Immune cell changes in swimmers : response to acute exercise and training

O treino competitivo envolve exercício intenso e prolongado, capaz de modular o número e actividade das células imunitárias. Quando demasiado exigente poderá induzir fadiga e aumentar a susceptibilidade a doenças. Esta dissertação apresenta três estudos desenvolvidos no âmbito da Imunologia do Exercício, considerando a análise da resposta celular imunitária sistémica aguda e crónica ao exercício aplicada em situações reais do treino competitivo de natação, controlando factores passíveis de influenciar esta resposta. Pretendeu-se avaliar a resposta imunitária a uma sessão de treino prolongada e intensa, durante as 24h de recuperação (Estudo 1) e a uma época de treino com sete meses (Estudo 2), e estudar a influência de um macrociclo de treino de quatro meses sobre a resposta imunitária à mesma sessão de treino e período de recuperação (Estudo 3), controlando sexo, fases do ciclo menstrual, maturidade, escalão, especialidade, performance, cargas de treino e sintomas respiratórios superiores (URS). A sessão de treino induziu a diminuição da vigilância imunitária adquirida imediatamente e, pelo menos nas 2h seguintes. Juvenis e seniores recuperaram totalmente 24h depois, mas não os juniores, reforçando a ideia da existência de uma janela aberta para a infecção após exercícios prolongados e intensos e sugerindo uma recuperação menos eficiente para os juniores. No período de treino mais intenso da época observou-se uma imunodepressão e maior prevalência de URS. No final da época, a imunidade inata diminuiu aparentando maior sensibilidade aos efeitos cumulativos da carga de treino, enquanto a imunidade adquirida parece ter recuperado após o taper. O macrociclo de treino atenuou a resposta imunitária à sessão de treino e aumentou o período de janela aberta às infecções (efeitos mais acentuados nos adolescentes). Os resultados evidenciam a importância de controlar alterações imunitárias durante a época competitiva, especialmente em períodos de treino intenso e quando se realizam sessões de treino intensas consecutivas com recuperações inferiores a 24h.

Successful vaccination against Boophilus microplus and Babesia bovis using recombinat antigens

Current methods for the control of the cattle tick Boophils microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the tick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed. This too is now undergoing pre-registration field trials.

Schistosomiasis vaccine development: approaches and prospects

Mounting evidence for acquired immunity to schistosomiasis in humans supports the case for immunological intervention. On the other hand, rapid reinfection poses a threat to younger age groups due to the slow maturation of natural resistance. However, rational approaches, based on advances in immunology and molecular biology, have substantially increased the odds of producing an effective vaccine. Since the parasite cannot replicate in the human host and serious morbidity generally occurs only after a relatively long period of heavy worm burden, complete protection against infection is not essential. The chances of success would increase if more than one of the various host/parasite interphases were targeted, for example reducing morbidity through decreased worm loads as well as through suppression of egg production. Several promising schistosome antigens have now reached an advanced phase of development and are currently undergoing independent confirmatory testing according to a standardized protocol. A few molecules are being contemplated for scaled-up production but, so far, only one has reached the stage of industrial manufacture and safety testing. Since schistosomiasis cannot realistically be controlled by a single approach, vaccination is envisaged to be implemented in conjunction with other means of control, notably chemotherapy.

Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis

For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

Sequential histological changes in Biomphalaria glabrata during the course of Schistosoma mansoni infection

Biomphalaria glabrata, highly susceptible to Schistosoma mansoni, were seen to shed less and less cercariae along the time of infection. Histological examination kept a close correlation with this changing pattern of cercarial shedding, turning an initial picture of no-reaction (tolerance) gradually into one of hemocyte proliferation with formation of focal encapsulating lesions around disintegrating sporocysts and cercariae, a change that became disseminated toward the 142nd day post miracidial exposure. Findings were suggestive of a gradual installation of acquired immunity in snails infected with S. mansoni.

Effects of corticosterone on innate and humoral immune functions and oxidative stress in barn owl nestlings.

