Browsing by Subject "Immunsystem"

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Diurnal and photoperiodic effects on the immune system and glucocorticoid signaling in domestic pigs
(2019) Engert, Larissa; Stefanski, Volker
Physiology and behavior of humans and animals display pronounced diurnal and seasonal rhythmic variations. Diurnal rhythms are controlled by daylight and seasonal rhythms are adjusted by the photoperiod, i.e., the relative span of light per day. Modern human life and housing conditions of livestock are often directed against natural daylight conditions and thus, may entail circadian disruption causing misalignment between the central circadian pacemaker and peripheral tissues. Thereby, glucocorticoids are regarded as a main link between these compartments. Circadian disruption might be detrimental for health, in particular affecting immune function, which was mainly investigated in humans and nocturnal rodents but underlying mechanisms are not clearly defined yet. Therefore, the main objective of the present thesis was to investigate diurnal and photoperiodic effects on the immune system and glucocorticoid signaling as well as potential underlying endocrine, behavioral, and molecular mechanisms of these effects in domestic pigs. Adult male castrated pigs, held under specific lighting schedules, were surgically catheterized to enable blood collection without disturbance of the animals. Initially, domestic pigs were held under standard 12L:12D-lighting conditions and blood samples were taken every 2 hours over periods of up to 50 hours. Cosinor analyses revealed pronounced diurnal rhythmicity in peripheral leukocyte numbers of various immune cell populations. These rhythms were mainly comparable to results in humans and nocturnal rodents in relation to their respective rest-activity cycles, with the exception of porcine neutrophils differing from both species. Moreover, the investigated diurnal rhythms in activity behavior and plasma cortisol concentration confirmed that domestic pigs under the applied experimental conditions are diurnally active like humans. Linear mixed model analyses revealed associations of immune cell counts with plasma cortisol concentration, which also resembles results from humans and rodents. Subsequently, photoperiodic effects on diurnal rhythms in peripheral immune cell numbers were investigated for the first time in any species. Domestic pigs were held either under long day conditions (LD) or under short day conditions (SD) and were sampled every 2 hours over periods of 50 hours. Distinct photoperiodic differences in relative amplitudes and peak times of cell counts in various porcine leukocyte types were found, whereas mesor values did not differ. Moreover, photoperiodic effects on diurnal rhythms in plasma cortisol concentrations and activity behavior were found, which is in agreement with human and primate studies. Generalized linear mixed model analyses again revealed associations of leukocyte counts with plasma cortisol concentration and with activity behavior as well. In summary, the results imply stronger rhythmicity of peripheral immune cell numbers in general under SD than under LD. Common intrinsic mechanisms seem to regulate diurnal rhythms in peripheral leukocyte numbers in most immune cell types in domestic pigs, except for neutrophils again. Finally, to investigate potential molecular differences in diurnal regulation between different immune cell types, glucocorticoid receptor (GR) number and affinity were examined in peripheral blood mononuclear cells (PBMC) and granulocytes of domestic pigs. Thereby, a greater number of GR sites per cell and a higher GR binding affinity in PBMC compared to granulocytes were found, pointing to differences in the molecular mechanisms of glucocorticoid signaling between leukocyte populations. The results of the present thesis project are subsequently discussed in regard to specific implications for immune function and health as well as animal husbandry and welfare. Moreover, a methodological assessment of the approaches used within the thesis project was carried out and finally, suggestions for future research directions were given. In conclusion, the present thesis revealed for the first time diurnal and photoperiodic effects on the immune system as well as glucocorticoid signaling in domestic pigs and uncovered potential underlying mechanisms of these effects. Hence, an additional diurnally active model species in chronoimmunology research was established. Moreover, conducting chronoimmunology research in the porcine species represents an innovative approach in agricultural science and provides entirely new opportunities to improve animal health and welfare. Thereby, future studies might investigate diurnal differences in immune function, clarify the role of different zeitgebers on immune rhythms, and assess potential consequences of stressor exposure at different times of the day.
