Systemic lupus erythematosus (SLE) is an autoimmune disease involving the inflammatory damages of multiple organs. Lupus nephritis (LN) as the manifestation in the kidney occurs in more than 50% of SLE patients and is a major cause of morbidity and mortality. Current treatments consist of immunosuppressants that always lead to compromised immune responses with increased risks of infections as the major side effect. To minimize this side effect, it is crucial to develop new treatments that are more natural and specific. Vitamin A, particularly in the form of its functional metabolite, retinoic acid, has shown some beneficial effects against LN in both lupus-prone mouse models and clinical cases. However, a more systemic evaluation of vitamin A treatment in lupus had not been investigated. In our study, we found paradoxical effects of all-trans-retinoic acid (tRA) on lupus-like disease in MRL/lpr lupus-prone mice. Starting at 6 weeks old when the inflammatory environment had been established in MRL/lpr mice, tRA administration reduced immune cell numbers in the secondary lymphoid organs and improved glomerulonephritis. However, circulating autoantibodies and inflammation in renal tubulointerstitium and other organs were increased. The detrimental effects of tRA were not present in MRL control mice, which didn't have an established inflammatory environment at 6 weeks old as shown in MRL/lpr mice, suggesting that the pro-inflammatory effects of tRA are dependent on the pre-existing inflammatory environment. Therefore, to successfully apply vitamin A-based treatment, it is important to avoid the detrimental effects of tRA on lupus by identifying and then specifically eliminating the critical pro-inflammatory immune cell types in lupus. As treatments usually start after the onset of apparent symptoms in patients at the effector stage of autoimmune responses, targeting the inflammatory contributors at this stage appears to be more practical and critical. Among different types of leukocytes, we chose to focus on dendritic cells (DCs), because they are highly diverse and critical in the immune responses as a bridge between the innate and adaptive immune systems. Plasmacytoid DCs (pDCs) as a candidate target have been demonstrated to be crucial for the initiation of lupus development by producing IFNα. However, we demonstrated that although pDCs produced a large amount of IFNα during disease initiation, those from late-stage lupus mice were found to be defective in producing IFNα, suggesting that pDC-targeted treatments should be performed at the initiation stage. This will depend on the progress in early diagnosis in the future. Besides pDCs, we identified a CD11c+ cell population absent at the early-stage but gradually accumulating at the late-stage in the kidneys of lupus mice. These cells have a phenotype of mature monocyte-derived DCs, with particularly high CX3CR1 expression on the surface. Consistent with their pathogenic cytokine profile, in vivo administration of anti-CX3CR1-saporin conjugates to dysfunction these cells in MRL/lpr mice significantly reduced proteinuria scores. Ex vivo activation of renal-infiltrating CD4+ T cells showed increased survival rate, proliferation and IFN-γ production of activated CD4+ T cells when they were cultured with these renal-infiltrating CD11c+ cells. These results suggest that the renal-infiltrating CD11c+ cells are pathogenic and promote inflammation in the kidney at the later effector stage of lupus by interacting with renal-infiltrating CD4+ T cells. In conclusion, although vitamin A showed anti-inflammatory effects on reducing glomerulonephritis, its use in lupus treatment should be guarded due to the other potential pro-inflammatory effects induced by the pre-existing inflammatory environment. IFNα-producing pDCs and CX3CR1highCD11c+ monocyte-derived DCs could be specific therapeutic targets to reduce the established inflammation at the early stage and late stage of LN, respectively. Therefore, it is worthwhile to further investigate the comprehensive effects of combination therapy on lupus, with vitamin A administration and pDCs-specific depletion at the early stage, and CX3CR1highCD11c+ monocyte-derived DCs-specific depletion at the late stage. / Ph. D. / Systemic lupus erythematosus (SLE) is an autoimmune disease involving the inflammatory damages of multiple organs. Lupus nephritis (LN) as the manifestation in the kidney occurs in more than 50% of SLE patients and is a major cause of morbidity and mortality in this disease. Current treatments consist of immunosuppressants that always lead to compromised immune responses with increased risks of infections as the major side effect. To minimize this side effect, it is crucial to develop new treatments that are more natural and specific. My first project was to determine whether vitamin A as a supplement could ameliorate SLE. It turned out to be effective at attenuating LN, but at the same time the nutrient caused massive inflammation in other peripheral organs