Ulm, Univ., Diss., 2008.

No description available.

As leishmanias são parasitas intracelulares obrigatórios que utilizam fagócitos (em especial os macrófagos), para sua sobrevivência e propagação. Estes parasitas são interiorizados via interação com diversos receptores da superfície celular, como o receptor CR3 (MAC-1; CD18/CD11b), componente da família das 2-integrinas. As B2-integrinas são importantes em diferentes mecanismos imunológicos, como a adesão e migração de células, fagocitose e modulação de vias de sinalização intracelulares. Estudos demonstram que a molécula CD18 (constituinte de todas as B2-integrinas) está envolvida na resposta imune na leishmaniose experimental, mas há controvérsias quanto a sua importância na defesa contra esta infecção. Leucotrienos e prostaglandinas são mediadores lipídicos envolvidos em diversos mecanismos da resposta imune inata e adaptativa. Na leishmaniose, os leucotrienos são considerados cruciais para a defesa do organismo por induzir mecanismos efetores celulares, como fagocitose e atividade microbicida. Por outro lado, as prostaglandinas (em especial PGE2) exercem efeito imunossupressor sobre estes mecanismos, podendo favorecer o desenvolvimento da doença. Entretanto, não há conhecimento sobre a relação entre a expressão das B2-integrinas e a produção destes mediadores lipídicos na leishmaniose e a importância destas moléculas na resposta imune contra esta infecção. Assim, o objetivo desta tese foi investigar o efeito da redução da expressão das B2-integrinas na produção de citocinas, quimiocinas e de medidores lipídicos, em modelo de infecção experimental por L. amazonensis. Para isso, camundongos da linhagem C57BL/6 selvagens (WT) e com baixa expressão de CD18 (CD18 Low), foram infectados com promastigotas de L. amazonensis na pata posterior, e o desenvolvimento da infecção foi acompanhado por 5 e 8 semanas após a inoculação. Com base nos resultados obtidos, os animais CD18 Low apresentaram maior carga parasitária nas patas, em comparação com os camundongos selvagens. Além disso, a maior suscetibilidade dos camundongos CD18 Low foi independente da produção de NO e de IL-10 e da redução de IL-12 mediada por IL-4. Estes animais também apresentaram reduzida produção de IFN-gama. Além disso, os camundongos CD18 Low tiveram alterações na produção de LTB4 e de PGE2 ,as quais podem ter reduzido as respostas efetoras das células no sítio da infecção. Também demonstramos que a migração de neutrófilos e de eosinófilos para o sítio de infecção ocorreu de modo independente da expressão de CD18. O maior edema e recrutamento de eosinófilos nas patas dos camundongos CD18 Low foi independente da produção de MCP-1 e parcialmente dependente de RANTES. Entretanto, a produção de LTC4 pareceu ter sido o principal responsável por estes efeitos, em conjunto com PGD2. Deste modo, nossos resultados mostram que a baixa expressão de CD18 favorece o desenvolvimento da infecção por L. amazonensis. / Leishmania are obligate intracellular parasites that use phagocytes (especially macrophages) for their survival and propagation. These parasites are internalized through interaction with several receptors of cellular surface, such as the CR3 receptor (Mac-1; CD18/CD11b), component of the B2-integrins family. B2- integrins are involved in important immune mechanisms, such as cell adhesion, cell migration, phagocytosis and modulation of intracellular signaling pathways. Studies have shown that the CD18 molecule (component of all B2-integrins), is involved in the immune response in experimental leishmaniasis, but its importance in the host defense against this infection is still controversial. Leukotrienes and prostaglandins are lipid mediators involved in various mechanisms of innate and adaptive immune response. In leishmaniasis, leukotrienes are considered critical for the host defense, once it induces cellular effector mechanisms such as phagocytosis and microbicide activity. On the other hand, prostaglandins (in particular PGE2), lead to immunosuppressive effects on these mechanisms and may favor development of disease. However, it is unknown whether there is an interaction between the B2 integrins expression and the production of these mediators in leishmaniasis and the role of these molecules in host defense against this disease. Thus, the aim of this thesis was to verify the effect of reduced expression of B2-integrins in cytokines, chemokines and lipid mediators production, in experimental model of L. amazonensis infection. For this purpose, wild type mice (WT) and mice with reduced CD18 expression (CD18Low) from C57BL/6 strain were infected with L. amazonensis promastigotes in the hind footpad. The development of the infection was assessed for 5 and 8 weeks after inoculation. Our results showed that the CD18Low mice had higher parasite load in footpad, compared to wild-type mice. Moreover, the higher susceptibility of CD18Low mice was independent of NO and IL-10 production and independent of IL-12 reduction mediated by IL-4. These mice also showed reduced IFN- gamma production. CD18Low mice had alterations in LTB4 and PGE2 levels in site of infection, which may have altered the effector cell responses. We also demonstrated that the migration of neutrophils and eosinophils to the site of infection was CD18-independent. Moreover, the higher edema and eosinophils recruitment in CD18Low mice was MCP-1-independent and partially dependent of RANTES. However, LTC4 production seemed to be the main factor for edema and migration of eosinophils, together with PGD2. Thus, our results show that the reduction on CD18 expression favors the development of L. amazonensis infection.

