The non-obese diabetic (NOD) mouse is widely used model Type 1 diabetes (T1D), a chronic inflammatory disease characterized by destruction of the insulin producing β cells in the islets of Langerhans by immune cells. The classical symptoms include increased glucose levels in urine and blood, frequent urination and enhanced thirst. The disease has a strong genetic component and is also influenced by the environment. NOD mice develop T1D spontaneously. The disease occurs in two phases; insulitis - the infiltration of immune cells in the islets of Langerhans and overt diabetes caused by the destruction of insulin producing β cells. Several disease associated gene regions or loci [termed insulin dependent diabetes (Idd) loci] have been associated with T1D development. Although, T1D is recognized as a T cell mediated disease in both mouse and man, many studies have shown the importance of B cells in the pathogenesis of the disease. Autoantibodies appear prior to islet infiltration and several molecular and cellular events precede this beta-cell autoimmunity. Although the pathogenesis of T1D is well characterized, less is known about the environmental and immunological factors that trigger the disease. In this thesis, we studied the contribution of B cell anomalies to the skewed immune response observed in the NOD mouse. In our studies covered in the thesis we observed that NOD mice display enhanced IgE in the serum already at one week of age. In addition, upon treatment of pre-diabetic NOD mice with anti-IgE antibodies, diabetes incidence was delayed. We hypothesize that the presence of IgE in the system may be explained due to enhanced class switching. Antibody feedback however, is an essential component of the immune response and can lead to either enhanced or dampened responses. Thus, increased IgE may provide positive feedback that might sustain an immune response. We also aimed to analyze the biological consequence of this feature. In vitro stimulation of B cells by the TACI ligand APRIL resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. In addition, TACI+ cells were observed in NOD germinal centers facilitating increased BAFF uptake and subsequent escape of low affinity antibody producing clones. NOD mice elicited an enhanced and prolonged immune response towards T-dependent antigens such as hen-egg lysozyme (HEL). Serum HEL-specific IgG level was significantly increased and was predominantly of the IgG1 isotype. Immunofluorescence analysis of NOD spleen revealed the presence of spontaneous germinal centers which others have perceived to provide a ready niche for the entry of naïve B cells that encountered novel antigen. Adoptive transfer experiments of purified B and T cells from NOD into NOD.Rag2-/- (NOD-RAG) mice illustrated the importance of B cell intrinsic defects in the reproduction of the original phenotype as observed in NOD.

Resumo: A reabsorcao do osso alveolar e uma das principais caracteristicas associadas a progressao da doenca periodontal. Apesar da enorme complexidade da microbiota envolvida, considera-se que bacterias Gram-negativas tenham um papel relevante em sua etiopatogenese. Um dos fatores de virulencia destes microrganismos e representado por um componente de sua parede externa denominado lipopolissacarideo (LPS). A presenca de LPS na proximidade dos tecidos periodontais e capaz de induzir a producao de diversos mediadores inflamatorios que levam a degradacao tanto do tecido conjuntivo quanto osseo. Atualmente acredita-se que a interacao do ligante do receptor-ativador do fator nuclear kappa-B (RANKL) com seu receptor (RANK) presente em precursores hematopoieticos e necessaria e suficiente para a inducao da diferenciacao de osteoclastos. Por outro lado, a ligacao de RANKL com seu falso-receptor, denominado osteoprotegerina (OPG), reduz sua biodisponibilidade e inibe, desta forma, a osteoclastogenese. Assim, a razao da expressao de RANKL e OPG e considerada como o principal determinante do "turnover" do tecido osseo. A producao de RANKL e OPG depende das vias de sinalizacao ativadas, as quais sao influenciadas pela natureza do estimulo extracelular. Atualmente, a familia de