such as the brain and lungs. This suggests that we need to be cautious when recommending vitamin A supplementation to lupus patients. In order to identify more specific targets in the treatment of SLE, my second and third projects focused on dendritic cells (DCs) that are essential for lupus pathogenesis. I found that plasmacytoid DCs (pDCs), known to be pathogenic in SLE, were in fact defective at promoting inflammation at the late stage of disease, suggesting that pDCs might not be a good target of intervention. In contrast, monocyte-derived conventional DCs turned out to be highly pathogenic especially for the development of LN and could be a potential therapeutic target. Altogether, my investigations have increased our understanding of the pathogenesis of SLE.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13630号 / 論医博第2321号 / 新制||医||1074(附属図書館) / (主査)教授 伊藤 能永, 教授 浅野 雅秀, 教授 濵﨑 洋子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Systemic lupus erythematosus (SLE) is a chronic multifactorial inflammatory autoimmune disease with heterogeneous clinical manifestations. Among different manifestations, lupus nephritis (LN) remains a major cause of morbidity and mortality. There are few FDA approved treatments for LN. In general, they are non-selective and lead to global immunosuppression with significant side effects including an increased risk of infection. In the past 60 years, only one new drug, belimumab was approved for lupus disease with modest efficacy in clinic and not approved for patients suffering for nephritis. Therefore, it is urgent to develop new treatments to replace or reduce the use of current ones. Histone deacetylase 6 (HDAC6) plays a variety of biologic functions in a number of important molecular pathways in diverse immune cells. Both innate and adaptive immune cells contribute to pathogenesis of lupus. Among those cells, B cells play a central role in pathogenesis of lupus nephritis in an anti-body dependent manner through differentiation into plasma cells (PCs). As a result, HDAC6 inhibitors represent an entirely new class of agents that could have potent effects in SLE. Importantly, the available toxicity profile suggests that HDAC6 inhibitors could be advanced into SLE safely. We have demonstrated previously that histone deacetylase (HDAC6) expression is increased in animal models of systemic lupus erythematosus (SLE) and that inhibition of HDAC6 decreased disease. ACY-738 is a hydroxamic acid HDAC6 inhibitor that is highly selective for HDAC6. In our current studies, we tested if an orally selective HDAC6 inhibitor, ACY-738, would decrease disease pathogenesis in a lupus mouse model with established early disease. Moreover, we sought to delineate the cellular and molecular mechanism(s) of action of a selective HDAC6 inhibitor in SLE. In order to define the mechanism by which HDAC6 inhibition decreases disease pathogenesis in NZB/W mice by using RNAseq to evaluate the transcriptomic signatures of splenocytes from treated and untreated mice coupled with applied computational cellular and pathway analysis. In addition, we sought to bridge between the transcriptomic data obtained from the HDAC6 treated mice and human gene expression information to determine the relevance to this target in possibly controlling human lupus. We treated 20-week-old (early-disease) NZB/W F1 female mice with two different doses of the selective HDAC6 inhibitor (ACY-738) for 4~5 weeks. As the mice aged, we determined autoantibody production and cytokine levels by ELISA, and renal function by measuring proteinuria. At the termination of the study, we performed a comprehensive analysis on B cells, T cells, and innate immune cells using flow cytometry and examined renal tissue for immune-mediated pathogenesis using immunohistochemistry and immunofluorescence. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. Our results showed a reduced germinal center B cell response, decreased T follicular helper cells and diminished interferon (IFN)-γ production from T helper cells in splenic tissue. Additionally, we found the IFN-α-producing ability of plasmacytoid dendritic cells was decreased along with immunoglobulin isotype switching and the generation of pathogenic autoantibodies. Renal tissue showed decreased immunoglobulin deposition and reduced inflammation as judged by glomerular and interstitial inflammation. The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. RNA sequence data showed that PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show selective HDAC6 inhibition decreased several parameters of disease pathogenesis in lupus-prone mice. The decrease was in part due to inhibition of B cell development and response. RNA sequence data analysis show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggests that a critical event might be modulation of cellular metabolism. / Doctor of Philosophy / Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease by which immune cells mistakenly attacks healthy self-cells in different organs. Kidney