As leishmanias são parasitas intracelulares obrigatórios que utilizam fagócitos (em especial os macrófagos), para sua sobrevivência e propagação. Estes parasitas são interiorizados via interação com diversos receptores da superfície celular, como o receptor CR3 (MAC-1; CD18/CD11b), componente da família das 2-integrinas. As B2-integrinas são importantes em diferentes mecanismos imunológicos, como a adesão e migração de células, fagocitose e modulação de vias de sinalização intracelulares. Estudos demonstram que a molécula CD18 (constituinte de todas as B2-integrinas) está envolvida na resposta imune na leishmaniose experimental, mas há controvérsias quanto a sua importância na defesa contra esta infecção. Leucotrienos e prostaglandinas são mediadores lipídicos envolvidos em diversos mecanismos da resposta imune inata e adaptativa. Na leishmaniose, os leucotrienos são considerados cruciais para a defesa do organismo por induzir mecanismos efetores celulares, como fagocitose e atividade microbicida. Por outro lado, as prostaglandinas (em especial PGE2) exercem efeito imunossupressor sobre estes mecanismos, podendo favorecer o desenvolvimento da doença. Entretanto, não há conhecimento sobre a relação entre a expressão das B2-integrinas e a produção destes mediadores lipídicos na leishmaniose e a importância destas moléculas na resposta imune contra esta infecção. Assim, o objetivo desta tese foi investigar o efeito da redução da expressão das B2-integrinas na produção de citocinas, quimiocinas e de medidores lipídicos, em modelo de infecção experimental por L. amazonensis. Para isso, camundongos da linhagem C57BL/6 selvagens (WT) e com baixa expressão de CD18 (CD18 Low), foram infectados com promastigotas de L. amazonensis na pata posterior, e o desenvolvimento da infecção foi acompanhado por 5 e 8 semanas após a inoculação. Com base nos resultados obtidos, os animais CD18 Low apresentaram maior carga parasitária nas patas, em comparação com os camundongos selvagens. Além disso, a maior suscetibilidade dos camundongos CD18 Low foi independente da produção de NO e de IL-10 e da redução de IL-12 mediada por IL-4. Estes animais também apresentaram reduzida produção de IFN-gama. Além disso, os camundongos CD18 Low tiveram alterações na produção de LTB4 e de PGE2 ,as quais podem ter reduzido as respostas efetoras das células no sítio da infecção. Também demonstramos que a migração de neutrófilos e de eosinófilos para o sítio de infecção ocorreu de modo independente da expressão de CD18. O maior edema e recrutamento de eosinófilos nas patas dos camundongos CD18 Low foi independente da produção de MCP-1 e parcialmente dependente de RANTES. Entretanto, a produção de LTC4 pareceu ter sido o principal responsável por estes efeitos, em conjunto com PGD2. Deste modo, nossos resultados mostram que a baixa expressão de CD18 favorece o desenvolvimento da infecção por L. amazonensis. / Leishmania are obligate intracellular parasites that use phagocytes (especially macrophages) for their survival and propagation. These parasites are internalized through interaction with several receptors of cellular surface, such as the CR3 receptor (Mac-1; CD18/CD11b), component of the B2-integrins family. B2- integrins are involved in important immune mechanisms, such as cell adhesion, cell migration, phagocytosis and modulation of intracellular signaling pathways. Studies have shown that the CD18 molecule (component of all B2-integrins), is involved in the immune response in experimental leishmaniasis, but its importance in the host defense against this infection is still controversial. Leukotrienes and prostaglandins are lipid mediators involved in various mechanisms of innate and adaptive immune response. In leishmaniasis, leukotrienes are considered critical for the host defense, once it induces cellular effector mechanisms such as phagocytosis and microbicide activity. On the other hand, prostaglandins (in particular PGE2), lead to immunosuppressive effects on these mechanisms and may favor development of disease. However, it is unknown whether there is an interaction between the B2 integrins expression and the production of these mediators in leishmaniasis and the role of these molecules in host defense against this disease. Thus, the aim of this thesis was to verify the effect of reduced expression of B2-integrins in cytokines, chemokines and lipid mediators production, in experimental model of L. amazonensis infection. For this purpose, wild type mice (WT) and mice with reduced CD18 expression (CD18Low) from C57BL/6 strain were infected with L. amazonensis promastigotes in the hind footpad. The development of the infection was assessed for 5 and 8 weeks after inoculation. Our results showed that the CD18Low mice had higher parasite load in footpad, compared to wild-type mice. Moreover, the higher susceptibility of CD18Low mice was independent of NO and IL-10 production and independent of IL-12 reduction mediated by IL-4. These mice also showed reduced IFN- gamma production. CD18Low mice had alterations in LTB4 and PGE2 levels in site of infection, which may have altered the effector cell responses. We also demonstrated that the migration of neutrophils and eosinophils to the site of infection was CD18-independent. Moreover, the higher edema and eosinophils recruitment in CD18Low mice was MCP-1-independent and partially dependent of RANTES. However, LTC4 production seemed to be the main factor for edema and migration of eosinophils, together with PGD2. Thus, our results show that the reduction on CD18 expression favors the development of L. amazonensis infection.

