O melanoma é a forma mais letal entre os cânceres de pele. Essa neoplasia freqüentemente apresenta-se resistente a abordagens terapêuticas. A angiogênese associada ao tumor representa um crítico passo da tumorigênese, resultado da ação de diferentes citocinas e fatores de crescimento como VEGF produzidos no microambiente tumoral. As galectinas extracelulares participam de múltiplos processos biológicos incluindo angiogênese tumoral e metástases, sua interação com as células presentes no microambiente tumoral pode ocorrer via receptores toll-like sugerindo seu envolvimento nos processos pro-inflamatórios e na secreção de citocinas. Recentemente mostramos que a ausência de gal-3 no estroma e parênquima tumoral diminui a angiogênese por interferir na resposta de macrófagos via VEGF e/ou TGFbeta1. Entretanto, o envolvimento de galectinas extracelulares na angiogênese e na modulação do sistema imune no microambiente tumoral ainda não está esclarecido. Assim, este estudo visa buscar respostas ao envolvimento das galectinas no crescimento tumoral e angiogênese contribuindo ao combate do melanoma maligno. Nossos resultados mostram a participação das galectinas 1 e 3 no crescimento tumoral e seu envolvimento com macrófagos via receptores toll-like, além de coordenarem a modulação do perfil de polarização de macrófagos derivados da medula óssea de camundongos wild-type. Dessa forma, podemos inferir que essas galectinas agem como coordenadoras de mudança de perfil dos macrófagos, uma vez que inibidas extracelularmente promovem uma diminuição do crescimento tumoral em camundongos wild-type, inoculados com células de melanoma murino e uma manutenção do perfil de macrófagos M1 in vitro. Assim, concluimos que as galectinas 1 e 3 extracelulares são importantes para o crescimento tumoral de melanomas murinos pois promovem o crescimento tumoral e são coordenadoras da mudança do perfil de macrófagos / Melanoma is the most aggressive form of skin cancer. This tumor often presents itself resistant to therapeutic approaches. The tumor-associated angiogenesis is a critical step in tumorigenesis and the result of the action of several cytokines and growth factors such as VEGF produced in the tumor microenvironment. The extracellular galectins participate in multiple biological processes including tumor angiogenesis and metastasis, their interaction with cells present in the tumor microenvironment may occur via toll-like receptors suggesting their involvement in pro-inflammatory processes and the secretion of cytokines. We have recently shown that the absence of Gal-3 the stroma and tumor parenchyma decreases angiogenesis by interfering with the macrophage response by VEGF and / or TGFbeta1. However, the involvement of extracellular galectins on angiogenesis modulation of the immune system in the tumor microenvironment is not yet clear. This study aims is to find answers to the involvement of galectins on tumor growth and angiogenesis contributing to the study of the malignant melanoma. Our results demonstrate the involvement of galectin 1 and 3 on tumor growth and its involvement in macrophage by toll-like receptors pathway, and coordinating the modulation of the polarization profile in wild-type mice bone marrow derived macrophages. Therefore, we show these galectins act as coordinators of macrophages profile change, since inhibited extracellularly promote a reduction in tumor growth in wild-type mice inoculated with murine melanoma cells and macrophages M1 maintenance of profile in vitro. Thus, we conclude that galectins 1 and 3 extracellular are important for tumor growth of murine melanomas because they promote tumor growth and are coordinators of change macrophages profile

