Most studies investigating the impact of space radiation on the hematopoietic system have been conducted using monoenergetic electron and gamma-ray beams. Exposure of rats to gamma rays was performed on board of the satellite Cosmos-690 along with a control group receiving matched dosing on Earth (88). Hematopoietic assessments demonstrated a significantly enhanced effect in rats irradiated in spaceflight, when compared to rats irradiated on Earth, with severe suppression of bone marrow hematopoiesis and thymopoiesis (88). Along these lines, the exposure of rat thymocyte suspensions to Co-60 gamma-rays induced severe apoptosis and distinct morphological and functional changes in thymocytes, assessed via electron microscopy, DNA fragmentation assays, and biochemical assays (89). Further mechanistic insights revealed an activation of intracellular and intranuclear proteases, typical of the extrinsic apoptotic pathway, leading to the degradation of mitochondria and the release of pro-apoptotic factors (90). However, a similar study on Cosmos-690 that measured the combined effects of microgravity and ionizing radiation from a Cs-137 source did not reveal significant changes in thymus weight and spleen after irradiation, although bone marrow hematopoiesis was affected (91). In two separate studies, exposure to Fe-56 particles, or C-12 (6+) ions induced severe spleen and bone marrow defects, as well as thymic involution in adult female C57BL/6 or King-Ming strain mice, respectively, demonstrating varying degrees of susceptibility for lymphocyte populations (92, 93). Collectively, these studies demonstrate the pleiotropic effects of diverse monoenergetic ion sources on thymic structure and function, although the precise mechanistic insights behind the observed variability are not fully understood. It should be noted however, that most research to evaluate health risks from space radiation has been historically performed via acute exposure to such monoenergetic single-ion beams, as outlined in the studies above. Nevertheless, it has now been established that such exposures do not faithfully recapitulate the intricacies of the galactic ray environment in our solar system (12), and as such, should be interpreted with caution.

To address such concerns, ground-based GCR simulators have been developed to expose experimental animals and cell cultures to “mission-relevant” radiation doses. These simulators incorporate diverse vehicle and shielding configurations, high design fidelity, precise material characterization, mission duration considerations, and realistic solar conditions (94–101). The most advanced, developed by the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory, delivers radiation doses comprising a mixture of protons (~65%-75%), helium ions (~10%-20%), and heavier ions (C, O, Si, Ti, Fe) (102). To more closely replicate the low-dose rates found in space, this system can additionally fractionate sequential field exposures over daily intervals for 2 to 6 weeks, allowing state-of-the-art cellular and animal model systems to be exposed to mission-relevant radiation (12). So far, sophisticated GCR simulation has been used to examine various organ system adaptations to space- and mission-relevant radiation doses, including the gastrointestinal, endocrine, cardiovascular, immune, ocular, and central nervous systems (103–111). To our knowledge however, there are currently no studies explicitly dedicated to evaluating thymus architecture and functions using GCR simulators.

Instead, most studies using GCR simulators have focused on the effects of mission-relevant GCR exposure on the bone marrow. For instance, mice exposed to mission-equivalent GCR doses showed increased osteoclast activity and trabecular bone loss, suggesting alterations in the endosteal niche (112, 113), which regulates hematopoiesis (114–116). Simulated SEP and GCR radiation also disrupted the ability of MSCs to support hematopoiesis and directly impaired human hematopoietic stem cell (HSC) functionality, inducing DNA damage and mutations. Sequential exposure to protons and iron ions, mimicking deep space radiation, was particularly harmful to HSC genome integrity. Notably, sequential exposure to protons and iron ions—mimicking the complexity of deep space radiation—proved significantly more harmful to HSC genome integrity and function than exposure to either particle type alone (117). These findings emphasize once again the importance of simulating the full spectrum of galactic cosmic radiation for accurate assessments. Collectively, these studies suggest that GCR may impact thymopoiesis indirectly by disrupting bone marrow hematopoiesis and the influx of early thymic progenitors. However, the possibility of direct effects of GCR/SEP on the thymus itself cannot be excluded, as indicated from astronaut observations and rodent experiments (25, 82, 118).

Development of ground-based models replicating microgravity is particularly challenging. Parabolic flights conducted on Earth on one side accurately replicate the gravitational conditions experienced in orbital spaceflight. Indeed, numerous experiments have explored the effects of microgravity on physiological systems using this approach (119). However, the duration of induced microgravity during parabolic flights is typically limited to several minutes, making it unsuitable for assessing long-term effects. Since the impact of microgravity on the thymus likely occurs over days, this model is likely inadequate for evaluating thymic responses.

