Almost 2 billion people world-wide are infected with parasitic helminths. These complex multicellular eukaryotic organisms are capable of establishing long-term infections even in the face of an intact immune response. Typically, in these settings regulatory components of the immune response, such as Foxp3+ T regulatory cells (Tregs), become dominant, limiting protective effector responses towards the parasite. Helminths are thought to have evolved mechanisms, including release of immunomodulatory molecules termed excretory-secretory products (ES), to sway the balance between the regulatory and effector arms of the immune response to favour their persistence. In this thesis both the development of a protective immune response toward, and the potential manipulation of the immune response by, the rodent gastrointestinal nematode Heligmosomoides polygyrus have been studied. Firstly, the effects of H. polygyrus ES (HES) on bone-marrow derived dendritic cells (DCs) were analysed. Although HES did not alter the phenotype of the DC it was found to be able to suppress the ability of the DC to respond to inflammatory stimuli. This activity was lost when HES was heat-inactivated (hiHES). After adoptive transfer, HES-pulsed DCs were able to induce a HESspecific T helper (Th)2-type response even if co-treated with an inflammatory stimulus. Th2-type responses are protective against H. polygyrus infection. Surprisingly, the ability of HES to generate a Th2-response in a co-treatment situation was not related to its anti-inflammatory properties; DCs co-treated with hiHES and an inflammatory stimulus were able to drive an equivalent Th2-response to HES in this situation. Next, making use of mouse strains with different susceptibility phenotypes to primary H. polygyrus infection, potential mechanisms of resistance were characterised. Development of granulomas in the gut wall were found to be associated with reduced worm burdens. Furthermore, in highly susceptible C57BL/6 mice, production of IL-23 was shown to be counter-regulatory to this process, as mice on the same background but deficient in this cytokine have increased numbers of granulomas and dramatically enhanced resistance. Susceptibility to H. polygyrus was also considered at the level of epigenetic regulation. A protein that binds specifically to methylated DNA, methyl-CpG binding domain protein (MBD)2, was found to affect the proportion of Foxp3+ Tregs within the CD4+ T cell population in vivo. Additionally, in vitro induction of Foxp3 in response to TGF-β was enhanced in MBD2-/- CD4+ T cells. MBD2-/- mice had a trend towards increased worm burdens when infected with H. polygyrus, suggesting that the difference in proportion of Tregs may limit generation of an effector response. Finally, the ability of HES to directly affect the regulatory arm of the immune response was focussed upon. It was found that HES was able to induce Foxp3 expression in naïve peripheral T cells, and that this was mediated by stimulation of the TGF-β pathway. The TGF-β mimic was of parasite origin as a pan-vertebrate TGF-β antibody was unable to block its effects but sera from H. polygyrus infected animals was competent to do this. Activity of this type was not limited to HES as ES from the ovine helminth Haemonchus contortus was found to have the same property. These data imply that some helminth parasites have evolved mechanisms to support generation of Foxp3+ Tregs, thus favouring the regulatory arm of the immune response and hence their own persistence.

