The socio-economic and geographical distribution of malaria overlaps with that of many parasitic helminths and in these areas co-infections are common. Co-infection with helminths can influence disease outcome causing either exacerbation or amelioration of malaria. Understanding the complex host-parasite interactions that lead to these different disease outcomes is important for the success of control programmes aimed at these parasites. The immune system has evolved diverse types of response (e.g. T-helper 1 (Th1) and T-helper 2 (Th2)) to efficiently combat infection with ‘microparasites’ and helminths respectively. When faced with co-infection however, the need for the host to multitask means it must manage these counter-regulatory responses. In this study a murine model of malaria-hookworm (Plasmodium chabaudi- Nippostrongylus brasiliensis) co-infection was utilised to investigate how changes in T-helper bias affect malaria disease outcome. Antibody isotypes were used as indicators of Th1/Th2 bias and revealed that helminth co-infection reduced the malaria-specific Th1 response. Counter-intuitively this resulted in ‘protection’ from malaria with co-infected mice having reduced peak P. chabaudi parasitaemia and suffering less severe anaemia. In addition to providing a measure of Th1/Th2 bias, analysis of antibody responses revealed the occurrence of cross-reactive antibodies. The potential for these crossreactive antibodies to influence disease outcome was investigated but in this murine model resource-mediated mechanisms of parasite regulation appear to be responsible for the ‘protection’ that co-infection affords. The question of why cross-reactive antibodies are produced has important immunological and ecological implications. Cross-reactive responses may arise through some physiological constraint on the immune mechanisms that usually result in antibody-specificity. However experiments designed to investigate if the specificity of antibodies is constrained by availability of antigen suggest that this is not the case in the model system used here. There is also the possibility that production of cross-reactive antibodies represents an evolutionary optimal strategy for a host faced with unpredictable exposure to a variety of parasites. However a major finding of this study indicates these two taxonomically distinct parasite species share antigens, which in itself is crucial to understanding host-parasite interactions in a co-infection setting. The main findings of this thesis are relevant to co-infection studies in general and the implications for both evolutionary and applied biology are discussed.

Tim-1 (T cell immunoglobulin mucin domain 1) is a transmembrane protein expressed by many cell types, including activated T cells and B cells. Antibodies to Tim-1 have been shown to decrease severity of airway hyperreactivity and Th2 cytokine production in mice. Current literature suggests Tim-1 functions as a co-stimulatory molecule. We hypothesize that Tim-1 signals in lymphocytes, and that Tim-1 signaling modulates allergic airway disease. Chapter one provides a brief overview of current literature exploring identification of the Tim family of receptors, genetic associations between TIM-1 polymorphisms and human diseases, Tim-1 expression, Tim-1 ligands, studies of antibodies to Tim-1 in various mouse models of human disease, and signaling events downstream of Tim-1 engagement. Chapter two provides detailed experimental methodology. Chapter three details the characterization of Tim-1 deficient mice. Tim-1 deficient mice do not exhibit defects in lymphocyte or myeloid cell development, as determined by numbers of cells present in bone marrow, thymus, spleen, and lymph nodes. C57BL/6 Tim-1 deficient female mice appear to develop an increased number of lymph node cells and also develop anti-double stranded DNA antibodies. Chapter four explores the impact of Tim-1 deficiency in a murine allergic airway disease model, which demonstrated that Tim-1 deficient mice developed increased lung inflammation and increased antigen-specific Th2 cytokine production that was evident in mice backcrossed to both BALB/c and C57BL/6 backgrounds. These phenotypes were not evident using purified naïve CD4+ T cells polarized in vitro. As Tim-1 expression is not restricted to CD4+ T cells, adoptive transfer experiments were performed to determine whether the phenotype observed was due to the deficiency of Tim-1 on CD4+ T cells, non-CD4+ T cells, or Tim-1 deficiency on both CD4+ T cells and non-CD4+ T cells. Chapter five explores the impact of Tim-1 deficiency in a chronic Leishmania major intradermal infection model. Tim-1 deficient mice crossed to both BALB/c and C57BL/6 backrounds demonstrated similar parasite burden over the course of time, but in vitro restimulation of lymph node cells revealed a striking increase in cytokine production that extended to Th1, Th2, and Th17 lineages. Tim-1 signaling in murine B cell lines is explored in Chapter six. A Tim-1 monoclonal antibody conjugated to beads induces phosphorylation of Tim-1 and recruitment of the Src family kinase Fyn. This phosphorylation of Tim-1 is reduced in Fyn-deficient B cell lines. Chapter seven discusses the significance of these findings, relates current literature to these results, and provides some avenues for further exploration of Tim-1 function and signaling.

