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Background: During deep-space travels, crewmembers face various physical and psychosocial stressors that could alter gut microbiota composition. Since it is well known that intestinal dysbiosis is involved in the onset or exacerbation of several disorders, the aim of this study was to evaluate changes in intestinal microbiota in a ground-based murine model mimicking psychosocial stressors encountered during a long-term space mission.Results: We demonstrate that 3 weeks of exposure to Chronic Unpredictable Mild Stress (CUMS) induce significant change in intracaecal β-diversity characterized by an important increase of the Firmicutes/Bacteroidetes ratio. These stress-induced alterations are associated with a decrease of Porphyromonadaceae, particularly of the genus Barnesiella that is a major member of gut microbiota in mice, but also in human, where it is described as having protective properties.Conclusions: These results raise the question of the impact of stress-induced decrease of beneficial taxa, support recent data obtained with in-flight experimentations or gravity change models, and emphasize the critical need for further studies exploring the impact of spaceflight on intestinal microbiota in order to propose strategies to countermeasure spaceflight-associated dysbiosis and its consequences on health.

International audience; During spaceflight, astronauts face radiations, mechanical, and socio-environmental stressors. To determine the impact of chronic socio-environmental stressors on immunity, we exposed adult male mice to chronic unpredictable mild psychosocial and environmental stressors (CUMS model) for 3 weeks. This duration was chosen to simulate a long flight at the human scale. Our data show that this combination of stressors induces an increase of serum IgA, a reduction of normalized splenic mass and tends to reduce the production of pro-inflammatory cytokines, as previously reported during or after space missions. However, CUMS did not modify major splenic lymphocyte sub-populations and the proliferative responses of splenocytes suggesting that these changes could be due to other factors such as gravity changes. Thus, CUMS, which is an easy to implement model, could contribute to deepen our understanding of some spaceflight-associated immune alterations and could be useful to test countermeasures.

Since mucosal surfaces represent major portals of entry for pathogens, its associated immune system is important to protect the organism. In this paper, we compared at the cellular and molecular levels intestinal leukocyte suspensions with their head kidney (HK) or peripheral blood (PBL) counterparts to highlight characteristics of intestinal immune functions in healthy rainbow trout. These studies show that intestinal phagocytes are less activated by yeast cells but when they are activated they can ingest as many yeast cells as their HK counterparts. A natural cytotoxic activity could be detected which is twice higher in intestinal than in HK leukocyte preparations. This natural cytotoxic activity is correlated with the expression of transcripts encoding the natural killer enhancement factor (NKEF). Intestinal leukocytes did not respond to an in vitro mitogenic stimulation performed under classical culture conditions. And finally, a high expression of CD8α transcripts was observed in gut leukocyte preparations, suggesting that the intestine could contain a high proportion of T cells expressing the αα homodimeric form of CD8. This kind of comparison on nonimmunized fish provides better knowledge on basal immune functions in the intestine to, analyze later on, immune responses induced by an antigenic stimulation.

Spaceflights are known to induce stress and immune dysregulation. Centrifugation, as hindlimb unloading, is a good ground based-model to simulate altered gravity which occurs during space missions. The aim of this study was to investigate the consequences of a long-term exposure to different levels of hypergravity on the stress response and the humoral immunity in a mouse model. For this purpose, adult C57Bl/6J male mice were subjected for 21 days either to control conditions or to 2G or 3G acceleration gravity forces. Corticosterone level and anxiety behavior revealed a stress response which was associated with a decrease of body weight, after 21-day of centrifugation at 3G but not at 2G. Spleen lymphocyte lipopolysaccharide (LPS) responsiveness was diminished by 40% in the 2G group only, whereas a decrease was noted when cells were stimulated with concanavalin A for both 2G and 3G groups (about 25% and 20%, respectively) compared to controls. Pro-inflammatory chemokines (MCP-1 and IP-10) and Th1 cytokines (IFNγ and IL2) were slightly decreased in the 2G group and strongly decreased in the 3G mouse group. Regarding Th2 cytokines (IL4, IL5) no further significant modification was observed, whereas the immunosuppressive cytokine IL10 was slightly increased in the 3G mice. Finally, serum IgG concentration was twice higher whereas IgA concentration was slightly increased (about 30%) and IgM were unchanged in 2G mice compared to controls. No difference was observed in the 3G group with these isotypes. Consequently, functional immune dysregulations and stress responses were dependent of the gravity level.

