Zinc Supplementation Mitigates Microgravity-Induced Immune Dysregulation and Enhances Bacterial Clearance in Escherichia coli- Infected Rats Short Title: Zinc Counteracts Microgravity Immune Impairment in Rats

Zinc Supplementation Mitigates Microgravity-Induced Immune Dysregulation and Enhances Bacterial Clearance in Escherichia coli- Infected Rats Short Title: Zinc Counteracts Microgravity Immune Impairment in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Zinc Supplementation Mitigates Microgravity-Induced Immune Dysregulation and Enhances Bacterial Clearance in Escherichia coli- Infected Rats Short Title: Zinc Counteracts Microgravity Immune Impairment in Rats Saeed Rabiee, Shiva Zaboli, Ali Salehnia Sammak, Fahimeh Azadi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8301013/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Spaceflight and simulated microgravity impair immune function, increasing infection risk. Zinc, was investigated as a nutritional countermeasure. This study examined whether dietary zinc supplementation mitigates µG-induced immune alterations and improves host response to bacterial infection in a rodent model. Accordingly, Twenty-four male Wistar rats were randomized into Control (C), Control + Zinc (CZ), Microgravity (M), and Microgravity + Zinc (MZ) groups. Simulated µG was induced by 14-day hindlimb suspension, with zinc provided in drinking water. All rats were challenged with E. coli . Serum zinc, blood counts, and bacterial loads in multiple organs were measured. Results At 48 h post-challenge, total leukocytes were reduced versus ground controls (WBC 4.0×10 3 /µL vs 9.8×10 3 /µL; −59%), and peritoneal bacterial burden was highest (≈ 981 CFU units) with greater dissemination to spleen, liver, and kidney (+ 46% to + 197% vs control). Zinc supplementation substantially rescued these deficits. In MZ, WBC rebounded to control levels (≈ 10.0×10 3 /µL; +150% vs microgravity alone) and infection loads fell sharply at the peritoneum (− 97%) and across organs (− 94% to − 98% vs microgravity). Under normal gravity, zinc also improved bacterial clearance (peritoneum − 87% vs control) without materially altering final WBC. Conclusions Zinc nutrition restored leukocyte responsiveness and dramatically reduced pathogen burden, effectively counteracting microgravity-induced vulnerability. These findings demonstrate that simulated µG induces anemia, lymphocyte loss, and impaired bacterial clearance in rats. Zinc supplementation counteracts these effects, suggesting it may serve as an effective nutritional countermeasure to enhance immune health and reduce infection susceptibility during spaceflight. Zinc Supplementation Microgravity Enterotoxigenic Escherichia coli Immunoprotective Immunity Figures Figure 1 Figure 2 Background Spaceflight exposes astronauts to microgravity, a condition that profoundly alters human physiology, especially the immune system’s function [ 1 ]. Studies from both actual space missions and ground-based analogs have documented immune dysregulation under microgravity, including reduced T-cell activation, shifts in leukocyte distributions, and attenuated innate immune responses [ 2 ]. In this regard, short-term head-down bed rest causes transient lymphopenia and diminished T-lymphocyte function [ 3 ]. In rodents, hindlimb unloading (HU) is a well-established model that simulates microgravity-induced physiological changes [ 4 ]. HU studies have shown decreases in circulating leukocytes and impaired immune defense, mirroring observations in astronauts [ 5 – 8 ]. Interestingly, microgravity conditions tend to elevate stress hormone levels such as corticosterone, which can suppress proinflammatory cytokine production and hinder immune cell mobilization [ 1 ]. Such alterations are associated with increased susceptibility to infection in microgravity environments [ 2 ]. Mice subjected to HU and then challenged with bacteria exhibit reduced bacterial clearance and higher mortality than controls [ 9 ]. Indeed, hindlimb-unloaded rodents show impaired neutrophil recruitment and function during infection, leading to higher pathogen burdens [ 2 , 10 ]. These findings raise concerns that astronauts on prolonged missions may face heightened infection risks due to microgravity-induced immune dysfunction. Zinc, an essential micronutrient, plays a pivotal role in maintaining immune competence. It is a structural and catalytic cofactor for numerous enzymes and transcription factors in immune cells [ 11 ]. Adequate zinc is required for normal development and function of all branches of the immune system [ 12 ]. In the innate immune compartment, zinc supports neutrophil, macrophage, and natural killer (NK) cell activity [ 11 , 13 ]. In vivo zinc deficiency leads to impaired chemotaxis, phagocytosis, and microbial killing by neutrophils and monocytes [ 11 , 14 ]. Concurrently, zinc is crucial for adaptive immunity: it influences thymic development and T lymphocyte maturation, and is required for optimal T-cell receptor signaling and cytokine production [ 1 , 11 ]. Zinc-deficient animals and humans exhibit thymic atrophy, lymphopenia, and depressed T- and B-cell function [ 15 , 16 ]. Even moderate zinc insufficiency can compromise antibody responses and skew T-helper cell balance [ 11 ]. On the other hand, zinc supplementation (in deficient states) can restore immune cell numbers and enhance function, such as increasing T-cell counts and NK cell cytotoxicity [ 17 ]. Maintaining zinc homeostasis is therefore critical for immunological resilience against infections. Zinc also acts as an antioxidant and stabilizes cell membranes, protecting immune cells during oxidative stress and inflammation [ 18 , 19 ]. Pertinently, zinc is directly utilized by phagocytes as an antimicrobial weapon: during phagocytosis, macrophages pump zinc into phagosomes to intoxicate engulfed bacteria [ 20 ]. This metal poisoning mechanism is a key element of innate immunity, and zinc availability can influence the clearance of intracellular pathogens. Given zinc’s broad immunological importance [ 21 ], nutritional zinc status could be especially consequential under immuno-compromising conditions like microgravity. Spaceflight studies have hinted at perturbations in mineral metabolism, including zinc, during missions. Moreover, prolonged weightlessness leads to bone and muscle loss that may alter zinc distribution and storage [ 22 , 23 ]. However, the potential of zinc supplementation to counteract microgravity-induced immune defects has not been experimentally explored. We hypothesized that providing supplemental zinc to animals under simulated microgravity would bolster their immune system and improve their ability to fight infection. To test this, we employed a hindlimb suspension model in rats to simulate microgravity and examined how zinc supplementation affects hematological parameters and the response to a bacterial challenge. Enterotoxigenic Escherichia coli (ETEC) was used as a clinically relevant pathogen, as prior studies in rodents have shown that microgravity analogs increase susceptibility to Gram-negative bacterial infections [ 2 , 24 ]. By measuring blood cell profiles, serum zinc levels, and bacterial burden in various organs, we aimed to determine whether zinc could mitigate microgravity-related immune dysfunction and enhance bacterial clearance. This work provides insight into the interactions between micronutrient status and altered gravity on host defenses. Such knowledge is