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Sarkar, R.; Pampaloni, F. In Vitro Models of Immune Dysfunction in Space. Encyclopedia. Available online: https://encyclopedia.pub/entry/21266 (accessed on 27 July 2024).
Sarkar R, Pampaloni F. In Vitro Models of Immune Dysfunction in Space. Encyclopedia. Available at: https://encyclopedia.pub/entry/21266. Accessed July 27, 2024.
Sarkar, Ryan, Francesco Pampaloni. "In Vitro Models of Immune Dysfunction in Space" Encyclopedia, https://encyclopedia.pub/entry/21266 (accessed July 27, 2024).
Sarkar, R., & Pampaloni, F. (2022, April 01). In Vitro Models of Immune Dysfunction in Space. In Encyclopedia. https://encyclopedia.pub/entry/21266
Sarkar, Ryan and Francesco Pampaloni. "In Vitro Models of Immune Dysfunction in Space." Encyclopedia. Web. 01 April, 2022.
In Vitro Models of Immune Dysfunction in Space
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This entry is adapted from the peer-reviewed paper 10.3390/biomedicines10040766

Spaceflight affects the body on every level. Reports on astronaut health identify bone marrow remodelling and dysfunction of the innate immune system as significant health risks of long-term habitation in space. Microgravity-induced alterations of the bone marrow induce physical changes to the bone marrow stem cell niche. Downstream effects on innate immunity are expected due to impaired hematopoiesis and myelopoiesis. To date, few studies have investigated these effects in real microgravity and the sparsely available literature often reports contrasting results. This emphasizes a need for the development of physiologically relevant in vitro models of the bone marrow stem cell niche, capable of delivering appropriate sample sizes for robust statistics.

3D cell culture bone marrow niche hematopoiesis hematopoietic progenitor cells innate immunity mesenchymal stem cells microgravity myelopoiesis

1. Hematopoiesis, Innate Immunity, and Spaceflight Conditions

The rapid pace of technological advancements in spaceflight has transformed the dynamic such that the physiology of the human body and its ability to withstand the long-term effects of space travel is now one of the greatest factors precluding further exploration into space [1][2]. Space travel presents individuals with numerous physical and mental challenges, as shown in Figure 1. Microgravity, the condition of apparent weightlessness, is a major environmental factor in space and exposes astronauts to physiological stress stemming from reduced mechanical loads on the weight-bearing structures of the body and a redistribution of bodily fluids [3]. Further health risks are posed by increased exposure to radiation (especially relevant for missions beyond low Earth orbit) and psychosocial changes from travel-associated long-term confinement [3][4].
/media/item_content/202204/624e395d172d6biomedicines-10-00766-g001.png
Figure 1. Overview of stressors and health challenges associated with spaceflight. The physical and psychological impacts of spaceflight are not restricted only to their specific stimuli but can also influence each other, resulting in a complex interplay that can have extensive health consequences for space travellers.
Given the demands of space travel, preserving the immune health of astronauts is of utmost importance. The immune system consists of two interlinked subsystems, the slow but specific adaptive immune system and the rapid, broadly responding innate immune system. The delayed response of adaptive immunity is antigen-specific, a trait resulting in the system maintaining a long-term memory [5]. In contrast, the innate immune system is non-specific and represents the first line of defence against pathogens, with key functions including the destruction of pathogens, induction of inflammation, and activation of the adaptive immune response [6][7]. This system relies on phagocytic cells recognizing conserved features of pathogens enabling their quick activation, as well as the release of cytokines and chemokines to facilitate immune cell recruitment [6]. That the adaptive and innate immune system are affected by microgravity and radiation has been well established, but the cellular mechanisms of long-term exposure are only beginning to be investigated [5][8][9][10][11][12][13].
Along with immune dysfunction, astronauts also experience bone marrow density loss and remodelling while in space, with the current literature suggesting these two issues are linked [5][14]. Accordingly, alterations to the hematopoietic stem cell (HSC) niche, located in the bone marrow, play a primary role. The HSC niche is crucial in the establishment and maintenance of innate immunity (Figure 2), and as such is likely a central aspect of any potential links between microgravity-induced bone marrow loss and the immune dysfunction experienced by astronauts [5][14][15][16][17][18]. Hematopoietic stem cells give rise to all blood cells through hematopoiesis. Consequently, bone marrow remodelling related to long-term exposure to microgravity and the associated impacts on hematopoietic function can lead to severe health consequences [19]. Yet the mechanisms of the relationship between the bone marrow remodelling in microgravity, its effect on hematopoietic progenitor cells, and immune dysfunction remains unknown. Studies performed on astronauts to further elucidate these relationships have been limited by their small sample size. While research on microgravity, radiation, and the immune system has also been performed on animal models, these too are often limited by small sample size. Furthermore, the reactions of animals are often not representative of human physiology, and their use precludes downstream applications in humans, thus further emphasizing the need for cellular models [8][20][21].
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Figure 2. Hematopoietic stem cells and mesenchymal stem cells form the two primary cell types of the bone marrow stem cell niche. Mesenchymal cells can differentiate into a variety of cell types while hematopoietic stem cells give rise to every component of the blood including immune cells via differentiation into myeloid or lymphoid progenitors. Myeloid progenitor cells differentiate into platelets, granulocytes, or monocytes, which themselves further differentiate into dendritic cells or macrophages. Lymphoid progenitors differentiate into T cells, B cells, and NK cells. The innate immune system is comprised of NK cells, granulocytes, monocytes, macrophages, and dendritic cells, which serve as messengers between the innate and adaptive immune systems.