Orientadores: Wagner José Fávaro, Patrick Vianna Garcia / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:25:33Z (GMT). No. of bitstreams: 1 Carniato_AmandaPocol_M.pdf: 2911906 bytes, checksum: 77e9b9f70f794e03c6fff7075745bfb0 (MD5) Previous issue date: 2014 / Resumo: Introdução: A alta recorrência do câncer de bexiga não-músculo invasivo (CBNMI) pode estar relacionada aos baixos efeitos das atuais terapias sobre os receptores Toll-like (TLRs) e a angiogênese. Atualmente, a terapia utilizada contra o CBNMI é a imunoterapia com BCG (Bacillus Calmette-Guerin) associada com a ressecção transuretral. Entretanto, a utilização do BCG está associada a efeitos colaterais, desde sintomas irritativos leves até reação sistêmica grave. Nesse contexto, uma nova perspectiva é representada pelo imunomodulador P-MAPA (agregado polimérico anidrídico fosfolinoleato-palmitoleato de amônio e magnésio). Objetivos: Os objetivos deste estudo foram caracterizar os efeitos da ativação dos mediadores das vias de sinalização dos TLRs 2, 4 (MyD88, IKK?, NF-kB, TNF-?, IL-6, TRIF, IRF-3, IFN-?), INOS e p53 na regulação do processo de angiogênese (VEGF e endostatina) e apoptose (BAX) frente ao uso do P-MAPA e BCG no CBNMI. Materiais e Métodos: Para a indução do CBNMI, 30 animais Fischer 344 induzidos quimicamente com uma dose intravesical de 1,5 mg/kg de N-metil-N-nitrosouréia (MNU) a cada 15 dias, totalizando 4 doses. Outros 10 animais Fischer 344 que não receberam MNU foram considerados como Grupo Controle. Duas semanas após a indução, os animais foram divididos em 3 grupos e tratados semanalmente por 6 semanas consecutivas via intravesical: Grupo Controle: 0,3 mL de solução fisiológica 0,9% ; Grupo MNU (Câncer): mesmo tratamento que o grupo Controle; Grupo MNU-BCG: 106 UFC ¿ 40 mg de BCG; Grupo MNU-P-MAPA: 5 mg/Kg de P-MAPA. Após tratamento os animais foram eutanasiados e as bexigas urinárias coletadas e submetidas às análises histopatológicas, imunohistoquímicas e Western Blotting. Resultados: O MNU demonstrou-se efetivo na indução do CBNMI, caracterizando carcinoma com invasão da lâmina própria (pT1), carcinoma papilífero (pTa), carcinoma in situ (pTis) e metaplasia escamosa. O grupo MNU-P-MAPA apresentou recuperação histopatológica em 100% dos animais, não apresentando alterações neoplásicas, enquanto que o grupo MNU-BCG recuperou apenas 60% dos animais. Os níveis proteicos para TLR2 e TLR4, dos intermediários da via de sinalização não-canônica (TRIF, IRF-3, IFN-?), p53 e BAX foram significativamente maiores no grupo MNU-P-MAPA em relação aos demais grupos experimentais. Sendo que para os intermediários da via de sinalização canônica (MyD88, IKK?, NF-kB, TNF-?, IL-6) foram maiores no grupo MNU-BCG. Os níveis proteicos de iNOS estiveram aumentados em ambos os tratamentos MNU-BCG e MNU-P-MAPA. E para VEGF, os níveis foram significativamente aumentados nos grupos MNU e MNU-BCG em relação aos demais grupos experimentais. Em contraste, a endostatina mostrou-se reduzida no grupo MNU e aumentada no grupo MNU-P-MAPA. Conclusão: Assim, pode-se concluir que a imunoterapia com P-MAPA promoveu distinta ativação do sistema imune inato mediada por TLRs 2 e 4 em relação ao BCG, resultando no aumento da via de sinalização para interferons, morte celular imunogênica, além da restauração dos níveis proteicos de p53 induzindo a apoptose e desaceleração do processo de angiogênese. A ativação da via de sinalização para interferons se correlacionou com a significativa melhora do CBNMI e ocorrência de apoptose / Abstract: Introduction: The high recurrence of non-muscle invasive bladder cancer (NMIBC) may be related to the low effect of the current therapies about receptors Toll-like (TLRs) and angiogenesis. Currently, the therapy used against the NMICB is the immunotherapy with BCG (Bacillus