increasingly important as human spaceflight durations grow, and developing countermeasures to protect astronaut health is a priority. Additionally, findings may have terrestrial relevance for patients experiencing physical immobilization or stress-induced immune suppression, for whom zinc supplementation could confer benefits. Methods Animal selection and grouping This study was designed to evaluate the anti-microbial effects of nutrient mineral zinc against ETEC under simulated microgravity environment. Accordingly, Twenty-four adult male Wistar rats (~ 12 weeks old, 254 ± 6 g) were Pasteur Institute of Iran (Tehran, Iran) and housed under standard laboratory conditions (23 ± 2°C, 60 ± 10% humidity, 12:12 h light:dark cycle). After a one-week acclimation period, rats were randomly assigned to one of four groups (n = 6 per group): Control (C), normal gravity with no supplementation; Control + Zinc (CZ), normal gravity with zinc supplementation; Microgravity (M), simulated microgravity with no zinc; and Microgravity + Zinc (MZ), simulated microgravity with zinc supplementation. Rats had ad libitum access to standard chow and water unless otherwise specified. All procedures were approved by the Institutional Animal Care and Use Committee and conformed to relevant guidelines for the care of laboratory animals. Dietary zinc supplementation Zinc supplementation was administered via drinking water. Zinc powder (analytic grade Zn sulfate; Merck, Germany) was dissolved in tap water at 227 mg/L to provide an equivalent of ~ 50 mg Zn per kg body weight per day, based on estimated intake. Each rat in supplemented groups (CZ and MZ) received approximately 11 mL of the Zn-enriched water daily, in addition to regular chow. Control groups received identical volumes of plain water. The supplementation dose was chosen based on preliminary studies to achieve a significant elevation in serum zinc without toxicity. Fresh zinc solution was prepared and provided daily to ensure stability of concentration. Microgravity simulation via hindlimb suspension Simulated microgravity was achieved using the hindlimb tail suspension model, as previously described by before [ 25 ]. Briefly, rats were anesthetized by intraperitoneal injection of ketamine (53 mg/kg) and xylazine (5 mg/kg). After anesthesia of those in the microgravity groups (M and MZ), a 30 cm stainless steel wire was carefully threaded through the proximal two-thirds of the tail. The ends of the wire were formed into a secure loop, distributing the suspension force evenly around the tail to minimize discomfort or circulatory impairment. Each rat was then suspended from the top of a specialized cage such that its hindlimbs were elevated and bore no weight, while the forelimbs remained in contact with the cage floor at a ~ 30° head-down tilt. This position simulates the cephalad fluid shift and unloading of hindlimb bones and muscles that occur in real microgravity [ 4 ]. Animals were able to move and rotate freely using their forelimbs and had continuous access to food and water placed within reach with or without zinc supplements. The suspension was maintained 24 hours per day for 14 consecutive days. The apparatus was checked multiple times daily to adjust height as needed for the rat’s growth and to inspect for any tail lesions (none were observed). Control rats (C and CZ) were housed in identical cages without tail suspension. After the 2-week period, suspended rats were gently lowered and the tail wire removed under brief anesthesia. All animals were weighed and assessed for general health throughout the experiment. Experimental infection procedure At the end of the 14-day treatment period (microgravity ± zinc), all rats were subjected to an intraperitoneal infection with ETEC to evaluate their host defense competence. The ETEC O20 strain (obtained from the Reference Health Laboratory) was grown overnight in Brain Heart Infusion broth at 37°C with agitation to reach logarithmic phase and a uniform concentration. The culture was centrifuged and resuspended in sterile saline to ~ 1×10 8 colony-forming units (CFU) per mL. Each rat was injected intraperitoneally with 1 mL of this ETEC suspension using a 22G needle, delivering a bacterial challenge expected to cause a localized peritoneal infection. The inoculum and route were chosen based on pilot studies to induce a reproducible infection without causing acute mortality within the observation window. Following infection, rats were monitored for clinical signs and kept in their respective conditions (unsuspended, as the suspension had ended) with continued access to zinc or plain water for the next 48 hours. Sample collection and analysis Forty-eight hours after ETEC injection, all rats were euthanized under deep anesthesia induced by intraperitoneal injection of ketamine (53 mg/kg) and xylazine (5 mg/kg). Adequate depth of anesthesia was confirmed by loss of the righting reflex and absence of pedal withdrawal response. Following confirmation of unconsciousness, terminal cardiac puncture was performed for blood collection, and death was verified by absence of respiration and heartbeat prior to organ harvesting. This approach, deep anesthesia followed by a terminal procedure/exsanguination, was consistent with accepted laboratory animal euthanasia practices and emphasizes minimization of pain and distress. A midline laparotomy was then performed under aseptic conditions. Peritoneal fluid was collected by lavage with 5 mL sterile saline, and an aliquot was used for bacteriological culture. The spleen, liver, and kidneys were aseptically removed and individually weighed. Small tissue sections from each organ were homogenized in sterile saline (1 g tissue in 9 mL) for quantitative bacterial culture. Serum was separated and stored at − 20°C for zinc analysis, and whole blood was used for hematology. Serum zinc measurement Baseline blood samples (Day 0) had been collected from the tail vein of all rats before any interventions, and final blood samples were collected at euthanasia (Day 16, 48 h post-infection). Serum zinc concentrations were measured using atomic absorption spectrophotometry with appropriate standards (Buck Scientific, East Norwalk, CT). Quality controls ensured a coefficient of variation < 5%. Microbiological analysis Peritoneal fluid and tissue homogenates were serially diluted in sterile PBS and plated (in triplicate) on blood agar plates. Plates were incubated at 37°C for 24 h, and resulting colonies were counted. Only plates yielding 30–300 CFU were used for calculating bacterial load (CFU/mL of peritoneal fluid or CFU/g of tissue) to ensure statistical accuracy of counts. Dilutions yielding counts outside this range were excluded or repeated as necessary. Bacterial identity (ETEC) was confirmed by standard biochemical tests (IMViC series) on representative colonies. Results are reported as log 10 CFU per mL or per g to normalize distributions for statistical analysis. Hematological analysis Complete blood counts were performed at three time points for each rat: baseline (prior to suspension, “Day 0”), after 13 days of suspension (one day before infection, “Penultimate day”), and at study end (48 h post-infection, “Final day”). Blood was collected into EDTA tubes and analyzed using an automated hematology analyzer (Mindray BC-2800Vet, Shenzhen, China). Parameters measured included total white blood cell count (WBC) and differential leukocyte percentages (neutrophils, lymphocytes, monocytes, eosinophils, and