The costs of coping with stressful situations are traded-off against other functions such as immune responses. This trade-off may explain why corticosterone secretion reduces immune reactions. Corticosterone differentially affects various immunity components. However, which component is suppressed varies between studies. It remains unclear whether the trade-off in energy, nutrition, autoimmunity or oxidative stress accounts for differential immunosuppression. In this study, we investigated whether corticosterone differentially affects the constitutive innate and humoral acquired immunity. We used barn owl nestlings, implanting 50% with a corticosterone-releasing pellet and the other 50% with a placebo pellet. To measure the effect on humoral immunity we vaccinated 50% of the corticosterone-nestlings and 50% of the placebo-nestlings with the antigens 'Tetravac' and the other 50% were injected with PBS. To assess the costs of elevated corticosterone, we measured body mass and resistance to oxidative stress. Administration of corticosterone increased corticosterone levels whereas vaccination induced the production of antibodies. Corticosterone reduced the production of antibodies, but it did not significantly affect the constitutive innate immunity. Corticosterone reduced body growth and resistance to oxidative stress. Under stressful conditions barn owl nestlings seem to keep the constitutive innate immunity, whereas elevated corticosterone levels negatively affected inducible immune responses. We found evidence that mounting a humoral immune reaction is not costly in terms of growth, but reduces the resistance to oxidative stress independently of corticosterone administration. We suggest that humoral immunity is suppressed because the risk of immunopathologies may be disproportionately high when mounting an antibody response under stressful situations.

The effect of treatment on the age-antibody relationship in children infected with Schistosoma mansoni and Schistosoma haematobium

The effect of praziquantel treatment on the age-antibody relationship was studied in 174 children aged between 6 and 17 years from a schistosome endemic area in Zimbabwe. The children were co-infected with Schistosoma mansoni and S. haematobium with infection prevalences of 74% and 53% respectively. Antibody levels for the isotypes IgA, IgE, IgM, IgG1, IgG2, IgG3 and IgG4, directed against soluble egg antigen were measured using an indirect ELISA assay. Treatment resulted in a significant increase in levels of IgG2 and IgG3 while levels of IgA decreased significantly. In untreated children there were significant decreases in levels of IgG4. Treatment also resulted in significant alteration in the age-antibody profiles for the isotypes IgE, IgM, IgG1 and IgG2 in treated children but not in untreated children. The results are discussed in the context of factors believed to give rise to the age-antibody relationship; i.e. age-related exposure patterns, age-related development of acquired immunity, age-related hormonal changes and age-related changes in innate susceptibility to infection.

Mechanisms of eosinophil cytokine release

Human eosinophils have been demonstrated to contain a multitude of cytokines and chemokines that exist pre-formed within these cells. This content of pre-formed cytokines, with diverse potential biologic activities, provides eosinophils with capabilities distinct from most other leukocytes. The localization of pre-formed cytokines within eosinophils is both within specific granules and associated with substantial numbers of morphologically distinct cytoplasmic vesicles. Stimulation for release of specific cytokines, such as IL-4, leads to a regulated signal transduction cascade, which is dependent on the formation of leukotriene C4 within eosinophils where it acts as an intracrine mediator. IL-4 release occurs selectively and is by means of vesicular transport. The capabilities of eosinophils not only to rapidly release pre-formed cytokines but also to differentially regulate which cytokines are released endow eosinophils with distinct abilities in innate and acquired immunity.

The Duffy binding protein as a key target for a Plasmodium vivax vaccine: lessons from the Brazilian Amazon

Plasmodium vivax infects human erythrocytes through a major pathway that requires interaction between an apical parasite protein, the Duffy binding protein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII) and DARC to P. vivax infection has motivated our malaria research group at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a number of immunoepidemiological studies to characterise the naturally acquired immunity to PvDBP in populations living in the Amazon rainforest. In this review, we provide an update on the immunology and molecular epidemiology of PvDBP in the Brazilian Amazon - an area of markedly unstable malaria transmission - and compare it with data from other parts of Latin America, as well as Asia and Oceania.

Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling.

