Immunology defines the study of all properties of the immune system including its structure and its function, the bodily distinction between self and nonself, innate and acquired immunity, and immunization. For the host defense in health and disease, it investigates especially the relationship between invading pathogens and the triggered immune responses of the organism. Immune effector mechanisms are altered in space by the unique conditions of this perilous environment (radiation, confinement, μG) together with stress hormone-related modifications. To understand the changes that occur during short- and long-term spaceflight and to counteract appropriately to prevent disease, in-flight immune monitoring will become pivotal.
General Principles of Human Host Defense
During everyday life, a human being encounters a multitude of microorganisms and potential pathogens. Disease, however, will manifest only rarely. An effective immune system maintains health by recognition and elimination of infectious agents, by building a memory for a fast immune response in case of a future re-encounter, as well as by autoregulation and self-tolerance to prevent from autoimmune aggression. The immune system consists of physical and chemical barriers, e.g., the skin, released signaling molecules, the chemokines or cytokines, and various types of circulating and resident cells, e.g., granulocytes and lymphocytes. Programmed first-line and more elaborate second-line effector mechanisms protect the body from becoming infected. The initial immune response system, called innate immunity, is capable to eliminate germs often without the outbreak of disease by directly recognizing a defined number of common pathogen features and to react hereon quickly and with a battery of dense tools. The second line that is initiated mostly within hours and in coordination with the innate immune responses is referred to as adaptive immunity. It represents a more advanced, highly specific and gradually regulated response and is able to reshape and newly design defense agents from scratch according to the needs.
Leukocytes: Cellular Sentinels of Immunity
Both innate and adaptive immunity rely on white blood cells (leukocytes) that derive from either common lymphoid or myeloid progenitor cells in the thymus (childhood) and in bone marrow. During the differentiation process, leukocytes develop highly specific features and become able to detect typical recognition patterns. Mature leukocytes may reside in specialized form within tissues and circulate in the bloodstream or the lymphatic system. From the common lymphoid progenitor, natural killer (NK) cells, B and T cells, and the smaller part of the dendritic cells develop in primary lymphoid organs bone marrow (B cells) and thymus (T cells). Maturation and activation occurs in secondary lymphoid organs (regional lymph nodes). The myeloid lineage gives rise to oxygen-carrying erythrocytes and platelets needed for clot formation as well as to leukocytes of the innate immune system: granulocytes, dendritic cells, monocytes/macrophages, and mast cells. Their differentiation can take place in the bone marrow, blood, and lymph nodes or in tissue, depending on the type of cell (Alberts 2014).
The evolutionary ancient and well-preserved innate immune system is constitutively active and exerts an immediate, nonspecific immune reaction to invading pathogens directly at the anatomical border to the environment (the skin, gut, lungs, eyes, nose, and oral cavity). It ranges from mechanical (epithelial tight junctions, tears, longitudinal air flow) to chemical (low pH, pulmonary surfactant) and microbial (normally inhabiting microbiota in the gut) barriers. Preformed antimicrobial enzymes and proteins (lysozyme, defensins), e.g., in saliva or skin, are capable of direct disintegration of the bacterial cell wall (Murphy 2012). The complement system, a very effective group of plasma proteins, tags pathogens (opsonization) for direct recognition and elimination by cells capable to eliminate pathogens by phagocytosis (phagocytes, e.g., macrophages). These can be tissue-specific residents, e.g., tissue macrophages in the lung, liver, or intestines, or circulating as precursors (monocytes) that get chemically attracted and migrate to the area of interest where they differentiate. All phagocytes are capable to detect distinct pathogen-associated molecular patterns (PAMPs) of most bacterial, viral, and fungal pathogens, not expressed on healthy host cells, via their pattern recognition receptors, e.g., the toll-like receptors (TLRs), which mediate a viral or bacterial inflammatory response as necessary (Murphy 2012). Once activated, macrophages create an inflammatory state by releasing small proteins (~25 kD) called cytokines and chemokines such as tumor necrosis factor-α (TNF-α), which lead to the activation of the vessel endothelium. Vessel permeability is enhanced and leads to local edema, which furthers leukocyte recruitment to the site of infection. Granulocytes, also called polymorphonuclear leukocytes (PMNLs) due to their irregularly shaped nuclei, are also phagocytes. PMNLs store different types of antimicrobial