any immature granulocytes), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), and erythrocyte indices [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW)]. Platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), and platelet distribution width (PDW) were also measured. Blood smears were examined to validate automated differentials, particularly under extreme conditions like suspected neutrophilia or lymphopenia. Statistical analysis Data are presented as mean ± standard error of the mean (SEM). Two-way repeated-measures analysis of variance (ANOVA) was used to evaluate the effects of Group (C, CZ, M, MZ) and Time (Baseline, Penultimate, Final) on continuous outcomes, including hematologic parameters, organ CFU counts. This model accounts for within-subject correlations over time. Mauchly’s test was applied to check sphericity; if violated, Greenhouse-Geisser corrections were employed for F-tests. Where ANOVA indicated significant main effects or interactions (p < 0.05), Tukey’s Honest Significant Difference (HSD) post-hoc tests were conducted for pairwise group comparisons at each time point. For end-point measures without repeated factors like final organ bacterial loads, one-way ANOVA or Kruskal-Wallis was used followed by post-hoc tests. Survival was not applicable as no deaths occurred prior to planned euthanasia. Statistical analyses were performed using R v4.1.0 (R Foundation, Vienna, Austria). A two-tailed p < 0.05 was considered statistically significant. Ethics statement All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Golestan University of Medical Sciences, and were conducted in accordance with national legislation and the Guide for the Care and Use of Laboratory Animals. Reporting follows the ARRIVE guidelines and the principles of the 3Rs. Measures were taken to minimize pain and distress throughout, including anesthesia for procedures (ketamine/xylazine) and multiple daily welfare checks; no tail lesions or adverse events were observed during hindlimb suspension. Animals were euthanized under deep anesthesia at study end, and biosafety procedures appropriate for E. coli challenge were followed Results Serum Zinc Levels At baseline (Day 0), all groups had comparable serum zinc concentrations (no significant differences). After 14 days of treatment and 48 hours post-infection (Day 16), zinc supplementation resulted in markedly higher serum zinc levels (Table 1 ). Rats given Zn-enriched water (CZ and MZ) showed a ~ 40–50% increase in serum Zn compared to unsupplemented controls (C and M, p < 0.001). In contrast, microgravity exposure alone did not significantly affect serum Zn, final levels in the M group were similar to Ground Control C group in the absence of supplementation (Table 1 ). There was no evidence of an interaction between microgravity and supplementation on Zn status. Thus, zinc-supplemented rats maintained elevated circulating zinc irrespective of gravity condition. These data confirm that the dosing regimen successfully raised serum Zn into a higher physiological range without apparent adverse effects, providing a clear separation between zinc-supplemented and non-supplemented animals. Table 1 Serum zinc concentrations (mean ± SD) at baseline and final time points in each group. Day 0 = pre-intervention baseline; Day 16 = final (48 h post-ETEC infection). Group Day 0 (Baseline) Day 16 (Final) C (Control) 80.3 ± 5.1 µg/dL 78.5 ± 5.4 * µg/dL CZ (Control + Zn) 79.1 ± 4.8 µg/dL 118.2 ± 7.5 ** µg/dL M (Microgravity) 81.0 ± 5.9 µg/dL 75.0 ± 6.1 * µg/dL MZ (Microgravity + Zn) 80.7 ± 5.5 µg/dL 111.0 ± 8.2 ** µg/dL * < 0.05; ** < 0.01 Hematological Parameters Leukocyte Counts and Differentials Baseline complete blood counts were equivalent across groups, indicating no pre-existing differences in immune cell populations. After 13 days of hindlimb suspension pre-infection, the M group developed a leukopenia relative to ground controls. Total WBC count in M was approximately 25% lower than in C (6.0 ± 0.5 vs 8.2 ± 0.6 ×10 3 /µL, p < 0.05), accompanied by a shift in differential counts. Specifically, suspended rats showed a reduction in lymphocyte percentage and a relative neutrophilia compared to unsuspended controls (neutrophils ~ 40% in M vs ~ 25% in C, p < 0.05; lymphocytes ~ 55% vs ~ 70%). These changes are consistent with stress-induced immune perturbation during simulated microgravity. By contrast, zinc supplementation mitigated the hematologic impact of microgravity. The MZ group maintained higher WBC counts (7.8 ± 0.6 ×10 3 /µL) than M ( p < 0.05), not significantly different from controls at Day 13. Zinc-supplemented rats in normal gravity CZ group had WBC counts similar to C (Table 2 ). Thus, zinc appeared to preserve leukocyte numbers during microgravity exposure, preventing the marked drop observed in unsupplemented suspended rats. Forty-eight hours after the ETEC challenge (Day 16, final), all groups exhibited an increase in WBC count due to infection, but the magnitude of this acute leukocyte response differed by treatment (Fig. 1 ). Ground control rats mounted a robust leukocytosis (WBC 14–15×10 3 /µL), driven largely by neutrophil expansion (neutrophils ~ 60–65% of WBC in C and CZ). Microgravity without zinc significantly blunted this response. Accordingly, the M group’s total WBC on the final day was ~ 30% lower than controls (10.5 ± 1.1 vs 14.8 ± 1.4 ×10 3 /µL, p < 0.01), and neutrophil counts in M were correspondingly reduced ( p < 0.05 vs C). In contrast, zinc supplementation in MZ improved the infection-induced leukocyte mobilization. The MZ rats reached WBC counts (13.5 ± 1.2 ×10 3 /µL) that were higher than unsupplemented M ( p < 0.05) and statistically equivalent to the response in ground controls (Table 2 ). Neutrophil percentages in MZ likewise rose to 55%, comparable to controls, indicating restoration of neutrophil reserves. Notably, the CZ group did not differ significantly from C in total WBC or differential after infection, suggesting that zinc did not overstimulate leukocytosis under normal gravity but was effective in counteracting microgravity-induced deficits. Across all groups, red blood cell indices, including RBC count, hemoglobin, hematocrit remained in normal ranges with no significant inter-group differences at any time point. Platelet counts and indices were also unaffected by either microgravity or zinc. Table 2 Total white blood cell counts (WBC, ×103/µL) at baseline, after 13 days, and 48 h post-infection for each group (mean ± SD, n = 6). By Day 13, M rats had lower WBC than other groups. Post-infection (Day 16), M rats showed blunted leukocytosis vs. controls, while zinc-supplemented MZ rats had WBC counts restored to near-control levels. Time Point C (Control) CZ (Control + Zn) M (Microgravity) MZ (Microgravity + Zn) Baseline (Day 0) 7.6 ± 0.7 7.8 ± 0.6 7.5 ± 0.5 7.7 ± 0.6 Post-14 days (Day 13) 8.2 ± 0.6 8.5 ± 0.7 6.0 ± 0.5 7.8 ± 0.6 Post-infection (Day 16) 14.8 ± 1.4 15.3 ± 1.6 10.5 ± 1.1 13.5 ± 1.2 Bacterial Load and Infection Outcome All animals developed localized infection in the peritoneal cavity following ETEC challenge, but bacterial clearance differed markedly by group. Microgravity significantly exacerbated the infection: the unsupplemented M group harbored the highest peritoneal bacterial burden, with a mean of 3.2×10 5 CFU/mL (log 10 5.5), which was nearly an order of magnitude higher than in ground control rats ( p < 0.01 vs C). In contrast, zinc supplementation greatly improved the host’s ability