receptores NLRs (nod-like receptors) foi identificada como receptor intracelular para componentes bacterianos e agentes moduladores de diferentes vias de sinalizacao. Considerando a relevancia do LPS bacteriano na patogenese da doenca periodontal, o papel do RANKL no processo de reabsorcao ossea e a possivel implicacao das proteinas Nod na transducao de sinais regulando a expressao de RANKL, o objetivo geral deste projeto foi estudar os mecanismos de regulacao da expressao de RANKL induzido por LPS bacteriano em celulas relevantes do periodonto (macrofagos, osteoblastos e fibroblastos). Os objetivos especificos propostos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bone resorption is one of the major characteristics of destructive periodontal disease. Despite the great number of different bacterial species in the dental biofilm, Gramnegative microorganisms were demonstrated to have a very important role on periodontal disease pathogenesis. Lipopolysaccharide (LPS) is a bacterial cell wall component, which is acknowledged as one of the main virulence factors of these microorganisms. The mere presence of LPS in proximity with the periodontal tissues initiates the expression and production of inflammatory mediators and other cytokines which can culminate in degradation of both soft and hard tissues. It is currently accepted that the interaction between receptor-activator of nuclear factor kappa-B ligand (RANKL) and its receptor (RANK) is both necessary and sufficient to induce osteoclast differentiation and activation. However, RANKL can interact with its soluble decoy receptor osteoprotegerin (OPG) inhibiting osteoclastogenesis by decreasing the bioavailability of RANKL. Production of RANKL/OPG is the result of the signaling pathways activated by external stimuli. Recently, the NLR (nod-like receptors) family was identified as cytosolic receptors for bacterial components and also, as capable of modulating different signaling pathways. Considering the relevance of LPS and RANKL in bone resorption and the possible implication of Nod proteins in signal transduction regulating RANKL expression, the aim of this study was to evaluate the influence of different intracellular signaling pathways on the regulation of RANKL expression induced by LPS in relevant cells of the periodontium (macrophages, osteoblasts and fibroblasts). The specific objectives proposed were to determine after LPS and interleukin-1 beta stimulation the role of MyD88-dependent and independent signaling pathways, Nod1 and Nod2 on the expression of RANKL, OPG, IL-10 and IFN-beta... (Complete abstract click electronic access below) / Orientador: Carlos Rossa Junior / Coorientador: Joni Augusto Cirelli / Banca: José Augusto Cezar Sampaio / Banca: Luis Carlos Spolidório / Banca: Luiz Carlos de Mattos / Banca: Cleni Mara Marzocchi Machado / Doutor

Utilization of tolerogenic dendritic cells (tolDCs) as a cell-based therapy represents a promising strategy in treatment of autoimmune diseases including type 1 diabetes (T1D). Numerous protocols have been established to generate tolDCs ex vivo and their therapeutic effect has been demonstrated in animal models of autoimmune diseases. In this thesis we compared three different variants of such protocols which are based on the combined treatment of bone marrow- derived DCs with vitamin D and dexamethasone applied at different time points of their maturation towards tolDCs. We assessed the efficiency of these protocols in regards of their effect on the expression of co-stimulatory molecules CD40, CD80, CD86, and MHC II and the chemokine receptor CCR7 on the surface of tolDCs. Then, we evaluated the migration pattern of antigen unloaded tolDCs in vivo as well as their effect on the induction of immune responses and cell proliferation of lymph node cells. This was achieved by labelling of tolDCs with membrane dye PKH26 and by following their migration path by flow cytometry after intraperitoneal (i.p) or subcutaneous (s.c.) injection into either left or right side of the body. On day 1, 3, 5, 7, and 9, the presence of PKH26+ tolDCs was examined in spleen, pancreatic, mesenteric, inguinal and axillary...