The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Plasmodium berghei ANKA infection model. CARD9 expression was upregulated in the brains of infected wild-type (WT) mice, suggesting a potential role for this pathway in ECM pathogenesis. However, P. berghei ANKA-infected Card9(-/-) mice succumbed to neurological signs and presented with disrupted blood-brain barriers similar to WT mice. Furthermore, consistent with the immunological features associated with ECM in WT mice, Card9(-/-) mice revealed (i) elevated levels of proinflammatory responses, (ii) high frequencies of activated T cells, and (iii) CD8(+) T cell arrest in the cerebral microvasculature. We conclude that ECM develops independently of the CARD9 signaling pathway.

Salmonella virulence factor SipB induces activation and release of IL-18 in human dendritic cells.

Interleukin-18 (IL-18) plays an important role in innate and acquired immunity, in particular against intracellular pathogens. However, little is known about the microbial factors that trigger IL-18 secretion by dendritic cells (DCs). To determine the influence of bacterial virulence factors on the activation and release of IL-18, we infected human monocyte-derived DCs with virulence mutants of the facultative intracellular pathogen Salmonella typhimurium. Our results show that infection by S. typhimurium causes caspase-1-dependent activation of IL-18 and triggers the release of IL-18 in human DCs. The secretion of IL-18 by the DCs was closely correlated with the ability of the S. typhimurium strains to induce apoptosis. We demonstrate that activation and release of IL-18 are blocked by mutations in the Salmonella sipB gene, which encodes a virulence factor that activates caspase-1 to induce apoptosis. These findings indicate that the activation and release of IL-18 induced by bacterial virulence factors may represent one component of innate immunity against the intracellular bacteria.

Immune and stress responses covary with melanin-based coloration in the barn swallow

Eumelanin and pheomelanin are the main endogenous pigments in animals and melanin-based coloration has multiple functions. Melanization is associated with major life-history traits, including immune and stress response, possibly because of pleiotropic effects of genes that control melanogenesis. The net effects on pheo- versus eumelanization and other life-history traits may depend on the antagonistic effects of the genes that trigger the biosynthesis of either melanin form. Covariation between melanin-based pigmentation and fitness traits enforced by pleiotropic genes has major evolutionary implications particularly for socio-sexual communication. However, evidence from non-model organisms in the wild is limited to very few species. Here, we tested the hypothesis that melanin-based coloration of barn swallow (Hirundo rustica) throat and belly feathers covaries with acquired immunity and activation of the hypothalamic-pituitary-adrenal (HPA) axis, as gauged by corticosterone plasma levels. Individuals of both sexes with darker brownish belly feathers had weaker humoral immune response, while darker males had higher circulating corticosterone levels only when parental workload was experimentally reduced. Because color of belly feathers depends on both eu- and pheomelanin, and its darkness decreases with an increase in the concentration of eu- relative to pheomelanin, these results are consistent with our expectation that relatively more eu- than pheomelanized individuals have better immune response and smaller activation of the HPA-axis. Covariation of immune and stress response arose for belly but not throat feather color, suggesting that any function of color as a signal of individual quality or of alternative life-history strategies depends on plumage region.

Novel therapeutic strategies targeting regulatory T cells and downstream TCR signaling in transplantation