Impact of dietary phosphorus and fermentable substrates on the immune system and the intestinal microbiota of the pig
(2016) Heyer, Charlotte Maria Elisabeth; Stefanski, Volker
Phosphorus (P) represents a crucial input for agriculture and food industries as a mineral present in ingredients used for livestock feeding as well as in mineral fertilisers. In the current systems, P is primarily derived from the finite mined phosphate rock resource. Thus, a critical challenge of global P scarcity is directly linked to future food security and sustainable resource management, especially in the European Union which is dependent on raw P from outside Europe. Apart from other future activities in animal nutrition, new dietary formulations of livestock diets emerged as a potential approach to increase the digestibility of plant P, phytate (myo inositol 1,2,3,4,5,6 hexakisphosphate, InsP6), and to reduce the supplementation with mineral phosphate. In non-ruminant animals, such as the pig, InsP6 hydrolysis is incomplete, as the small intestine lacks sufficient enzymes such as endogenous mucosal phytase and phosphatase. As a consequence, there is rising scientific interest to improve the understanding of InsP6 degradation in the digestive tract as well as the effects on nutritional factors and finally animal performance and health. The aim of the present thesis was to investigate the impact of dietary P, InsP6 and InsP6 hydrolysis products in combination with different fermentable substances (protein, carbohydrate) on the porcine immune system, the intestinal microbiota and animal health. First, a comprehensive literature overview describes the impact of P on the immune system and the microbiota along the gastrointestinal tract (GIT), including potential effects on host health with special focus on the pig. Secondly, an in vivo study with growing pigs was conducted to examine the effects of diets with varying mineral calcium-phosphorus (CaP) levels as well as different fermentable substrates on intestinal CaP concentration, InsP6 hydrolysis, the intestinal microbial ecosystem, and the peripheral and gut-associated immune system. In 2 consecutive experiments, 31 growing pigs (55 ± 4 kg) were allotted to a 2 × 2 factorial arrangement with 4 treatment groups, fed either a corn-soybean meal or a corn-pea based diet, each with 2 different CaP levels (low, 66% of the CaP requirement; high, 120% of the CaP requirement) supplemented with monocalcium phosphate and calcium carbonate. After 3 weeks of adaptation to the diets, all pigs were immunized twice with keyhole limpet hemocyanin (KLH). Blood and faeces samples were taken. After slaughtering, immunological tissue (jejunal, ileal mesenteric lymph nodes, spleen) as well as jejunal, ileal, caecal and colonic digesta were taken. Faecal and digesta samples were examined for P, Ca, inositol phosphate (InsP) isomers and for the marker titanium dioxide. The number of different leukocyte subpopulations analysed by flow cytometry, mitogen-induced lymphocytes proliferation in vitro were assessed. In addition, concentrations of plasma anti KLH IgM and plasma anti-KLH IgG analysed by ELISA and haematological parameters analysed by an automated hematology system have been measured in blood and tissue samples. In digesta samples, bacterial 16S rRNA gene copy numbers were determined by quantitative real-time PCR. The concentration of short chain fatty acids (SCFA) and ammonia was assessed. In addition, the use of terminal restriction fragment length polymorphism has been proven to characterize the structure of porcine gut microbiota. Results of the current study demonstrated that CaP and fermentable substrates had a distinct effect on the peripheral and gut-associated immune system, as well as on microbial composition and activity in growing pigs. High dietary CaP concentrations and the corn-pea diets increased P net absorption. Almost no InsP6 degradation could be observed in the GIT, and mainly myo inositol pentakisphosphate (InsP5) isomers were measured in jejunal, caecal digesta and faecal samples. In particular, the high CaP diets showed higher InsP6 and InsP5 concentrations, indicating a reduction of the initial steps of P release from InsP6 and a further breakdown of InsP5 isomers. The low CaP content might cause an impaired first line of defence and activation of the cellular and humoral adaptive immune response. As an example, the high CaP content affected the outcome of the adaptive immune response including a higher number of antigen experienced T-helper cells in the blood as well as higher plasma anti-KLH IgG concentrations. The reactivity of blood and mesenteric lymph node lymphocytes to Concanavalin A in these pigs was impaired, indicating modulating effects of other origin such as migration patterns or activity of antigen-presenting cells. Since results of the present study suggest contradictory effects of CaP level on immune cell numbers and lymphocyte reactivity in vitro and in vivo, further studies are needed to determine effects on cell signalling such as cytokine production profiles. Moreover, the high CaP content and the soybean meal diets increased the number of butyrate-producing bacteria, such as Eubacterium rectale and Roseburia spp. and increased the concentration of various SCFA in the small and large intestine, thereby contributing to improve gut health. Potentially harmful bacteria, such as Enterobacteriaceae and Bacteroides Prevotella Porphyromonas, were increased by the low CaP level and pea diets, indicating a less healthy microbiota. Results demonstrated that both, CaP supply and the amount of fermentable substrates, may beneficially affect gut health due to modulations of the composition and activity of the intestinal microbiota. Further studies should evaluate the impact of CaP on specific pathogenic bacteria known to produce toxic products creating a direct link to the immune system and animal health. Although most parameters of the present study indicate a positive effect of the high CaP diet, not all values showed a consistent effect on animal health, such as immune cell numbers and lymphocyte proliferation in vitro. In conclusion, variations in P availability and the formation of individual InsPs have to be considered when formulating diets in support of a stable intestinal microbial ecosystem and immune functions of the host.