A desnutrição é uma condição nutricional que pode afetar muitos aspectos da resposta imunológica, como alterações na migração celular, na fagocitose, na resposta bactericida, mudanças na produção de radicais livres e espécies de nitrogênio e na produção de citocinas pró-inflamatórias. Logo, indivíduos desnutridos apresentam maior susceptibilidade a infecções. Visto que a glutamina é um aminoácido de extrema importância para a funcionalidade de diversas células do sistema imune e que as mesmas apresentam aumento da utilização desse aminoácido durante processos infecciosos, investigou-se, neste trabalho, quais os efeitos da glutamina sobre alguns aspectos da mobilização, migração e sinalização celular em um modelo experimental de desnutrição proteica. Para tanto, utilizou-se camundongos da linhagem BALB/c machos, os quais foram divididos em dois grupos, Controle e Desnutrido, que passaram a receber dietas isocalóricas contendo 12% (normoproteica) e 2% de caseína (hipoproteica), respectivamente, durante 5 semanas. Para as avaliações in vivo, animais de ambos os grupos receberam por via endovenosa 100µL de solução contendo 1,25µg de LPS e após 1 hora 0,75mg/Kg de L-glutamina (GLUT). Após o período de desnutrição ou de indução ao processo inflamatório, os animais foram eutanasiados e as amostras biológicas coletas. Foram avaliados nos animais estimulados in vivo hemograma, mielograma, as citocinas IL-10 e TNF-α circulantes e a expressão de CD11b/CD18 nos granulócitos do sangue periférico. Foi avaliado, in vitro, a capacidade migratória, a expressão de CD11b/CD18 de polimorfonucleados da medula óssea e do sangue periférico, bem como a síntese de citocinas IL-1α, IL-6, IL-10, IL-12 e TNF-α e a expressão de NF-κB e IκBα em células cultivadas em meio com 0; 0,6; 2 e 10 mM de GLUT. Os animais desnutridos apresentaram anemia, leucopenia, hipoplasia medular e diminuição na concentração sérica de proteínas, albumina e pré-albumina. A GLUT, in vitro, apresentou capacidade de reduzir a produção de IL-1α e IL-6, bem como a ativação da via do NF-κB. No modelo in vivo a GLUT, em animais estimulados com LPS, alterou a cinética de migração neutrofílica e reduziu a expressão de CD18, bem como diminuiu os níveis de TNFα circulantes. / Malnutrition is a nutritional condition that can affect many aspects of immune responses, affecting cell migration, phagocytosis, bactericidal response and changing free radicals production as nitrogen species and production of proinflammatory cytokines. Therefore, malnourished individuals are more susceptible to infections. Once glutamine is an amino acid of extreme importance to the functionality of various immune cells and those cells exhibit increased use of this amino acid during infectious processes. In this work was investigated, the effects of glutamine in some aspects of mobilization, cell migration and signaling in an experimental model of protein malnutrition. For this purpose, we used BALB/c mice, which received isocaloric diets, normoproteic or hypoproteic, containing respectively, 12% (Control group) and 2% (Malnourished group) of protein for a period of 5 weeks. The animals in both groups, for in vivo evaluations, received intravenous 100 µl of a solution containing 1.25µg of LPS and after 1 hour 0.75mg/kg of L-glutamine (GLUT). After the malnutrition period or the inflammatory process induction, the animals were euthanized and biological samples were collected. Were evaluated blood count, bone marrow, the cytokines IL-10 and TNF-α circulating and expression of CD11b/CD18 in granulocytes from peripheral blood of animals stimulated in vivo. In vitro were evaluated the migratory capacity, the expression of CD11b/CD18 polymorphonuclear bone marrow and peripheral blood, as well as the cytokines synthesis IL-1α, IL-6, IL-10, IL-12 and TNF-α and the expression of NF-κB and IκBα in cultured cells in media with 0; 0.6; 2 and 10 mM GLUT. Malnourished animals presented anemia, leukopenia, marrow hypoplasia and lower serum proteins, albumin and prealbumin. The GLUT in vitro has the capacity to reduce IL-1α and IL-6 as well as the activation of the NF-κB. In in vivo model, the GLUT altered neutrophil migration kinetics and reduced the expression of CD18, as well as decreased levels of circulating TNF-α in animals stimulated with LPS.