2. Response of the Innate Immune System to Spaceflight Conditions

The previous decades of space exploration have provided a wealth of evidence associating spaceflight with immune system dysfunction [5]. Studies on astronauts have demonstrated that neutrophil count, phagocytic function, and oxidative function are affected by spaceflight [22][23][24]. Other studies reported that long duration spaceflight impairs function of Natural Killer (NK) cells in astronauts [25]. In a study of 11 astronauts to fly on the space shuttle, 7 were found to have reduced levels of monocytes [26]. Consequently, microgravity-induced immune dysfunction currently presents one of the greatest barriers to long-distance space travel, affecting both the adaptive and innate immune systems comprehensively [5]. As most cells of the innate immune system do not divide but are instead the product of hematopoietic stem cells (Figure 2), increasing the present understanding of the impact of microgravity on hematopoiesis remains of utmost importance.

2.1. Response of Innate Immune Cells to Microgravity and Ionizing Radiation

2.1.1. Hematopoietic Stem Cells

The study of hematopoiesis, whether on Earth or in space, revolves around discussion of hematopoietic progenitor cells. Crucial characteristics of progenitor cells are their self-renewal and their capacity to differentiate into diverse types of cells with diverse functions. As all types of immune cells originate from hematopoietic progenitor cells, damage linked to their integrity can affect all downstream immune responses.
The capacity of HSC to proliferate and differentiate has been widely studied. Early studies investigating the mechanisms and signalling pathways involved in HSC proliferation and differentiation under microgravity showed that CD34+ HSC (CD34 being a typical marker of HSC progenitors) maintained at 1 g proliferate three-fold faster than HSC exposed to simulated microgravity in a RWV [27]. In fact, HSC under microgravity left the G0/G1 phase slower than the cells at 1 g. Furthermore, the hematopoietic potential of the HSC in microgravity was higher and more persistent in time compared to the 1 g condition. However, this study did not investigate the molecular mechanism at the base of the observed phenotype. A follow-up study by the same group found that following three days of culture in the RWV, HSC downregulated the expression of stromal cell–derived factor 1 (SDF-1α) and F-actin [28]. This induces a reduction in the migratory ability of HSC that correlates with a reduced tendency of the cells to differentiate to mature progenitors. These results provided a first mechanistic explanation of the effect of microgravity on HSC proliferation and differentiation through HSC mobilization pathways.
In another experiment featuring two space flights, Wang et al. found murine HSC to have proliferated significantly less and express significantly lower levels of the proliferation marker Ki67 following 12 days of spaceflight in comparison to ground controls [29]. This was also observed when the experiment was repeated in simulated microgravity using an RWV, with the cell cycle primarily being blocked at the G1/S transition. A similar study performed on human CD34+ HSC isolated from adult bone marrow samples showed decreased cell proliferation in a RWV when compared to 2D controls, although in this case the primary point of cell cycle arrest was the G2/M phase [30]. The results of both studies coincide with those of Davis et al., who found human HSC in co-culture with endothelial cells to exhibit a 57–84% decrease in proliferation after 11–13 days of real microgravity exposure aboard the Space Shuttle in comparison to ground-based controls [31]. Additionally, different cell types had different responses to microgravity with myeloid progenitors expanding less in space than in normal gravity while the erythroid progenitor population in the space shuttle actually shrank in contrast to the expansion seen in ground controls [31].