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Obesity is characterized by hyperinsulinemia and chronic inflammation, contributing to insulin resistance and type 2 diabetes (T2D) risk. Pattern recognition receptors (PRRs) of the innate immune system, including Toll-like Receptors (TLRs) and Nod-like Receptors (NLRs), have been identified as propagators of metabolic inflammation. Circulating bacterial components exert distinct effects on inflammation and insulin sensitivity via TLRs and NLRs. Specific types of bacterial peptidoglycan engage NOD1 and NOD2. Activators of NOD1 increase inflammation and insulin resistance, while activators of NOD2 promote immune tolerance and insulin sensitivity. NOD1 and NOD2 use the common downstream adaptor RIPK2 to drive immune responses, but the role of RIPK2 in glucose homeostasis was unknown. RIPK2 is positioned to mediate effects of xenobiotics and microbial components on blood glucose. For example, tyrosine kinase inhibitors (TKIs) are being investigated for diabetes treatment. Improvements in blood glucose control have been observed in diabetic cancer patients receiving TKI therapy but the mechanism underlying these changes remains unclear. Several TKIs inhibit RIPK2. We sought to understand if TKIs that inhibit RIPK2 block inflammatory and metabolic consequences of NOD signalling. We hypothesized inhibition of inflammation via NOD1-RIPK2 by certain TKIs contributes to lowered blood glucose/improved insulin sensitivity in pre-clinical models of obesity. We showed that RIPK2 was required for acute glycemic consequences of NOD1 and NOD2 activation, and RIPK2-specific TKIs attenuated these glycemic effects. We found TKI-mediated improvements in blood glucose are independent of NOD-RIPK2 signalling during diet-induced obesity. However, RIPK2 mediated the effects of certain TKIs on blood insulin. Finally, we tested if RIPK2 mediated the effects of bacterial components derived from commensal microbiota. We found injection of upper intestinal microbe components lowered blood glucose via NOD2-RIPK2 signalling. These findings demonstrate that modulation of RIPK2 signalling by xenobiotic or microbial factors is an important contributor to blood glucose and insulin homeostasis. / Thesis / Doctor of Philosophy (PhD) / Obesity increases the risk of chronic diseases, including Type 2 Diabetes (T2D). Obesity stops insulin from working properly, leading to the inability to lower blood glucose. Obesity and T2D are linked to chronic, low-grade activation of the immune system. The immune system normally defends the body against microbes by inducing a pro-inflammatory response. Inflammation can also be activated or inhibited by drugs (xenobiotics), and different aspects of inflammation can increase or decrease blood glucose and insulin. A major unanswered question was how certain cancer drugs and bacterial components interact with the immune system to change blood glucose or insulin. This work tested how an innate immune pathway that detects bacterial cell wall components is influenced by cancer drugs and alters blood glucose and insulin in pre-clinical models. This work is targeted at understanding how new prebiotics or existing drugs can be tasked as therapeutic strategies for prediabetes and T2D.

The innate immune system houses cellular signaling proteins called pattern recognition receptors (PRRs) that are responsible for recognizing highly-conserved molecular patterns associated with pathogens or damage to elicit an immune response. However, NLRX1 is a unique PRR in the NOD-like receptor (NLR) family that instead functions to attenuate pro-inflammatory pathways that are activated by other PRRs, such as NF-κB and type-1 interferon signaling which both have implications in cancer. NLRX1 can regulate additional cancer-associated pathways, such as MAPK and AKT, and cancer-associated functions like metabolism and reactive oxygen species (ROS) production. Interestingly, depending on the type and subtype of cancer, NLRX1 can either be tumor promoting or tumor suppressing. Here, we investigate the role of NLRX1 in two