A fibrose hepática é uma patologia que acompanha outras doenças crônicas do fígado como a cirrose e o hepatocarcinoma. As células estreladas hepáticas (HSC, do inglês hepatic stellate cells) compõem uma população celular heterogênea que se caracteriza por transitar entre dois fenótipos. As células com fenótipo quiescente possuem a capacidade de armazenar vitamina A em gotas lipídicas. Os insultos ao fígado desencadeiam uma resposta inflamatória que gera estímulos parácrinos e autócrinos mediados por citocinas e espécies reativas. Neste contexto, as HSC assumem um fenótipo ativado fibrogênico e tornam-se responsáveis pela cicatrização hepática. Danos crônicos ao fígado levam a uma deposição de matriz extracelular exagerada que configura o estado patológico da fibrose. O resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-estilbeno) é uma fitoalexina produzida por algumas espécies de plantas. Inúmeros efeitos benéficos à saúde são atribuídos ao RSV por causa do seu potencial antioxidante, antiinflamatório e pró-apoptótico. Estudos anteriores mostraram que tratamento da GRX, uma linhagem murina de HSC ativadas, com concentrações de RSV próximas as biodisponíveis (0,1 a 1 μM) resultou em parada do ciclo na fase S com consequente inibição de proliferação celular, um efeito associado à citotoxicidade e que pode favorecer a resolução da fibrose hepática. Neste estudo, por técnicas espectrofotométricas, foi demonstrado que tratamento da GRX por 24 horas com concentrações entre 0,1 a 50 μM de RSV promoveu um efeito pró-oxidante que causa uma citotoxicidade dependente da dose, bastante aumentada no grupo tratado com a concentração mais alta. Os efeitos citotóxicos atenuados encontrados nas células tratadas por 120 horas sugerem que a GRX pode se tornar resistente a estes efeitos. O potencial pró-oxidante do RSV foi o ponto de partida para investigar a possibilidade de que esta fitoalexina provocasse uma alteração no metabolismo mitocondrial da GRX. Para isso, os efeitos do RSV (1 a 50 μM) na função mitocondrial, na indução de morte mediada por estas organelas e na autofagia/mitofagia foram investigados por técnicas de espectrofotometria, de imunocitoquímica, de citometria de fluxo, de microscopia confocal e de microscopia eletrônica de transmissão em GRX tratadas por 24 e 120 horas. Foi demonstrado que todas as concentrações de RSV promovem apoptose por meio da ativação de caspases, alteram a dinâmica/função mitocondrial e induzem o aumento de autofagia/mitofagia na GRX. No entanto, o RSV provocou biogênese de mitocôndrias nos grupos tratados com 1 e 10 μM, enquanto que o tratamento com 50 μM causou dano celular evidente na GRX, sem induzir biogênese de mitocôndrias. Desta forma, é possível que a citotoxicidade “dose-dependente” do RSV, que causa a morte celular e dano oxidativo em 24 horas de tratamento, esteja relacionada com o desequilíbrio entre a indução concomitante de apoptose mediada por dano mitocondrial, autofagia/mitofagia e biogênese de mitocôndrias. Por fim, foi investigada a liberação de TNF-α, Interleucina-6 e Interleucina-10 pela GRX tratada por 24 e 120 horas com RSV (0,1 a 50 μM), considerando o papel antiinflamatório do RSV e o papel das HSC ativadas na sinalização autócrina que contribui para a modulação fenotípica destas células. Foi demonstrado que o tratamento da GRX com RSV por 24 e 120 horas induziu a redução da liberação de Interleucina-6; enquanto que a liberação de TNF-α e Interleucina-10 foi aumentada. Estes resultados confirmam um efeito antiinflamatório do RSV que deve contribuir na prevenção da ativação ou da perpetuação do estado ativado das HSC por meio de sinalização autócrina. Ainda que a concentração do RSV seja importante para efetivamente induzir a morte das HSC ativadas, o tratamento com esta fitoalexina pode ser promissor para a resolução da fibrose hepática por diminuir a população de células ativadas e, possivelmente, prevenir a perpetuação do estado fenotípico ativado. Estudos avaliando indicadores de quiescência em células tratadas são ainda necessários para desvendar completamente os efeitos do RSV quanto às possibilidades de inibição da perpetuação ou reversão fenotípica das HSC ativadas. / Liver fibrosis is a disease that accompanies other hepatic chronic diseases such as cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSC) are a heterogeneous cell population characterized by transiting between two phenotypes. Cells with a quiescent phenotype are able to store vitamin A into lipid droplets. Damage to the liver trigger an inflammatory response that generates paracrine and autocrine stimulation mediated by cytokines and reactive species. In this context, HSC assume an activated and fibrogenic phenotype responsive for hepatic wound-healing. Chronic insults to the liver lead to an excessive deposition of extracellular matrix that configures the pathological state of fibrosis. Resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-stilbeno) is a phytoalexin produced by some species of plants. Several beneficial effects are attributed to this molecule due to its antioxidant, antiproliferative and pro-apoptotic potential. Previous studies showed that treatment with bioavailable concentrations of RSV (0.1 to 1 μM) promoted an arrest cycle at the S phase in GRX, a murine activated HSC model, leading to cell proliferation inhibition, a cytotoxic effect that contributes to the liver fibrosis resolution. In this study, it was shown by spectrophotometric techniques that GRX treatment for 24 hours at concentrations between 0.1 to 50 μM of RSV promoted a fairly clear pro-oxidant effect that causes a dose-dependent cytotoxicity that was higher in the group treated with 50 μM. The attenuated cytotoxicity found after 120 hours of GRX treatment suggest that these cells became resistant to this effect. The pro-oxidant potential of RSV was the starting point for investigating the possibility that this phytoalexin would cause a change in the GRX mitochondrial metabolism. Thus, the effects of RSV (1 to 50 μM) on altering the mitochondrial function, on inducing mitochondrial-mediated cell death, and autophagy/mitofagia were investigated in GRX treated for 24 and 120 hours by spectrophotometric techniques, immunocytochemistry, flow cytometry, confocal microscopy, and transmission electron microscopy. All the RSV concentrations promote cell apoptosis through caspases activation, alter the mitochondrial dynamics and function, and induce an increase of autophagy/mitofagia. Curiously, only 1 and 10 μM of RSV induced mitochondrial biogenesis in GRX, while the highest concentration caused an evident cell damage without inducing mitochondrial biogenesis. Thus, it is possible that the "dose-dependent" cytotoxicity of RSV, which causes cell death and oxidative damage in 24 hours of treatment, is related to an imbalance between the concomitant induction of mitochondrial-mediated apoptosis, autophagy/mitofagia, and mitochondrial biogenesis. Finally, it was investigated the release of TNF-α, Interleukin-6 and Interleukin-10 by GRX treated for 24 and 120 hours with RSV (0.1 to 50 μM), considering the anti-inflammatory role of RSV and the autocrine signalling role of HSC that contributes to the perpetuation of its activated phenotype. It was demonstrated that GRX treatment with RSV for 24 and 120 hours reduced the release of Interleukin-6 in the culture medium; whereas the release of TNF-α and Interleukin-10 was increased. These results confirm the anti-inflammatory properties of RSV and may contribute to the prevention of HSC activation through autocrine signalling. Although RSV concentration is important to effectively induce activated HSC death, cells treatment with this phytoalexin may be promising for liver fibrosis resolution through decreasing the population of activated cells or through preventing the perpetuation of activated state of HSC. Future studies evaluating the quiescence indicators of GRX under RSV treatment are still needed to fully unravel the effects of this phytoalexin on inhibiting the perpetuation of activated HSC or reversing its activated phenotype.

