Submitted by Erika Demachki (erikademachki@gmail.com) on 2016-08-04T19:10:35Z No. of bitstreams: 2 Dissertação - Lucas Luiz de Lima Silva - 2016.pdf: 2502108 bytes, checksum: 9af274c5556e225e1478c978f79254ad (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2016-08-04T19:11:54Z (GMT) No. of bitstreams: 2 Dissertação - Lucas Luiz de Lima Silva - 2016.pdf: 2502108 bytes, checksum: 9af274c5556e225e1478c978f79254ad (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-04T19:11:54Z (GMT). No. of bitstreams: 2 Dissertação - Lucas Luiz de Lima Silva - 2016.pdf: 2502108 bytes, checksum: 9af274c5556e225e1478c978f79254ad (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-05-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / American Tegumentary Leishmaniasis (ATL) is a disease caused by Leishmania protozoan, belonging to the subgenus Viannia and Leishmania. In Brazil, the most common and prevalent species is L. (V.) braziliensis. In patients with ATL, it was detected the expression of interleukin 32 (IL-32) in skin or mucosal lesions caused by L. Viannia spp. However, the role of IL-32 on ATL is still unclear. It has been shown that IL-15 induces IL-32 and also IL-15 leads to L. infantum control. This study aimed to investigate the effects of IL-32 and IL-15 in the production of cytokines and microbicidal activity of primary human macrophages infected with L. (V.) braziliensis. For this, human peripheral blood monocytes were derived into macrophages and infected with metacyclic forms of L. (V.) braziliensis; evaluation of the infection index (4 h, phagocytosis, 48 h, microbicidal activity) in the absence or presence of rIL-32, rIL-15 or gamma interferon (rIFN) and lipopolysaccharide (LPS); in culture supernatants, IL-32, tumor necrosis factor (TNF) and IL-10 were measured by enzime-linked immunoassay. The addition of rIL-32 to macrophages did not significantly altered phagocytosis of the parasites or microbicidal activity of macrophages. Classical activation of macrophages with rIFN plus LPS decreased the infection index. rIL-32 em high concentration (200 ng/mL) was able to induce TNF in uninfected or infected macrophages, and IL-10 was not induced. Parasites induced lower amounts of intracellular IL-32 as well as rIFN0.1 ng / ml), but there was a synergism between the activation signals provided by the parasites and rIFN (0.1 ng / ml). In the opposite, rIFN in higher concentration (10 ng/mL) induced higher amounts of IL-32, but its activity was partially inhibited by parasites. The rIL-15 was able to induce IL- 32 and TNF in macrophages, but not IL-10 in both non-infected or infected macrophages. The rIL-15 also decreased phagocytosis of parasites by macrophages and increased the microbicidal activity of these cells. The data suggest that IL-15 induces IL-32 and TNF which can contribute to control of the infection. To evaluate the leishmanicidal mechanism pathways induced by IL-15 and IL-32 can help in the development of new therapies for the control of ATL. / A leishmaniose tegumentar americana (LTA) é uma doença infectoparasitária causada por protozários Leishmania, pertencentes aos subgêneros Viannia e Leishmania. No Brasil, a espécies mais comum e prevalente é L. (V.) braziliensis. Em pacientes com LTA, foi detectada a expressão da citocina interleucina 32 (IL-32) nas lesões cutâneas ou mucosas causadas por L. Viannia spp. No entanto, o papel da IL-32 na LTA ainda não foi esclarecido. Foi demonstrado que a IL-15 induz IL-32, além disso a IL-15 leva à morte de L. infantum. O presente estudo teve como objetivo investigar os efeitos da IL-32 e IL-15 na produção de citocinas e na atividade microbicida de macrófagos humanos primários infectados com L. (V.) braziliensis. Para isto, foi realizada a derivação de macrófagos humanos a partir de monócitos do sangue periférico de doadores não infectados e infecção com formas metacíclicas de L. (V.) braziliensis; avaliação do índice de infecção (4 h, fagocitose; 48 h, atividade microbicida), na ausência ou presença de tratamento com rIL-32, rIL-15 ou interferon gama (rIFN) e lipopolissacarídeo (LPS); e avaliação da produção de citocinas IL-32, fator de necrose tumoral (TNF) e IL-10, por ensaio imunoenzimático. A adição de rIL-32 às culturas de macrófagos primários infectados com L. (V.) braziliensis não alterou significantemente a fagocitose ou a atividade microbicida das células. A ativação clássica dos macrófagos com rIFN e LPS resultou em uma diminuição do índice de infecção. A rIL-32 em elevada concentração foi capaz de induzir TNF nos macrófagos não infectados ou infectados pelos parasitos L. (V.) braziliensis, sendo que não foi induzida produção de IL- 10. Os parasitos L. (V.) braziliensis induziram baixas quantidades de IL-32 intracelular, assim como rIFN0,1 ng/mL), porém houve sinergismo entre os sinais de ativação fornecidos pelos parasitos e os do rIFN (0,1 ng/mL). O rIFN em uma concentração mais elevada (10 ng/mL) induziu maiores quantidades de IL-32, porém esta indução foi parcialmente inibida pela infecção com os parasitos. A rIL-15 foi capaz de induzir IL-32 nos macrófagos e, além disso, induziu TNF e não induziu a produção de IL-10, tanto nos macrófagos infectados quanto não infectados. A rIL-15 também diminuiu a fagocitose dos parasitos pelos macrófagos e aumentou a atividade microbicida destes. Os dados sugerem que a IL-15 induz IL-32 e TNF que podem contribuir para o controle da infecção. Avaliar as vias de ativação de mecanismos leishmanicidas induzidos pelas IL- 15 e IL-32 podem auxiliar no desenvolvimento de novas terapias para o controle da LTA.