deadly cancers: triple-negative breast cancer (TNBC) and pancreatic cancer. In a murine mammary tumor model that highly mimics TNBC, we discovered that NLRX1 is protective against disease burden in vivo when NLRX1 is expressed in healthy host cells. NLRX1 exerts its protection through limiting the recruitment of eosinophils to the tumor, suppressing epithelial-mesenchymal transition (EMT), and attenuating the formation of the metastatic niche. Conversely, when NLRX1 is instead expressed by the mammary tumor cells, NLRX1 promotes cancer-associated characteristics in vitro and disease burden in vivo by promoting EMT. This indicates that the role of NLRX1 in TNBC is highly dependent on cellular context. Conversely, in murine pancreatic cancer cells, we found that NLRX1 expression by the tumor cells is protective against cancer-associated characteristics in vitro, and that this is likely driven by NF-κB, MAPK, AKT, and inflammasome signaling with a potential to also limit immune evasion. Together, this research indicates that the role of NLRX1 can be highly variable based on the cell and tumor type and identifies the underlying mechanisms through which NLRX1 functions in these two cancer models. This is critical information for drug development initiatives so therapies can be developed that target NLRX1 in the appropriate cell type and in the appropriate disease. / Doctor of Philosophy / Inflammation, which is characterized by redness, heat, pain, swelling, and sometimes loss of function, is a critical way in which our bodies fight infections and repair tissue damage. However, chronic inflammation occurs when our bodies are unable to turn inflammation off and can result in cancerous mutations. Therefore, the successful resolution of inflammation is critical to maintaining inflammatory balance and has previously been dubbed the "Goldilocks Conundrum". The immune system houses a class of cellular signaling proteins called pattern recognition receptors (PRRs), which often function to turn inflammation on. However, a unique PRR in the NOD-like receptor (NLR) family called "NLRX1" functions to turn inflammation off and therefore plays an important role in preventing damaging chronic inflammation. NLRX1 has historically been studied in the context of infectious diseases, but because NLRX1 is involved in inflammation and because inflammation is a critical factor of cancer, its role as a tumor suppressor or tumor promoter has recently become an area of interest. NLRX1 has also been found to regulate biological pathways beyond inflammation that are also important for cancer initiation and progression. Interestingly, depending on the type and subtype of cancer, NLRX1 can either be tumor promoting or tumor suppressing. Here, we investigate the role of NLRX1 in two deadly cancers: triple-negative breast cancer (TNBC) and pancreatic cancer. In a mouse mammary tumor model that highly mimics TNBC, we discovered that NLRX1 is protective against disease burden when NLRX1 is expressed in healthy, non-tumor cells. NLRX1 exerts its protection through impacting the immune cells recruited to the tumor, limiting the ability of the tumor cells to leave the original tumor and spread throughout the body in the process known as metastasis, and suppressing the formation of a favorable tumor metastasis environment in the lung. Conversely, when NLRX1 is instead expressed by the mammary tumor cells, NLRX1 promotes disease burden by helping tumor cells leave the original tumor and spread throughout the body. This indicates that the role of NLRX1 in TNBC is highly dependent on cellular context, including if the cell is healthy or cancerous. Conversely, in mouse pancreatic cancer cells, we found that NLRX1 expression by the tumor cells is protective against cancer-associated characteristics. Together, this research indicates that the role of NLRX1 can be highly variable based on the cell and tumor type. This is critical information for drug development initiatives so therapies can be developed that turn NLRX1 on or off in the appropriate cell type and in the appropriate disease.