The aim of this study was to investigate the effects of an early olfactory deprivation on novelty-seeking behavior and lymphoid organs during post-natal development in rats. Therefore, we performed a bilateral nasal obstruction on 8-day-old rats and we studied its effects at 21 days of age. Animals exposed to nasal obstruction presented an increased plasma corticosterone level, an adrenal hypertrophy more marked in females (+68% in females and +29% in males compared to sham group), a reduced growth of olfactory bulbs only in females (-43% compared to sham group) and an impaired novelty-seeking behavior, which seemed also more marked in females. Moreover, thymus specific weight and ConA-induced proliferative activity of thymocytes were diminished after induction of nasal obstruction in females but not in males. At last, spleen specific weight was not affected by nasal obstruction, although mitogen-induced proliferative activity of splenocytes was diminished in both sexes. These data showed that early olfactory deprivation by nasal obstruction was associated with some behavioral, endocrine and immunological alterations, which may have serious consequences for the health and the social status of the individuals.

International audience; Analysis of bacterial DNA from fecal samples of mice is commonly performed in experimental studies. Although DNA extraction is a critical step in various molecular approaches, the efficiency of methods that may be used for DNA extraction from mice fecal samples has never been evaluated. We compared the efficiencies of six widely used commercial kits (MasterPure (TM) Gram Positive DNA Purification Kit, QIAamp (R) DNA Stool Mini Kit; NucliSENS (R) easyMAG (R), ZR Fecal DNA MiniPrep (TM), FastDNA (R) SPIN Kit for Feces and FastDNA (R) SPIN Kit for Soil) and a non-commercial method for DNA isolation from mice feces and cecal contents. DNA quantity and quality were assessed by fluorometry, spectrophotometry, gel electrophoresis and qPCR. Cell lysis efficiencies were evaluated by qPCR targeting three relevant bacteria in spiked specimens. For both feces and intestinal contents, the most efficient extraction method was the FastDNA (R) SPIN Kit for Soil.

International audience; Extended space missions are known to induce stress and immune dysregulation. Hindlimb unloading is a ground-based model used to reproduce most spaceflight conditions. The aim of this study was to better characterize the consequences of prolonged exposure to hindlimb unloading on murine splenic lymphocyte sub-populations. To ensure that the observed changes were not due to tail restraint but to the antiorthostatic position, three groups of mice were used: control (C), orthostatic restrained (R) and hindlimb unloaded (HU). After 21 days of exposure, no difference in serum corticosterone levels nor in thymus and spleen weights were observed between HU mice and their counterparts, revealing a low state of stress. Interestingly, flow cytometric analyses showed that B cells were drastically reduced in HU mouse spleens by 59% and, while the T cells number did not change, the Th/Tc ratio was decreased. Finally, the use of a fluorescent dye monitoring lymphoproliferation demonstrated that lymphocyte response to mitogen was reduced in Th and Tc populations and to a greater extent in B cells. Thus, we showed for the first time that, even if restraint has its own effects on the animals and their splenic lymphocytes, the prolonged antiorthostatic position leads, despite the absence of stress, to an inversion of the B/T ratio in the spleen. Furthermore, the lymphoproliferative response was impaired with a strong impact on B cells. Altogether, these results suggest that B cells are more affected by hindlimb unloading than T cells which may explain the high susceptibility to pathogens, such as gram-negative bacteria, described in animal models and astronauts.