inflammation occurs in nearly 50% of patients with lupus resulting in kidney damage leading to end stage renal disease. Lupus nephritis (LN) is major cause of morbidity and mortality associated with SLE. Current treatments for LN consist primarily of immunosuppressants that block the immune response and leave the patients with unwanted side effects including an increased risk of infection. To circumvent the unwanted side effects, we explored a novel mechanism to target the immune response. My project was to determine whether histone deacetylase 6 (HDAC6) inhibition would suppress the autoimmune inflammatory response in lupus. We found that inhibition of HDAC6 was effective at attenuating early LN, probably by down-regulating innate immune response, which suppressed subsequent adaptive immune responses downstream. HDAC6 inhibition affected the innate immune response by inhibiting type I interferon production by plasmacytoid dendritic cells. HDAC6 inhibition affected the cell mediated immune response by decreasing T helper cell and B cell activation. To determine the mechanism by which HDAC6 inhibits immune cells activation, we used RNAseq to reveal HDAC6 inhibition on multiple signaling events associated with the induction of lupus disease. These results suggest that HDAC6 could be a potential therapeutic target in the early stage of LN.

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. Despite years of study, the etiology of SLE is still unclear. Both genetic and environmental factors have been implicated in the disease mechanisms. Gut microbiota as an environmental factor and the immune system interact to maintain tissue homeostasis, but whether this interaction is involved in the pathogenesis of SLE is unclear. In a classical model of lupus nephritis, MRL/lpr, we found decrease of Lactobacillales but increase of Lachnospiraceae in the gut microbiota. Increasing Lactobacillales in the gut by suppling a mixture of 5 Lactobacillus strains improved renal function of these mice and prolonged their survival. Further studies revealed that MRL/lpr mice possessed a "leaky" gut, which was reversed by increased Lactobacillus colonization. Inside the kidney, oral Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. To remove Lachnospiraceae that was higher in lupus-prone mice than controls, we administered vancomycin orally to MRL/lpr mice after disease onset from 9 to 15 weeks of age. Vancomycin functions by removing Gram-positive bacteria such as Lachnospiraceae but sparing Lactobacillus spp. The treatment during active lupus reshaped the gut microbiota and significantly ameliorated systemic autoimmunity and kidney histopathology at 15 weeks of age. However, when vancomycin treatment was initiated from a very early age, the beneficial effect was not observed. Strikingly, mice given vancomycin only at the young age exhibited an even worse disease outcome. Indeed, regulatory B (Breg) cells were found to be reduced after the vancomycin treatment at young age. Importantly, adoptive transfer of Breg cells at 6-7 weeks of age rescued the beneficial effect, which indicates that Breg cells, inducible by vancomycin-sensitive gut microbiota, plays an important role in suppressing lupus disease initiation and progression. Finally, we demonstrated that bacterial DNA from the gut microbiota might be the inducer of Breg cells, as bacterial DNA administration at young age reproduced the beneficial effect seen in the Breg adoptive transfer experiment. Future studies are required to examine the clinical efficacy of targeting gut microbiota as a novel treatment against SLE. / Ph. D. / Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. SLE affects over 5 million people worldwide, especially women of childbearing age. Lupus nephritis is a manifestation of SLE occurring in the kidney which affects more than 50% of SLE patients and is a major cause of morbidity and mortality in SLE. Current treatments for lupus nephritis are primarily nonselective immunosuppressants, which can cause a higher incidence of severe infections. There is an imperative need for the development of new therapeutic strategies against SLE. Our research team was the first to describe the dynamics of gut microbiota in a mouse model of SLE. My dissertation research studying the role of gut microbiota in the pathogenesis of lupus-like disease in mice showed that there were both pathogenic and beneficial bacteria co-existing in the gut microbiota of lupus-prone mice. My studies revealed not only the effects of different bacteria on lupus pathogenesis, but also the immunological mechanisms by which they exert the effects. The results suggest that modulation of the gut microbiota through diet, probiotics, and/or prebiotics to selectively enhance the abundance and activity of beneficial bacteria may be an attractive strategy for disease prevention and treatment of SLE patients. Nevertheless, studies on human samples and clinical trials are required to confirm the translational application of this strategy.