Genotype-phenotype analyses continue to be the primary goal for genome analyses in livestock and poultry breeding. Essential to accomplish this goal is the need to identify variation at the genomic level. To test the hypothesis that DNA sequence variations in inflammatory response genes are associated with phenotypic differences in the heritage turkey, the primary objective of this project was to search for single nucleotide polymorphisms (SNPs) in candidate inflammatory response genes. A minor objective was to develop a system for inducing inflammatory response in the turkey using a microbe-based lipopolysaccharide (LPS), an approach previously described for the chicken. A total of 18 SNPs was identified in the three genes screened in this project: Interleukin 6 (IL6) and 8 (IL8), and NLRX1. Mortality data from the LPS challenge were not significantly different among the strains. Further gene expression analyses will be part of future work. The SNP data represent the first extensive analyses of candidate inflammatory response genes in the turkey. Combined with the protocols developed for inflammation assessment in the turkey the SNPs described here will be valuable resources for future inflammation:genotype evaluation in the turkey / MS / Though progress has been made in the genome analyses of the turkey, Meleagris gallopavo, our understanding of the genotype: phenotype relationships continue to lag those of agriculturally important animal species. Among the phenotypes for which genetic understanding can be useful is inflammation, a complex trait that is influence by many interdependent response mechanisms. These mechanisms, regarding differences across heritage turkeys, has been mildly investigated. Since Nucleotide Polymorphism (SNP) screening is a common method used to comprehend the robust effects these differences have on genotype and phenotype. Here, we report initial investigations in our lab of the genetics of inflammation in the turkey using comparative information from the chicken NOD like receptor X1 (NLRX1), turkey interleukin 6 (IL6), and Interleukin 8 (IL8). These genes were screened for nucleotide variants that may be informative for future studies that will investigate the turkey’s response to Salmonella derived lipopolysaccharide that can induce inflammation. The rationale for selecting these three genes is that IL8, IL6, and NLRX1 have pro inflammatory and/or anti-inflammatory functions that respond to maintain homeostasis. Primers were designed and investigated using DNA from Broad Breasted White (BBW), and Broad Breasted Bronze (BBB), Blue slate (SL), Spanish Black (SBL), Midget White (MW) and Royal Palm (RP). The birds were also challenged with 1.5 mg/kg Lipopolysaccharide (LPS) intra-abdominally to collect tissue post LPS challenge. Tissues was collected from the thymus, spleen, and bursa of fabricius: organs identified to effect inflammation. A total of 2,239 bp for IL8, 2,439 bp for IL 6, and 572 bp for NLRX1 were screened for SNPs. SNP analysis revealed 16 SNPs in the inflammatory response genes mentioned.

O biofilme bacteriano é o agente etiológico primário no desenvolvimento da resposta inflamatória observada na doença periodontal. Os receptores do tipo NOD (NLRs) são proteínas citosólicas que reconhecem componentes microbianos presentes no citoplasma liberados por bactérias invasoras. Sabendo que bactérias periodontopatogênicas têm a capacidade de invadir e colonizar diversas células do tecido periodontal, o presente projeto tem o objetivo de estudar a participação dos receptores do tipo NOD (NOD1 e NOD2) no reconhecimento das bactérias Porphyromonas gingivalis, na modulação da resposta imune e no processo de reabsorção óssea. Camundongos isogênicos machos da linhagem C57BL/6 (WT) e camundongos deficientes para o receptor NOD1 (NOD1-/-) e receptor NOD2 (NOD2-/-) foram infectados com Phorphyromonas gingivalis utilizando um modelo experimental de doença periodontal. O grau de reabsorção óssea foi avaliado por análise morfométrica macroscópica e histológica da maxila, além da quantificação do número de osteoclastos na crista óssea