For in vitro experiments, devices such as the clinostat and magnetic levitation are commonly used to simulate microgravity. These models are primarily restricted to cultured cell studies, thus posing an impediment to recapitulate the complex microenvironment of lymphoid organs. However, fetal thymic organ cultures, typically derived from E14 to E16 mouse embryos, can be maintained in vitro, and physiologically recapitulate stromal-thymocyte interactions (120), potentially enabling the study of simulated microgravity effects on thymocyte development. Indeed, a clinostat study demonstrated a reduction in CD4⁺CD8⁺ thymocytes after a 12-day fetal thymic organ culture (121). Nonetheless, these findings should be interpreted with caution, as the cellular composition of the fetal thymus differs significantly from the adult one, highlighting the need for further studies to understand the effects of microgravity on adult thymic function.

The hindlimb unloading (HU) model, also known as the tail suspension model, is frequently used to simulate weightlessness in rodents (122). This model removes weight-bearing from the hindlimbs, impacting the musculoskeletal system, and causing a redistribution of body fluids towards the head, analogous to the fluid shifts observed in humans under microgravity conditions. Besides musculoskeletal ramifications, short-term (2-day) HU in mice resulted in reduced thymic mass, with CD4⁺CD8⁺ thymocytes being particularly sensitive (123). The total number of mature single-positive (CD4⁺CD8^- or CD4^-CD8⁺) thymocytes was markedly reduced and accompanying TUNEL assays indicated an increase in apoptotic cells in the thymus (123). Combined with steroid receptor blocking experiments, these findings also suggested that corticosterone-dependent apoptosis is responsible for thymic cell reduction during short-term HU. Another study revealed that osteopontin is involved in HU-induced thymic apoptosis by regulating corticosterone levels during a 3-day HU (124). Subsequent studies have demonstrated that circulatory osteopontin can interfere with the hypothalamus-pituitary-adrenal (HPA) axis, thus regulating steroid hormone production and modulating stress responses (125), although the precise mechanisms behind this regulation have not been elucidated. In contrast, long-term HU does not selectively reduce CD4⁺CD8⁺ thymocytes, despite a decrease in overall thymic mass (126). Instead, long-term HU led to significant decline in medullary thymic epithelial cells (TECs), particularly those expressing high levels of CD80 and the autoimmune regulator (AIRE). Consistent with this, the expression of tissue-specific antigens was downregulated in the thymus of long-term HU-loaded mice (126). Together, these findings indicate that the effects of HU are distinctly time-dependent: short-term HU selectively induces corticosterone-driven apoptosis in CD4⁺CD8⁺ thymocytes, while long-term HU impacts all thymocytes in a non-selective manner, along with the AIRE⁺ mTEC (i.e., mTEC^hi) population, likely through apoptosis-independent mechanisms, despite the persistently elevated corticosterone levels in either condition.

Several studies have indicated that spaceflight induces thymic involution in mice with similar lesions to those observed in the HU model (74, 75, 86, 87, 118, 126), suggesting that hindlimb unloading may effectively replicate some aspects of spaceflight conditions. However, a key limitation of the HU model is that it not only simulates weightlessness, but also induces psychological stress in mice, which can act as a model for depression (127). This introduces complexity in interpreting results, because it becomes challenging to differentiate whether thymic atrophy is due to stress, musculoskeletal changes, fluid redistribution, or a combination of these factors. All these conditions are present under both microgravity and hindlimb unloading environments.

Circadian rhythms control many aspects of human physiology, affecting daily variations in body temperature, blood pressure, and hormone levels and coordinate function across different organ systems, including neurological, metabolic, endocrine, cardiovascular, and immune (128). Circadian rhythmicity in the body is entrained by photic cues and a tight network of central and peripheral clocks enabled by a neural pacemaker directly responsive to environmental and behavioral states such as the sleep-wake cycles, feeding, metabolic cues, and secretion of hormones (particularly glucocorticoids) (129–131).

Circadian derailment is considered a risk factor during space missions by NASA. During space flight, astronauts are exposed to changes in microgravity, which impose pathophysiological effects on circadian rhythmicity, leading to derailment as a consequence of disturbed sleep, wakefulness, and feeding patterns (30, 31). Astronauts working at the ISS experience 16 sunrises and sunsets within a 24-hour period, impairing the 24-hour diurnal cycle experienced on earth. Even more so, the profound workload during space missions, which requires astronauts to complete highly complex tasks during long periods of time, contributes to the disruption of sleep-wakefulness cycles that collectively affect the body’s physiological diurnal rhythms (132–135). Derailment of circadian rhythm affects human health as increased occurrence of cardiovascular disease (CVD) (136), metabolic disorders (137), and cancer (138–140) were reported to be associated with shift work or frequent time zone travel. Coupled with other hazards of spaceflight, derailment of circadian oscillations during space missions may result in considerable risk to astronaut health, including not only sleep deprivation and diminished alertness, loss of cognitive abilities, depression, and anxiety (141, 142), but also the development of metabolic syndrome, CVD and cancer.