Os antimaláricos, cloroquina e hidroxicloroquina, têm sido usados há décadas com bons resultados terapêuticos para o tratamento do lupus eritematoso e são considerados medicações seguras, muito embora, haja preocupação em relação à retinotoxicidade, notadamente com a cloroquina. O objetivo deste trabalho foi avaliar a ocorrência dos efeitos adversos desencadeados pelo tratamento com 250mg/d de difosfato de cloroquina em doentes com lupus eritematoso, dando ênfase à retinotoxicidade. Foram estudados 350 doentes e reavaliados seus respectivos prontuários, que datavam de 1980 a 2003. Os doentes foram acompanhados no ambulatório de colagenoses da Divisão de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. A ocorrência dos efeitos adversos foi de 35,7%, sendo que 17,4% decorreram de alterações oculares. Em 12% dos doentes ocorreu pigmentação retiniana sugestiva de retinopatia antimalárica, em 3,1% depósitos corneanos e, em 2,3%, sintomas visuais agudos. Em 10% dos doentes houve alterações gastrointestinais: epigastralgia (6%), náuseas e vômitos (3,7%) e diarréia (0,3%). Alterações dermatológicas ocorreram em 3,4% dos doentes: rash cutâneo no início do tratamento (2%), exacerbação de quadro de psoríase pré-existente (0,3%) e pigmentação cutânea (1,1%). Ocorreram ainda cefaléia (2,9%), alterações neuromusculares (1,7%) com quadro gripal símile no início do tratamento (1,1%), neuropatia sensitiva (0,3%) e miopatia compatível com miastenia (0,3%) e, sintomas neuropsiquiátricos (0,3%). A droga foi suspensa devido aos efeitos adversos em 22,9% dos doentes, principalmente, em decorrência de alterações oculares, gastrointestinais e dermatológicas. A reavaliação oftalmológica de 12% dos doentes com pigmentação retiniana, confirmou a retinopatia antimalárica em apenas 2,6%, o que demonstrou uma tendência à valorização de alterações retinianas inespecíficas, discretas e unilaterais, com indicação desnecessária da suspensão da droga em 9,4% dos doentes. Não ocorreram casos de retinopatia antimalárica avançada com lesão do tipo bull-eye. Não houve associação estatisticamente significativa entre a ocorrência de efeitos adversos e alterações retinianas com dose diária de difosfato de cloroquina por quilo de peso e com o tipo clínico do lupus eritematoso. As alterações retinianas foram estatisticamente significativas nos doentes acima de cinqüenta anos quando comparado ao grupo abaixo dos cinqüenta anos, possivelmente pela dificuldade em diferenciar as alterações iniciais da retinopatia antimlárica daquelas decorrentes da degeneração macular senil. O controle oftalmológico foi realizado em intervalo médio de 10,5 meses, demonstrando que o controle anual foi eficaz para o acompanhamento dos doentes. Nove doentes foram expostas durante o primeiro trimestre gestacional, não ocorrendo casos de mal formação fetal / Antimalarial agents, chloroquine and hydroxichloroquine, have been used for decades leading to good therapeutic outcomes at treatment approach for lupus erythematosus and are considered safe medication; however, the main concern is retina toxicity, especially with chloroquine. The purpose of the present study was to conduct analysis of the occurrence of adverse effects, triggered by use of 250 mg/d of chloroquine diphosphate at treatment for lupus erythematosus, especially retina toxicity. We analyzed 350 patients and reviewed their medical charts, from 1980 to 2003. The patients were followed up by the outpatient unit of collagenosis, Division of Dermatology, Hospital das Clinicas, Medical School, University of São Paulo. The occurrence of adverse effects was 35.7%, and eye affections were detected in 17.4% of patients. Impairment of retina pigmentation suggestive of antimalarial retinopathy occurred in 12%, cornea deposits in 3,1%, and acute visual symptoms in 2.3%. Gastrointestinal