Review describes hazards present as humans explore space and focuses on the effects of spaceflight on immunity. This year, we celebrate the 40th birthday of the first landing of humans on the moon. By 2020, astronauts should return to the lunar surface and establish an outpost there that will provide a technical basis for future manned missions to Mars. This paper summarizes major constraints associated with a trip to Mars, presents immunological hazards associated with this type of mission, and shows that our current understanding of the immunosuppressive effects of spaceflight is limited. Weakening of the immune system associated with spaceflight is therefore an area that should be considered more thoroughly before we undertake prolonged space voyages.

We investigated the effect of exogenously administered 20-hydroxyecdysone (20E) on sexual behavior and on contact sex signals of adult females of the spider Tegenaria atrica. In untreated or control females cannibalism occurred in 36–47% of encounters, whereas no cannibalism at all was observed with 20E-treated females. The frequency of sexual receptivity was low (42%) in untreated or control females and high (100%) in 20E-treated females. Variability of female sexual behavior could be related to quantitative changes in web and cuticular compounds. We showed that 20E altered the production of polar contact sex pheromones on web and cuticle: methylesters and fatty acids are produced in higher amounts in 20E-treated females. Bioassays demonstrated clearly that methylesters and fatty acids of webs and cuticles were involved in attraction of males. Moreover, bioassays with synthetic substances showed that four fatty acids (myristic, pentadecanoic, hexadecanoic, and oleic acids) and one methylester (methyl stearate) are particularly effective in eliciting sexual behavior in males. In T. atrica, 20E inhibits cannibalism during sexual activity and changes sex pheromone production.

Spaceflight weaken the immune system. The aims of this thesis were to determine the effects of exposure to simulated microgravity, hypergravity or chronic ultra-mild stress on murine humoral immunity, using ground-based models. We were able to show that the anti-orthostatic suspension, that mimics some of the effects of microgravity, caused a 59% decrease of the number of splenic B cells and an inversion of the ratio between B and T lymphocytes. A decrease in B lymphopoiesis, as evidenced by the decrease of lymphoid progenitors and pre-B cells is likely the cause. Furthermore, we showed that the anti-orthostatic position, in the absence of stress, leads to a decreased in vitro response of B cells to LPS, more important than the one of T cells to ConA, as did hypergravity exposure. To determine the mechanisms responsible of the decreased response of B cells from hypergravity mice, transcripts encoding genes involved in the TLR-4 signaling pathway, the LPS receptor on B cells, have been quantified. The expression of several genes of the MyD88-dependent pathway was increased after 21 days of hypergravity, but no change was observed in B cells stimulated with LPS. Finally, the contribution of chronic ultra-mild stresses was evaluated. The proportions of splenic lymphocytes were not affected by these stresses. However, the levels of serum IgA were increased and those of Th1 pro-inflammatory cytokines were decreased, as in astronauts. Thus, these stresses do not fully explain the negative impact of spaceflight conditions on humoral immunity. These researches are important to identify the cause(s) of spaceflight-associated impaired immunity in order to test/develop effective countermeasures to strengthen the immune system of astronauts, but also of stressed or elderly people, Les vols spatiaux affaiblissent le système immunitaire. Les objectifs de cette thèse étaient de déterminer, à l’aide de modèles terrestres, les conséquences d’une exposition à la micropesanteur simulée, l’hypergravité ou des stress chroniques ultra légers sur l’immunité humorale murine. Nous avons ainsi montré que la suspension anti-orthostatique, qui mime certains effets de la micropesanteur, induit une diminution de 59% du nombre de lymphocytes B spléniques et une inversion du rapport entre lymphocytes B et T. L’affaiblissement de la lymphopoïèse B mis en évidence par la diminution du nombre de progéniteurs lymphoïdes et des cellules pré-B en est probablement la cause. De plus, la position anti-orthostatique entraîne, en l’absence de stress, une diminution de la réponse in vitro des lymphocytes B au LPS, plus importante que celle des lymphocytes T à la ConA, comme en hypergravité. Afin de déterminer les mécanismes responsables de la diminution de la réponse des lymphocytes B chez les souris hypergravitaires, les transcrits de gènes impliqués dans la voie TLR-4, le récepteur du LPS, ont été quantifiés. L’expression de plusieurs gènes de la voie MyD88-dépendante est augmentée après 21 jours d’hypergravité, mais aucune modification n’a été observée dans les lymphocytes B stimulés avec du LPS. Enfin, la contribution de stress chroniques ultra légers a été évaluée. Les proportions de lymphocytes spléniques ne sont pas affectées par ces stress. Par contre, le taux d’IgA sériques s’est révélé augmenté et une baisse des cytokines pro-inflammatoires de type Th1 a été observée comme chez les astronautes. Ainsi, ces stress n’expliquent pas complètement l’impact négatif des vols spatiaux sur l’immunité humorale. Ces recherches sont importantes pour identifier la/les cause(s) de l’altération de l’immunité humorale en vol, afin de tester/développer des contremesures efficaces pour renforcer le système immunitaire des astronautes, mais également de personnes stressées ou âgées