Experimental evidence from previous studies demonstrates that mechanical forces play a crucial role in determining HSC fate [32][33]. HSC lineage differentiation depends on the stiffness of the surrounding microenvironment: HSCs in contact with a soft substrate (0.71 KPa) display a round shape, lack of polarity in the actin cytoskeleton, and quiescence, while HSCs cultured on a stiff (196 KPa) matrix show formation of cell protrusions, cell polarity, and fast decision to a specific cell fate [34]. From these results, it follows that the mechanical unloading sensed by HSC under microgravity, mediated by the actin cytoskeleton, would impair their polarization, migration ability, and proliferation potential [34]. Microgravity-responsive genes were identified at a systemic level by RNAseq transcriptomics analysis of murine HSC cultured on the Tianzhou-1 orbital cargo ship over 12 days [29]. GO-biological process enrichment analysis found that genes belonging to apoptosis, hypoxic stress, hematopoietic capacity, and inflammatory response processes were upregulated. In contrast, a downregulation of cell proliferation pathways and blockage of the G0/G1 phase were detected, in agreement with Plett et al., 2001 [27][29]. Notably, the expression of the c-KIT SCF (Stem Cell Factor) receptor, involved in HSC quiescence and maintenance, was significantly downregulated under microgravity. SCF is a pivotal cytokine promoting proliferation, differentiation, and migration of HSC, which activates HCS proliferation through the PI3K/AKT and MEK/ERK [29][35]. Interestingly, the ERK signalling is one of the main regulators of cell motility [36]. Thus, perturbation of the ERK pathway is a possible mechanism linking mechanical unloading of bones under microgravity and the low proliferation and differentiation of HSC.
An ISS experiment on human blood-derived stem cells determined that microgravity results in a more pronounced loss of pluripotency compared to controls on the ground, as well as increased osteogenic differentiation [37]. Conversely, RWV culture of human CD34+ HSC cells found simulated microgravity to inhibit differentiation when compared to static control cultures [30]. The origin of the cells may also play a role, with Davis et al. observing human HSC in space to differentiate towards the macrophage lineage, while Wang et al. observed no microgravity-mediated effects on differentiation in murine HSC [29][31]. As stated earlier, previous research has highlighted biomechanical forces as important factors to consider in differentiation [33]. A study on embryonic hematopoiesis demonstrated the importance of shear stress and mechanical loading in HSC differentiation, maturation, and colony-forming potential—such factors would be affected in space studies, such as those of Wang et al. and Davis et al., as fluid dynamics and mechanical forces are strongly reduced in microgravity when compared to 1 g [32].
The typical microgravity HSC phenotype consisting of low proliferation and low differentiation directly affects the innate immune system. In fact, the circulating immune cells have a short life span and a continuous and sustained replenishment of blood immune cells is needed for a healthy immune system [38]. The perturbation of this homeostatic mechanism by microgravity leads to an immediate, albeit transient, effect on the innate immune response
Collectively, the effects of microgravity on HSC generally involve a decrease in the rate of proliferation and population expansion of progenitor cells. Differentiation has also observed to be affected both in real and simulated microgravity. Given the cell types descended from HSC, these effects could cause the impairment of the innate system function in astronauts.