alveolar. O grau de inflamação local foi realizado por contagem do número total de bactérias orais, quantificação de neutrófilos no tecido gengival (MPO) e avaliação dos mediadores inflamatórios por ELISA. Foi também avaliada a expressão de marcadores osteogênicos e osteoclastogênicos no tecido gengival pela técnica de qPCR. Animais NOD1-/- e NOD2-/- apresentaram menor delta de reabsorção óssea quando comparados aos animais WT. Animais NOD2-/- infectados apresentaram debilitado controle bacteriano quando comparados aos animais WT infectados. Animais NOD1-/- e NOD2-/- infectados apresentaram baixa expressão de Cxcl1 e MPO quando comparados aos animais WT infectados. Além disso, foi observado que animais NOD2-/- infectados apresentaram reduzida produção de TNF-α e elevada produção de IL-10 quando comparados aos animais WT infectados. Não foi detectado diferença estatística na expressão de fatores osteogênicos, Runx2 e osteocalcina, entre animais WT, NOD1-/- e NOD2-/- infectados. Embora não houve diferença no número de células TRAP positivas presentes na crista óssea alveolar entre os grupos no tempo avaliado, animais WT infectados apresentaram elevada expressão gênica de RANKL/OPG quando comparados aos animais NOD2-/- infectados. Além disso, a expressão de marcadores de atividades dos osteoclastos, catepsina k e matrix metaloproteinase-9, foi significantemente baixa nos animais NOD1-/- e NOD2-/- infectados quando comparados aos animais WT infectados. Esses resultados permitem sugerir que independente das variáveis observadas, verificamos que a ausência dos dois receptores impede a rápida progressão da reabsorção óssea alveolar observada na periodontite, portanto os receptores NOD1 e NOD2 contribuem para progressão da reabsorção óssea no modelo experimental de periodontite. / The bacterial biofilm has been identified as an etiological agent in the pathogenesis of periodontal disease. The NOD-like receptors (NLRs) are cytoplasmic proteins that sense microbial by products released by invasive bacteria. Since periodontopathogenic bacteria are able to invade and colonize some periodontal tissue cells, the purpose of this study is to determine the role of NOD1 and NOD2 receptors in the recognition of invasive periodontopathogenic bacteria such as Porphyromonas gingivalis, modulating the immune response and bone resorption. Isogenic strain C57BL/6 males (WT), NOD1 (NOD1-/-) and NOD2 (NOD2-/-) knockout mice were infected with Porphyromonas gingivalis in an experimental model of periodontal disease. Alveolar bone resorption was evaluated by macroscopic and histological morphometric analysis, quantification of osteoclasts numbers in bone crest alveolar. Imune inflammatory response was evaluated by, bacterial load, neutrophils quantification and inflammatory mediators levels by ELISA. We also evaluated the osteogenic and osteoclastogenic markers expression in gingival tissue by Real time PCR techniques. NOD1-/- and NOD2-/- animals showed lower bone resorption when compared to WT animals. NOD2-/- infected animals expressed higher bacterial load compared to WT infected ones. NOD1-/- and NOD2-/- infected animals presented lower Cxcl1 and MPO levels compared to WT infected animals. In addition, NOD2-/- infected animals presented lower level of TNF-α and higher level of IL-10 when compared to WT infected animals. There was no significant difference in the osteogenic factors expression, Runx2 and osteocalcin, when compared NOD1-/- and NOD2-/- infected animals to WT infected ones. Although there was no difference in TRAP-positive cells number evaluated in the alveolar bone crest among the studied groups, WT infected animals showed elevated ratio RANKL/OPG when compared to NOD2-/- infected animals. Moreover, the expression of osteoclasts activity markers, cathepsin k and matrix metalloproteinase-9, was significantly lower in NOD1-/- and NOD2-/- infected animals compared to WT infected ones. These results suggest that NOD1 and NOD2 receptors contribute to progression of bone resorption in experimental model of periodontitis, since the lack of NOD like receptors impair the bone resorption.