Calmette-Guerin) associated with the transurethral resection. However, the use of BCG is associated to side effects, since symptoms irritative mild to severe systemic reaction In this context, a new perspective is represented by the immunomodulatory P-MAPA (polymeric aggregate anidrídico phospholinoleate-palmitoleate ammonium and magnesium). Aims: The aims of this study were characterize the effects of activation of mediators of signaling pathways of TLRs 2, 4 (MyD88, IKK?, TNF-?, IL-6, TRIF, IRF-3, IFN-?, iNOS) and p53 in regulating the process of angiogenesis (VEGF, HIF, Endostatin) and apoptosis (BAX) compared to using P-MAPA and BCG in the NMIBC. Materials and Methods: For NMIBC induction, 30 Fischer 344 animals were chemically induced with intravesical dose of 1.5 mg / kg of N-methyl-N-nitrosourea (MNU) every 15 days, totaling 4 doses. Another 10 Fischer 344 animals that not received MNU were considered as Control Group. Two weeks after induction, the animals were divided into 3 groups and treated intravesically for 6 consecutive weeks: Control Group: 0.3 ml of 0.9% saline; MNU Group (Cancer): same treatment as the Control group; BCG-MNU Group: 106 UFC - 40 mg of BCG; MNU-P-MAPA group: 5 mg/kg P-MAPA. After treatment, the animals were euthanized and the urinary bladders collected and subjected to histopathological, immunohistochemical and Western blotting analyzes. Results: The MNU demonstrated to be effective in inducing NMIBC, presenting undifferentiated tumor, featuring carcinoma with invasion of the lamina propria (pT1), papillary (pTa), carcinoma in situ (PTis) and squamous metaplasia. The MNU-P-MAPA group presented histopathological recovery in 100% of animals showing no neoplasic alterations, while MNU-BCG group recovered only 60% of animals. Protein levels of TLR2 and TLR4, the intermediary of non-canonical signaling pathway (TRIF, IRF-3, IFN-?), p53 and BAX were significantly higher in MNU-P-MAPA group compared to other experimental groups. Whereas the intermediate canonical signaling pathway of TLRs (MyD88, IKK?, TNF-?, IL-6) were higher in MNU-BCG group. Protein levels of iNOS were high in both MNU-BCG and MNU-P-MAPA treatment groups. Protein levels for VEGF and HIF were significantly increased in MNU and MNU-BCG group compared to the other experimental groups. In contrast, endostatin was reduced in MNU group and increased in the MNU-P-MAPA group. Conclusion: Thus, it can be concluded that immunotherapy with P-MAPA promoted distinct activation of the innate immune system mediated by TLRs 2 and 4 compared to BCG, resulting in increased signaling pathway for interferon, immunogenic cell death, in addition to restoration of p53 protein levels that leads to apoptosis and slowing the process of angiogenesis. The activation of the signaling pathway for interferon correlated with significant improvement NMIBC and the occurrence of apoptosis / Mestrado / Biologia Tecidual / Mestra em Biologia Celular e Estrutural

Orientadores: Ubirajara Ferreira, Wagner José Fávaro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T12:56:11Z (GMT). No. of bitstreams: 1 Stopiglia_RafaelMamprin_D.pdf: 6417556 bytes, checksum: 52b7e5f09e9aa811c8597b3dbd50aae3 (MD5) Previous issue date: 2015 / Resumo: Em diferentes tipos de cânceres uma população muito pequena de células tem sido reconhecida como células-tronco, por apresentarem capacidade de auto-renovação e diferenciação. Para o seu mapeamento, nos tumores uroteliais, diversos marcadores de superfície celular são utilizados para caracterizar tanto células-tronco normais (CTU) quanto cancerosas (CTC), destacando-se entre eles os antígenos de superfície CD44, CD117 e CD133 e o transportador de membrana do tipo ATP Binding Cassette (ABCG2). Com relação a sinalização dos receptores