proteins inside their cytoplasmic granules and are divided into neutrophils, eosinophils, and basophils according to their staining patterns. Neutrophils own the strongest phagocytic activity and are well known for the respiratory burst reaction, where they produce oxygen-free radicals for the elimination of pathogens. Their G-protein coupled fMet-Leu-Phe (fMLP) receptor has high affinity for bacterial proteins that typically start with N-formylmethionine (fMet) residues. Once activated, hydrolytic enzymes and reactive oxygen species (ROS) such as superoxide (O2−), H2O2, and many other highly toxic molecules are generated subsequently. These reactive compounds can cause considerable damage not only to engulfed pathogens inside the cell but also to the tissue if they enter the extracellular space. Neutrophils and macrophages are capable to scavenge microbes also independently without opsonization. Dendritic cells, another group of phagocytes, are disintegrating microbes and presenting their foreign proteins on the surface in order to activate T cells, thus bridging the gap and signaling information between innate and adaptive immunity (Murphy 2012; Alberts 2014). Other leukocytes involved in innate immunity are NK cells. Cytokines like interferon (IFN)-α or IFN-ß that are released by macrophages or dendritic cells can activate NK cells. They express invariant receptors on their surface and carry cytotoxic granules containing the pore-forming protein perforin, which enables the deposition of proteases inside the target cell that induces programmed cell death (Murphy 2012; Alberts 2014).
The Adaptive Immune System
The most evolved, “highest” level of host defense has only been detected in vertebrates and is executed by the cells and functions of the adaptive immune system. It comprises of B and T lymphocytes that carry an individual antigen receptor (B- or T-cell receptor, respectively) on their surface. Due to genetic rearrangement of variable protein structures at the receptor gene segments and clonal selection of the needed specific type of receptor, a remarkably diverse collection of lymphocytes are on hand for recruitment. This delayed effector mechanism is highly specific, based on antibody-mediated (humoral) and cellular-mediated effector mechanisms, and will eventually generate an immunologic memory (circulating memory B and T cells), which enables a more rapid and stronger immune response when the same pathogen is encountered again (Murphy 2012). An antigen is any substance that triggers a response from the adaptive immune system. At the site of infection, tissue-based dendritic cells pick up antigens, degrade them internally, and present them after processing on their surface, therefore called antigen-presenting cells (APCs), to T lymphocytes in regional lymph nodes. The presented antigen binds to the T-cell receptor (TCR) and promotes activation and differentiation to fully functional effector T cells that enter the circulation equipped with a uniquely specific, membrane-bound antigen receptor for the ongoing infection. APCs present antigens via different receptors called major histocompatibility complex (MHC) class II. Additionally, T cells express co-receptors (e.g., CD4, CD8), which are essential for activation and recognition of MHCs together with the TCR (CD3). APCs activate CD4+ helper T cells via MHC II that regulate adaptive immunity via secretion of cytokines and paracrine activation of neighboring cells. MHC class I activates CD8+ cytotoxic T cells that in turn directly eliminate the target cell in a similar way like NK cells. Most eukaryotic cells can express MHC class I and present antigens; however, only professional APCs can stimulate a distinct CD4+ T helper cell response. There are several subtypes of CD4+ T helper cells (Th) that act on different pathogens. The Th1 response mostly targets intracellular pathogens and is characterized by the release of IFN-gamma-stimulating opsonization of microorganisms by macrophages and B cells. A Th2 response against extracellular pathogens and helminths involves cytokines interleukin (IL)-4 and interleukin-5. Th17 cells are a special T-cell subset that involves IL-17 signaling and are particularly known to be associated with wound healing and autoimmune diseases. CD4+ regulatory T (Treg) cells modulate inflammation via suppressive cytokines as the transforming growth factor-ß (TGF-ß) and IL-10, hence preventing an overshoot immune response and autoimmunity. Although B cells can detect unprocessed antigens directly via their receptors, they often require co-stimulation by T cells. B cells as effector cells or in their short-lived active form (plasma cells) show large amounts of rough endoplasmic reticulum for the production of different classes of antibodies called immunoglobulins (Ig) that can be implemented into the membrane as B-cell receptor (BCR) and bind to the respective antigens. A standing memory of the previously acquired immunity against a pathogen, stored as soluble antibodies and preserved in memory cells, prevents from recurrent infections and is the mechanism that vaccines are based on (Murphy 2012; Alberts 2014).