O biofilme bacteriano é o agente etiológico primário no desenvolvimento da resposta inflamatória observada na doença periodontal. Os receptores do tipo NOD (NLRs) são proteínas citosólicas que reconhecem componentes microbianos presentes no citoplasma liberados por bactérias invasoras. Sabendo que bactérias periodontopatogênicas têm a capacidade de invadir e colonizar diversas células do tecido periodontal, o presente projeto tem o objetivo de estudar a participação dos receptores do tipo NOD (NOD1 e NOD2) no reconhecimento das bactérias Porphyromonas gingivalis, na modulação da resposta imune e no processo de reabsorção óssea. Camundongos isogênicos machos da linhagem C57BL/6 (WT) e camundongos deficientes para o receptor NOD1 (NOD1-/-) e receptor NOD2 (NOD2-/-) foram infectados com Phorphyromonas gingivalis utilizando um modelo experimental de doença periodontal. O grau de reabsorção óssea foi avaliado por análise morfométrica macroscópica e histológica da maxila, além da quantificação do número de osteoclastos na crista óssea alveolar. O grau de inflamação local foi realizado por contagem do número total de bactérias orais, quantificação de neutrófilos no tecido gengival (MPO) e avaliação dos mediadores inflamatórios por ELISA. Foi também avaliada a expressão de marcadores osteogênicos e osteoclastogênicos no tecido gengival pela técnica de qPCR. Animais NOD1-/- e NOD2-/- apresentaram menor delta de reabsorção óssea quando comparados aos animais WT. Animais NOD2-/- infectados apresentaram debilitado controle bacteriano quando comparados aos animais WT infectados. Animais NOD1-/- e NOD2-/- infectados apresentaram baixa expressão de Cxcl1 e MPO quando comparados aos animais WT infectados. Além disso, foi observado que animais NOD2-/- infectados apresentaram reduzida produção de TNF-α e elevada produção de IL-10 quando comparados aos animais WT infectados. Não foi detectado diferença estatística na expressão de fatores osteogênicos, Runx2 e osteocalcina, entre animais WT, NOD1-/- e NOD2-/- infectados. Embora não houve diferença no número de células TRAP positivas presentes na crista óssea alveolar entre os grupos no tempo avaliado, animais WT infectados apresentaram elevada expressão gênica de RANKL/OPG quando comparados aos animais NOD2-/- infectados. Além disso, a expressão de marcadores de atividades dos osteoclastos, catepsina k e matrix metaloproteinase-9, foi significantemente baixa nos animais NOD1-/- e NOD2-/- infectados quando comparados aos animais WT infectados. Esses resultados permitem sugerir que independente das variáveis observadas, verificamos que a ausência dos dois receptores impede a rápida progressão da reabsorção óssea alveolar observada na periodontite, portanto os receptores NOD1 e NOD2 contribuem para progressão da reabsorção óssea no modelo experimental de periodontite. / The bacterial biofilm has been identified as an etiological agent in the pathogenesis of periodontal disease. The NOD-like receptors (NLRs) are cytoplasmic proteins that sense microbial by products released by invasive bacteria. Since periodontopathogenic bacteria are able to invade and colonize some periodontal tissue cells, the purpose of this study is to determine the role of NOD1 and NOD2 receptors in the recognition of invasive periodontopathogenic bacteria such as Porphyromonas gingivalis, modulating the immune response and bone resorption. Isogenic strain C57BL/6 males (WT), NOD1 (NOD1-/-) and NOD2 (NOD2-/-) knockout mice were infected with Porphyromonas gingivalis in an experimental model of periodontal disease. Alveolar bone resorption was evaluated by macroscopic and histological morphometric analysis, quantification of osteoclasts numbers in bone crest alveolar. Imune inflammatory response was evaluated by, bacterial load, neutrophils quantification and inflammatory mediators levels by ELISA. We also evaluated the osteogenic and osteoclastogenic markers expression in gingival tissue by Real time PCR techniques. NOD1-/- and NOD2-/- animals showed lower bone resorption when compared to WT animals. NOD2-/- infected animals expressed higher bacterial load compared to WT infected ones. NOD1-/- and NOD2-/- infected animals presented lower Cxcl1 and MPO levels compared to WT infected animals. In addition, NOD2-/- infected animals presented lower level of TNF-α and higher level of IL-10 when compared to WT infected animals. There was no significant difference in the osteogenic factors expression, Runx2 and osteocalcin, when compared NOD1-/- and NOD2-/- infected animals to WT infected ones. Although there was no difference in TRAP-positive cells number evaluated in the alveolar bone crest among the studied groups, WT infected animals showed elevated ratio RANKL/OPG when compared to NOD2-/- infected animals. Moreover, the expression of osteoclasts activity markers, cathepsin k and matrix metalloproteinase-9, was significantly lower in NOD1-/- and NOD2-/- infected animals compared to WT infected ones. These results suggest that NOD1 and NOD2 receptors contribute to progression of bone resorption in experimental model of periodontitis, since the lack of NOD like receptors impair the bone resorption.