Bei IL-10 handelt es sich um ein antiinflammatorisches Zytokin, dessen immunmodulatorische Effekte bereits in zahlreichen Studien aufgezeigt werden konnten. Ziel dieser Studie war, die Unterschiede in der systemischen Inflammation und Organdysfunktion an Mäusen zu untersuchen, die nach Induktion eines hämorrhagischen Schocks, entweder inhalativ oder systemisch, mit IL-10 behandelt wurden. Männliche C57/BL6 Mäuse (6 Tiere pro Gruppe) wurden für 1.5 Stunden blutdruckkontrolliert in einen hämorrhagischen Schock versetzt. Nach anschließender Volumensubstitution wurde ihnen inhalativ oder intraarteriell rekombiniertes Maus - IL-10 verabreicht. Nach einer Gesamtversuchsdauer von 6 - bzw. 24 Stunden erfolgte die Tötung der Tiere. Die Ergebnisse der Studie zeigen, dass die lokale und systemische Verabreichung von IL-10 das Zytokinprofil der systemischen Inflammationsantwort unterschiedlich beeinflusst. Die Lunge kann durch inhalative Gabe von IL-10 geschützt werden, ohne die systemische Inflammationsantwort zu beeinflussen.

No description available.

T cells play a pivotal role in controlling viral infection; however, the precise mechanisms responsible for regulating T‐cell differentiation and function during infections are incompletely understood. In this study, we demonstrated an expansion of myeloid‐derived suppressor cells (MDSC s), in particular the monocytic MDSC s (M‐MDSC s; CD 14+ CD 33+ CD 11b+ HLA ‐DR −/low), in patients with chronic hepatitis C virus (HCV ) infection. Notably, HCV ‐induced M‐MDSC s express high levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT 3) and interleukin‐10 (IL ‐10) compared with healthy subjects. Blocking STAT 3 signalling reduced HCV ‐mediated M‐MDSC expansion and decreased IL ‐10 expression. Importantly, we observed a significant increase in the numbers of CD 4+ CD 25+ Foxp3+ regulatory T (Treg) cells following incubation of healthy peripheral blood mononuclear cells (PBMC s) with MDSC s derived from HCV ‐infected patients or treated with HCV core protein. In addition, depletion of MDSC s from PBMC s led to a significant reduction of Foxp3+ Treg cells developed during chronic HCV infection. Moreover, depletion of MDSC s from PBMC s significantly increased interferon‐γ production by CD 4+ T effector (Teff) cells derived from HCV patients. These results suggest that HCV ‐induced MDSC s promote Treg cell development and inhibit Teff cell function, suggesting a novel mechanism for T‐cell regulation and a new strategy for immunotherapy against human viral diseases.