affections were detected in 10% of patients: epigastralgia (6%), nausea and vomiting (3.7%) and diarrhea (0.3%). Dermatological affections occurred in 3.4% of patients: skin rash in the beginning of treatment (2%), exacerbation of preexisting psoriasis (0.3%) and skin pigmentation (1.1%). We also detected headache (2.9%), neuromuscular disorders (1.7%) with flu-like episode at the beginning of treatment (1,1%), sensitive neuropathy (0,3%) and myopathy compatible with myasthenia (0.3%) and neuropsychiatric symptoms (0.3%). Discontinuation of drugs owing to side effects occurred in 22.9% of the patients, being that the main affections were eye, gastrointestinal and dermatological occurrences. Ophthalmologic reevaluation of retina pigmentation affections occurred in 12% of the patients, but we confirmed antimalarial retinopathy only in 2.6%, detecting a tendency to value nonspecific, discreet and unilateral affections, which generated unnecessary recommendations for discontinuation of drug in 9.4% of the patients. There were no cases of advanced retinopathy with bull-eye type lesion. There was no statistically significant association between occurrence of adverse effects and retina affections with daily dose per kg of chloroquine diphosphate and the differents types of lupus erythematosus. In patients over the age of 50, there was statistically significant increase in number of retina affections when compared to the group aged below 50 years, possibly owing to difficulty to differentiate between initial affections in antimalarial retinopathy from those resultant from senile macular degeneration. Ophthalmologic control was conducted on average after 10.5 months, showing that annual follow-up was effective to keep track of patients. Nine of the patients were exposed during the first gestational trimester and there were no cases of fetal malformations

Os antimaláricos, cloroquina e hidroxicloroquina, têm sido usados há décadas com bons resultados terapêuticos para o tratamento do lupus eritematoso e são considerados medicações seguras, muito embora, haja preocupação em relação à retinotoxicidade, notadamente com a cloroquina. O objetivo deste trabalho foi avaliar a ocorrência dos efeitos adversos desencadeados pelo tratamento com 250mg/d de difosfato de cloroquina em doentes com lupus eritematoso, dando ênfase à retinotoxicidade. Foram estudados 350 doentes e reavaliados seus respectivos prontuários, que datavam de 1980 a 2003. Os doentes foram acompanhados no ambulatório de colagenoses da Divisão de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. A ocorrência dos efeitos adversos foi de 35,7%, sendo que 17,4% decorreram de alterações oculares. Em 12% dos doentes ocorreu pigmentação retiniana sugestiva de retinopatia antimalárica, em 3,1% depósitos corneanos e, em 2,3%, sintomas visuais agudos. Em 10% dos doentes houve alterações gastrointestinais: epigastralgia (6%), náuseas e vômitos (3,7%) e diarréia (0,3%). Alterações dermatológicas ocorreram em 3,4% dos doentes: rash cutâneo no início do tratamento (2%), exacerbação de quadro de psoríase pré-existente (0,3%) e pigmentação cutânea (1,1%). Ocorreram ainda cefaléia (2,9%), alterações neuromusculares (1,7%) com quadro gripal símile no início do tratamento (1,1%), neuropatia sensitiva (0,3%) e miopatia compatível com miastenia (0,3%) e, sintomas neuropsiquiátricos (0,3%). A droga foi suspensa devido aos efeitos adversos em 22,9% dos doentes, principalmente, em decorrência de alterações oculares, gastrointestinais e dermatológicas. A reavaliação oftalmológica de 12% dos doentes com pigmentação retiniana, confirmou a retinopatia antimalárica em apenas 2,6%, o que demonstrou uma tendência à valorização de alterações retinianas inespecíficas, discretas e unilaterais, com indicação desnecessária da suspensão da droga