The Effects of Space Travel on the Human Immune System
Alterations in the immune system of astronauts have been observed and investigated since the late 1960s during the Apollo era. During pre- or postflight, 50–60% of the crewmembers reported symptoms of an infectious illness. This overwhelming incidence of disease led to the implementation of the NASA Health Stabilization Program (HSP) in 1971 prior to Apollo 14 (Pits 1985; Crucian and Choukèr 2012; Crucian 2015). The limitation of public contact prior to spaceflight reduced the incidence of reported diseases in subsequent Apollo missions. Albeit all efforts are undertaken to further reduce risks for infection (selection of candidates for flight, improvement of food quality, etc.) and with the HSP ongoing, the incidence of infectious diseases continued to be high in the direct pre- and postflight setting (Barratt and Pool 2008; Crucian 2015). Experimental proof together with clinical reports from shuttle, MIR, and space station missions has established the knowledge that spaceflight strongly affects the immune system (Busby 1968; Barrat and Pool 2008; Feuerecker 2012; Crucian 2015; Yi et al. 2016). These observations arise even during short-term missions and persist during long-term flights. Among the witnessed changes are altered numbers of peripheral blood leukocytes, reduced T-cell mitogenesis and function, a cytokine signaling shift toward the Th2 cell population, and dormant virus reactivation (Crucian and Sams 2012; Feuerecker 2012). Most of the data, however, has been acquired during pre- and postflight sampling that can only inaccurately reflect the in-flight changes that seem to occur. Ground-based spaceflight analogues (Antarctica winter-over, parabolic flight, 6° head-down tilt bed rest, immersion, and isolation studies) have also been used to extend our knowledge but may only provide the investigation of partial aspects of the multifactorial setting that the human system encounters on board the international space station (ISS). Contributing factors to the immune alterations observed during spaceflight are psychological stress, confinement and isolation, microgravity, radiation, circadian misalignment, and alterations in microbial load and virulence (Busby 1968; Barrat and Pool 2008; Yi et al. 2016). Given the ongoing planning of long-term and interplanetary missions, mechanistic insights into the cause for the described immune alterations in the innate and adaptive system will prove to be elementary to maintain crew health and to ascertain appropriate countermeasures for the prevention of disease. Although both cellular- and animal-based models provide the advantage of more controlled conditions, genetic and pharmacological modelling, and tissue sample collections hereby enabling new mechanistic insights and smaller laboratory efforts, the complexity of the environmental factors contributing to immunological changes in human space explores cannot be reflected as such (Clément et al. 2011; Feuerecker 2012; Wotring 2012). Despite the organizational challenges of onboard experimentation as mentioned, there are also very few studies that cover samples from animals directly in-flight but rather retrieve data from postflight sampling. The transfer of in vitro and animal data to humans seems therefore limited at this stage. Hence this chapter will focus on data retrieved from studies involving humans.