toll-like (TLRs) no câncer, ainda é uma questão controvertida, pois dados conflitantes os apontam como reguladores negativos ou positivos da carcinogênese. Assim, os objetivos principais do presente estudo foram caracterizar e comparar os perfis das potenciais células-tronco uroteliais e correlacioná-los com a expressão e sinalização dos TLRs 2 e 4 no câncer da bexiga urinária. Para isso, foram selecionados tecidos de bexiga humana sem lesões uroteliais e outras com tumores não-músculo invasivos e músculo invasivos e separadas 30 (trinta) amostras obtidas de homens na faixa etária de 50 a 80 anos (média de 61 anos). Dez dessas amostras de bexiga foram provenientes de necropsia de pacientes sem diagnóstico de lesão urotelial ou doença urológica. As outras 20 amostras vesicais foram obtidas de pacientes submetidos à ressecção transuretral (RTU) e cistectomia radical. Em seguida, divididos em 3 grupos com 10 amostras cada e assim denominados: Grupo Normal, Grupo Câncer Não-Músculo Invasivo e Grupo Câncer Músculo Invasivo de bexiga. E por fim, as amostras foram processadas e submetidas às análises histopatológicas e imunohistoquímicas. Os resultados observados são os seguintes: As imunorreatividades para CD44 e CD133 foram significativamente intensas no grupo câncer músculo invasivo quando relacionadas aos demais grupos. O biomarcador ABCG2 apresentou intensa imunorreatividade nos grupos não-músculo e músculo invasivos, enquanto que no grupo normal a imunorreatividade para esse biomarcador foi ausente. Fraca imunorreatividade para o biomarcador CD117 foi verificada tanto no grupo normal quanto nos grupos não-músculo e músculo invasivos. Assim, as potenciais CTC apresentaram positividade para CD44/CD133/ABCG2 e ocorreram somente nos grupos tumores não-músculo e músculo invasivos. Com relação à caracterização das células-tronco uroteliais normais (CTU), estas foram positivas para CD44/CD133/CD117 e ocorreram com maior frequência no grupo normal em relação aos grupos não-músculo e músculo invasivos. Portanto, os 4 receptores analisados foram reconhecidos como possíveis identificadores de células tronco normais e cancerosas na bexiga. Com relação a análise dos receptores toll-like, as imunorreatividades para TLR2 e TLR4 foram significativamente reduzidas no grupo de pacientes com câncer não-músculo e músculo invasivos, sendo que a imunorreatividade para TLR2 foi ausente neste último. Assim, verificamos que no desenvolvimento dos tumores vesicais a partir de células tronco cancerosas, essas puderam ser identificadas com os marcadores propostos neste estudo e também que a expressão dos receptores TLR 2 e 4 nesta população celular foi mínima ou ausente, atuando, provavelmente, como receptores negativos na carcinogênese urotelial. Como os TLR2 e 4 provavelmente atuaram como reguladores negativos da carcinogênese urotelial pode-se concluir que possivelmente a ocorrência das CTC foi sensível ao decréscimo das imunorreatividades para os TLR2 e TLR4 / Abstract: In different cancers a very small population of cells has been recognized as stem cells , since they have the capacity for self - renewal and differentiation. For their mapping in urothelial tumors, several cell surface markers are used to characterize both normal stem cells ( CTU ) and cancer ( CTC ) , foremost among them the surface antigens CD44 , CD117 and CD133 and membrane transporter ATP Binding Cassette type ( ABCG2 ) . Relationship with the signaling of toll-like receptors ( TLRs ) in cancer is still a controversial issue because conflicting data indicate them as negative or positive regulators of carcinogenesis . Thus, the main objectives of this study were to characterize and compare the profiles of urothelial stem cells and correlates them with the expression