Impact of housing environment and rearing condition on the immune system and welfare of laying hens
(2021) Hofmann, Tanja Melanie; Stefanski, Volker
In recent years, consumers’ interest in modern farm animal husbandry and, above all, criticism of intensive production systems that can impair animal welfare, have increased sharply. From hatch, chickens are confronted by a wide range of environmental and social stressors that may confound homeostasis and impair their health and welfare. Not being able to deal with the environment can lead to an activation of the stress system, resulting in a release of neuroendocrine signals like glucocorticoids. These have the potential to modify the immune system and alter species-specific behavior with possible detrimental impacts on animals’ health and welfare. In this context, early-life conditions play a special role as they were shown to have long-term consequences for physiology and behavior in the later life. High stocking densities which activate the hypothalamic-pituitary-adrenal-axis are said to be one of the largest welfare concerns in the poultry industry. Previous research has primarily focused on the effects of stocking density on broilers and adult laying hens, but few studies have focused explicitly on layer pullets, and none of them investigated long-term effects on the immune system. The understanding of short- and long-term consequences of environmental conditions during rearing on the immune system and behavior of layer pullets is essential for shaping an environment that avoids allostatic overload, thus promoting animal health and welfare. This doctoral thesis aimed to identify and evaluate potential immunomodulating factors in the housing environment of chickens. In order to achieve this, it summarizes and reviews important aspects of housing environments for the immune system in chickens. Furthermore, an existing flow cytometric method for the enumeration of leukocytes and discrimination of lymphocyte subsets in blood was adapted to lymphatic tissues. Moreover, short- and longterm consequences of stocking density during rearing on the immune system, stress hormone level and behavior in layer pullets were evaluated. The results are described in detail in three peer-reviewed manuscripts published in international journals. The literature analysis identified several housing conditions as immunomodulating factors. Light management was marked as an effective tool to modulate the immune response in chickens, as long light phases were demonstrated to lower adaptive cellular and humoral immune responses while certain light colors, especially of shorter wavelengths, promote some immune functions. Moreover, particularly high ammonia and hydrogen sulfide concentrations pose a threat to chickens’ health by dampening the adaptive immune response and promoting inflammation. However, the interaction between housing environment and management is complex. Depending on the type of housing form, chickens are confronted with different social and environmental challenges, which in turn can have an impact on the immune and stress system. An understanding of immunosuppressive risk factors is essential for successful poultry management aiming to optimize health, welfare and economic efficiency. To broaden the assessment of immune status for scientific purposes, new staining- and gating strategies for avian lymphatic tissues were adapted to an existing flow cytometric method for blood. The adaptation process showed that the application of already established protocols to other tissues is possible, but has to be examined carefully as tissue-specific effects or cell-preparative procedures might lead to differences in possible combinations of antibody-fluorochrome conjugates. The results of the experimental study within this doctoral thesis demonstrate for the first time that stocking density during rearing has not only short- but also long-term impacts on the immune system in laying hens. Pullets reared at high stocking densities showed stress-related alterations in immune cell numbers and higher incidences of feather pecking that manifest in the laying period. This highlights the significance of early-life conditions for immunocompetence throughout the whole production cycle. Of particular interest is that the number of gamma-delta T cells was lower in the high-density group, which has not been investigated in any stress-related studies with chickens before. The present doctoral thesis provides a significant contribution to the improvement of health and welfare in poultry farming and forms a basis for further research about long-lasting consequences of rearing conditions on the immune system, especially in laying hens.
