Spaceflight can pose novel challenges to the health of astronauts. For instance, physiological aging occurs significantly faster as a result of spaceflight, when measured by muscle wasting, loss of bone density, and immune dysfunction (1, 2). Processes regulated by redox imbalance may contribute to these adverse outcomes (3–9). Redox imbalance results from a disproportionate increase in reactive oxygen species (ROS) produced by the mitochondria (10) compared to antioxidants in the cell. Elevated ROS is also a product of the oxidative burst response of neutrophils (11). In response to stimuli, terminally differentiated neutrophils in circulation become activated and engage the oxidative burst response, producing inflammatory mediators (11). If left unchecked, elevated ROS can cause cellular damage that potentiates inflammation both on Earth and during spaceflight (4, 12). Therefore, it is necessary to maintain tight regulation of the oxidative burst response to limit inflammation (13) and regulate immunity during prolonged spaceflight.

Neutrophils are granulocytes that constitute approximately 50–70% of the total leukocyte population in humans. Neutrophils are the first responders to infection or injury and are typically short-lived in blood circulation under homeostatic conditions (14, 15). Lymphocytes are an important group of white blood cells involved in both innate and adaptive immunity. They constitute 20–50% of total leukocytes in circulation and consist of natural killer, natural killer T cells, innate lymphoid cells, T cells, and B cells (16, 17). On Earth, elevated neutrophil-to-lymphocyte ratio (NLR) is a useful biomarker to measure subclinical inflammation in humans (18). Chronic, persistent inflammation can be a major pre-existing cause of disease development (19, 20) and can be monitored by the expression of blood-based biomarkers. For example, elevated NLR predicts poor prognosis in some cancers (21–24), positively correlates with age (25), and reflects chronic stress in mice (26). Although, elevated human NLR (>3.53) (27) has been implicated in clinical settings to identify heightened inflammation (18), this biomarker has not yet been recognized for spaceflight-induced inflammation. Spaceflight raises circulating white blood cell (WBC) counts, primarily granulocytes, may reduce lymphocyte counts (12, 28, 29), and impairs immune cell functions (6, 12, 30, 31). Although, the spaceflight environment elevates circulating blood granulocytes counts in astronauts (29), the underlying molecular mechanisms remain elusive. Currently, there are no well-established biomarkers for astronauts on long-duration, deep space missions, where medical intervention will be limited. Thus, identifying biomarkers to monitor in-flight astronaut health and developing countermeasures that reverse these adverse outcomes are necessary for successful future missions to the lunar surface and Mars. Therefore, we propose that an elevated NLR may be a useful prognostic indicator or diagnostic biomarker to assess astronaut immune status during long-duration missions.

To test this, we analyzed both spaceflight-treated, and ground-based simulated microgravity-treated, samples to determine if NLR was elevated. Analyses of complete blood count (CBC) leukocyte differentials revealed spaceflight caused a progressive increase of granulocyte-to-lymphocyte (GLR) in astronauts and NLR in rodents. To simulate microgravity using established methods, human leukocytes were cultured in high-aspect rotating wall vessels (HARV-RWV) in vitro and mice were hindlimb unloaded (HU) in vivo (32, 33). HARV-RWV is a bioreactor allowing 3D-spatial freedom for cells and can model microgravity. It has two unique aspects similar to the spaceflight-associated microgravity environment, (1) a state of constant suspension, and (2) a quiescent surrounding without any shear or turbulent forces. Previous studies have determined leukocyte responses utilizing HARV-RWV produce similar responses as leukocytes cultured post-landing or ex vivo in flight (34–42). Hindlimb unloading (HU) is a ground-based model mimicking spaceflight-associated microgravity in rodents. The hindlimbs of rodents are elevated to produce 30–40 degree head-down tilt, inducing a cephalad fluid shift and preventing weightbearing of hindlimbs (43). The HU model can lead to immune, bone, and musculoskeletal alterations, some of which have also been observed in International Space Station (ISS) crew (44, 45). In our study, functional outputs of neutrophils in response to simulated microgravity (sµg) revealed elevated ROS and proinflammatory myeloperoxidase (MPO) expression in activated neutrophils. Interestingly, this effect could be mitigated with antioxidant treatment. Furthermore, sµg HU wildtype (Wt) mice displayed elevated neutrophils, NLR and marginal inflammation, which was dampened in mitochondrial catalase (mCAT) transgenic mice, known to show reduced oxidative stress responses. Our findings demonstrated that, albeit distinct mechanisms, both sµg models (in vitro HARV-RWV and in vivo HU), displayed elevated oxidative stress and NLR, that could be mitigated by antioxidants. Therefore, modifying mechanisms involved in ROS-driven inflammation (46) may provide a promising avenue to limit chronic inflammation and maintain homeostatic immunity during long-duration missions.