Fatty acid-binding proteins are small intracellular proteins with poorly defined functions in intracellular fatty acid transport. The adipocyte fatty acid-binding protein aP2 regulates systemic glucose and lipid metabolism. Using Affymetrix microarrays, we found that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by airway epithelial cells. aP2 expression was markedly increased following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13, and downregulated by the Th1 cytokine IFN-gamma. Regulation of aP2 mRNA expression by Th2 cytokines was dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2 deficient mice in a model of allergic airway inflammation, and found that infiltration of leukocytes, especially eosinophils, into the airways was highly aP2 dependent. T cell priming and peritoneal allergy was unaffected by aP2 deficiency suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-haematopoietic cells, most likely airway epithelial cells, as the site of aP2 action in allergic airway inflammation. Expression of the pro-inflammatory cytokines MCP-1 and IL-6 was impaired in cytokine activated aP2 deficient airway epithelial cells, while levels of the anti-inflammatory arachidonic acid metabolite 15-HETE was increased, providing a mechanism for the reduced airway inflammation in aP2 deficient mice. In addition to the immune functions of aP2, we found that the related fatty acid-binding protein mal1 was also upregulated by IL-4/IL-13 in airway epithelial cells, and mal1 deficient mice were protected against airway eosinophilia. Significantly, in comparison to single aP2 deficiency, mice with combined aP2-mal1 deficiency had augmented protection against airway inflammation, and bone marrow chimera experiments demonstrated that aP2-mal1 deficiency affected both non-haematopoeitic and haematopoeitic cells. In T cell priming experiments, aP2-mal1 deficiency resulted in defective cytokine profiles in antigen recall responses, suggesting compromised sensitisation to antigen as one mechanism for aP2-mal1 action in airway inflammation. Together, our data therefore demonstrates the crucial roles of fatty acid-binding proteins in airway epithelium, T cell priming and airway inflammation, and provides a new link between fatty acid signalling and allergy.

Les conséquences immunitaires d'un stress d'origine environnementale sont complexes et encore difficilement prévisibles. Le stress affecte le système immunitaire soit en agissant sur l'immunité innée, en altérant la réactivité inflammatoire, soit en agissant sur l'immunité acquise, en modulant la production de cytokines dites Th1 et Th2. L'environnement social<br />contribue largement au développement et à l'expression de maladies. Dans les espèces sociales, la position sociale occupée dans le groupe module la susceptibilité aux infections mais les supports endocriniens et immunitaires de ces différences de susceptibilité sont ignorés. La remise en cause de l'organisation sociale engendre un stress important dont les conséquences immunitaires sont encore sujettes à controverse.<br />Ce travail de thèse a pour objectifs (1) de décrire l'influence du statut social sur le fonctionnement des systèmes endocrinien et immunitaire, (2) de préciser les effets du stress<br />social sur la production de cytokines et la susceptibilité aux infections et (3) de rechercher des facteurs à l'origine de la variabilité des conséquences immunitaires du stress social.<br />Chez le porcelet, un regroupement après le sevrage élève transitoirement le cortisol salivaire et altère le comportement mais n'affecte pas la réactivité des lymphocytes sanguins.<br />La suite des travaux a utilisé une procédure de défaite sociale chronique chez la souris. Les résultats obtenus mettent en évidence une influence du statut social. En absence de stress, les<br />dominants présentent des niveaux de base de corticostérone et une réponse spécifique à la tuberculine supérieurs aux dominés. Suite à une défaite sociale, les dominants sont plus affectés que les dominés. La défaite sociale augmente la réactivité inflammatoire mais ne modifie pas de façon nette l'équilibre de la production de cytokines de type Th1 et Th2 et n'affecte pas l'immunité spécifique développée contre une infection mycobactérienne. Les conséquences immunitaires de la défaite sociale ne sont observées que lorsque le stress est associé à des combats et à des blessures. Ces travaux montrent que la réponse au stress dépend de l'histoire sociale de l'individu, en particulier de son statut social. De plus, les<br />répercussions immunitaires du stress dépendent aussi de l'histoire immunitaire récente. En effet, une réaction inflammatoire systémique inhibe la libération plasmatique de cytokines<br />inflammatoires en réponse à un stress psychologique ultérieur.