Avec plus de 100000 transplantations d'organes solides (TOS) par année dans le monde, la transplantation d'organes reste actuellement l'un des meilleurs traitements disponibles pour de nombreuses maladies en phase terminale. Bien que les médicaments immunosuppresseurs couramment utilisés soient efficaces dans le contrôle de la réponse immune engendrant le rejet aigu d'une greffe, la survie du greffon à long terme ainsi que la présence d'effets secondaires indésirables restent un enjeu considérable en clinique. C'est pourquoi il est nécessaire de trouver de nouvelles approches thérapeutiques innovantes permettant de contrôler la réponse immunitaire et ainsi d'améliorer les résultats à long terme. L'utilisation des lymphocytes T régulateurs (Treg), suppresseurs naturels de la réponse inflammatoire, a fait l'objet de nombreuses études ces dix dernières années, et pourrait être considérée comme un moyen intéressant d'améliorer la tolérance immunologique de la greffe. Cependant, l'un des obstacles de l'utilisation des Treg comme agent thérapeutique est leur nombre insuffisant non seulement en conditions normales, mais en particulier lors d'une forte réponse immune avec expansion de cellules immunitaires alloréactives. En raison des limitations techniques connues pour l'induction des Treg ex-vivo ou in vitro, nous avons dédié la première partie du travail de thèse à la détermination de l'efficacité de l'induction des Treg in vivo grâce à l'utilisation d'un complexe protéique IL-2/JES6-1 (IL2c). Nous avons montré que l'expansion des Treg par IL2c permettait d'augmenter la survie du greffon sur un modèle murin de transplantation de peau avec mismatch entre le donneur et le receveur pour le complexe majeur d'histocompatibilité (CMH). De plus, nous avons vu qu'en combinant IL2c à une inhibition à court terme de la voie de co-stimulation CD40L-CD40 (anti-CD154/MRl, administré au moment de la transplantation) pour empêcher l'activation des lymphocytes T, il est possible d'induire une tolérance robuste à long terme. Finalement, nos résultats soulignent l'importance de cibler une voie de co-stimulation bien particulière. En effet, l'utilisation d'IL2c combinée au blocage de la co-stimulation CD28-B7.1/2 (CTLA-4 Ig) n'induit qu'une faible prolongation de la survie de la greffe et n'induit pas de tolérance. L'application chez l'humain des traitements induisant la tolérance dans des modèles expérimentaux murins ou de primates n'a malheureusement pas montré de résultats probants en recherche clinique ; une des principales raisons étant la présence de lymphocytes B et T mémoires provenant du systeme d immunité acquise. C est pourquoi nous avons testé si la combinaison d'IL2c et MR1 améliorait la survie de la greffe dans des souris pré¬sensibilisées. Nous avons trouvé qu'en présence de lymphocytes B et T mémoires alloréactifs, l'utilisation d'IL2c et MR1 permettait une amélioration de la survie de la greffe de peau des souris immunocompétentes mais comparé aux souris receveuses naïves, aucune tolérance n'a pu être induite. Toutefois, l'ajout d'un traitement anti-LFA-1 (permettant de bloquer la circulation des lymphocytes T activées) a permis d'améliorer de manière significative la survie de la greffe. Cependant, le rejet chronique, dû à la présence de lymphocytes B activés/mémoires et la production d'anticorps donneur-spécifiques, n'a pas pu être évité. Cibler l'activation des lymphocytes T est la stratégie immunothérapeutique prépondérente après une TOS. C'est pourquoi dans la deuxième partie de cette thèse nous nous sommes intéressés au système de signalisation d'un récepteur des lymphocytes T qui dépend de la paracaspase Malti en tant que nouvelle stratégie immunosuppressive pour le contrôle des lymphocytes T alloréactifs. Nous avons montré que bien que l'inhibition de la signalisation du lymphocyte T en aval de Malti induise une tolérance envers un greffon de peau avec incompatibilités antigéniques mineures, cela ne permet cependant qu'une régulation partielle de l'alloréponse contre des antigènes du CMH. Nous nous sommes aussi intéressés spécifiquement à l'activité protéolytique de Malti. L'inhibition constitutive de l'activité protéolytique de Malti chez les souris Malti-ki s'est révélée délétère pour l'induction de la tolérance car elle diminue la fonction des Treg et augmente l'alloréactivité des cellules Thl. Cependant, lors de l'utilisation d'un inhibiteur peptidique de l'activité protéase de Malti in vitro, il a été possible d'observer une atténuation de l'alloéactivité des lymphocytes T ainsi qu'un maintien de la population des Treg existants. Ces résultats nous laissent penser que des études plus poussées sur le rôle de la signalisation médiée par Malti seraient à envisager dans le domaine de la transplantation. En résumé, les résultats obtenus durant cette thèse nous ont permis d'élucider