The impact of nutrition on rainbow trout intestinal immunity, a farmed fish with high economic value, remains unclear. Consequently, the objectives of this thesis were to better characterize the intestinal immune system of that fish and to determine if it is possible to modulate its intestinal immune response by dietary free nucleotides. Our results show that intestinal phagocytes are less activated by yeast cells but when they are activated they can ingest as many yeast cells as their head kidney (HK) counterparts. We noted that the natural cytotoxic activity of intestinal leukocytes is twice higher than the one of HK leukocytes. This natural cytotoxic activity is correlated with an increase of transcripts encoding the natural killer enhancement factor (NKEF). Intestinal leukocytes did not respond to an in vitro mitogenic stimulation. This lack of response is not due to apoptosis. We also observed a high expression of CD8a and CD3 transcripts in gut leukocytes, suggesting that the intestine could contain a high proportion of T cells expressing the aa homodimeric form of CD8. Finally, we observed that dietary free nucleotides stimulate the spontaneous proliferation and the phagocytic activity of intestinal leucocytes in vitro. However, they did not modulate natural cytotoxicity activity nor did they affect the amounts of transcripts encoding specific markers of T and B lymphocytes and inflammatory cytokines. In the future, it will be interesting to repeat these experiments using infected fish in order to study the effect of nucleotides on the inflammatory and specific immune responses, L'impact de la nutrition sur l'immunité intestinale de la truite arc-en-ciel est encore mal connu. C'est pourquoi cette thèse avait pour objectifs de mieux caractériser son système immunitaire intestinal et d'évaluer les possibilités de modulation de la réponse immune intestinale par l'ajout de nucléotides libres dans son alimentation. Nos résultats indiquent que les phagocytes intestinaux présentent une activité de phagocytose plus faible que ceux du rein antérieur. La cytotoxicité naturelle mesurée au niveau intestinal est deux fois plus élevée que celle du rein antérieur et cette observation est corrélée à une augmentation du transcrit codant NKEF (Natural Killer Enhancement Factor). Nous avons également montré que les lymphocytes intestinaux ne répondent pas à une stimulation mitogénique in vitro et que ceci n'est pas due à l'apoptose des cellules. Une forte expression des transcrits codant CD8a et CD3 a été détectée dans les leucocytes intestinaux, ce qui suggère une importante proportion de lymphocytes T exprimant la forme homodimérique aa de CD8 dans ce tissu. Enfin, nous avons montré que l'ajout de nucléotides libres à l'alimentation de truites saines stimule la prolifération spontanée ainsi que la phagocytose des leucocytes intestinaux in vitro. Par contre, aucune modulation de la cytotoxicité naturelle ou de l'expression des transcrits codant les marqueurs spécifiques des lymphocytes T et B et les cytokines inflammatoires n'a été observée. Il serait à présent intéressant de renouveler ces essais en utilisant des poissons infectés afin de pouvoir observer l'effet des nucléotides sur la réponse inflammatoire et sur la réponse spécifique