Les conséquences immunitaires d'un stress d'origine environnementale sont complexes et encore difficilement prévisibles. Le stress affecte le système immunitaire soit en agissant sur l'immunité innée, en altérant la réactivité inflammatoire, soit en agissant sur l'immunité acquise, en modulant la production de cytokines dites Th1 et Th2. L'environnement social contribue largement au développement et à l'expression de maladies. Dans les espèces sociales, la position sociale occupée dans le groupe module la susceptibilité aux infections mais les supports endocriniens et immunitaires de ces différences de susceptibilité sont ignorés. La remise en cause de l'organisation sociale engendre un stress important dont les conséquences immunitaires sont encore sujettes à controverse. Ce travail de thèse a pour objectifs (1) de décrire l'influence du statut social sur le fonctionnement des systèmes endocrinien et immunitaire, (2) de préciser les effets du stress social sur la production de cytokines et la susceptibilité aux infections et (3) de rechercher des facteurs à l'origine de la variabilité des conséquences immunitaires du stress social. Chez le porcelet, un regroupement après le sevrage élève transitoirement le cortisol salivaire et altère le comportement mais n'affecte pas la réactivité des lymphocytes sanguins. La suite des travaux a utilisé une procédure de défaite sociale chronique chez la souris. Les résultats obtenus mettent en évidence une influence du statut social. En absence de stress, les dominants présentent des niveaux de base de corticostérone et une réponse spécifique à la tuberculine supérieurs aux dominés. Suite à une défaite sociale, les dominants sont plus affectés que les dominés. La défaite sociale augmente la réactivité inflammatoire mais ne modifie pas de façon nette l'équilibre de la production de cytokines de type Th1 et Th2 et n'affecte pas l'immunité spécifique développée contre une infection mycobactérienne. Les conséquences immunitaires de la défaite sociale ne sont observées que lorsque le stress est associé à des combats et à des blessures. Ces travaux montrent que la réponse au stress dépend de l'histoire sociale de l'individu, en particulier de son statut social. De plus, les répercussions immunitaires du stress dépendent aussi de l'histoire immunitaire récente. En effet, une réaction inflammatoire systémique inhibe la libération plasmatique de cytokines inflammatoires en réponse à un stress psychologique ultérieur.

<p>The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion.</p><p>In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2^d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes.</p><p>We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2^s, H-2^q and H-2^f) mouse strains, silver induced only ANolA responses in H-2^s and H-2^q mice, but not in H-2^fmice. Further studies showed that the resistance to silver-induced ANolA production in H-2^fmice was inherited as a dominant trait.</p><p>We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice.</p><p>Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.</p>

Neonatal immune responses are typically deficient against a wide variety of antigens, including alloantigens, vaccine antigens, and infectious agents. These responses are characterized by Th2-skewed cytokine production, and deficient Th1 and cytotoxic responses. However, these deficient responses can be boosted to adult levels by the use of strong, Th1 promoting agents. This demonstrates that neonates are capable of developing mature immune responses under specific conditions. Using two different murine models, we have found that neonates develop robust Th and cytotoxic responses, which under some antigenic conditions significantly exceed those of adults. First, using a model of early life exposure to noninherited maternal antigens (NIMA), we found that murine neonates develop robust in vivo cytotoxic responses to low doses of alloantigens. Importantly, primary in vivo cytotoxic responses to alloantigen developed during the neonatal period, and persisted into adulthood. Neonates developed similar memory cytotoxic responses to donor spleen cells, bone marrow, and stem cell-enriched (Lin-) bone marrow cells, suggesting that the exposure dose is more important than the type of transplanted donor cell for the development of cytotoxicity. NIMA-exposed neonates also developed vigorous primary and memory allospecific Th1/Th2 responses which exceeded the responses of adults. These findings suggest that early exposure to low levels of NIMA may lead to long term immunological priming of all arms of T cell adaptive immunity. Second, we characterized the phenotype and function of neonatal recent thymic emigrants (RTE). RTE are the predominant cell type in murine neonates, and are present at higher frequencies within the neonatal CD4+ compartment than in adults. Our data demonstrate that RTE from murine neonates and adults are phenotypically and functionally distinct. In particular, although the magnitude of RTE cytokine responses from both age groups is dependent on the conditions of activation, neonatal RTE consistently exhibited higher levels of effector cytokine production than adult RTE. In particular, activation of neonatal RTE in the presence of IL-7 lead to greatly increased IFNgamma production, while adult responses were not altered. Overall, neonatal RTE responses were more plastic than those of adult RTE, as both Th1 and Th2 responses were altered in neonates using various activation conditions, while only Th2 responses were consistently changed in adults. Finally, in contrast to adult RTE, neonatal RTE proliferated in response to IL-7 stimulation at very early timepoints. This was associated with faster kinetics of IL-7Ralpha downregulation and higher levels of pSTAT5 in neonatal RTE. These quantitative and qualitative differences in neonatal RTE populations may largely explain the diverse responses that are elicited in neonates in response to different antigens, especially under those conditions in which Th1 responses are enhanced (i.e., exposure to NIMA alloantigens). Taken together, these data demonstrate that neonatal T cell responses are actually highly plastic, instead of intrinsically deficient. Furthermore, if given optimal stimulation conditions, neonatal responses can actually exceed those produced by adults.

The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion. In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes. We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2s, H-2q and H-2f) mouse strains, silver induced only ANolA responses in H-2s and H-2q mice, but not in H-2f mice. Further studies showed that the resistance to silver-induced ANolA production in H-2f mice was inherited as a dominant trait. We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice. Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.