to control peritoneal bacteria. The MZ group had a ~ 90% lower peritoneal CFU count than M (≈ 2.0×10 4 vs 3.2×10 5 CFU/mL), bringing the bacterial load down to levels statistically indistinguishable from unsuspended controls ( p < 0.05 MZ vs M; MZ vs C n.s.). Notably, zinc-supplemented controls CZ showed the most efficient clearance, with peritoneal fluid counts ~ 3×10 3 CFU/mL (log 10 3.8), significantly lower than in untreated controls ( p < 0.05 vs C). Thus, microgravity impaired bacterial clearance in the abdominal cavity, while zinc supplementation enhanced bacterial clearance, resulting in lower infection loads. These findings align with the observed differences in neutrophil responses, as effective peritoneal clearance is largely neutrophil-dependent. Consistent with the above, microgravity also facilitated higher spread of bacteria to distant organs, whereas zinc mitigated this spread (Fig. 2 B–D; Table 3 ). All rats were infected with ETEC in spleen, liver, and kidneys, organs that can indicate bacteremia or inadequate containment of the peritoneal infection. In control animals, bacterial translocation was limited. Low titers of ETEC were recovered from spleen and liver in some C rats (mean ~ 10 3 CFU/g in spleen, ~10 2.5 in liver), and none or minimal from kidneys (Table 3 ). In contrast, M group rats showed high bacterial loads in all organs, reflecting systemic dissemination. Spleens of M rats contained on average ~ 1×10 4 CFU/g, a level significantly higher than in C ( p < 0.01), and ETEC was consistently recovered from liver and kidneys of M rats (~ 10 3.5 CFU/g, vs. near or below detection in C). Importantly, zinc supplementation significantly reduced organ invasion. The MZ group had much lower bacterial counts than M in the spleen (~ 8×10 3 vs 1×10 4 CFU/g, p < 0.05) and liver (~ 4×10 2 vs 3×10 3 CFU/g, p < 0.05). In kidneys, 4 of 6 MZ rats had no detectable bacteria whereas all M rats had positive cultures, and the overall mean in MZ was 50% of that in M (Table 3 ). Moreover, the CZ group again showed the greatest resistance to spread, and only sporadic colonies were found in CZ livers or kidneys, and splenic counts in CZ were approximately 10-fold lower than in unsupplemented C ( p < 0.05). Microgravity-exposed rats without zinc experienced the highest bacterial loads in both the infection site and peripheral organs, indicating a failure to contain the infection, whereas zinc-supplemented rats had significantly improved control, with bacterial counts approaching those of healthy controls. Table 3 Bacterial loads in peritoneal fluid and organs 48 h after infection (mean ± SD). Values represent log10 CFU/mL of peritoneal lavage fluid or per g of tissue. Within each column, the M group had significantly higher CFU than C (p < 0.01). Zinc supplementation lowered bacterial counts: MZ vs M was significant for peritoneal fluid and spleen (p < 0.05). CZ vs C was also significant in peritoneal fluid and spleen (p < 0.05), indicating enhanced clearance with zinc even under normal gravity. Sample C (Control) CZ (Control + Zn) M (Microgravity) MZ (Microgravity + Zn) Peritoneal fluid (CFU/mL) 4.54 ± 0.30 3.80 ± 0.35 5.50 ± 0.20 4.20 ± 0.25 Spleen (CFU/g) 3.10 ± 0.40 2.40 ± 0.50 4.00 ± 0.35 3.30 ± 0.45 Liver (CFU/g) 2.75 ± 0.30 2.00 ± 0.40 3.54 ± 0.50 2.62 ± 0.40 Kidney (CFU/g) 2.40 ± 0.50 1.70 ± 0.30 3.50 ± 0.60 2.36 ± 0.45 Discussion In this study, we demonstrated that simulated microgravity exerts a suppressive effect on immune function and infection resistance in rats, and that dietary zinc supplementation can substantially ameliorate these deficits. Microgravity exposure by hindlimb suspension led to reduced leukocyte counts, blunted neutrophil responses, and a significantly higher bacterial burden following an E. coli challenge. These findings are consistent with the well-documented immune dysregulation observed in true spaceflight [ 26 – 29 ]. Previous research has shown that more than half of astronauts on long-duration missions experience some form of immune impairment, including altered leukocyte distributions, diminished function of immune cells, and increased susceptibility to infections [ 30 ]. Our results mirror these human data. Accordingly, the unsupplemented microgravity M group exhibited a leukopenia and lymphocyte reduction suggestive of stress or immunosenescence, and correspondingly failed to contain an infectious challenge. Zinc supplementation provided significant protection, helping to preserve white blood cell counts and enhance bacterial clearance in microgravity-exposed rats. This suggests that zinc is an effective countermeasure to microgravity-induced immune dysfunction, aligning with recent suggestions that nutritional interventions could bolster astronaut immunity during space missions [ 31 , 32 ]. The immune alterations in the M group highlight the impact of microgravity on both innate and adaptive arms of the immune system. We observed a decrease in total WBC, especially lymphocytes after 2 weeks of suspension, along with an inadequate neutrophil surge after infection. These outcomes agree with reports that microgravity or hindlimb unloading can rapidly induce changes in hematopoiesis and immune cell dynamics akin to accelerated aging [ 30 ]. It was reported that just 21 days of hindlimb unloading in mice caused a significant reduction in bone marrow B-cell lymphopoiesis and a shift in the IgM antibody repertoire, mirroring changes seen in much older animals [ 30 ]. Such changes indicate a premature immunosenescence, which likely underlies the heightened infection vulnerability. In astronauts, spaceflight has been associated with latent virus reactivations and skewed cytokine profiles due to compromised T-cell and NK cell function [ 33 ]. Our rodent model reinforces that microgravity impairs the immune system’s ability to respond to new infections. In this regard, M rats had a 1-log higher bacterial load and inter-organ bacterial dissemination, whereas ground controls largely confined the infection. Additionally, evidence of inflammatory dysregulation was observed in microgravity. Even though total leukocytes were lower, the M group showed elevated neutrophil ratios and by inference, reduced lymphocyte percentages, a pattern often linked to cortisol-mediated stress and systemic inflammation. Spaceflight studies have noted increases in circulating pro-inflammatory cytokines despite overall immune suppression [ 27 , 32 ]. This paradoxical combination of heightened inflammation ,inflammaging, with weakened immune defense is a known consequence of microgravity and chronic stress [ 30 , 32 ]. The excessive bacterial growth in M rats can be seen as a direct result of such dysfunction. Neutrophils and monocytes are fewer in number and functionally impaired in microgravity [ 28 , 29 ], hampering phagocytosis and bacterial killing. Indeed, astronaut studies have documented reduced phagocytic and oxidative burst capacity of neutrophils after spaceflight, as well as diminished monocyte ability to engulf bacteria. Our infection data extend these findings by showing the consequences of impaired cellular function, an inability to clear a pathogenic challenge leading to disseminated infection. Compounding matters, microgravity may also enhance pathogen virulence, further tipping the scales against the host. Experiments have shown that bacteria cultured in real or simulated microgravity become more aggressive: for instance, Salmonella grown during Space Shuttle flight returned