Spaceflight causes immune dysfunction that can lead to health risks for astronauts. Health risks that arise from immune dysfunction are complex and include an inability to defend against pathogens, altered tolerance to self-antigens resulting in potential autoimmunity development, chronic inflammation, and immune senescence. Therefore, mitigation during spaceflight will likely require selective targeting of specific immune cell types and/or developmental stages. Elevated NLR may be a prospective biomarker candidate to identify immune deviations that can cause disease. Currently, elevated NLR is used as a clinical biomarker to detect sub-clinical inflammation in humans and predicts poor prognosis in cancer (18). In this study, a spike in GLR in humans and NLR in rodents was observed at landing and elevated GLR and NLR were observed during spaceflight ( Figure 1 ). In humans, GLR was elevated at 180-days in-flight, suggesting prolonged exposure to spaceflight caused leukocyte differential changes. This change in immune differentials may be in response to elevated inflammation experienced in-flight (4, 6, 12), since elevated, chronic inflammation is often coupled with immune dysfunction and disease development (13, 19, 20, 67–70). Therefore, monitoring distinct biomarkers, such as elevated NLR, can determine when countermeasures can intervene to avert immune dysfunction, promote immune recovery, and prevent disease development.

Elevated numbers of granulocytes and neutrophils were observed in vitro following 20 h of HARV-RWV modeled microgravity in human peripheral blood ( Figure 2 and Figure S1 ). However, since mature neutrophils are terminally differentiated in blood circulation, i.e., banded or segmented neutrophils, we estimate that this increased percentage is due to increased scatter properties indicative of cellular activation. Indeed, elevated cell surface expression of CD66b and CD11b per granulocyte, which are activation markers for neutrophils (58, 59), were increased in sµg ( Figure 3 and Figure S1 ). Further studies revealed elevated ROS and MPO expression ( Figure 3 ), confirming HARV-RWV sµg resulted in granulocytes, and in particular, neutrophil activation. Physiological effects of elevated active granulocytes or neutrophils in circulation intensifies sterile inflammation (70–72), promotes edema, and non-specific tissue damage (73), and can threaten astronaut health if not adequately controlled. Interestingly, MPO gene expression ( Figure 5D ) was elevated in HU, suggesting immature neutrophil entry into blood circulation, compared to neutrophil activation observed in vitro in the HARV-RWV. MPO gene synthesis only occurs in bone marrow early in neutrophil development, i.e. immature neutrophils (myeloblasts, promyelocytes, and myelocytes). MPO gene expression ends once neutrophils differentiate into metamyelocytes (74) and synthesized MPO protein is packaged into granules released during neutrophil activation (75). Typically, immature neutrophils are not released into blood circulation unless the body is in a diseased or inflammatory state (74, 75). However, since MPO gene expression was elevated in HU mice blood, this suggests the potential for myelocyte/immature neutrophil infiltration and may also serve as a biomarker for elevated inflammation during spaceflight. To our knowledge no measurements have been recorded for elevated immature neutrophils in blood circulation in-flight; however, elevated neutrophils were identified in blood from 9 of 16 astronauts at landing (6, 76, 77). As compared to the rodent spaceflight results (in-flight day 14), elevated neutrophils were also observed at days 14 and 30 of HU in mice ( Figures 4C and 5A ), suggesting the physical effects of fluid-shifting experienced during spaceflight and HU may stimulate the release of neutrophils into circulation; however this requires further investigation.