Innate Immunity Changes with Spaceflight
The innate immune system plays the role of a sentinel in terms of host defense. Alterations leading to a loss of proper function in space would permit microbial breach and could result in deleterious diseases. The knowledge on changes of the innate immune system is however limited. The isolated contemplation of cell numbers cannot describe functionality; therefore, cell counts and functional tests need to be correlated. Although results differ depending on duration (long term, short term) of mission and type of study (animal, human), analysis of peripheral leukocyte counts in blood samples in general shows an increased number of granulocytes and reduced numbers of NK cells and monocytes (Feuerecker 2012; Morukov et al. 2012; Yi et al. 2016). Since most data is retrieved from postflight time points, it is possible that the observed granulocytosis is at least in part due to a stimulated release from the bone marrow by the elevated stress hormones epinephrine and norepinephrine due to the landing maneuver. Whereas shorter (5 day) missions seem to have no influence, missions lasting 9–10 days resulted in significant reduction of the neutrophils’ ROS production during the oxidative burst reaction and phagocytosis activity when challenged in vitro. Similar results were obtained in neutrophils after a 6-month mission when stimulated with fMLP. Monocytes reduced in number also evidenced reduced phagocytic capability toward E. coli bacteria. When monocytes were stimulated with the strong toxin of gram-negative bacteria lipopolysaccharide (LPS), typical pro-inflammatory cytokines IL-6 and IL-1 were downregulated indicating that the IL-1-dependent activation of adaptive immune responses might be weakened. Exposure of whole blood samples after 6 months of spaceflight to distinct viral or fungal antigens in vitro shows an impaired response to viral but a higher immunologic response to fungal antigens (Feuerecker 2012; Yi et al. 2016). The reason for the distinct response remains to be clarified; however, the obvious impairment of viral defense tallies nicely with the previously observed reactivation of latent viral infections such as herpes virus, Epstein-Barr virus (EBV), or varicella zoster virus (VZV). It appears that the findings of increased antifungal activity might be also due to prolonged exposure, as it seems there is considerable fungal colonization of the ISS (Crucian and Choukèr 2012; Crucian 2015; Feuerecker 2012). NK cells have been studied since 1980 in cosmonauts on Salyut-6, 7, MIR, and ISS. Mission durations varied between 65 and 438 days. Although results showed a high individual variability, overall more than 60% of the crewmembers on Salyut evidenced a reduced cytotoxic NK cell activity postflight. In seven cosmonauts, cytotoxicity was even close to nonexistent, and 2 weeks after landing, cytotoxicity continued to be decreased in about 30% of all cosmonauts included in the study. Interestingly, correlation of data with the duration of the mission showed a ceiling effect for NK cell depression, when increasing the stay from few to 14 months did not further reduce NK cell activity. Moreover, repeated long-term spaceflight seemed to weaken the suppressing effects on NK cells. Subsequently, a full program of countermeasures was started involving physical exercises, salt additives, loading suits, and a well-balanced diet. In data retrieved from MIR and ISS cosmonauts, NK cell suppression was detectable also for the different NK subsets (CD56bright and CD56dim) but to a much lesser degree (Morukov et al. 2012; Busby 1968; Barratt and Pool 2008). These results seem to go in line with the later discovered fact that moderate regular physical exercise, together with slightly elevated plasma epinephrine levels, positively stimulates NK cell functions (Morukov 2012; Yi et al. 2016). Moreover, the previously mentioned viral reactivation might also contribute in some way to the activation of NK cells. So far, the evidence on spaceflight-induced changes of the innate immune system is scarce and the cause and mechanism for these changes not fully understood. Moreover, the interaction between the innate and the adaptive immune system via APCs remains to be elucidated.