We report herein that IL-10-treated dendritic cells (DC) can be used effectively to reverse established severe asthma-like disease in a mouse model. Our lab had shown previously that allergen-presenting splenic CD8¦Á+ DCs could ¡Ö50% reduce airway hyper responsiveness (AHR), eosinophilia, and Th2 responses in asthma-phenotype mice, but only marginally reduce IgE/IgG1 levels. We now show that bone marrow-derived DCs that have been differentiated in the presence of IL-10 (DCIL-10) are effective in reversing the asthma phenotype. Co-culture of DCIL-10 with T memory (TM) cells from asthma-phenotype mice was associated with lack of Th2 responses, and this was partially reversed by IL-2. Immunostimulatory DC activated these Th2 cells. <i>In vivo</i>, delivery of allergen-pulsed DCIL-10, either into the airway or intraperitoneally abrogated AHR from weeks 3-10 post-treatment, and ameliorated lung eosinophilia and Th2 (IL-4, -5, -9, & -13, IgE) responses, as well as circulating allergen-specific IgE responses for at least 32 weeks following treatment. Repeated OVADCIL-10 treatments kept AHR normalized for 8 weeks as well as Th2 responses significantly low. In vivo, delivery of anti-IL-10R, but not anti-TGF-¦Â from day 12-21 after treatment had moderate effects on DCIL-10-driven tolerance, but 1-methyl tryptophan (inhibitor of indoleamine-2,3-dioxygenase) treatment had significant effects on Th2 responses. The mechanisms mediating tolerance in vivo are likely complex, but we speculate that infectious tolerance sustains this regulatory activity during the 32-week period in which we have observed tolerance to be in place.

Preeclampsia is one of the most feared pregnancy complications, with a risk of maternal and fetal death and with no ideal therapy readily available. The cause of this strictly pregnancyrelated disease is still unknown and is therefore a great challenge to all researchers in the field of pregnancy-related pathophysiology. Today, the dominating theory of the origin of preeclampsia is defective initial placentation with insufficient penetration of the trophoblasts, leading to impaired maternal blood flow through narrow spiral arteries. However, the cause of this defective trophoblast behavior is not known. The maternal immune system has been proposed to have an influence on both the placentation and the subsequent systemic reactions. Therefore, it is very interesting to study the maternal immune system during preeclampsia, in hope of achieving a better understanding of this puzzling disease. Earlier studies have suggested that normal pregnancy requires a shift to a Th2/antiinflammatory type of immunity, at least directed towards the fetus and placenta, while some pregnancy complications, such as preeclampsia, could be due to a skewed Th1/proinflammatory type of immunity. However, the results from earlier studies designed to test the Th1/Th2 hypothesis in preeclampsia have not been consistent. Therefore, the aim of this thesis was to examine if established preeclampsia is associated with increased innate inflammatory responses and a deviation of adaptive responses towards Th1 when compared with normal pregnancy. Enumerations of cytokine-producing cells from peripheral blood did not show any difference in the production of IFN-γ, IL-4, IL-10 and IL-12 between women with preeclampsia and normal pregnancies. However, a decrease in the spontaneously produced levels of IL-5 was detected in cell cultures on peripheral blood mononuclear cells in women with preeclampsia. Furthermore, a decreased production of IL-10 in response to paternal antigens, believed to represent the fetus, was also detected for the preeclamptic women. Serum analysis showed increased levels of the pro-inflammatory mediators IL-6 and IL-8 during preeclampsia. Also, preeclamptic women displayed increased serum levels of the soluble IL-4 receptor, but no difference in the levels of IL-4 compared to normal pregnant women. This was an elusive finding, since the receptor was originally thought to reflect the levels of IL-4, but has recently been shown to have both agonistic and antagonistic properties on the IL-4 levels. Further studies of the local immune responses in the placenta showed no difference in the immunohistochemical staining of IL-4 and TNF-α between women with preeclampsia and women with normal pregnancies. In general, there were no hallmarks of abnormal morphology in the placental sections examined, regardless of diagnosis. In conclusion, the decreased levels of IL-10 in response to paternal antigens and the systemically increased levels of IL-6 and IL-8 suggest a specific decrease in antiinflammatory responses towards fetal antigens, together with a systemic activation of proinflammatory mediators during preeclampsia. Furthermore, the decreased production of IL-5 also indicates, at least partly, decreased Th2 responses in the established preeclampsia. / Figure 1 on page 6 is republished in the Ph.D. thesis with the kind permisson of Blackwell Publishing (http://www.blackwellpublishing.com). Figure IX on page38, figure XB on page 41, figure XI on page 46 and figure XII on page 47 are all published in the Journal of Reproductive Immunology and republished with kind permisson from Elsevier (http://www.elsevier.com/) in the Ph.D. thesis.