2.1.2. Peripheral Blood Mononuclear Cells

The peripheral blood mononuclear cells (PBMC) are the main immune cell group isolated from the blood cells and comprise cells such as Natural Killer (NK) cells, dendritic cells, monocytes, lymphocytes, and macrophages. These cells possess a round nuclei in comparison to the multi-lobed nuclei found in granulocytes.
Moreno-Villanueva et al. subjected the entire PBMC population to varying degrees of ionizing radiation while incubated in an RWV [39]. Although increased background radiation is another major environmental factor of space travel, very few studies have examined the effect of simultaneous microgravity and radiation exposure. Ionizing radiation, such as that used in this experiment, is what humans are generally exposed to on Earth. Radiation from beyond Earth’s orbit is quite different, however, as it contains high-energy protons, solar particles, and charged particles [1]. Unfortunately, current facilities are unable to replicate this nonionizing radiation on Earth, limiting its study to space. Accordingly, studies such as this one by Moreno-Villanueva et al. are important for providing a preliminary understanding of the biological effects of spaceflight conditions, despite being limited to ionizing radiation.
When PBMC were subjected to an absorbed exposure of 0.8 Gy radiation, the rate of apoptosis significantly increased in 1 g controls but, interestingly, no change was observed in cells in simulated microgravity. Additionally, radiation induced more double-strand breaks in RWV cells. Radiation was only seen to induce cytokine release under simulated microgravity, which it did in a dose-dependent manner, with higher radiation inducing a greater cytokine release. To mitigate the combined effects of microgravity and radiation, the authors treated the cells with the sympathomimetic drug isoproterenol. This treatment would induce the release of stress hormones which influence immune regulation, DNA repair, and bone homeostasis, thereby reducing the effects of microgravity [39]. Based on their results, the authors concluded that isoproterenol did prevent most microgravity-mediated effects. In contrast, at a level of 2 Gy, radiation isoproterenol treatment was much less effective in cells incubated in the RWV compared to 1 g controls.
As space is characterized not only by microgravity but also elevated background radiation, this study is valuable in establishing a knowledge base into the combined effects of radiation and microgravity on PBMC.

