important finding is that 4BAFF is an important component of the efficacy of regulatory B cell activity. Our work introduces an entirely novel concept in biology suggesting that a human cytokine gene can be transcriptionally regulated by the activity of one of its own splice variants.We have also tried to understand the complexity of the various forms of BAFF. We observed that epithelial cells expressed BAFF-receptor (BR3) and produce BAFF suggesting autocrine properties. Blocking BR3 results in nuclear translocation of PKC promoting epithelial cell apoptosis.Furthermore, only some forms of BAFF are required for epithelial cell survival. Finally, we studied the consequences of the expression of TLR9 on the B cell surface and demonstrated that TLR9 acts as a co-receptor of the B cell receptor to influence B cell fate independently of CpG binding. We show that CpG activation of B cells, acting synergistically with BCR signals, was inhibited by anti-TLR9 stimulation. Induction of CD25 expression and proliferation of B cells were thus down-regulated by engagement of cell surface TLR9. Overall, our results indicate that TLR9 expressed on B cell plasma membrane might be a negative regulator of endosomal TLR9, and could provide a novel control by which activation of autoreactive B cells is restrained. All these findings contribute to a better understanding on immunopathology of autoimmune diseases with potential applications in therapy.