certains mécanismes immunologiques propres à de nouvelles stratégies thérapeutiques potentielles dont le but est d'induire une tolérance lors de TOS. De plus, ces résultats nous ont permis de souligner l'importance d'utiliser des modèles davantage physiologiques contenant, notamment en tenant compte des lymphocytes B et T mémoires alloréactifs. -- Organ transplantation remains the best available treatment for many forms of end-stage organ diseases, with over 100,000 solid organ transplantations (SOT) occurring worldwide eveiy year. Although the available immunosuppressive (IS) drugs are efficient in controlling acute immune activation and graft rejection, the off-target side effects as well as long-term graft and patient survival remain a challenge in the clinic. Hence, innovative therapeutic approaches are needed to improve long-term outcome across immunological barriers. Based on extensive experimental data obtained over the last decade, it is tempting to consider immunotherapy using Treg; the natural suppressors of overt inflammatory responses, in promoting transplantation tolerance. The first hurdle for the therapeutic use of Treg is their insufficient numbers in non- manipulated individuals, in particular when facing strong immune activation and expanding alloreactive effector cells. Because of the limitations associated with current protocols aiming at ex-vivo expansion or in vitro induction of Treg, the aim of the first part of this thesis was to determine the efficacy of direct in vivo expansion of Treg using the IL-2/JES6- 1 immune complex (IL2c). We found that whilst IL2c mediated Treg expansion alone allowed the prolonged graft survival of fìlli MHC-mismatched skin grafts, its combination with short-term CD40L-CD40 co-stimulation blockade (anti-CD 154/MR1) to inhibit T cell activation administered at the time of transplantation was able to achieve long-term robust tolerance. This study also highlighted the importance of combining Treg based therapies with the appropriate co-stimulation blockade as a combination of IL2c and CD28-B7.1/2 co- stimulation blockade (CTLA-4 Ig) only resulted in slight prolongation of graft survival but not tolerance. The translation of tolerance induction therapies modelled in rodents into non-human primates or into clinical trials has seldom been successful. One main reason being the presence of pre-existing memory T- and B-cells due to acquired immunity in humans versus laboratory animals. Hence, we tested whether IL2c+MRl could promote graft survival in pre-sensitized mice. We found that in the presence of alloreactive memory T- and B-cells, IL2c+MRl combination therapy could prolong MHC-mismatched skin graft survival in immunocompetent mice but tolerance was lost compared to the naïve recipients. The addition of anti-LF A-1 treatment, which prevents the trafficking of memory T cells worked synergistically to significantly further enhance graft survival. However, late rejection mediated by activated/memory B cells and persistent donor-specific alloantibodies still occurred. Immunotherapeutic strategies targeting the activation of T cells are the cornerstone in the current immunosuppressive management after SOT. Therefore, in the next part of this thesis we investigated the paracaspase Malti-dependent T-cell receptor signalling as a novel immunosuppressive strategy to control alloreactive T cells in transplantation. We observed that although the inhibition of Malti downstream T signalling lead to tolerance of a minor H- mismatch skin grafts, it was however not sufficient to regulate alloresponses against MHC mismatches and only prolonged graft survival. Furthermore, we investigated the potential of more selectively targeting the protease activity of Malti. Constitutive inhibition of Malti protease activity in Malti-ki mice was detrimental to tolerance induction as it diminished Treg function and increased Thl alloreactivity. However, when using a small peptide inhibitor of Malti proteolytic activity in vitro, we observed an attenuation of alloreactive T cells and sparing of the pre-existing Treg pool. This indicates that further investigation of the role of Malti signalling in the field of transplantation is required. Collectively, the findings of this thesis provide immunological mechanisms underlying novel therapeutic strategies for the promotion of tolerance in SOT. Moreover, we highlight the importance of testing tolerance induction therapies in more physiological models with pre-existing alloreactive memory T and B cells.

Gene expression in IFN-gamma-activated murine macrophages

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Rôle des neutrophiles dans l'inflammation allergique associée au souffle chez le cheval, un modèle naturel d'asthme

Réalisé en cotutelle avec le Dr James G Martin de l'Université McGill (Meakins-Christie laboratories)