2.1.3. Monocytes and Macrophages

Of the multiple cell types comprising PBMC, monocytes, and their descendants, macrophages, are some of the most widely studied. Non-polarized M0 Macrophages can be polarized into either the pro-inflammatory M1 phenotype or the pro-healing/anti-inflammatory M2 phenotype [40]. The three phenotypes have been shown to have differing responses to simulated microgravity. A study by Ludtka, Moore, and Allen reported a RWV clinorotation-mediated shift from the initial singular phenotypes to mixed populations of cells co-expressing M1 and M2 specific genes [40]. Another study using murine HSC progenitors found that both spaceflight and RWV-simulated microgravity reduced macrophage differentiation from HSC progenitors, the quantity of macrophages, and their functional polarization [41]. Further analysis with qPCR revealed that key genes relating to macrophage proliferation and differentiation were downregulated in both spaceflight and RWV-simulated microgravity. Interestingly, stimulation of the RAS, ERK, and NFκB signalling pathways of cells cultured in the RWV was found to partially rescue the effects of simulated microgravity, perhaps suggesting potential targets for space medicine. Further research into the underlying mechanisms of real and simulated microgravity on the macrophage phenotype could elucidate the effect of microgravity on innate immunity.
Reporting on phenotypic changes, researchers have conducted experiments on primary human macrophages in real microgravity on sounding rockets. The macrophages have been observed responding to microgravity in a matter of seconds, exhibiting cytoskeletal changes in addition to increases in the volume and surface area of both cells and nuclei [42]. However, following the initial swelling period, cell volume and surface area shrank significantly below the starting values. Despite the shrinking, upon return to Earth, no structural changes were observed, indicating a recovery of the original cytoskeletal organization.
The structure of macrophages plays an important role in determining their function, with specific defined features corresponding to their status [43]. M1 macrophages, for example, are typified by lamellipodia and filopodia, with the actin cytoskeleton distributed throughout the cell [43]. In contrast, M2 macrophages exhibit a rounded shape, with actin concentrated around the nucleus [44]. Questions over whether structural changes in microgravity affect macrophage function have led to studies examining their oxidative burst reaction. Notably, 2D clinostat-simulated microgravity reduced ROS production in NR8383 rat macrophages, while parabolic flight resulted in ROS release to increase and decrease in hyper- and microgravity, respectively [45][46]. These results indicate a gravisensitive step in signalling, ultimately identified as Syk phosphorylation [46]. Other studies demonstrated that NR8383 cells adapt to real microgravity within 1 min, but also rapidly re-adapt to 1 g conditions [47]. This adaptability was also seen in human cells during longer-term research aboard the ISS, which found primary human macrophages to exhibit no structural changes in the actin and vimentin cytoskeletons after 11 days of spaceflight when compared to 1 g controls [48]. The transient nature of the structural changes in macrophages and the rapid cellular response observed in these studies suggest the establishment of an adapted steady state by the macrophages based on their gravitational condition.
In contrast, U937 myelomonocytic cells exhibited decreased proliferation, actin expression, and cytoskeletal disorganization after 72 h of RWV culture [49]. These results coincide with those of Paulsen et al., where U937 cells exhibited disruption of the actin cytoskeleton and disorganization of tubulin following a five-day spaceflight [50]. These cells also had reduced expression of CD18, CD36, and MHC-II. Such a phenotype, the authors report, would be incapable of migration, recognizing pathogens, attacking pathogens, or activating the adaptive immune system. Further research found macrophage-like differentiated U937 cells to express higher levels of intracellular adhesion molecule 1 (ICAM-1) in 2D clinostat-simulated microgravity, parabolic flight, and spaceflight [51]. These effects were also observed in primary human M2 macrophages yet undifferentiated U937 showed no microgravity-related changes in expression of ICAM-1. Conversely, spaceflight was found to decrease the levels of ICAM-1 expression in human primary M1 macrophages, potentially hindering the migration ability of the cell [48]. These varying results could arise from differences in macrophage polarization or be cell-type dependent. A study by Moser et al. found that in microgravity during parabolic flight, PBMCs close to the ICAM-1 coated substrate were moved to the centre of the chamber, thus rendering binding impossible and likely preventing immune activation [52]. These results raise the additional question as to what extent decreased ICAM-1 expression impairs the ability of the cell to migrate in microgravity versus an inability to even reach a substrate.
Finally, osteocytes present an additional factor to consider as a result of the role they play in macrophage activation. Representing more than 90% of the bone cell population, osteocytes can sense and transmit mechanical stimuli over long distances through the bone via their elongated cell processes, which build an extended network of cell–cell and cell–matrix contacts with other bone cell types and the bone extracellular matrix. Osteocytes react to mechanical cues by releasing inflammatory mediators including nitric oxide and prostaglandin E2, which favours macrophage maturation and activation [53]. Compelling evidence suggests that macrophages are pivotal cells for the maintenance of the bone marrow HSC niche and for the modulation of osteogenesis [54]. Activated (M1) macrophages induce bone resorption by releasing the pro-inflammatory cytokines TNFα, IL-1β, and IL-6. IL-1 induces bone loss by the NF-kB/RANKL pathway-induced osteoclast formation. IL-6 activates the osteocyte-mediated osteoclasts’ formation through the JAK2/RANKL pathway [55].