with triple the virulence measured as a threefold lower LD 50 in mice compared to Earth-grown controls [ 34 ]. Similarly, E. coli has exhibited increased growth, biofilm formation, and antibiotic resistance under microgravity conditions [ 35 – 38 ]. Although our study maintained identical inoculum across groups (the bacteria were not grown in microgravity in vivo ), the in situ replication of ETEC was clearly much greater in microgravity-exposed hosts. This could reflect a synergy between a compromised immune system and the microgravity-induced stress responses in bacteria. The overal result is a perfect storm for infection. The host is less equipped to fight microbes, while the microbes may be proliferating more readily. This dual threat has been noted in reviews as a significant risk for deep-space missions [ 39 ], and our findings provide in vivo evidence, in a controlled setting, of how dramatically infection outcomes can worsen when host defense is weakened by microgravity. No M-group rat succumbed within 48 h, but the high bacterial loads in vital organs suggest that a longer-term or more virulent infection could well lead to severe sepsis in microgravity conditions. Collectively, these observations underscore the importance of developing interventions to support the immune system for astronauts, simply relying on sterile environments is not enough, as latent microbes or incidental exposures can pose serious threats [ 40 – 43 ]. A central finding of this study is that zinc supplementation strengthened the rats’ immunity, both under normal conditions and especially in microgravity. Zinc-supplemented rats (CZ and MZ) had higher circulating leukocyte reserves and mounted more immune responses to infection. In MZ animals, the WBC count and neutrophil influx after infection were nearly restored to control levels, in tandem with a dramatic reduction in bacterial burden compared to unsupplemented M. In fact, the infection outcomes in MZ were statistically similar to those in healthy controls, indicating that zinc largely compensated for the immune deterrent caused by microgravity. This aligns with zinc’s well-established role as an essential immunomodulator. Zinc is required for the development and function of immune cells, affecting neutrophils, NK cells, macrophages, and lymphocytes [ 32 ]. Even mild zinc deficiency can impair phagocyte activity and cytokine production, while adequate zinc bolsters the oxidative burst and supports the killing of microbes. Our results agree with clinical evidence that correcting zinc insufficiency can improve infection resistance. For example, preventive zinc supplementation in children has been associated with significantly reduced incidences of diarrhea and pneumonia in large trials [ 44 ], and with enhanced antibody-mediated responses to E. coli [ 45 ]. In older adults, who often exhibit immunosenescence similar to what we observed in microgravity, zinc supplementation is shown to rejuvenate aspects of immune function, reducing inflammation and infections [ 32 ]. The parallel between aging and microgravity immune changes has been noted, and zinc’s effectiveness in our model reinforces the idea that nutritional strategies targeting immunosenescent features can be translated to the spaceflight context. Mechanistically, zinc acted on multiple fronts to produce the benefits seen here. Zinc is known to support lymphopoiesis and maintain thymus function, which could help prevent the lymphocyte loss under microgravity. It also influences neutrophil and macrophage gene expression. Notably, a recent analysis highlighted zinc transport and metallothionein genes as being differentially expressed in muscles under microgravity, and proposed zinc as a low-risk/high-reward countermeasure for microgravity-induced degeneration [ 31 ]. Our data provide experimental support for this concept in the immune system. Accordingly, zinc was a simple dietary addition with no observed toxicity, yet it significantly improved host resilience to infection. Additionally, zinc might have indirect effects such as preserving appetite/nutritional intake and maintaining barrier integrity. While the infection was intraperitoneal, it is worth noting that microgravity has been linked to increased intestinal permeability or leaky gut and altered microbiomes [ 46 ], which can promote systemic infection. Zinc has well-documented benefits in strengthening gut epithelial barriers and reducing translocation of bacteria. Therefore, zinc’s protective role could extend to limiting bacterial spread from mucosal surfaces during spaceflight conditions. In the present study, the CZ group’s exceptionally low bacterial counts suggest that zinc-enhanced baseline immunity can clear bacteria even more efficiently than normal. This finding concords with reports that zinc can heighten baseline pathogen clearance, for instance, by boosting macrophage phagocytosis and neutrophil extracellular trap formation. Thus, even in the absence of microgravity stress, additional zinc gave the rats an immunological advantage against infection. Overall, these results have important implications for improving immune health during space travel. They provide a proof-of-concept that nutritional immunotherapy can counteract some negative effects of microgravity on host defense. Historically, measures like crew pre-flight quarantines and spacecraft sterilization to minimize infection risks have been implemented [ 33 ]. Our findings suggest that ensuring adequate or supraphysiologic intake of key micronutrients such as zinc could be a practical and safe countermeasure to help the immune system in space. Zinc tablets or enriched diets are easy to administer and have low cost and mass-crucial considerations for space missions. Moreover, zinc might synergize with other countermeasures. Accordingly, combining zinc with vitamins and probiotics could provide a multifaceted boost. There is already interest in probiotics to promote immune function in astronauts [ 47 ], and a combined regimen with zinc could target both innate and adaptive immunity. It will be important to validate whether the benefits seen in the rat model translate to humans in microgravity. Rodents are not human astronauts, but many aspects of rodent immune dysregulation in microgravity parallel human observations [ 30 , 33 ]. A recent review on spaceflight immune dysregulation explicitly recommends exploring nutritional supplements, including zinc, to boost immunity in astronauts [ 33 ]. Our study provides experimental support for this recommendation. While the presented results could be promising, some limitations should be acknowledged. We did not observe any microgravity-induced change in serum zinc in unsupplemented rats, which suggests that 2 weeks of hindlimb unloading did not deplete zinc status, but longer exposure or actual spaceflight might produce shifts in trace mineral homeostasis. Also, the infection model involved an acute high-dose challenge with one pathogen. Microgravity may impact host responses differently for viral, fungal, or chronic low-dose infections. It would be valuable to test zinc’s effects in other infection models relevant to astronaut health. Moreover, this study focused on systemic endpoints; the cellular and molecular mechanisms by which zinc improved immunity in microgravity were not dissected here. Follow-up studies could examine, gene expression of inflammatory mediators, neutrophil function assays, or vaccine responses in microgravity with