em 9,4% dos doentes. Não ocorreram casos de retinopatia antimalárica avançada com lesão do tipo bull-eye. Não houve associação estatisticamente significativa entre a ocorrência de efeitos adversos e alterações retinianas com dose diária de difosfato de cloroquina por quilo de peso e com o tipo clínico do lupus eritematoso. As alterações retinianas foram estatisticamente significativas nos doentes acima de cinqüenta anos quando comparado ao grupo abaixo dos cinqüenta anos, possivelmente pela dificuldade em diferenciar as alterações iniciais da retinopatia antimlárica daquelas decorrentes da degeneração macular senil. O controle oftalmológico foi realizado em intervalo médio de 10,5 meses, demonstrando que o controle anual foi eficaz para o acompanhamento dos doentes. Nove doentes foram expostas durante o primeiro trimestre gestacional, não ocorrendo casos de mal formação fetal / Antimalarial agents, chloroquine and hydroxichloroquine, have been used for decades leading to good therapeutic outcomes at treatment approach for lupus erythematosus and are considered safe medication; however, the main concern is retina toxicity, especially with chloroquine. The purpose of the present study was to conduct analysis of the occurrence of adverse effects, triggered by use of 250 mg/d of chloroquine diphosphate at treatment for lupus erythematosus, especially retina toxicity. We analyzed 350 patients and reviewed their medical charts, from 1980 to 2003. The patients were followed up by the outpatient unit of collagenosis, Division of Dermatology, Hospital das Clinicas, Medical School, University of São Paulo. The occurrence of adverse effects was 35.7%, and eye affections were detected in 17.4% of patients. Impairment of retina pigmentation suggestive of antimalarial retinopathy occurred in 12%, cornea deposits in 3,1%, and acute visual symptoms in 2.3%. Gastrointestinal affections were detected in 10% of patients: epigastralgia (6%), nausea and vomiting (3.7%) and diarrhea (0.3%). Dermatological affections occurred in 3.4% of patients: skin rash in the beginning of treatment (2%), exacerbation of preexisting psoriasis (0.3%) and skin pigmentation (1.1%). We also detected headache (2.9%), neuromuscular disorders (1.7%) with flu-like episode at the beginning of treatment (1,1%), sensitive neuropathy (0,3%) and myopathy compatible with myasthenia (0.3%) and neuropsychiatric symptoms (0.3%). Discontinuation of drugs owing to side effects occurred in 22.9% of the patients, being that the main affections were eye, gastrointestinal and dermatological occurrences. Ophthalmologic reevaluation of retina pigmentation affections occurred in 12% of the patients, but we confirmed antimalarial retinopathy only in 2.6%, detecting a tendency to value nonspecific, discreet and unilateral affections, which generated unnecessary recommendations for discontinuation of drug in 9.4% of the patients. There were no cases of advanced retinopathy with bull-eye type lesion. There was no statistically significant association between occurrence of adverse effects and retina affections with daily dose per kg of chloroquine diphosphate and the differents types of lupus erythematosus. In patients over the age of 50, there was statistically significant increase in number of retina affections when compared to the group aged below 50 years, possibly owing to difficulty to differentiate between initial affections in antimalarial retinopathy from those resultant from senile macular degeneration. Ophthalmologic control was conducted on average after 10.5 months, showing that annual follow-up was effective to keep track of patients. Nine of the patients were exposed during the first gestational trimester and there were no cases of fetal malformations

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 5 uppsatser.