No description available.

Previous studies have demonstrated that the interaction of CD40 on monocytes with CD40 ligand, present on activated CD4+ T cells, induces monocyte inflammatory cytokine synthesis and rescues monocytes from apoptosis. These findings suggest a role for CD40 signaling of monocyte activation in the maintenance and/or exacerbation of nonseptic (e.g., autoimmune) inflammatory responses. In the present study the effects of the modulatory cytokines IL-4 and IL-10 on CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis were examined. Both IL-4 and IL-10 decreased CD40-dependent IL-1beta synthesis in a dose-dependent manner individually and synergized in this effect when used concurrently, with minimal effect on CD40 surface expression. CD40 signaling of IL-1beta synthesis was shown to be dependent on the induction of protein tyrosine kinase (PTK) activity, and both IL-4 and IL-10 diminished CD40-mediated tyrosine phosphorylation of monocyte cellular proteins. However, IL-4, but not IL-10, blocked CD40-mediated rescue from apoptosis, an event that we have demonstrated previously to be dependent on PTK activity as well. Together these results suggest that in monocytes 1) both IL-4 and IL-10 target CD40-induced PTK activity in the down-regulation of IL-1beta synthesis; and 2) IL-4 and IL-10 have divergent effects on the CD40 signaling pathway, in that these cytokines are synergistic with respect to their abilities to inhibit CD40-mediated IL-1beta synthesis and differ in their abilities to block CD40-mediated rescue from apoptosis.

Weaning piglets are susceptible to the invasive Streptococcus (S.) suis infection, which can result in septicemia. The aim of this study was to investigate the cytokine profile induced upon S. suis infection of blood, to determine the cellular sources of those cytokines, and to study the potential effects of the induced cytokines on bacterial killing. We measured TNF-α, IL-6, IFN-γ, IL-17A and IL-10 after an experimental intravenous infection with S. suis serotype 2 in vivo, and analyzed whole blood, peripheral blood mononuclear cells (PBMC) and separated leukocytes to identify the cytokine-producing cell type(s). In addition, we used a reconstituted whole blood assay to investigate the effect of TNF-α on bacterial killing in the presence of different S. suis-specific IgG levels. An increase in IL-6 and IL-10, but not in IFN-γ or IL-17A, was observed in two of three piglets with pronounced bacteremia 16 to 20 h after infection, but not in piglets with controlled bacteremia. Our results confirmed previous findings that S. suis induces TNF-α and IL-6 and could demonstrate that TNF-α is produced by monocytes in vitro. We further found that IL-10 induction resulted in reduced secretion of TNF-α and IL-6. Rapid induction of TNF-α was, however, not crucial for in vitro bacterial killing, not even in the absence of specific IgG.

No description available.

Interleukin-10 (IL-10) is an important regulator of immune responses secreted by different cell types. Previous results from our group suggested that the biological effects of this cytokine critically depend on its cellular source. Recent studies reported IL-10 dependent immunosuppressive functions of a specialized subset of regulatory B cells and mast cells. These results relied on adoptive cell transfers, a technique which can potentially introduce artifacts. Therefore, we aimed to readdress these questions in independent models using IL-10 transcriptional reporter mice and various conditional IL-10 mutant mice. Findings in IL-10 reporter system suggested prominent IL-10 transcription in regulatory B cells upon LPS administration. Exposure of mice to contact allergen revealed robust reporter expression in CD8 T cells, moderate to mild reporter expression in CD4 T cells and dendritic cells (DC) respectively, and lack of reporter expression in B cells, mast cells and NK cells in allergen challenged ears. We generated cell-type specific IL-10 mutants by Cre/LoxP-mediated conditional gene inactivation. Efficiency and specificity of Cre-mediated recombination was demonstrated by Southern blot and PCR methods. Various immunogenic challenges in conditional IL-10 mutants did not reveal a role for B cell-derived IL-10 in restraining innate TLR or T cell-dependent inflammatory responses. Likewise, mice with selective inactivation of the il10 gene in mast cells exhibited normal CHS responses and unaltered immune response to CpG oligodeoxynucleotides. On the other hand, DC-specific IL-10 mutants developed excessive inflammatory responses to contact allergens, while innate responses to TLR ligands were not altered. This indicates a non-redundant role for DC-derived IL-10 in contact allergy. Thus, the conditional IL-10 ‘‘knockout’’ mice combined with the novel transcriptional IL-10 reporter system can serve as ideal tools to understand the cell-type specific contributions to IL-10-mediated immune regulation.

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.