Made available in DSpace on 2014-06-11T19:33:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-06Bitstream added on 2014-06-13T19:44:15Z : No. of bitstreams: 1 leite_frm_dr_arafo.pdf: 1468835 bytes, checksum: e5bbe1112c569efdf39842ed3eb0c03b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A reabsorcao do osso alveolar e uma das principais caracteristicas associadas a progressao da doenca periodontal. Apesar da enorme complexidade da microbiota envolvida, considera-se que bacterias Gram-negativas tenham um papel relevante em sua etiopatogenese. Um dos fatores de virulencia destes microrganismos e representado por um componente de sua parede externa denominado lipopolissacarideo (LPS). A presenca de LPS na proximidade dos tecidos periodontais e capaz de induzir a producao de diversos mediadores inflamatorios que levam a degradacao tanto do tecido conjuntivo quanto osseo. Atualmente acredita-se que a interacao do ligante do receptor-ativador do fator nuclear kappa-B (RANKL) com seu receptor (RANK) presente em precursores hematopoieticos e necessaria e suficiente para a inducao da diferenciacao de osteoclastos. Por outro lado, a ligacao de RANKL com seu falso-receptor, denominado osteoprotegerina (OPG), reduz sua biodisponibilidade e inibe, desta forma, a osteoclastogenese. Assim, a razao da expressao de RANKL e OPG e considerada como o principal determinante do “turnover” do tecido osseo. A producao de RANKL e OPG depende das vias de sinalizacao ativadas, as quais sao influenciadas pela natureza do estimulo extracelular. Atualmente, a familia de receptores NLRs (nod-like receptors) foi identificada como receptor intracelular para componentes bacterianos e agentes moduladores de diferentes vias de sinalizacao. Considerando a relevancia do LPS bacteriano na patogenese da doenca periodontal, o papel do RANKL no processo de reabsorcao ossea e a possivel implicacao das proteinas Nod na transducao de sinais regulando a expressao de RANKL, o objetivo geral deste projeto foi estudar os mecanismos de regulacao da expressao de RANKL induzido por LPS bacteriano em celulas relevantes do periodonto (macrofagos, osteoblastos e fibroblastos). Os objetivos especificos propostos... / Bone resorption is one of the major characteristics of destructive periodontal disease. Despite the great number of different bacterial species in the dental biofilm, Gramnegative microorganisms were demonstrated to have a very important role on periodontal disease pathogenesis. Lipopolysaccharide (LPS) is a bacterial cell wall component, which is acknowledged as one of the main virulence factors of these microorganisms. The mere presence of LPS in proximity with the periodontal tissues initiates the expression and production of inflammatory mediators and other cytokines which can culminate in degradation of both soft and hard tissues. It is currently accepted that the interaction between receptor-activator of nuclear factor kappa-B ligand (RANKL) and its receptor (RANK) is both necessary and sufficient to induce osteoclast differentiation and activation. However, RANKL can interact with its soluble decoy receptor osteoprotegerin (OPG) inhibiting osteoclastogenesis by decreasing the bioavailability of RANKL. Production of RANKL/OPG is the result of the signaling pathways activated by external stimuli. Recently, the NLR (nod-like receptors) family was identified as cytosolic receptors for bacterial components and also, as capable of modulating different signaling pathways. Considering the relevance of LPS and RANKL in bone resorption and the possible implication of Nod proteins in signal transduction regulating RANKL expression, the aim of this study was to evaluate the influence of different intracellular signaling pathways on the regulation of RANKL expression induced by LPS in relevant cells of the periodontium (macrophages, osteoblasts and fibroblasts). The specific objectives proposed were to determine after LPS and interleukin-1 beta stimulation the role of MyD88-dependent and independent signaling pathways, Nod1 and Nod2 on the expression of RANKL, OPG, IL-10 and IFN-beta... (Complete abstract click electronic access below)