and signaling of TLRs in cancer of the urinary bladder . Tissues of human bladder urothelial lesions and no other separate tumors with invasive and invasive non- muscle and muscle were selected thirty (30 ) samples of men aged 50-80 years (mean 61 years). Ten of these samples were derived from autopsy bladder of patients without a diagnosis of urothelial injury or urologic disease. The other 20 bladder samples were obtained from patients undergoing transurethral resection (TURP ) and radical cystectomy. After this, divided into 3 groups with 10 samples each and named as follows: Normal Group , Group Non- Muscle Invasive Cancer Group and Muscle Invasive Bladder Cancer . Then, the samples were processed and subjected to histological and immunohistochemical analysis. The observed results are as follows: The imunorreatividades for CD44 and CD133 were significantly intense in group Muscle Invasive Cancer as related to the other groups. The ABCG2 biomarker groups showed intense immunoreactivity in Non- Muscle Invasive and muscle, while in the Normal group immunoreactivity was absent for that biomarker . Weak immunoreactivity for CD117 biomarker was observed both in the Normal group and the groups Non-Muscle Invasive and Muscle. Thus, CTC were positive for CD44/CD133/ABCG2 and occurred only in tumors groups Non- Muscle Invasive and Muscle. Regarding the characterization of normal urothelial stem cells ( CTU ), these were positive for CD44/CD133/CD117 and occurred more frequently in the Normal group in relation to groups and Non-Muscle Invasive Muscle. Therefore, the four receptors were analyzed for possible identifiers recognized as normal stem cells and bladder cancer . Regarding the analysis of toll-like receptors, TLR2 and TLR4 to imunorreatividades were significantly reduced in patients with Cancer and Non- Muscle Invasive Muscle, and the TLR2 immunoreactivity was absent in the latter . Thus, we see that the development of bladder tumors from stem cells cancerous, these cells could be identified with markers and also proposed that the expression of TLR receptors 2 e 4 this cell population was minimal or absent, acting probably as receptors negative in urothelial carcinogenesis. As TLR2 and 4 probably acted as negative regulators of urothelial carcinogenesis can be concluded that most likely the occurrence of the CTC was sensitive to the decrease in imunorreactivities for TLR2 and TLR4 / Doutorado / Fisiopatologia Cirúrgica / Doutor em Ciências

Toll like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play a central role in host cell recognition and responses to pathogens. Primarily they are responsible for induction and regulation of the innate and adaptive immune responses whereby the effector function is executed chiefly by differentiated myeloid cells. Somewhat unexpectedly, TLRs have been also shown to be involved in direct pathogen sensing by bone marrow-derived hematopoietic stem cells (HSCs) and hematopoietic progenitors when, under inflammatory conditions, the rapid generation of innate immune effector cells that effectively combat the infection is of utmost priority. While it has been recognized that the release of inflammatory cytokines from inflamed tissues along with the changes in proportions of differentiating cells in the bone marrow (BM) as well as the BM niche can nudge the differentiation of adult BM-derived cells towards myeloid cells and granulocytes, a direct role of TLRs expressed by HSCs in this process has been demonstrated only recently. However, whether a similar mechanism operates also during embryonic hematopoiesis is unknown. Here we show that TLRs and their adaptor proteins are functionally expressed during early stages of embryogenesis by short-lived maternally-transferred...