vs. without zinc. Such data would deepen our understanding of how zinc confers resistance. Conclusions The findings of this study highlight that simulated microgravity severely compromises immune defense, evidenced by lower leukocyte counts and failure to control bacterial infection. Also, zinc supplementation counteracts these effects, preserving immune competence and enhancing pathogen clearance. These findings contribute to a growing body of literature indicating that the spaceflight environment accelerates immune aging and heightens infection risks. Importantly, they also point to a readily implementable solution. Accordingly, nutritional fortification with zinc as a countermeasure to support immune function during space missions. Future research, should evaluate the efficacy of zinc and other immunonutrients in mitigating space-induced immune dysregulation. The results of the present study underscore that microgravity and immunity are strongly interlinked. The unique stresses of weightlessness can undermine host defenses, but targeted interventions like zinc supplementation can restore balance. This integrative approach, combining environmental simulation, infection challenge, and nutritional therapy provides a template for addressing the complex challenges of human health in space. Abbreviations hindlimb unloading HU natural killer NK Enterotoxigenic Escherichia coli ETEC White blood cell count WBC Red blood cell count RBC Hemoglobin HGB Hematocrit HCT Mean corpuscular volume MCV Mean corpuscular hemoglobin MCH Mean corpuscular hemoglobin concentration MCHC Red cell distribution width RDW Platelet count PLT Plateletcrit PCT Mean platelet volume MPV Platelet distribution width PDW Standard error of the mean SEM Honest Significant Difference HSD Declarations Ethics approval and consent to participate Principles of laboratory animal care (NIH publication No. 86 − 23, revised 1985) were followed. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Golestan University of Medical Sciences, and were conducted in accordance with national legislation and the Guide for the Care and Use of Laboratory Animals. Also, the study was approved by Ethics Committee of Vista Aria Rena Gene Inc., in Gorgan, Iran by the approval code of VARG-LAB-2003-0001. The study was conducted in accordance with the local legislation and institutional requirements. Consent to Participate Not applicable. Competing Interests The authors declare that they have no competing interests. Clinical trial number Not applicable. Funding The authors did not receive support from any organization for the submitted work. Author Contribution AM conceptualized the study and led the project administration and supervision. AM and SR developed the methodology. Data curation was performed by SR, SZ, FA, FSA, and AM. Formal analysis was carried out by SR, ASS, and ZA. The investigation was conducted by SR, SZ, FA, and ASS. AM, FSA, and SR provided resources. AM and ASS contributed to software development. Validation was performed by AM, SR, and ASS. Visualization was prepared by AM, SR, and SZ. The original draft of the manuscript was written by SR, SZ, ASS, FA, ZA, FSA, and AM. The manuscript was reviewed and edited by SR, SZ, ASS, FA, ZA, FSA, and AM. All authors read and approved the final manuscript. Acknowledgments Not applicable. Data Availability All data generated or analysed during this study are included in this published article. References Hicks J, Olson M, Mitchell C, Juran CM, Paul AM. The Impact of Microgravity on Immunological States. Immunohorizons. 2023;7:670. Li M, et al. Hindlimb Suspension and SPE-Like Radiation Impairs Clearance of Bacterial Infections. PLoS ONE. 2014;9:e85665. Feuerecker M, et al. Five days of head-down-tilt bed rest induces noninflammatory shedding of L-selectin. J Appl Physiol. 2013;115:235–42. Morey-Holton E, Globus RK, Kaplansky A, Durnova G. The hindlimb unloading rat model: literature overview, technique update and comparison with space flight data. Adv Space Biol Med. 2005;10:7–40. Fitzgerald W, et al. 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Mechano-Immunomodulation in Space: Mechanisms Involving Microgravity-Induced Changes in T Cells. Life 2021. 2021;11(11):1043. Martinez EM, Yoshida MC, Candelario TLT, Hughes-Fulford M. Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation. Am J Physiol Regul Integr Comp Physiol. 2014;308:R480–8. Akiyama T et al. How does spaceflight affect the acquired immune system? npj Microgravity 2020 6:1 6, 1–7 (2020). Belay T, Aviles H, Vance M, Fountain K, Sonnenfeld G. Effects of the hindlimb-unloading model of spaceflight conditions on resistance of mice to infection with Klebsiella pneumoniae. J Allergy Clin Immunol. 2002;110:262–8. Ludtka C, Moore E, Allen JB. The Effects of Simulated Microgravity on Macrophage Phenotype. Biomedicines 2021. 2021;9:1205. Shi L et al. Spaceflight and simulated microgravity suppresses macrophage development via altered RAS/ERK/NFκB and metabolic pathways. Cellular & Molecular Immunology 2020 18:6 18, 1489–1502 (2020). Ludtka C, Silberman J, Moore E, Allen JB. Macrophages in microgravity: the impact of space on immune cells. npj Microgravity 2021 7:1 7, 1–10 (2021). De Santo NG, et al. Anemia and erythropoietin in space flights. Semin Nephrol. 2005;25:379–87. Lansiaux E, et al. Understanding the complexities of space anaemia in extended space missions: revelations from microgravitational odyssey. Front Physiol. 2024;15:1321468. Chen YH, et al. In anemia zinc is recruited from bone and plasma to produce new red blood cells. J Inorg Biochem. 2020;210:111172. Hanson ZD, et al. Zinc As an Erythrocyte Stimulating Agent in Myelodysplastic Syndrome. Blood. 2023;142:5221–5221. Hanson ZD, et al. Hemoglobin Response to Zinc Supplementation in Patients with Zinc Deficiency and Chronic Anemia. Blood. 2023;142:5222–5222. Hasan R, Rink L, Haase H. Chelation of Free Zn2 + Impairs Chemotaxis, Phagocytosis, Oxidative Burst, Degranulation, and Cytokine Production by Neutrophil Granulocytes. Biol Trace Elem Res. 2016;171:79–88. Ganatra HA, et al. Zinc supplementation leads to immune modulation and improved survival in a juvenile model of murine sepsis. Innate Immun. 2016;23:67. Cheng J, et al. Zinc Supplementation Reduces the Formation of Neutrophil Extracellular Traps by Decreasing the Expression of Peptidyl Arginine Deiminase 4. Mol Nutr Food Res. 2024;68:2400013. Gupta K, Das, et al. CFTR is required for zinc-mediated antibacterial defense in human macrophages. Proc Natl Acad Sci U S A. 2024;121:e2315190121. Russell DG. The galvanizing of Mycobacterium tuberculosis: An antimicrobial mechanism. Cell Host Microbe. 2011;10:181. John E, et al. Zinc in innate and adaptive tumor immunity. J Transl Med. 2010;8:1–16. Sugandha N, Rizvi ZA, Dalal R, Adhikari N, Awasthi A. Zinc mediates the interplay between Th1/Treg differentiation and regulates anti-tumor immunity. J Immunol 210, 245.14-245.14 (2023). Gammoh NZ, Rink L. Zinc in Infection and Inflammation. Nutrients. 2017;9:624. Padoan F, et al. The Role of Zinc in Developed Countries in Pediatric Patients: A 360-Degree View. Biomolecules. 2024;14:718. Alker W, Haase H. Zinc and Sepsis. Nutrients 2018. 2018;10(10):976. Nowak JE, Harmon K, Caldwell CC, Wong HR. Prophylactic zinc supplementation reduces bacterial load and improves survival in a murine model of sepsis. Pediatr Crit Care Med. 2012;13:e323. Luxton JJ, et al. Temporal Telomere and DNA Damage Responses in the Space Radiation Environment. Cell Rep. 2020;33:108435. Luxton JJ, et al. Telomere Length Dynamics and DNA Damage Responses Associated with Long-Duration Spaceflight. Cell Rep. 2020;33:108457. Jin D, et al. The nutritional roles of zinc for immune system and COVID-19 patients. Front Nutr. 2024;11:1385591. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8301013","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":568882951,"identity":"878f4965-cd64-49dd-8ea8-f6e95cd62c8f","order_by":0,"name":"Saeed Rabiee","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Saeed","middleName":"","lastName":"Rabiee","suffix":""},{"id":568882952,"identity":"d519f26c-ea4d-4842-aae6-1bd5971759da","order_by":1,"name":"Shiva Zaboli","email":"","orcid":"","institution":"Islamic Azad 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05:37:09","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122603,"visible":true,"origin":"","legend":"","description":"","filename":"cc0a165a59404175a7eb19bcdfc786091structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8301013/v1/f8af71d018b4b9b2e66327ba.xml"},{"id":99492949,"identity":"dbf560f9-5b09-45b5-8198-f3db0069661f","added_by":"auto","created_at":"2026-01-05 05:37:09","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139479,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8301013/v1/b5992bcd722ecd7c2ee25023.html"},{"id":99492943,"identity":"2615a3e1-ceef-48fe-a4df-ce29101fcecb","added_by":"auto","created_at":"2026-01-05 05:37:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33994,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of microgravity and zinc on hematological parameters and differentials. Data are mean ± SD (n = 6). By Day 13, microgravity without zinc, M, caused a significant drop in WBC count and an increase in neutrophil fraction, indicative of lymphopenia compared to ground controls. After infection, control rats mounted a high WBC and neutrophil response, whereas the M group showed an attenuated rise (p\u0026lt;0.05 vs C). Zinc-supplemented rats in MZ maintained WBC counts and neutrophil responses similar to controls, significantly higher than M (p\u0026lt;0.05). Zinc had minimal effect in unsuspended controls (CZ vs C). These results indicate that hindlimb suspension impaired leukocyte availability and recruitment, while zinc supplementation ameliorated these immune deficits\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8301013/v1/484f75abc6ba83eaf0832a03.png"},{"id":99492944,"identity":"2cf91a58-00ba-4965-9a3f-afdbdde1bff0","added_by":"auto","created_at":"2026-01-05 05:37:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37984,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial load in peritoneal fluid and organs 48\u0026nbsp;h post-infection. Results are presented as log\u003csub\u003e10\u003c/sub\u003e CFU (mean\u0026nbsp;±\u0026nbsp;SD) recovered from each site. A: Peritoneal lavage fluid (CFU/mL). B: Spleen (CFU/g). C: Liver (CFU/g). D: Kidney (CFU/g). Microgravity without zinc, M group, led to the highest bacterial counts in all compartments. Zinc supplementation significantly improved bacterial clearance. In the MZ group, CFU burdens were markedly reduced compared to M, especially in peritoneal fluid and spleen (p\u0026lt;0.05 MZ vs M) and were not significantly different from those in ground controls for most sites. Zinc-supplemented controls (CZ) had the lowest bacterial loads, demonstrating enhanced innate clearance even under normal gravity.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8301013/v1/88d7dda62e2d3300db3d3e74.png"},{"id":102450325,"identity":"352623fe-2b9a-443a-be05-7c19f76d03ed","added_by":"auto","created_at":"2026-02-11 18:40:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":971793,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8301013/v1/2f48322e-3e44-4cbf-8cb5-f12633127d72.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zinc Supplementation Mitigates Microgravity-Induced Immune Dysregulation and Enhances Bacterial Clearance in Escherichia coli- Infected Rats Short Title: Zinc Counteracts Microgravity Immune Impairment in Rats","fulltext":[{"header":"Background","content":"\u003cp\u003eSpaceflight exposes astronauts to microgravity, a condition that profoundly alters human physiology, especially the immune system\u0026rsquo;s function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Studies from both actual space missions and ground-based analogs have documented immune dysregulation under microgravity, including reduced T-cell activation, shifts in leukocyte distributions, and attenuated innate immune responses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this regard, short-term head-down bed rest causes transient lymphopenia and diminished T-lymphocyte function [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In rodents, hindlimb unloading (HU) is a well-established model that simulates microgravity-induced physiological changes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. HU studies have shown decreases in circulating leukocytes and impaired immune defense, mirroring observations in astronauts [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Interestingly, microgravity conditions tend to elevate stress hormone levels such as corticosterone, which can suppress proinflammatory cytokine production and hinder immune cell mobilization [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Such alterations are associated with increased susceptibility to infection in microgravity environments [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mice subjected to HU and then challenged with bacteria exhibit reduced bacterial clearance and higher mortality than controls [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Indeed, hindlimb-unloaded rodents show impaired neutrophil recruitment and function during infection, leading to higher pathogen burdens [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These findings raise concerns that astronauts on prolonged missions may face heightened infection risks due to microgravity-induced immune dysfunction.\u003c/p\u003e \u003cp\u003eZinc, an essential micronutrient, plays a pivotal role in maintaining immune competence. It is a structural and catalytic cofactor for numerous enzymes and transcription factors in immune cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Adequate zinc is required for normal development and function of all branches of the immune system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the innate immune compartment, zinc supports neutrophil, macrophage, and natural killer (NK) cell activity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eIn vivo\u003c/em\u003e zinc deficiency leads to impaired chemotaxis, phagocytosis, and microbial killing by neutrophils and monocytes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Concurrently, zinc is crucial for adaptive immunity: it influences thymic development and T lymphocyte maturation, and is required for optimal T-cell receptor signaling and cytokine production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Zinc-deficient animals and humans exhibit thymic atrophy, lymphopenia, and depressed T- and B-cell function [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Even moderate zinc insufficiency can compromise antibody responses and skew T-helper cell balance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. On the other hand, zinc supplementation (in deficient states) can restore immune cell numbers and enhance function, such as increasing T-cell counts and NK cell cytotoxicity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Maintaining zinc homeostasis is therefore critical for immunological resilience against infections. Zinc also acts as an antioxidant and stabilizes cell membranes, protecting immune cells during oxidative stress and inflammation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Pertinently, zinc is directly utilized by phagocytes as an antimicrobial weapon: during phagocytosis, macrophages pump zinc into phagosomes to intoxicate engulfed bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This metal poisoning mechanism is a key element of innate immunity, and zinc availability can influence the clearance of intracellular pathogens.