New Zealand Black/White F₁ hybrid (NZB/W) and MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop a Systemic Lupus Erythematosus-like autoimmune disease. While the primary immunologic defect in the NZB/W is due to B cells, in the MRL/lpr it is a result of T cell abnormalities. Diethyldithiocarbamate (DTC), an agent suggested to enhance T cell function, was used to treat both strains. Weekly treatment of NZB/W mice with 25 mg/kg DTC had no significant effect upon survival or autoantibody levels but did induce changes in cell surface antigen expression. MRL/lpr mice treated with DTC displayed normalization of cell surface antigen expression (particularly increased expression of Lyt-2, macrophage markers and Lyt-2⁺/L3T4⁺ thymocytes), decreased lymphoproliferation and thymic atrophy, decreased serum autoantibody levels and kidney deposition of C3 and IgM, restored responses to mitogens and significantly prolonged survival. To determine both the influence of MRL background and lpr genes and to better understand on what cell populations DTC effects, changes in cell surface antigen expression were examined in DTC treated MRL-+/+, Balb/c, and Balb/lpr strains. The only consistent similarities observed between all strains tested were DTC induced changes in Mac-1 splenocyte surface antigen expression. In vitro studies showed DTC to have variable effects upon the mitogenic responses of lymphoid cells to phytohemagluttinin, but DTC alone stimulated both MRL/lpr and Balb/lpr lymphocytes. DTC stimulated the null cell population that predominates in lpr gene-bearing mice, but all observed in vitro effects of DTC were dependent upon the adherent cell population included in culture. DTC had no apparent direct effects upon adherent cells alone however. These studies have shown that DTC is capable of positive effects upon one autoimmune murine strain, the MRL/lpr, but not the NZB/W. DTC appears to affect macrophages, but other cell populations are required to obtain full activity of this compound. The variable effects of DTC emphasize the need to define the immunopathology of individual patients with autoimmune disease before initiating treatment with immunomodulative therapy.

Les récepteurs Toll-like (TLRs) détectent des structures conservées exprimées par différentes classes de microorganismes, jouant ainsi un rôle majeur dans la réponse immunitaire. Les TLRs localisés dans les endosomes (TLR3, 7, 8 et 9) reconnaissent principalement des acides nucléiques dérivés de microbes. Cependant, ils peuvent également être responsables de la reconnaissance d’acides nucléiques endogènes et contribuer au développement d’auto immunité. A la différence du TLR8humain, le TLR8 murin n’induit pas de réponse à l’ARN simple brin et a ainsi été considéré comme non fonctionnel. Le but de cette étude est d’étudier le rôle du TLR8 murin dans l’immunité. Nous avons montré que les cellules dendritiques déficientes en TLR8 surexpriment le TLR7 et présentent une réponse accrue à une stimulation de TLR7. Chez la souris, la déficience en TLR8 entraine une augmentation des taux d’anticorps circulant (IgM, IgG, IgG2a), des autoanticorps, et au niveau rénal la présence de dépôts de complexes immuns. A l’inverse des souris TLR8-/-, les souris TLR7/8-/- sont protégées de tout symptôme. Nos résultats indiquent donc que chez la souris le TLR8 joue un rôle primordial dans la modulation de l’expression de TLR7, et cette régulation est cruciale dans le contrôle du développement d’autoimmunité spontanée. / Toll-like receptors (TLRs) detect conserved molecular products of microorganisms and play anessential role in the induction of immune responses. Endosomal TLRs (TLR3, 7, 8 and 9) sensenucleic acids derived from microbes. However they can also recognize self nucleic acids and thus beinvolved in the development of autoimmunity. Unlike human TLR8, murin TLR8 does not respond tosingle-stranded RNA suggesting that it could be not functional. In the current study, we investigatedthe role of murine TLR8 signaling in immunity. We found that TLR8-/- dendritic cells overexpressTLR7 and are hyperesponsive to various TLR7 ligands. In mice, TLR8 deficiency leads to increasedlevels of IgM, IgG, IgG2a circulating antibodies, autoantibodies and in the kidney to higher depositionof immunocomplexes while double TLR7/8-/- mice are protected from autoimmune features. Thesedata provide evidence for a pivotal role of murine TLR8 in the regulation of murine TLR7 expressionand this control is critical for the prevention of spontaneous autoimmunity development.