Infections with parasitic helminths are counted as neglected tropical diseases; they infect millions of people worldwide, causing high morbidity and economic loss. Many parasites establish long lasting infections in the host by blocking immune recognition, activation and effector pathways. To allow in depth research on their modes of immune evasion, several mouse models for parasitic helminth infections have been established. Heligmosomoides polygyrus for example is a gastrointestinal nematode of rodents exhibiting a wide spectrum of immunomodulatory effects, mediated in part by soluble molecules released by adult worms in vitro, the excretory/secretory products (HES). HES is a potent inhibitor of dendritic cell (DC) activation by Toll-like receptor (TLR) ligands, completely abolishing LPS induced IL-12 production and reducing the upregulation of cell surface activation markers. As of now, neither the modulatory molecule nor its mechanism of action are known. Here, the effect of HES on TLR ligand induced DC maturation was characterized in considerably more detail compared to previous publications. It could be shown to inhibit DC maturation induced by various TLR ligands, on both protein and mRNA levels. These effects were comparable in both C57BL/6 and BALB/c derived cells; in contrast to this HES differentially affected alternative activation of BMDC from these two mouse strains. Although for most of the experiments GM-CSF differentiated BMDC were used, HES also inhibited LPS induced activation of splenic CD11c+ cells as well as the activation of all three populations described in Flt3-L differentiated BMDC - pDCs, CD11b+ cDCs and CD24+ cDCs. Furthermore, it could be shown here that HES also inhibits LPS induced maturation in human monocyte derived DCs. In the search for the component in HES responsible for its inhibition of TLR ligand induced DC maturation, exosome depleted HES rather than exosomes was inhibitory, and the effect was heat labile. This lead to the conclusion that the modulatory molecule has a protein component which is indispensable for its effect; following this reasoning HES was subjected to fractionation, with subsequent analysis of the fraction protein contents by mass spectrometry. The top nine candidate proteins were expressed recombinantly; however, the recombinants were not able to inhibit LPS induced DC activation. In parallel, experiments to elucidate the mechanism by which HES inhibits TLR ligand induced DC maturation were performed. This led to the conclusion that HES induces changes in the cells that, while not affecting the induction of signalling downstream of TLRs, do impair its maintenance. As a complement to these experiments, the transcriptomes of LPS and LPS+HES treated cells eight hours after LPS stimulation were compared. This revealed that transcripts encoding a number of transcription factors inducing the expression of activation markers after TLR ligation were reduced upon treatment of cells with HES, as were the transcript levels of IRAK2, a kinase necessary for persistent signalling. In addition, HES increased the transcript levels for several factors known to negatively regulate DC maturation, including ATF3. Furthermore, this analysis revealed changes in transcript levels of factors like HIF-1a, indicating an even greater reliance on aerobic glycolysis if cells were treated with HES, in addition to hints at increased ER and oxidative stress. In conclusion, this work narrows down the list of potential DC modulators in HES, gives a first insight into changes in DC metabolism induced by HES and sheds light on the role of a number of signalling pathways with important roles in DC activation as targets of DC inhibition by HES.

(English): Trichobilharzia regenti is a neurotropic parasite of birds from the family Schistosomatidae. Cercariae, the invasive stages of these trematodes actively penetrate the host skin employing excretory- secretory products (ESPs), which contain proteolytic enzymes able to disrupt host tissues and thus reach the successful transmission. The most abundant secreted enzyme responsible for cercarial penetration of the human schistosome S. mansoni is a cercarial elastase. This serine peptidase is well known for the degradation of skin proteins such as elastin, keratin, collagen or laminin. However, the active expression of the orthologue of this enzyme has never been found in the genus Trichobilharzia. For this reason, it was firmly believed, that cercaria of T. regenti uses mainly cysteine peptidases for the invasion of the host, particularly cathepsins, which were repeatedly identified in this life stage. To strengthen this hypothesis, we incubated T. regenti cercariae in the apparatus with the excised duck skin stimulating the release of their glands' content. The collected ESPs were further analysed by shotgun mass-spectrometry and for the first time, the protein form of cercarial elastase was identified. Unfortunately, we failed to produce its active recombinant protein in yeast and bacterial...

In this study, we examined the immunomodulatory effect of excretory/secretory products, crude adult extracts and crude larvae extracts from Hymenolepis diminuta on the intestinal epithelilal cell line from a rat. For determination of the immunomodulation effect of all H. diminuta extracts was used relative gene expression of TNFa, IL-17re and IL-33 from epithelial cells and it was tested using real-time PCR. Our result showed that excretory/secretory products had the strongest antiinflammatory effect on the epithelial cells. We assume that crude adult extracts play an important role in increase of gene expression of IL-33 and also in the immunomodulatory ability of H. diminuta in the host organism.

Giardia intestinalis is a unicellular protozoan parasite responsible for 280 million gastrointestinal infections every year. When colonizing its host, Giardia interacts closely with the small intestine epithelium by attaching to enterocytes and releasing multiple proteins to the extracellular environment. Some of the released proteins have been shown to aid the parasite’s survival in the intestine by disrupting various host defense mechanisms. Here, we attempt to characterize the specific localization of five proteins after their secretion by Giardia. In parallel we aim to produce and identify parasite’s molecules potentially working as triggers of the immune response built during infection. To study the localization of specific secreted proteins during in vitro interactions with differentiated Caco-2 cells, we started by creating transgenic parasites expressing the ADI, EF1α and G3PD proteins with a downstream detectable tag. To identify candidate proteins from Giardia, thought by our lab to be involved in immune system activation, we established a mammalian expression system for the production of recombinant versions of the selected candidate giardial PAMPs. We achieved the expression of the VSP1267 protein, natively present on the parasite’s surface. However, we found that this protein was not secreted after expression, thus complicating its purification and later use in TLR-activation experiments. In the future, we aim to localize the tagged proteins, expressed by the produced transgenic trophozoites, and optimize the mammalian expression system in order to identify candidate immune triggers during giardiasis.