Indeed, elevated MPO during sµg may contribute to immune dysfunction. MPO catalyzes hydrogen peroxide into reactive intermediates that can damage proteins, lipids, and DNA (67). Excess MPO impairs phagocytic function (67–69) and triggers neutrophil degranulation, causing inflammatory tissue damage (78) in cardiovascular disease (62, 67, 79). Pathologically this is relevant during spaceflight, as cardiovascular disease is a prominent risk factor associated with returned astronauts (80). Furthermore, elevated NLR is currently used as a predictor of cardiovascular disease risk on Earth (81), thereby highlighting the clinical relevance of monitoring NLR during spaceflight. Neutrophil oxidative burst responses and elevated ROS can induce cellular death (82), including lymphocyte apoptosis (83), and suppression of T lymphocyte function (84). HARV-RWV sµg induced ROS in granulocytes, indicating HARV-RWV sµg activated granulocytes and triggered the oxidative burst response. Thus HARV-RWV sµg can serve as a valuable model to study ROS-induced inflammation (72, 85). Indeed, redox imbalance occurs in humans and cell cultures exposed to spaceflight (4–6) and in vitro ground-based sµg models (8, 9, 86). The cause of elevated ROS in sµg may be due to: (1) cell death factors or other unknown stimulators of the oxidative burst response, (2) a mechanosensitive stress receptor in phagocytes that triggers redox imbalance (87), and/or (3) a combination of these effects. In our study, HARV-RWV sµg induced active Caspase 3/7 expression in lymphocyte populations ( Figure 2D ), indicating lymphocyte apoptosis and shifting of immune differentials to favor higher NLR and GLR. Further analyses of our in vivo HU studies revealed no difference in lymphocyte percent or absolute count populations ( Figures 4 and 5 , and Figures S2 and 3 ) compared to HARV-RWV sµg studies, suggesting elevated lymphocyte recovery or unknown ROS-quenching mechanisms that limit lymphocyte apoptosis, both of which require further studies.

Therefore, the two sµg models, HARV-RWV and HU, displayed different mechanisms towards generating an elevated NLR. The HARV-RWV microgravity model appears to display robust lymphocyte turnover, i.e. elevated lymphocyte apoptosis, and most likely immune function that may differ from astronauts in-flight. However, this does not rule out that lymphocyte apoptosis does not occur in vivo HU, as turnover of lymphocytes to replace loss most likely occurs, albeit apoptosis may occur at a slower rate than in vitro HARV-RWV. Furthermore, ROS concentration within each sµg model may be drastically different. For example, ROS levels in HARV-RWV may be much higher in the absence of in vivo ROS quenchers compared to 14- or 30-day HU, which would affect the rate of lymphocyte apoptosis (88, 89). Indeed, concentration and exposure time of ROS determines cellular responses. Homeostatic levels of ROS can promote cell survival, while elevated ROS (oxidative stress) can induce cellular death (82). In line with this, the timeline of measurements of lymphocyte counts (20 h HARV-RWV versus 14- and 30-days HU) differ between the two models; therefore direct comparisons cannot be assumed. Finally, the HARV-RWV model cultured human blood samples, which have different leukocyte percentages compared to mice leukocytes in the HU model; therefore the kinetics of apoptosis across the two microgravity models would also be affected. Crucian et al. showed there is an elevation of granulocytes in blood circulation, while no differences are observed in lymphocyte absolute counts, suggesting lymphocytes may not undergo apoptosis in spaceflight; rather there may be release of more granulocytes into blood circulation (29). Controversially however, multiple reports have indicated lymphocytes and lymphocyte-like cell lines undergo apoptosis during spaceflight/microgravity conditions (36–42), albeit measurements were either reported post-landing or from ex vivo cell cultures in flight. In fact, the role of apoptosis in lymphocyte depression (ROALD) experiment that was part of the BIO-4 mission and comprised of ESA, Energia, and NASA agencies, was performed with the goal to understand how microgravity affects lymphocyte apoptosis. The results showed after 48 h on-board the ISS ex vivo cultures of lymphocytes displayed increased DNA fragmentation, PARP protein expression, and elevated p53 expression, compared to ground controls (36). Due to this, we believe the in vivo HU microgravity model, although having its own limitations, may be a better representative ground-based model for spaceflight. Nonetheless, additional studies are required to better understand the degree of lymphocyte turnover during in vivo HU that is comparable to spaceflight.