A desnutrição é uma condição nutricional que pode afetar muitos aspectos da resposta imunológica, como alterações na migração celular, na fagocitose, na resposta bactericida, mudanças na produção de radicais livres e espécies de nitrogênio e na produção de citocinas pró-inflamatórias. Logo, indivíduos desnutridos apresentam maior susceptibilidade a infecções. Visto que a glutamina é um aminoácido de extrema importância para a funcionalidade de diversas células do sistema imune e que as mesmas apresentam aumento da utilização desse aminoácido durante processos infecciosos, investigou-se, neste trabalho, quais os efeitos da glutamina sobre alguns aspectos da mobilização, migração e sinalização celular em um modelo experimental de desnutrição proteica. Para tanto, utilizou-se camundongos da linhagem BALB/c machos, os quais foram divididos em dois grupos, Controle e Desnutrido, que passaram a receber dietas isocalóricas contendo 12% (normoproteica) e 2% de caseína (hipoproteica), respectivamente, durante 5 semanas. Para as avaliações in vivo, animais de ambos os grupos receberam por via endovenosa 100µL de solução contendo 1,25µg de LPS e após 1 hora 0,75mg/Kg de L-glutamina (GLUT). Após o período de desnutrição ou de indução ao processo inflamatório, os animais foram eutanasiados e as amostras biológicas coletas. Foram avaliados nos animais estimulados in vivo hemograma, mielograma, as citocinas IL-10 e TNF-α circulantes e a expressão de CD11b/CD18 nos granulócitos do sangue periférico. Foi avaliado, in vitro, a capacidade migratória, a expressão de CD11b/CD18 de polimorfonucleados da medula óssea e do sangue periférico, bem como a síntese de citocinas IL-1α, IL-6, IL-10, IL-12 e TNF-α e a expressão de NF-κB e IκBα em células cultivadas em meio com 0; 0,6; 2 e 10 mM de GLUT. Os animais desnutridos apresentaram anemia, leucopenia, hipoplasia medular e diminuição na concentração sérica de proteínas, albumina e pré-albumina. A GLUT, in vitro, apresentou capacidade de reduzir a produção de IL-1α e IL-6, bem como a ativação da via do NF-κB. No modelo in vivo a GLUT, em animais estimulados com LPS, alterou a cinética de migração neutrofílica e reduziu a expressão de CD18, bem como diminuiu os níveis de TNFα circulantes. / Malnutrition is a nutritional condition that can affect many aspects of immune responses, affecting cell migration, phagocytosis, bactericidal response and changing free radicals production as nitrogen species and production of proinflammatory cytokines. Therefore, malnourished individuals are more susceptible to infections. Once glutamine is an amino acid of extreme importance to the functionality of various immune cells and those cells exhibit increased use of this amino acid during infectious processes. In this work was investigated, the effects of glutamine in some aspects of mobilization, cell migration and signaling in an experimental model of protein malnutrition. For this purpose, we used BALB/c mice, which received isocaloric diets, normoproteic or hypoproteic, containing respectively, 12% (Control group) and 2% (Malnourished group) of protein for a period of 5 weeks. The animals in both groups, for in vivo evaluations, received intravenous 100 µl of a solution containing 1.25µg of LPS and after 1 hour 0.75mg/kg of L-glutamine (GLUT). After the malnutrition period or the inflammatory process induction, the animals were euthanized and biological samples were collected. Were evaluated blood count, bone marrow, the cytokines IL-10 and TNF-α circulating and expression of CD11b/CD18 in granulocytes from peripheral blood of animals stimulated in vivo. In vitro were evaluated the migratory capacity, the expression of CD11b/CD18 polymorphonuclear bone marrow and peripheral blood, as well as the cytokines synthesis IL-1α, IL-6, IL-10, IL-12 and TNF-α and the expression of NF-κB and IκBα in cultured cells in media with 0; 0.6; 2 and 10 mM GLUT. Malnourished animals presented anemia, leukopenia, marrow hypoplasia and lower serum proteins, albumin and prealbumin. The GLUT in vitro has the capacity to reduce IL-1α and IL-6 as well as the activation of the NF-κB. In in vivo model, the GLUT altered neutrophil migration kinetics and reduced the expression of CD18, as well as decreased levels of circulating TNF-α in animals stimulated with LPS.