La résistance aux lactones macrocycliques (LM) constitue une préoccupation croissante dans le contrôle des nématodes parasitaires, notamment l'Haemonchus contortus chez les ruminants. Parmi les mécanismes étudiés dans la résistance aux LM chez les nématodes d’importance en santé animale, il y a les pompes ABC, principalement les glycoprotéines-p, connues pour leur rôle dans la détoxification des LM chez les strongles. Il n'existe toutefois aucune étude sur l'extrusion des lipides par les pompes ABC en tant que produits excrétoires/sécrétoires provenant d'H. contortus (Hc-PES). Nous émettons l’hypothèse que les pompes ABC chez H. contortus sont à la fois impliquées dans l’extrusion de LM (contribuant à la résistance aux antihelminthiques) et dans l’efflux de lipides secrétés par le parasite. Notre objectif était de caractériser le rôle des pompes ABC chez H. contortus dans le contexte de la résistance aux LM et de l'extrusion des lipides. L'efficacité de l'ivermectine, un membre de LM, a été évaluée dans 8 fermes étudiées par un test de réduction de la numération des œufs dans les selles (TRNOS). Les niveaux d'expression des pompes ABC ont été évalués dans des isolats de champ d’H. contortus avec des résultats TRNOS faibles (présumé souches résistantes). D’ailleurs, des vers adultes d’H. contortus ont été incubés avec trois inhibiteurs de pompes ABC, dont le Fumitremorgin C, le Kétoconazole et le Mk-571 à concentrations différentes. Les lipides ont été identifiés par CL/SM dans les milieux de culture récupérés à 2 h, à 4 h et à 8 h après l'incubation d’H. contortus dans les groupes contrôle et traités. L'expression des gènes Hco-pgp-2 et Hco-pgp-3 était augmentée chez les isolats de champ d’H. contortus. Nous avons identifié 1045 lipides appartenant à diverses catégories. L'extrusion des lipides en Hc-PES a changé en présence d'inhibiteurs de pompes ABC, en particulier pour les lipides composés de structures correspondant à celles pour le transport par les pompes ABC. Nous avons donc conclu que les pompes ABC chez H. contortus représentent un système de multi-extrusion et sont impliquées dans la sécrétion de lipides avec importance dans l’interaction avec l’hôte, mais aussi dans la résistance aux LM chez le nématode. / Macrocyclic lactones (MLs) resistance is a growing concern in controlling parasitic nematodes, particularly Haemonchus contortus in the ruminants’ industry. ABC transporters are known to participate in translocating various lipophilic molecules, including MLs and lipids. Some ABC transporters, mostly P-glycoproteins are known to be involved in MLs detoxification in parasitic nematodes; but there is no data about extrusion of lipids by ABC transporters as Excretory/Secretory Products in H. contortus (Hc-ESP). We hypothesize that ABC transporters in H. contortus have a dual role participating in the efflux of MLs, thus contributing to anthelmintic resistance, and in the extrusion of lipids out of the parasite. This study aimed to characterize the role of H. contortus ABC transporters in the context of ML resistance and the extrusion of lipids. Ivermectin (a member of MLs) efficacy was evaluated in 8 studied farms by the fecal egg count reduction test (FECRT). The expression levels of ABC transporters were evaluated in field isolates of H. contortus with low FECRT results (suspected of resistance). H. contortus adult worms were incubated with three ABC inhibitors, such as Fumitremorgin C, Ketoconazole and Mk-571 with different concentrations. Lipids were identified by LC/MS in culture media at 2h, 4h and 8h post incubation with H. contortus in control and treated groups. Hco-pgp-2 and Hco-pgp-3 were found upregulated in H. contortus field isolates. We identified 1045 lipid molecules belonging to different categories. Interestingly, the lipid profile in Hc-ESP was altered in the presence of ABC transporter inhibitors, which shows structural features compatible as substrates for nematode transporters’ activity. Therefore, ABC transporters in H. contortus participate in extrusion of lipids and also may help in detoxification of MLs, becoming a multipurpose pumping system involved in ML resistance and secretion of lipids at the interplay with the host and among nematodes.