Due to the complex nature of cellular communication networks with differentially orchestrated stimuli and response patterns, monitoring adaptive immune cell functions requires different approaches ranging from enumerative to functional cell- or receptor-specific stimulation assays (Murphy 2012; Alberts 2014). Comprehensive monitoring of cell-mediated immunity (CMI) in whole blood reflecting this orchestrated action is tested toward antigens that the immune system has previously encountered. In functional cell assays, T cells are stimulated and activated with distinct antigens (e.g., viral, bacterial, or fungal) resulting in different cytokine responses. Emanating from the primed CD4+ T cell, pathogen-specific Th subsets and the respective cytokine communication platform develop according to the stimulus and the type of infection. Disease occurs, when adequate T-cell direction is impaired or excessive (Murphy 2012). The early phase of T-cell activation is characterized by the expression of preformed CD69 on the surface. After 24 h of activation, CD25 (IL-2 receptor) can be detected. Short-term flight resulted in reduced numbers of CD69+ and CD69+/CD25+ T cells following bacterial antigen stimulation. Antibodies directed to CD3/CD28 surface receptors directly prime and activate T cells via their TCR, a method used for characterizing T cell-specific responses without the possible bias of non-fully functional APCs (Crucian and Sams 2012; Crucian 2015; Murphy 2012; Alberts 2014). In-flight data revealed that the direct, CD3/CD28-driven T-cell response analyzed by the secreted type II interferon IFN-γ, IL-10, and IL-17 was significantly decreased. Mitogenic stimulation is another way to further and evaluate the proliferation of peripheral blood lymphocytes. The term mitogen roughly summarizes agents with different properties that stimulate the proliferation of a target cell. The plant-derived concanavalin A (Con A) is a lectin, which specifically binds α-d-mannosyl and α-d-glucosyl residues. Among other features, Con A is a potent lymphocyte mitogen and frequently used to analyze the stimulus-driven proliferation capacity of peripheral blood lymphocytes (Cogoli et al. 1989; Alberts 2014). On T cells, Con A acts via binding and cross-linking the T-cell receptor. Ex vivo sample analysis and in vitro cultures during the first space shuttle flights evidenced that in microgravity, the human lymphocyte response to Con A is reduced by 90% when compared to ground-based or in-flight 1 g control samples. Related ground experiments conducted under 10 g conditions revealed and increased T-cell proliferation rate (Cogoli et al. 1984, 1989). Mechanistic insight as to how microgravity impairs T-cell activation derived from follow-up in vitro experiments on sounding rockets. It was shown that Con A still binds to the surface of lymphocytes and that cell-cell interaction was still preserved (Cogoli et al. 1984, 1989; Cogoli-Greuter et al. 1997). Nevertheless, the intracellular production of IL-2 and IL-2 receptor (IL-2R), both crucial for T-cell activation and the proliferation of different T-cell subsets, was found to be strongly impaired. The effects on the in vitro sample can be regarded as mostly physical in nature; nevertheless, they are evidencing that lymphocytes show gravity sensing properties (Cogoli et al. 1984, 1989; Cogoli-Greuter et al. 1997). And although in the ex vivo sample, which was blood drawn from astronauts, physical as well as psychological and neuroendocrine effects are influencing lymphocyte responses, similar results were observed.