The hematopoietic and immune systems are particularly sensitive to circadian derailment. Mobilization and trafficking of leukocytes and hematopoietic stem and progenitor cells (HSPCs) between lymphoid organs and other tissues in the body is tightly regulated by central and peripheral clocks (34, 143–145). Innate immune cells (including granulocytes, monocytes, and macrophages) and T and B cells exhibit strong circadian oscillations in peripheral blood, peaking during the behavioral rest phase (daytime in rodents, and at night in humans) (146–148). Oscillation of blood lymphocytes was demonstrated to depend on glucocorticoids, catecholamines, and hypoxia-inducible factor 1a (HIF-1a) (149–151) that mediate rhythmic expression of chemokine receptors (e.g., CXCR4, CXCR5, CCR7, CX₃CR1) that oscillate in phase with tissue-specific chemokines (e.g., CXCL12 in bone marrow and lung and CCL21 in lymph nodes) and endothelial adhesion molecules, including P and E-Selectin, Intercellular adhesion molecule-1 (ICAM-1), ICAM-2, and vascular cell adhesion molecule-1 (VCAM-1) across lymphoid and other organs, (including liver, skin, gut and lung) (146). Besides leukocyte trafficking and recruitment into tissues, recent studies have demonstrated that innate and adaptive immune responses depend on circadian rhythmicity, including response to pathogens, B cell development, and T cell differentiation. Circadian control of immune response is not the scope of this review; a detailed summary of this topic can be found elsewhere (34, 152).

Whether and how derailment of circadian rhythmicity affects thymic function and T cell development is less known. Although it has been shown that loss of intrinsic circadian rhythms by deletion of the master clock regulator Brain and Muscle Arnt-like protein-1 (Bmal1) in thymocytes does not affect T cell development (153), CD4⁺ single positive (SP4) thymocyte emigration from the thymus was shown to be regulated by circadian rhythms, as well as rhythmic expression of emigration related-molecules sphingosine 1-phosphate receptor (S1PR1) and C-C chemokine receptor 2 (CCR2) (154). As spaceflight and altered microgravity were shown to induce thymic involution (75, 87), it is yet to be determined to what extent the derailment of circadian rhythmicity contributes to thymic dysfunction. Future studies utilizing ground-based models of acute and chronic jet lag will directly test this question and determine how circadian derailment affects thymic structure and functionality. However, as spaceflight is associated with microgravity disruption, which can also contribute to impaired circadian rhythmicity (155–158), a combination of jet lag with hindlimb unloading may be necessary to properly simulate spaceflight conditions that derail circadian rhythmicity. Furthermore, it will be important to show to what extent derailment of circadian rhythmicity during spaceflight contributes to the development of CVD and cancer, as defective immune response contributes to the pathogenicity of either condition.

During prolonged space missions, astronauts are exposed to extreme environments for extended durations, potentially leading to adverse physical and mental health effects, such as depression and cognitive impairment. The concept of “long-term spaceflight composite stress” (LSCS) encapsulates the multifaceted sources of stress encountered in space (142). Among these, psychosocial stress stands out as a significant contributor, distinct from well-known hazards like cosmic radiation, microgravity, and circadian disruptions (142). Instead, it arises from factors such as social isolation, confinement in cramped and crowded spaces, cultural differences and conflicts among crew members, homesickness, performance anxiety, and persistent noise from the onboard equipment (e.g., fans, exercise machines, life-support systems) (142). However, studying the isolated effects of psychosocial stressors on normal physiology, and the immune system, in ground-based rodent models presents significant challenges. The multifactorial nature of these stressors is inherently difficult to replicate in controlled laboratory settings (159–164). Additionally, fundamental differences between humans and rodents further complicate such models: the human brain, with its unparalleled complexity and advanced cognitive and emotional capacities, processes psychosocial stimuli in ways that are not easily mirrored in rodent counterparts (165).

The effects of LSCS have been previously studied, although psycho-social factors have not been isolated from other spaceflight-associated stressors. For example, a 42-day simulation combining microgravity, isolation, noise, circadian rhythm disturbances, and low pressure demonstrated significant weight loss, anxiety, memory deficits, and depression in rats. These behavioral changes correlated with reduced postsynaptic density thickness and synaptic interface curvature, indicating impaired synaptic plasticity in the hippocampus of LSCS-exposed rats (166–168). While a connection between depression and immune system dysregulation is loosely supported (169), direct evidence linking LSCS to immunological and thymic functions is still lacking.