2.1.4. Osteocytes

Studies aimed at understanding the modulation of the hematopoietic microenvironment by bone cells, a vital research field known as osteoimmunology, found that osteocytes (which represent 90–95% of all the bone cell mass) and the bone extracellular matrix are closely interconnected with the HSC niche [56]. Osteocytes control the HSC through both soluble factors and direct cell–cell communication with HSC via intermediate cells of the stem cell niche, such as osteoblasts and osteoclasts [57]. Osteocytes are mechanosensory cells and sense the shear and compressive forces in the bone extracellular matrix, such as the mechanical unloading occurring in microgravity. Microgravity impairs the formation of the osteocyte network and subsequently reduces the level of osteocyte-secreted Granulocyte Colony-Stimulating Factor (G-CSF) [58]. G-CSF directly stimulates the production of leukocytes from the HSC niche, therefore, low levels would be concordant with immune dysfunction [58].

2.1.5. Natural Killer Cells

Natural killer (NK) cells are key effectors of the innate immune system mediating anti-tumour and anti-viral responses [59]. Spaceflight is known to impair the anti-viral capabilities of the immune system, with over half of all astronauts demonstrating herpes virus reactivation suggesting microgravity-induced NK cell dysfunction [25][60]. Spaceflight has also been shown to significantly reduce the number and proportion of NK cells in astronauts, although levels were observed to recover within 10 days after landing [61].
Short-term experiments aboard the ISS with primary NK cells and K562 tumour cells in co-culture found the level of NK cytotoxicity and interferon production to be unaffected by microgravity [62][63][64]. Microgravity simulated by a 2D clinostat was also observed to have no effect, leading the authors to conclude that microgravity bears no effect on critical NK function. In contrast, Li et al. performed a study of ex vivo expanded primary NK cells in a horizontally-rotating RWV and found cytotoxicity to significantly decrease after 48 h and even further after 72 h when compared to 1 g and vertical rotation controls [65]. Additional analysis of these cells observed increased apoptosis, necrosis, decreased secretion of IFN-γ and perforin, and downregulated cell surface receptor expression to be potential contributors to the loss of cytotoxicity. Interestingly, after removal from the RWV, NK cells recovered quickly, within only three days at 1 g. Another study observed NK cell viability dropping significantly after only 12 h in a RWV [66]. This discrepancy could originate from differences in cell type but also from the difference in culture conditions. Li et al. used NK cells expanded ex vivo in monoculture in the RWV compared to the freshly harvested cells of Mylabathula et al., where all PBMC were cultured together in the RWV with the NK cells being isolated afterwards [65][66]. More recent work has suggested that downregulation of the NKG2D receptor induced by simulated microgravity is the cause of this NK cell dysfunction [67]. Contrary to the work of Li et al., following exposure to RWV-simulated microgravity, Mylabathula et al. observed no change in the cell surface phenotype of NK cells, though they acknowledge this may be due to the limited length of the experiment necessitated by the cytokine-free culture [65][66]. In both works, the NK cells and K562 cells were cultured together in 1 g after exposure to simulated microgravity to facilitate contact between the cells, but as noted by Mylabathula et al., this potentially allows the NK cells the chance to recover from their treatment. Although not completely in concordance, both papers provide insight into the factors contributing to the impaired function of NK cells in microgravity environments.

2.1.6. Granulocytes

Granulocytes are a family of white blood cells including neutrophils, eosinophils, basophils, and mast cells. The latter three play key roles in allergic inflammation while neutrophils, also called polymorphonuclear leukocytes due to their lobed nuclei, are noted for their immediate and non-specific destruction of invading pathogens [68][69]. Research on microgravitational effects in granulocytes is lacking with the few published works investigating neutrophils.
A study by Paul et al. used the Neutrophil-to-Lymphocyte ratio (NLR) as a marker to determine the status of the immune system in experiments performed in real and simulated microgravity as astronauts had previously demonstrated elevated granulocyte-to-lymphocyte ratios [70]. Treatment in the RWV increased the NLR and the number of granulocytes in human leukocytes in vitro, in concordance with what the authors observed when examining previous data from experiments on astronauts. RWV-simulated microgravity also elevated the release of ROS and induced neutrophil activation. Notably, these effects could be reduced by treatment with the antioxidant N-acetyl cysteine. In contrast, an earlier study found that RPM-simulated microgravity does not affect the oxidative burst reaction in neutrophils, though this discrepancy could stem from the differing experimental platforms [71].