Stress hormone-induced immunomodulation and interplay between immune cells and bacteria in response to stress hormones in domestic pigs
(2020) Reiske, Lena; Stefanski, Volker
The two main endocrine systems involved in the regulation of stress reactions are the HPA axis, leading to the synthesis of glucocorticoids like cortisol or corticosterone, and the SAM axis, whose activation is associated with the release of the catecholamines adrenaline and noradrenaline. These stress hormones modulate the function of the immune system. Although pigs in modern husbandry systems face many stressors, the consequences of elevated plasma stress hormone levels on porcine immune cell numbers and functionality are insufficiently resolved. While some research on glucocorticoid effects has been conducted, data on many parameters are missing and catecholamines have not been studied systematically in the pig, yet. It is known that stress can negatively affect pigs’ resistance to infections like salmonellosis, but the underlying mechanisms are still subject to intense research efforts, with new perspectives arising since the discovery of interkingdom-signalling and microbial catecholamine perception. The aim of this thesis was to determine the effects of cortisol, adrenaline and noradrenaline on porcine immune cell functionality and the blood numbers of different leukocyte subsets. Furthermore, the interplay of immune cells and Salmonella Typhimurium under the influence of catecholamines was investigated. Adult male castrated pigs were surgically equipped with indwelling catheters to enable stress-free blood collection and intravenous application of hormones. In an initial experiment, the effects of in vitro stress hormone treatment on lymphocyte proliferation and the production of the proinflammatory cytokine TNFa were described. Cortisol reduced both proliferation and number of TNFa producers. Both catecholamines caused an increased lymphocyte proliferation at low concentrations whereas noradrenaline drastically decreased proliferation at high concentrations. While noradrenaline had no impact on TNFa producers, they were reduced in gd T cells and monocytes upon adrenaline addition. Overall, the effects were comparable to humans in terms of direction and dose but there were some disparities regarding adrenaline. In the second part of the project, the impact of in vivo stress hormone administration on immune cell numbers and functionality was examined by infusion for 48h. Cortisol and noradrenaline led to a decreased lymphocyte proliferation but to a variable extent and all three hormones promoted phagocytic function of innate immune cells. Cortisol caused a marked increase of neutrophil numbers while almost all other cell types declined strongly. For most cell types, noradrenaline exerted similar effects but solely after 2h whereas cortisol-induced alterations lasted the whole treatment period. Adrenaline effects were mostly reduced to CD8- T cells, which were reduced at first but increased after 24h. A sharp peak in NK cell numbers after 2h adrenaline infusion is particularly noteworthy and resembles findings from rodent and human studies. Overall, both hormone groups led to a shift from adaptive to innate immunity, underpinning the picture of a promotion of fast and unspecific defence systems to respond to threats in stressful situations. In a third study, S. Typhimurium was grown in the presence of catecholamines to determine the effects of supernatants from these cultures on porcine immune cell function. Both lymphocyte proliferation and TNFα production were hampered substantially, as opposed to the findings on catecholamine effects in the first experiment. It was demonstrated that these effects were not caused by catecholamines or their oxidation products and the formation of a so-far unknown immunosuppressive substance by catecholamine-primed bacteria was assumed. The results contribute to a better understanding of the increased susceptibility to infection in stressed animals and reveal a new dimension of cross-species communication. Finally, the results of the present thesis were discussed regarding their comparability to studies in humans and rodents and previous stress experiments in pigs. Furthermore, the effects of acute and chronic stress as well as different coping styles that are characterised by a SAM or HPA predominance on animal welfare and pig health were discussed, based on the endocrine mechanisms investigated in the present thesis. Possible implications of enhanced glucocorticoid and catecholamine levels for practical pig husbandry were given. Lastly, suggestions for future research to further elucidate the impact of stress hormones on the porcine immune system and the interplay with pathogenic bacteria were made.