Monocytes were significantly reduced following HARV-RWV ( Figure 2B ); however no differences were observed in HU mice ( Figures S2D, E ), further indicating the variability between the two simulated microgravity models. Yet, inconsistency with these cell types in terms of population differentials have also been noted across spaceflight literature (77, 90, 91), which may be a factor of sampling timepoints. However, consensus suggests phagocytic function of monocytes following spaceflight is impaired (92, 93). Phagocytic impairment of monocytes can directly affect clearance of neutrophils from circulation, inflammation resolution (94), and can impact NLR. Therefore, further research into the function and distribution of monocytes following simulated microgravity are currently underway.

Transgenic mice expressing the human antioxidant gene catalase reversed HU-induced elevation of NLR and dampened inflammatory gene expression ( Figure 5 ), suggesting redox imbalance caused leukocyte differential changes. In line with this, mice deficient in apolipoprotein E (ApoE), a protein with antioxidant activity, display elevated ROS expression and activated neutrophils (95). In our study, HARV-RWV sµg of human leukocytes induced neutrophil activation that was reversed in the presence of the antioxidant NAC ( Figure 3D ), further suggesting antioxidants can suppress inflammation. Indeed, in vivo NAC treatment successfully ameliorated acetic acid-induced colitis by reversing pro-inflammatory mediators TNF-α, IL-6, and MPO in rats (96). Collectively, these results indicate antioxidants as viable countermeasures to regulate spaceflight-induced inflammation and immune dysfunction.

Clinically, elevated NLR (>3.53) (27) is a prognostic indicator for cancer development, cardiovascular disease, inflammation, and infectious conditions (18, 21–23, 25), but no NLR standard has been established for astronaut immunity. Our results revealed elevated GLR in astronauts at landing (GLR = 3.6, Figure 1B ), compared to clinically relevant GLR (>2.24) (97), may result in biological significance for astronaut health. Although restoration to normal GLR occurred at landing on Earth, landing on the lunar surface and/or Mars, where gravity is less than Earth’s, may pose a significant risk to astronaut immune recovery. Therefore, monitoring and developing countermeasures to mitigate elevated NLR, GLR, and inflammatory neutrophil phenotypes for future long-duration and long-distance space travel are essential for mission success.

In summary, we identified increased GLR and NLR in both human and rodent spaceflight samples and ground simulations of microgravity. Our results in vitro indicated that leukocytes shift in favor of elevated activated inflammatory neutrophils, which may amplify disease development in vivo. Further, antioxidants may be useful countermeasures to ameliorate these outcomes in sµg, as NAC treatment inhibited activated inflammatory human neutrophils in HARV-RWV and catalase partially mitigated elevated NLR in HU mice. Based on these findings, we suggest monitoring both in-flight and landing NLR to assess astronaut immune status. We further advocate the investigation of antioxidants as future countermeasures to mitigate immune deviations, including elevated NLR and inflammation, to safeguard astronaut health on future missions.

The data analyzed in this study are subject to the following licenses/restrictions: NASA Life Sciences Data Archive (LSDA) is an active repository for astronaut health datasets. Requests for dataset access is required. Requests to access these datasets should be directed to https://lsda.jsc.nasa.gov.

The studies involving human participants were reviewed and approved by NASA Ames Institutional Review Board (IRB, 201791646CTO-02, HR-357 and HR-358) with informed consent from each blood donor. Astronaut and rodent CBC data sets were approved for use by the electronic (e)IRB/Life Sciences Data Archive (LSDA) advisory board (#11028). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by NASA Ames Research Center Institutional Animal Care and Use Committee (IACUC, NAS-17-001-Y2).

AMP conceived/performed majority of the experiments and wrote/prepared the manuscript. SDM performed experiments and contributed to manuscript preparation. EC provided human blood samples and edited the manuscript. A-SS, CGTT, and RKG performed the HU animal experiments, provided mouse blood samples, and edited the manuscript. SA assisted with statistical testing and edited the manuscript. RG, BEC, and SB edited the manuscript and provided intellectual advice. SB provided funding for the study. BEC provided human LSDA-sourced data sets and edited the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported in part by Universities Space Research Association (USRA) and NASA’s Space Biology Program post-doctoral fellowship (to AMP) and NASA’s Space Biology Grant # NNX15AB42G (to SB).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.