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Les progresseurs lents du VIH-1 sont de rares sujets asymptomatiques pendant plusieurs années sans thérapie antirétrovirale. Parmi ces sujets à progression lente vers le SIDA, il est possible qu’un sous-groupe perde le contrôle de leur infection après plusieurs années de contrôle. Notre laboratoire a analysé l’expression différentielle de différentes protéines et voies moléculaires associées à la perte de contrôle de l’infection: l’interleukine-32 (IL-32) est une cytokine pro-inflammatoire dont le niveau des isoformes alpha et delta a significativement diminué chez les progresseurs lents lors de la perte de contrôle. Par ailleurs, des études antérieures ont attribué, de façon intrigante, à l’IL-32 aussi bien des propriétés anti-VIH-1 que des propriétés immunosuppressives induisant un environnement propice à la réplication du VIH-1. Ce projet de maitrise s’est penché sur l’implication de l’IL-32 dans la progression de l’infection à VIH-1 avec un accent particulier sur les progresseurs lents. Nous avons principalement mesuré les niveaux d’IL-32 des sujets séropositifs comparativement aux sujets VIH négatif et estimé les fonctions de cette cytokine à travers des études longitudinales et de corrélation. Nous avons observé que l’IL-32 total demeure plus élevé chez les séropositifs comparativement aux sujets VIH négatif. Également, l’infection par le VIH-1 entraine une augmentation du niveau d’IL-32 total. De plus, après une année de thérapie antirétrovirale, les taux plasmatiques d’IL-32 total demeurent significativement plus élevés que ceux des sujets VIH négatif. Comme attendu, le taux d’IL-32 total augmente lors de la perte de contrôle de l’infection chez les progresseurs lents. Une forte concentration plasmatique d’IL-32 total coïncide avec: 1) une augmentation du taux plasmatique de sCD14 et de la cytokine pro-inflammatoire IL-6, 2) une baisse du compte cellulaire CD4 et une augmentation de la charge virale. Un taux plasmatique élevé de CCL5 pourrait prédire une faible concentration d’IL-32 total. L’isoforme alpha de l’IL-32 est plus élevée dans le plasma des sujets VIH négatif tandis que l’IL-32 gamma semble induire un environnement pro-inflammatoire et immunosuppressif. Il ressort à l’issue de ces observations que l’augmentation de l’IL-32 total est associée à la progression de l’infection à VIH-1 et pourrait constituer un biomarqueur permettant d’apprécier le pronostic de cette infection. / HIV-1 slow progressors constitute a rare population of subjects who remain asymptomatic for many years without antiretroviral therapy. Among this population, some individuals will lose control of their infection after several years of immunological control. Our laboratory has analyzed the differential expression profile of various proteins and molecular pathways associated with the loss of control of HIV infection. The pro-inflammatory cytokine interleukin-32 alpha and delta isoforms significantly decreased in slow progressors as they were losing control of their infection. Furthermore, previous studies have attributed to IL-32 both antiviral property against HIV-1 and immunosuppressive properties that can induce an environment conducive to HIV-1 replication. This project addresses the role of IL-32 in HIV-1 disease progression with a particular emphasis on slow progressors. We compared the levels of IL-32 in HIV-1 positive versus HIV-1 negative subjects and evaluated the role of this cytokine using longitudinal studies. We observed that levels of IL-32 remains higher in HIV-positive compared to HIV-negative subjects. Also, HIV-1 infection leads to increased level of IL-32. In addition, after one year of antiretroviral therapy, IL-32 plasma levels remain significantly higher than those of HIV-negative subjects. As expected, the levels of IL-32 increased as slow progressors lost control of their infection. A high plasma concentration of IL-32 predicts: 1) an increase in plasma levels of sCD14 as well as pro-inflammatory cytokine IL-6, 2) a decrease in CD4 cell count and an increase in viral load. High plasma CCL5 predicted a low concentration of IL-32. The alpha isoform of IL-32 is elevated in the plasma of HIV negative subjects while IL-32 gamma appears to induce a pro-inflammatory and immunosuppressive environment. We conclude that increased IL-32 levels are associated with progression of HIV-1 disease and could be used as a biomarker for assessing HIV-1 prognosis.