Chronic inflammation is essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration/accumulation of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages in damaged tissue. The mechanism of macrophage migration and subsequent accumulation is still not fully understood. Currently, the amoeboid adhesion-independent motility is considered essential for leukocyte migration in the three-dimensional environment. We challenge this hypothesis by studying the contribution of leukocyte adhesive receptors, integrins αMβ2, and αDβ2, to three-dimensional migration of M1-polarized, M2-polarized, and resident macrophages. Both integrins have a moderate expression on M2 macrophages, while αDβ2 is upregulated on M1 and αMβ2 demonstrates high expression on resident macrophages. The level of integrin expression determines its contribution to macrophage migration. Namely, intermediate expression supports macrophage migration, while a high integrin density inhibits it. Using in vitro three-dimensional migration and in vivo tracking of adoptively-transferred fluorescently-labeled macrophages during the resolution of inflammation, we found that strong adhesion of M1-activated macrophages translates to weak 3D migration, while moderate adhesion of M2-activated macrophages generates dynamic motility. Reduced migration of M1 macrophages depends on the high expression of αDβ2, since αD-deficiency decreased M1 macrophage adhesion and improved migration in fibrin matrix and peritoneal tissue. Similarly, the high expression of αMβ2 on resident macrophages prevents their amoeboid migration, which is markedly increased in αM-deficient macrophages. In contrast, αD- and αM-knockouts decrease the migration of M2 macrophages, demonstrating that moderate integrin expression supports cell motility. The results were confirmed in a diet-induced diabetes model. αD deficiency prevents the retention of inflammatory macrophages in adipose tissue and improves metabolic parameters, while αM deficiency does not affect macrophage accumulation. Summarizing, β2 integrin-mediated adhesion may inhibit amoeboid and mesenchymal macrophage migration or support mesenchymal migration in tissue, and, therefore, represents an important target to control inflammation.

Neutrophil-macrophage interplay is a fine-tuning mechanism that regulates the innate immune response during infection and inflammation. Cell surface receptors play an essential role in neutrophil and macrophage functions. The same receptor can provide different outcomes within diverse leukocyte subsets in different inflammatory conditions. Understanding the variety of responses mediated by one receptor is critical for the development of anti-inflammatory treatments. In this study, we evaluated the role of a leukocyte adhesive receptor, integrin αDβ2, in the development of acute inflammation. αDβ2 is mostly expressed on macrophages and contributes to the development of chronic inflammation. In contrast, we found that αD-knockout dramatically increases mortality in the cecal ligation and puncture sepsis model and LPS-induced endotoxemia. This pathologic outcome of αD-deficient mice is associated with a reduced number of monocyte-derived macrophages and an increased number of neutrophils in their lungs. However, the tracking of adoptively transferred fluorescently labeled wild-type (WT) and αD−/− monocytes in WT mice during endotoxemia demonstrated only a moderate difference between the recruitment of these two subsets. Moreover, the rescue experiment, using i.v. injection of WT monocytes to αD-deficient mice followed by LPS challenge, showed only slightly reduced mortality. Surprisingly, the injection of WT neutrophils to the bloodstream of αD−/− mice markedly increased migration of monocyte-derived macrophage to lungs and dramatically improves survival. αD-deficient neutrophils demonstrate increased necrosis/pyroptosis. αDβ2-mediated macrophage accumulation in the lungs promotes efferocytosis that reduced mortality. Hence, integrin αDβ2 implements a complex defense mechanism during endotoxemia, which is mediated by macrophages via a neutrophil-dependent pathway.

Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDß2 (CD11d/CD18) and αMß2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDß2-transfected HEK293 cells, WT, and αD-/- mouse M1- polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDß2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDß2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDß2- recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.

No description available.