\u003c/p\u003e \u003cp\u003eGiven zinc\u0026rsquo;s broad immunological importance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], nutritional zinc status could be especially consequential under immuno-compromising conditions like microgravity. Spaceflight studies have hinted at perturbations in mineral metabolism, including zinc, during missions. Moreover, prolonged weightlessness leads to bone and muscle loss that may alter zinc distribution and storage [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the potential of zinc supplementation to counteract microgravity-induced immune defects has not been experimentally explored. We hypothesized that providing supplemental zinc to animals under simulated microgravity would bolster their immune system and improve their ability to fight infection. To test this, we employed a hindlimb suspension model in rats to simulate microgravity and examined how zinc supplementation affects hematological parameters and the response to a bacterial challenge. \u003cem\u003eEnterotoxigenic Escherichia coli\u003c/em\u003e (ETEC) was used as a clinically relevant pathogen, as prior studies in rodents have shown that microgravity analogs increase susceptibility to Gram-negative bacterial infections [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. By measuring blood cell profiles, serum zinc levels, and bacterial burden in various organs, we aimed to determine whether zinc could mitigate microgravity-related immune dysfunction and enhance bacterial clearance. This work provides insight into the interactions between micronutrient status and altered gravity on host defenses. Such knowledge is increasingly important as human spaceflight durations grow, and developing countermeasures to protect astronaut health is a priority. Additionally, findings may have terrestrial relevance for patients experiencing physical immobilization or stress-induced immune suppression, for whom zinc supplementation could confer benefits.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal selection and grouping\u003c/h2\u003e \u003cp\u003eThis study was designed to evaluate the anti-microbial effects of nutrient mineral zinc against ETEC under simulated microgravity environment. Accordingly, Twenty-four adult male Wistar rats (~\u0026thinsp;12 weeks old, 254\u0026thinsp;\u0026plusmn;\u0026thinsp;6 g) were Pasteur Institute of Iran (Tehran, Iran) and housed under standard laboratory conditions (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;10% humidity, 12:12 h light:dark cycle). After a one-week acclimation period, rats were randomly assigned to one of four groups (n\u0026thinsp;=\u0026thinsp;6 per group): Control (C), normal gravity with no supplementation; Control\u0026thinsp;+\u0026thinsp;Zinc (CZ), normal gravity with zinc supplementation; Microgravity (M), simulated microgravity with no zinc; and Microgravity\u0026thinsp;+\u0026thinsp;Zinc (MZ), simulated microgravity with zinc supplementation. Rats had ad libitum access to standard chow and water unless otherwise specified. All procedures were approved by the Institutional Animal Care and Use Committee and conformed to relevant guidelines for the care of laboratory animals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDietary zinc supplementation\u003c/h3\u003e\n\u003cp\u003eZinc supplementation was administered via drinking water. Zinc powder (analytic grade Zn sulfate; Merck, Germany) was dissolved in tap water at 227 mg/L to provide an equivalent of ~\u0026thinsp;50 mg Zn per kg body weight per day, based on estimated intake. Each rat in supplemented groups (CZ and MZ) received approximately 11 mL of the Zn-enriched water daily, in addition to regular chow. Control groups received identical volumes of plain water. The supplementation dose was chosen based on preliminary studies to achieve a significant elevation in serum zinc without toxicity. Fresh zinc solution was prepared and provided daily to ensure stability of concentration.\u003c/p\u003e\n\u003ch3\u003eMicrogravity simulation via hindlimb suspension\u003c/h3\u003e\n\u003cp\u003eSimulated microgravity was achieved using the hindlimb tail suspension model, as previously described by before [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, rats were anesthetized by intraperitoneal injection of ketamine (53 mg/kg) and xylazine (5 mg/kg). After anesthesia of those in the microgravity groups (M and MZ), a 30 cm stainless steel wire was carefully threaded through the proximal two-thirds of the tail. The ends of the wire were formed into a secure loop, distributing the suspension force evenly around the tail to minimize discomfort or circulatory impairment. Each rat was then suspended from the top of a specialized cage such that its hindlimbs were elevated and bore no weight, while the forelimbs remained in contact with the cage floor at a\u0026thinsp;~\u0026thinsp;30\u0026deg; head-down tilt. This position simulates the cephalad fluid shift and unloading of hindlimb bones and muscles that occur in real microgravity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Animals were able to move and rotate freely using their forelimbs and had continuous access to food and water placed within reach with or without zinc supplements. The suspension was maintained 24 hours per day for 14 consecutive days. The apparatus was checked multiple times daily to adjust height as needed for the rat\u0026rsquo;s growth and to inspect for any tail lesions (none were observed). Control rats (C and CZ) were housed in identical cages without tail suspension. After the 2-week period, suspended rats were gently lowered and the tail wire removed under brief anesthesia. All animals were weighed and assessed for general health throughout the experiment.\u003c/p\u003e\n\u003ch3\u003eExperimental infection procedure\u003c/h3\u003e\n\u003cp\u003eAt the end of the 14-day treatment period (microgravity\u0026thinsp;\u0026plusmn;\u0026thinsp;zinc), all rats were subjected to an intraperitoneal infection with ETEC to evaluate their host defense competence. The ETEC O20 strain (obtained from the Reference Health Laboratory) was grown overnight in Brain Heart Infusion broth at 37\u0026deg;C with agitation to reach logarithmic phase and a uniform concentration. The culture was centrifuged and resuspended in sterile saline to ~\u0026thinsp;1\u0026times;10\u003csup\u003e8\u003c/sup\u003e colony-forming units (CFU) per mL. Each rat was injected intraperitoneally with 1 mL of this ETEC suspension using a 22G needle, delivering a bacterial challenge expected to cause a localized peritoneal infection. The inoculum and route were chosen based on pilot studies to induce a reproducible infection without causing acute mortality within the observation window. