Introduction: Space is an extreme environment exposing astronauts to microgravity and cosmic radiation resulting in immune dysfunction. To overcome the complex challenges of studying astronauts in space, bed rest studies represent an alternative model simulating microgravity exposure on Earth. We sought to characterize the steady state transcriptome changes in leukocytes isolated from two microgravity models: (1) participants to 60 days of bed rest and (2) astronauts to ~6 months of spaceflight. Methods: The bed rest study recruited twenty healthy men receiving a nutritional supplement or not; the spaceflight study had fourteen male and female astronauts participate. For both studies, ten blood samples were collected over three study phases, leukocytes were isolated, and transcriptomes were quantified using high throughput RNA-sequencing. My pipeline of data analysis applied differential expression (DE) methods and functional enrichment to identify gene expression changes and pathways responding to the altered gravity environments of both bed rest and spaceflight models. Results: Temporal differential expression identified transcriptome modulation reflecting multisystem shifts and immune dysregulation in response to the transitions to and from bed rest (2,415 DE genes) and spaceflight (247 DE genes). Interestingly, later bed rest and in-flight timepoints trended towards stable RNA levels with no differential expression. The bed rest study found the nutritional intervention had no mitigating effects on transcriptome changes (0 DE genes), and the spaceflight study revealed down-regulation in response to spaceflight followed by an opposite up-regulation upon return to Earth. Conclusion: The altered gravity environments of bed rest and spaceflight significantly modulated leukocyte transcriptome compositions revealing immune dysfunction at the molecular level. Future analyses utilizing the higher quality bed rest dataset is required to isolate the effect of microgravity from other space stressors and apply validation experiments to develop gene biomarkers indicative of immune deconditioning.

In Southeast Asia, the family Pangasiidae is important for commercial fisheries and aquaculture. Pangasianodon hypophthalmus (striped catfish) is the most economically important species farmed in Vietnam, with a total export value of 1.7 billion USD in 2012. Intensive aquaculture can lead to problems with major outbreaks of disease and Edwardsiella ictaluri and Aeromonas hydrophila represent two important bacterial pathogens in P. hypophthalmus aquaculture. Immunostimulants have proven to be a very useful food additive for the aquaculture industry, since they can be easily fed to fish to enhance their immune response at times of stress and to improve resistance to disease. The immune system of pangasius catfish has not been fully described, despite the recent growth in aquaculture for this species, and little is known about the effects of immunostimulants on disease resistance. Understanding the immune response is very important in order to evaluate the health status of the fish and assist in control of disease (including prevention) so that production levels by the aquaculture industry can be sustained. The aims of this thesis were to develop and standardise methods to elucidate and measure immune responses in P. hypophthalmus and then to use these with relevant disease models (A. hydrophila and E. ictaluri) and immunomodulators (β-glucans from different sources and at different doses) to determine if bacterial diseases can be controlled, and which functional immune responses and immune genes could be correlated with disease resistance. As a variety of different species from family Pangasiidae are economically important for aquaculture, initial work focused on the characterisation of the immunoglobulin IgM molecule in these species, and anti-P. hypophthalmus IgM mAbs were tested to determine if they cross-reacted between different Pangasiidae species (Chapter 2). Although affinity purification of IgM from the different fish species resulted in a purer preparation ammonium sulphate precipitation (14% w/w), the latter proved faster and easier to perform. The heavy (H) and light (L) chains of IgM from P. hypophthalmus were estimated to be 70-72 kDa and 25-26 kDa, respectively, using SDS-PAGE (12.5%). The L chains of IgM in the other Asian fish species examined were similar in molecular weight to P. hypophthalmus, while the H chains varied (P. gigas and P. larnaudii 76kDa, P. sanitwongsei 69kDa, H. filamentus 73kDa, P. borcoti and H. wyckioides 75kDa, C. bactracus 74kDa, C. macrocephalus 73kDa and C. carpio 70kDa), as did the native IgM molecules. Sedimentation velocity ultracentrifugation was used to determine the molecular weight of the whole IgM molecule from P. hypophthalmus as an alternative to the more commonly used native gels that are run under non-denaturing conditions, although this technique proved more complex. Anti–P. hypophthalmus IgM monoclonal antibodies (mAbs) cross reacted with all of the Pangasiidae species and were successfully applied in an enzyme-linked immunosorbent assay (ELISA) using mAb 23 to measure serum antibody response of P. hypoophthalmus following experimental infection with A. hydrophila by interperitoneal (I.P.) injection in Chapter 3 and E. ictaluri by immersion in Chapter 4. As P. hypophthalmus is a relatively new aquaculture species, there are few reports evaluating its immune response to pathogens. Thus, functional assays were standardised to evaluate both innate and adaptive immune responses of this species and then these assays used to compare immune response following stimulation with live and killed A. hydrophila. (Chapter3). Four treatment groups of 40 fish per group (53.2 ± 14.8g.) consisting of an untreated control group, a group injected I.P. with adjuvant (Montanide ISA 760 VG) only, a group injected with heat-killed A. hydrophila (1 x109 cfu ml-1 mixed with adjuvant), and a group injected with a subclinical dose of live A. hydrophila 2.7 x105 cfu ml-1 were used in the study. Samples were collected 0, 1, 3, 7, 14 and 21 days post injection (d.p.i.) to assess the immune response of fish. The results indicated that challenge with live or/and dead bacteria stimulated the immune response in P. hypophthalmus significantly above control groups with respect to specific antibody titre, lysozyme activity, phagocytosis and plasma peroxidase at 7 or/and 14 d.p.i. Moreover, on 21 d.p.i. total IgM, specific antibody titre and lysozyme activity from both live and dead A. hydrophila challenge groups were significantly different to the control groups. Differential immune responses between live and dead bacterial challenges were also observed as only live A. hydrophila significantly stimulated WBC counts and plasma peroxidase at 3 d.p.i. with the greatest increase in WBC counts noted at 21 d.p.i. and in phagocytosis at 14 d.p.i. By 21 d.p.i. only the macrophages from fish challenged with dead A. hydrophila showed significantly stimulated respiratory burst activity. Immunostimulants are food additives used by the aquaculture industry to enhance the immune response, and β-glucan is now commonly used for this purpose in aquaculture. In Chapter 4 the effect of the prebiotic β-glucan on the immune response and disease resistance of P. hypophthalmus was evaluated. The fish (60.3 ± 11.7 g.) were fed with a basal diet (control) or diets supplemented with fungal derived β-glucan at concentrations of 0.05 %, 0.1 %, or 0.2 % g/kg for four weeks. Fish fed 0.1 % commercial yeast derived β-glucan were also included as a positive control group. Samples were collected from fish on Days 0, 1, 3, 7, 14, 21 and 28. The results showed that fish fed with the highest two levels of fungal derived β-glucan had enhanced immune responses compared to the control group, with respiratory burst activity on all days examined and lysozyme activity on 7 days post feeding (d.p.f.) being significantly elevated (P<0.05) in the group fed with 0.2 % fungal derived β-glucan, while plasma anti-protease activity on 21 d.p.f., natural antibody titre on 3 d.p.f. and complement activity 7 d.p.f. and 14 d.p.i. were significantly enhanced (P<0.05) in the group fed 0.1 % fungal derived β-glucan. The lowest dose of fungal derived β-glucan (0.05 %) appeared insufficient to effectively stimulate the fish’s immune response. WBC count, respiratory burst, lysozyme activity and complement were useful as an early indication of immunostimulation (1 to 7 days). Four weeks after feeding with the different diets, the fish were experimentally infected with E. ictaluri by immersion using 8 x104 cfu ml-1 for 1 h and mortalities were monitored for 14 days. There was a great deal of variation in the level of mortalities within the four replicate tanks for each dietary group. Although the in vivo challenge results showed no statistical differences between the groups fed on the different diets, the highest mortalities were observed in group fed with the control diet and the lowest mortalities were observed in the groups fed with commercial yeast derived β-glucan and 0.2 % fungal derived β glucan. Immune gene expression following stimulation with β-glucan and challenge with E. ictaluri was investigated in Chapter 5.

Early innate response of intestinal epithelial cells is the first line defense against enteric pathogens. Toxoplasma gondii infections acquired naturally via the peroral route, encounter intestinal epithelial cells early post-infection. Although the population structure of T. gondii is known to be highly clonal, clinical strains of T. gondii have been classified into three genotypes based on their virulence. In this study we investigated whether human intestinal epithelial cell immune response to T. gondii is virulence dependent. We demonstrated distinct virulence of the three T. gondii genotype strains evaluated in human intestinal epithelial cells by their capacity to replicate and induce host cell cytotoxicity. The early host innate mechanisms such as activation of signaling pathways and induction of innate effectors were likewise differentially elicited by the three T. gondii strains. Low levels of TLR dependent NF-kB activation and a failure to rapidly up-regulate innate cytokine and chemokine genes was observed after virulent Type I strain infection. In contrast, early innate response to the less virulent Type II strain was rapid, efficient and led to high levels of IL-8 and IL-6 secretion, whereas response to Type III parasites was intermediate. Early expression of b-defensin 2 gene was suppressed specifically by virulent Type I strain and its activation prior to infection in intestinal epithelial cells led to decreased parasite viability. These findings provide evidence for T. gondii strain virulence dependent down-modulation of early human intestinal epithelial cell innate responses and highlight the importance of these cells in host defense against this infection.

Early innate response of intestinal epithelial cells is the first line defense against enteric pathogens. Toxoplasma gondii infections acquired naturally via the peroral route, encounter intestinal epithelial cells early post-infection. Although the population structure of T. gondii is known to be highly clonal, clinical strains of T. gondii have been classified into three genotypes based on their virulence. In this study we investigated whether human intestinal epithelial cell immune response to T. gondii is virulence dependent. We demonstrated distinct virulence of the three T. gondii genotype strains evaluated in human intestinal epithelial cells by their capacity to replicate and induce host cell cytotoxicity. The early host innate mechanisms such as activation of signaling pathways and induction of innate effectors were likewise differentially elicited by the three T. gondii strains. Low levels of TLR dependent NF-kB activation and a failure to rapidly up-regulate innate cytokine and chemokine genes was observed after virulent Type I strain infection. In contrast, early innate response to the less virulent Type II strain was rapid, efficient and led to high levels of IL-8 and IL-6 secretion, whereas response to Type III parasites was intermediate. Early expression of b-defensin 2 gene was suppressed specifically by virulent Type I strain and its activation prior to infection in intestinal epithelial cells led to decreased parasite viability. These findings provide evidence for T. gondii strain virulence dependent down-modulation of early human intestinal epithelial cell innate responses and highlight the importance of these cells in host defense against this infection. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Adaptation of host receptor system to optimal detection of infection-related structures is one of the key evolutionary challenges of immunity in host-pathogen interactions. Toll-like receptors (TLRs) are genetically variable molecules of vertebrate innate immunity that recognise danger signals, e.g. pathogenic molecules. Examination of genetic variation in TLRs may reveal mechanisms of host immunity adaptation to pathogenic pressure at molecular level. Trans-species polymorphism (TSP) is a phenomenon which assumes that several identical alleles or allelic lineages are inherited from ascendant to descendant species and these may be subsequently maintained over a long period of time in a polymorphic state. Whereas in adaptive immune genes the concept of TSP is well understood, little is presently known about TSP in innate immune genes such as TLRs. In this thesis I describe genetic polymorphism in functionally-relevant regions of TLR4 and TLR5 in 192 individuals representing 20 species Paridae family (tits, chickadees and titmice). These two receptors bind mainly bacterial ligands (TLR4 detects lipopolysaccharide and TLR5 detects flagellin), being among the first ones to trigger immune response to bacterial pathogens. To differentiate presumed TSP from gene flow among species, intron sequences of six...