2.2. Response to Microgravity of the Innate Immune System in Animal Models

In addition to in vitro experiments, numerous animal experiments have also been flown to space to study the effects of microgravity on the immune system in vivo. Mice represent one of the most commonly utilized animal models in space experiments. In 2013, Blaber et al. reported significant bone resorption of the trabecular endosteal surface in the femoral head of mice after 15 days of spaceflight, thereby enlarging the bone marrow cavity [72]. Cells isolated from the femoral head also exhibited reduced expression of markers for early hematopoietic and mesenchymal differentiation. The differentiation potential of bone marrow MSC (mesenchymal stromal progenitor cells) was found to increase upon reloading, while microgravity was found to predispose HSC progenitors to differentiate towards the osteoclast lineage upon reloading at 1 g, with the authors suggesting an accumulation of undifferentiated progenitors occurring as a result of exposure to microgravity. That stem cell differentiation is affected by microgravity was also observed in in vitro experiments [31][37]. Blaber et al. suggest that a reduction in mechanical load, as experienced in microgravity, may inhibit the differentiation of stem cells, potentially leading to detrimental effects on regenerative health [72]. While this study provides valuable insight into bone remodelling under microgravity, it is important to note that only eight mice were used.
Much like the devices used to simulate microgravity for cell cultures, platforms also exist for the simulation of microgravity for animal experiments. Recently, the European Space Agency has unveiled a Random Positioning Machine able to subject aquatic model organisms to simulated microgravity [73]. The facility also contains cages for Hindlimb Unloading (HU), a widely used method of simulating weightlessness in rodents. The method is based on elevating the hindlimbs of rodents inducing a 30° head-down tilt that results in a cephalad fluid shift [74][75]. This forms a more cost-effective method of identifying biological systems affected by microgravity and can be used to study specific biological systems under microgravity [75]. Such a model has the potential to incite stress in the organism potentially impacting studies on the stress response or related systems, such as immunity. However, with proper control of environmental and physiological factors, markers of stress in HU mice have been shown to return to normal levels within a few days [75].
A study examining mice under HU for 28 days found the frequency of HSC to be significantly lower in the bone marrow when compared to controls, although this discrepancy was not observed in mice that subsequently underwent 28 days of hindlimb reloading (HR), indicating this decline recovers after a short-term [61]. In contrast, the neutrophil count in the bone marrow of HU mice was increased and, after 28 days of HR, showed only a slight decrease, demonstrating that increased granulopoiesis does not completely recover in the short term. Examination of NK cells found their frequency to be significantly reduced in both peripheral blood and bone marrow in HU mice, with levels failing to return to normal after 28 days recovery, results consistent with those the authors had obtained from samples taken from astronauts [61]. Frequency of erythrocyte precursors was significantly decreased in the bone marrow of HU mice compared to controls but, unlike the NK cells, was unchanged after 28 days of HR, showing that the decline in hematopoiesis recovers over a short term. Peripheral blood of HU mice also showed decreased counts of white blood cells and lymphocytes in comparison to controls, but this was shown to recover quickly, with no difference being seen after seven days of HR. The increase in neutrophils, coupled with a decrease in lymphocytes, as reported by Cao et al., would infer an elevated NLR which concurs not only with the in vitro experiments of Paul et al. but also with their in vivo experiments that also were conducted on HU mice [61][70]. Analysis of the HU mice in Cao et al. showed that, regardless of reloading, HSC population was elevated in any mice that underwent HU [61]. However, the proliferation of these cells was not seen to have increased, indicating an inhibition of apoptosis. Further analysis of the HSC showed that in non-HR mice, the HSCs were found to have severely impaired functionality. The declines in hematopoiesis and white blood cell count, in conjunction with the impairments of HSC function and differentiation, are factors that could contribute to immune dysfunction. As an in vivo experiment, this study provides great insight into the potential mechanisms of the impact of microgravity on immune condition.

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