A large body of evidence supports the fact that spaceflight induces the reactivation of latent EBV, VZV, and cytomegalovirus (CMV) measured in blood and saliva samples. The clinical relevance remains to be clarified; however, the probability of occurrence seems to correlate with the duration of the mission. Furthermore, it has been established for saliva samples that the detection of VZV-DNA does reliably reflect the presence of live and potential infectious viral particles. Viral antigen stimuli result in the activation of virus-specific CD8+T-cell phenotypes. Intracellular amounts of IFN-γ were found to be reduced in EBV- and CMV-specific CD8+ T cells after short-term flight, though their number remained constant (Crucian and Sams 2012; Crucian 2015). The detection of this phenomenon does support the other findings that a competent innate and adaptive immune system is challenged and suppressed by spaceflight. In the past, a functional in vivo CMI skin test was applied to monitor the T-cell-mediated delayed-type hypersensitivity reaction in-flight. A variety of antigen stimuli were injected subcutaneously, and the resulting delayed-type hypersensitivity (DTH) response could be measured in form of a resulting skin erythema after 48 h of exposure. Diminished CMI responses were observed during long-term spaceflight when compared to preflight values. Since this skin test was phased out of the market in 2002, current strategies involve the development of a new ex vivo DTH test for application in space (Crucian and Sams 2012; Feuerecker 2012; Crucian 2015; Yi et al. 2016). Another approach involves the analysis of large plasma cytokine panels to describe the overall cellular cross talk within an inflammatory environment. These studies may help to understand distinct shifts toward a certain inflammatory or anti-inflammatory state and allow understanding the communication between innate and adaptive immunity (Feuerecker 2012) during long-term flight. Recent evidence shows elevated blood levels for TNFα, IL-8, IL-1 receptor antibody (IL-1ra), thrombopoietin (Tpo), vascular endothelial growth factor (VEGF), and mediators of leukocyte recruitment indicating a prolonged stimulated immunologic state mediating complex tissue remodeling such as angiogenesis. Cytokine panel analysis in astronauts that showed viral shedding evidenced high levels for a broad spectrum of signaling molecules, among them IL-6 and IL-4 and IFN-γ. Interestingly, the differential analysis of results showed a much stronger expression of IL-4 compared to IFN-γ indicating a shift away from typical Th1 cytokines (IL-2, IL-12, IFN-γ) toward a Th2-mediated and Th2-communicated immune response (IL-4, IL-5, IL-10) postflight (Crucian and Choukèr 2012; Crucian and Sams 2012; Feuerecker 2012; Crucian 2015; Yi et al. 2016). Neuroendocrine modulations of immunity influencing adaptive immunity are mostly due to higher stress hormone levels such as cortisol especially in long-duration flights. The anti-inflammatory profile of glucocorticoids is capable to suppress IL-12 and further IL-10 levels supporting the Th2-orchestrated immune response (Feuerecker 2012; Crucian 2015). Results from larger in-flight studies such as the recently completed “Integrated immune” (NASA) and the ongoing “Functional Immune” (NASA) and “Immuno-2” (ESA/RSA) study will provide answers to the occurring cross talk between physiological and psycho-endocrine changes that modulate our immune system in space. Data on humoral immunity show no alterations of immunoglobulins during short-term shuttle flights or long-duration spaceflight on board the ISS, whereas B-cell numbers are often elevated (Crucian 2015; Yi et al. 2016).
So far, technical restrictions make it impossible to study immune cells from blood samples under standardized conditions live on board the ISS, and as previously mentioned, in-flight studies are unfortunately still not common. It will be the task of future studies to develop new techniques to monitor these alterations directly on board in such a way that the crew can treat and even prevent disease autonomously and sustain a healthy team.
Human space exploration involves short- and long-term stays in an extreme, life-endangering environment triggering distinct immune responses. Most of the available data has been gathered from postflight samples. The more relevant in-flight evidence is still constricted due to technical limitations and the considerable efforts necessary for conducting an in-flight study. Our knowledge on long-term spaceflight immune alterations scarcely extends beyond a 6-month stay in space and a larger number (>20) of subjects enrolled per study. Furthermore, this knowledge is often only descriptive and lacks the complete mechanistic insight. Nevertheless, current evidence base shows that immune alterations are a fact, and the so-called human factor might interfere with the success of a mission. The future will involve exploration class missions that are far more challenging than traveling around the lower Earth orbit. Deep space missions aggravate further the enormous psycho-endocrine stressors when live communication with mission control, Earth’s protective magnetic shield, the possibility of direct emergency evacuation to Earth, and access to extensive emergency medical treatment become unavailable. In regard to future long-term interplanetary missions and in order to secure safe travel and return, it is imperative to understand the immune changes that occur. Only then, adequate detection measures such as the establishment of new laboratory methods for in-flight monitoring as well as prevention and treatment of illness can be provided to future space explorers.
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