Objetivo: Avaliar a prevalência das manifestações clinicas, alterações laboratoriais e tratamento em um estudo de coorte multicêntrico incluindo 847 pacientes com diagnóstico de lúpus eritematoso sistêmico juvenil (LESJ) com ou sem hemorragia pulmonar (HP) assim como parâmetros de gravidade. Métodos: HP foi definida de acordo com a presença de pelo menos três sintomas/sinais respiratórios associados à imagem de infiltrado intersticial/alveolar difuso em radiografia de tórax e/ou tomografia computadorizada e queda aguda dos níveis de hemoglobina. Analise estatística foi realizada utilizando-se correção de Bonferroni (p < 0,0022). Resultados: HP foi observada em 19/847 (2,2%) dos pacientes com LESJ. Tosse, dispneia, taquicardia e/ou hipoxemia ocorreram em todos os pacientes com HP. Parâmetros de gravidade associados observados foram: ventilação mecânica em 14/19 (74%), hemoptise 12/19 (63%), síndrome de ativação macrofágica 2/19 (10%) e óbito 9/19 (47%). Análises de pacientes com LESJ ao diagnóstico de HP comparando-os com 76 pacientes controles com LESJ sem hemorragia pulmonar e com mesmo tempo de doença [3 (1-151) vs. 4 (1-151) meses, p=0,335], revelaram maior frequência de envolvimento constitucional (74% vs. 10%, p < 0,0001), serosite (63% vs. 6%, p < 0,0001) e sepse (53% vs. 9%, p < 0,0001) no grupo com HP. A mediana de escore de atividade de doença (SLEDAI-2K) foi significantemente maior em pacientes com LESJ com HP [18 (5-40) vs. 6 (0-44), p < 0,0001] assim como a mediana da dose de corticoide em uso em mg/Kg [1,4 (0,3-2) vs. 05 (0,03-3), p < 0,0001]. A frequência de plaquetopenia (53% vs. 12%, p < 0,0001), uso de metilprednisolona endovenosa (95% vs. 16%, p < 0,0001) e ciclofosfamida endovenosa (47% vs. 8%, p < 0,0001) também foi significantemente maior em pacientes com HP. Conclusões: Pacientes com LESJ apresentaram baixa prevalência de HP. Pacientes com LESJ e HP tiveram uma doença ativa grave, sistêmica, associada a envolvimento constitucional, serosite, plaquetopenia, sepse, dose maior de manutenção de corticoide, e principalmente tratada com pulsoterapia de corticoide e ciclofosfamida endovenosa / Objective: To evaluate prevalence, clinical manifestations, laboratory abnormalities and treatment in a multicenter cohort study including 847 childhood-onset systemic lupus erythematosus (cSLE) patients with and without diffuse alveolar hemorrhage (DAH), as well as concomitant parameters of severity. Methods: DAH was defined as the presence of at least three respiratory symptoms/signs associated with diffuse interstitial/alveolar infiltrates on chest x-ray or high-resolution computer tomography and sudden drop in hemoglobin levels. Statistical analysis was performed using Bonferroni correction (p < 0.0022). Results: DAH was observed in 19/847 (2.2%) cSLE patients. Cough/dyspnea/tachycardia/hypoxemia occurred in all cSLE patients with DAH. Concomitant parameters of severity observed were: mechanical ventilation in 14/19 (74%), hemoptysis 12/19 (63%), macrophage activation syndrome 2/19 (10%) and death 9/19 (47%). Further analysis of cSLE patients at DAH diagnosis compared to 76 cSLE control patients without DAH with same disease duration [3 (1-151) vs. 4 (1-151) months, p = 0.335], showed higher frequencies of constitutional involvement (74% vs. 10%, p < 0.0001), serositis (63% vs. 6%, p < 0.0001) and sepsis (53% vs. 9%, p < 0.0001) in the DAH group. The median of disease activity score(SLEDAI-2 K) was significantly higher in cSLE patients with DAH [18 (5-40) vs. 6 (0-44), p < 0.0001]. The frequencies of thrombocytopenia (53% vs. 12%, p < 0.0001), intravenous methylprednisolone (95% vs. 16%, p < 0.0001) and intravenous cyclophosphamide (47% vs. 8%, p < 0.0001) were also significantly higher in DAH patients. Conclusions: cSLE patients have shown low prevalence of DAH. Patients with both cSLE and DAH had serious, active systemic disease, associated with constitutional involvement, serositis, thrombocytopenia, sepsis, higher doses of maintenance corticotherapy, and mostly treated with corticoid pulse therapy and intravenous cyclophosphamide