Indiana University-Purdue University Indianapolis (IUPUI) / Embryonic stem cells (ESCs) are unique in that they have potential to give rise to every cell type of the body. Little is known about stimuli that promote mouse (m)ESC differentiation and proliferation. Therefore the purpose of this study was to determine the role of Toll Like Receptor (TLR) ligands in mESCs proliferation, survival, and differentiation in the presence of Leukemia Inhibitory Factor (LIF). We hypothesized that TLRs are expressed and functional, and when activated by their ligand will induce survival, proliferation, and prevent differentiation. In this study, mESC line E14 was used to determine the expression of TLRs at the mRNA level and three mESC lines, R1, CGR8, and E14, were used to determine cell surface protein levels. We found expression of TLRs 1, 2, 3, 5, and 6 at the mRNA level, but no expression of TLRs 4, 7, 8, and 9 in the E14 mESC line. We confirmed the presence of TLR-2 but not of TLR-4, protein on the cell surface using flow cytometric analysis for all three cell lines. We focused our studies mainly on TLR-2 using the E14 cell line. Pam3Cys, is a synthetic triacyl lipoprotein and a TLR-2 ligand, which induced a significant increase in mESC proliferation on Days 3, 4, and 5 and enhanced survival of mESC in a dose dependent manner in the context of delayed addition of serum. All the latter experiments were performed in triplicate and student T-test was performed to establish significant differences. Next, we demonstrated functionality of TLR-2 via the MyD88/IKK pathway, where MyD88 was expressed and IKKα/β phosphorylation was enhanced. This was associated with increased NF-κB nuclear translocation upon activation by Pam3Cys. Finally, we showed that there were no changes in expression of mESCs markers Oct-4, KLF-4, Sox-2, and SSEA-1, thus illustrating that the mESCs may have remained in a pluripotent state after activation with the TLR-2 ligand in the presence of LIF. These results demonstrate that mESCs can respond to microbial products, such as Pam3Cys, and can induce proliferation and survival of the mESCs. This finding expands the role of TLRs and has some implications in understanding embryonic stem cell biology.

Cardiovascular disease is a leading cause of death in the United States. Toll-like receptor (TLR)-mediated pathways have been demonstrated to play a role in myocardial ischemia/reperfusion (I/R) injury. We and others have shown that PI3K/Akt signaling is involved in regulating cellular survival and protecting the myocardium from I/R induced injury. In this dissertation, we provide compelling evidence that miR-125b serves to “fine tune” TLR mediated NF-kB responses by repressing TNF-a and TRAF6 expression. We constructed lentiviral expressing miR-125b, delivered it into the myocardium. The data showed that delivery of lentivirus expressing miR-125b significantly reduces myocardial infarct size and improves cardiac function in I/R hearts. Mechanistic studies demonstrated that miR-125b negatively regulates TLR mediated NF-kB activation pathway by repressing TNF-a and TRAF6 expression in the myocardium. We also observed that transfection of the myocardium with lentivirus expressing miR-214 markedly attenuates I/R induced myocardial infarct size and cardiac dysfunction. We demonstrated that miR-214 activates PI3K/Akt signaling by targeting PTEN expression in the myocardium. We also investigated the role of TLR3 in neonatal heart repair and regeneration following myocardial infarction (MI). Wild type (WT) neonatal mice showed fully cardiac functional recovery and small infarct size, while TLR3 deficient mice exhibited impaired cardiac functional recovery and large infarct area after MI. Poly (I:C), a TLR3 ligand, administration significantly enhances glycolysis, YAP1 activation and the proliferation of WT neonatal cardiomyocytes. 2-deoxyglucose (2-DG), a glycolysis inhibitor treatment abolished cardiac functional recovery and YAP1 activation in neonatal mice after MI. In vitro either inhibition of glycolysis by 2-DG or inhibition of YAP1 activation prevents Poly (I:C) induced YAP1 activation and neonatal cardiomyocyte proliferation. Importantly, YAP1 activation increases miR-152 expression, leading to cardiomyocyte proliferation through suppression P27kip1 and DNMT1 expression. We conclude that microRNAs play an important role in TLR modulation induced protection against myocardial I/R injury by increasing the activation of PI3K/Akt signaling pathway, decreasing TLR/NF-kB mediated inflammatory response, and suppressing activation of apoptotic signaling following myocardial I/R injury. In addition, TLR3 is an essential for neonatal heart repair and regeneration after myocardial infarction. TLR3 modulation could be a novel strategy for heart regeneration and repair.

No description available.

Doctor of Philosophy / Department of Anatomy and Physiology / Chris R. Ross / The immediate antiviral defense residing in the innate immune system of multicellular organisms critically determines the outcome of viral infection. This dissertation presents a study of the "effectors" and "receptors" of porcine innate immunity in infection caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is the most devastating pathogen impacting the swine industry. In the first investigation, eleven novel porcine host defense peptides (HDPs), [Beta]-defensins (pBDs), were identified and characterized. All of these peptides have a consensus [Beta]-defensin motif and phylogenetically are similar to orthologs from other species. A differential expression pattern for these 11 newly identified genes was found. For example, pBD-2 and pBD-3 were expressed in bone marrow, lung, skin and other lymphoid tissues. pBD-2 and pBD-3 were further characterized for their gene structure, and antimicrobial activity of synthetic peptides. The second study was conducted to evaluate PRRSV-induced differential expression of porcine HDPs and direct antiviral activity of selected HDPs against PRRSV. In vitro incubation of PRRSV with synthetic pBD-3 or protegrin-4 (PG-4) significantly inhibited viral infectivity. Using nine protegrin-derived peptides, it was determined that cyclization of PG-4 increased anti-PRRSV activity and mutation of some residues in PG-4 diminished some of the activity. These findings suggest the potential role of porcine HDPs as a group of innate antiviral effectors. In the third and fourth investigations, porcine Toll-like receptor (TLR) 3 and TLR7 were identified and functionally expressed. Increased expression of TLR3 was observed in PRRSV-infected porcine lungs. Stimulation of porcine alovelar macrophages with poly (I:C), a synthetic TLR3 ligand, increased expression of interferon-[Beta] and suppressed PRRSV infectivity. Activation of porcine TLR3 overexpressed in a PRRSV-sensitive cell line, elicited antiviral responses to PRRSV infection. Partial silencing of TLR3 in PAMs resulted in increased PRRSV infection. In summary, these data provide molecular information on porcine TLR3 and TLR7, and their involvement in PRRSV pathogenesis, which may elicit new strategies to prevent this costly swine disease.

Ce travail de thèse s’est attaché à comprendre le lien étroit entre les dérégulations métaboliques et les dérégulations du système immunitaire, dans le cadre du modèle de diabète auto-immun de la souris NOD. Nous avons constaté que les bezafibrates protègent les souris NOD du diabète. Nous avons mis en évidence que ceci était lié à une diminution de l’infiltration des ilots beta du pancréas par les lymphocytes et cellules présentatrice d’antigen (CPA). La diminution d’IL-6 dans le sérum des souris traitées suggère une diminution générale de l’inflammation. Aussi, nous avons évalué l’implication du traitement en dehors du système immunitaire, en l’occurrence sur le stress cellulaire et la survie des cellules beta des ilots de Langherans, en utilisant une méthode de streptozotocine à faible dose, et nous avons observé une protection des souris traitées par les bezafibrates. Dans une deuxième partie de l’étude, nous avons observé qu’il existait au niveau du système immunitaire inné une surexpression des Toll-like récepteurs (TLR) 1, 2 et 6 dans les souris NOD comparées aux souris contrôles C57BL/6, ceci au niveau transcriptionnel ainsi qu’au niveau traductionnel. Nous avons ensuite démontré que cette surexpression avait une légitimité fonctionnelle. En effet, les CPA sécrètent plus d’Interleukine 6 (IL-6), et les cellules B, plus d’Immunoglobuline M (IgM). Nous avons poursuivi notre étude en modulant le contenu sérique en lipides dans nos souris NOD, en utilisant un régime gras pour augmenter la lipidémie et des bezafibrates hypolipidémiantes. Nous n’avons pas observé de modulation majeure de l’expression de TLR2 et TLR6, mais il est à noter que les CPA issues des souris soumises au régime gras sont plus susceptible à la réponse au ligand synthétique FSL-1 de TLR 2/6, qui sécrètent alors plus d’IL-6 que les NOD contrôles ou traitées aux bezafibrates. Nous avons pu ainsi souligner l’importance des liens entre le métabolisme et le système immunitaire, dans l’apparition de la maladie auto-immune que constitue le diabète de type 1. / In this thesis we sought to understand the link between metabolic deregulation and immune deregulation in the context of the NOD mouse autoimmune model. We observed that bezafibrates protect NOD mice from type 1 diabetes (T1D). We showed that pancreas beta islet infiltration by lymphocytes and APCs was diminished. IL-6 decrease in bezafibrate treated mice serum suggested a general dampening down of inflammation. Besides we evaluated the effect of bezafibrate out of the immune system by using a low-dose streptozotocin method and we observed that bezafibrate mice treated were protected. In a second part of this study we observed an overexpression of Toll-like receptors (TLR) 1, 2, 6 in NOD mice compared to C57BL/6 controls. This was noticeable at the transcription and translation level. We showed that this overexpression had a functional role. Indeed APCs from NODs secreted more IL-6, and B cells secreted more IgM when stimulated with corresponding ligands. We then modified the lipid content of NOD mice by using bezafibrates to decrease lipidemia, and a high fat diet (HFD) to increase lipidemia. No modulation of TLR2 and TLR6 expression was observed. However, APCs from HFD mice were more susceptible to TLR2/6 ligand FSL-1 stimulation by secreting more IL-6 than control or bezafibrate treated NODs. We here highlight the important links existing between metabolism immunity in the autoimmune T1D onset

Le lupus érythémateux disseminé (LED) est une maladie chronique auto-immune caractérisée par la production d'autoanticorps dirigés contre les antigènes nucléaires. Des nombreuses études indiquent un rôle des récepteurs Toll-like (TLR). Des études antérieures de notre laboratoire ont révélé que le TLR8 murin contrôle la fonction de TLR7 dans les cellules dendritiques et est aussi impliqué dans le lupus. TLR9 contrôle également le lupus dépendant de TLR7. Mon projet de thèse avait deux objectifs dont le premier était de comprendre comment le TLR8 et TLR9 contribuent au lupus dépendant de TLR7. En outre, nous avons révélé que TLR8 contrôle l'expression de TLR7 dans les cellules dendritiques, tandis que le TLR9 contrôle la fonction de TLR7 dans les cellules B. Le deuxième objectif était d'étudier l'implication du répresseur transcriptionnel Gfi-1 dans la signalisation des TLR et le développement de lupus en utilisant des souris Genista qui portent une mutation ponctuelle dans le gène Gfi-1. Nous avons constaté que les souris Genista développent un lupus dépendant de TLR7 et que Gfi-1 agit comme un répresseur de la transcription en aval de TLR7 et contrôle l'expression d'Interféron de type I dépendante des TLR. Ainsi, le déséquilibre des interactions entre TLR ainsi que les facteurs transcriptionnels en aval de ces TLR peuvent conduire à des mécanismes d'inflammation et d'auto-immunité qu'il est important de prendre en compte dans le développement d'approches thérapeutiques nouvelles ciblant les TLRs. / Systemic lupus erythematosus is a chronic autoimmune disease characterized by production of autoantibodies against nuclear antigens. Many studies indicate a role for Toll-like receptors (TLRs) in the initiation and establishment of systemic lupus erythematosus (SLE). Previous studies in the lab revealed that murine TLR8 controls TLR7 function in dendritic cells (DCs) and is implicated in SLE. TLR9 also controls TLR7-mediated lupus. My thesis had two aims: the first was to understand how the cooperation of TLR8 and TLR9 contributes to TLR7-mediated lupus. By studying double TLR8/9-deficient mice we found that TLR8 and TLR9 have an additive effect on controlling TLR7-mediated lupus, where TLR8 controls TLR7 function on DCs, while TLR9 restrains TLR7 responses in B cells. The second aim of my thesis was to investigate the implication of Gfi-1 in lupus and TLR signaling by studying Genista mice that carry a hypomorphic mutation of Gfi-1. We found that Genista mice develop TLR7-dependent lupus and that Gfi-1 acts as a transcriptional repressor downstream of TLR7 and controls type-I IFN expression. Thus, unbalancing TLR-interactions and transcription factors downstream of TLRs can lead to inflammation and autoimmunity and these mechanisms have to be taken into account when novel therapeutic approaches are developed that target TLRs.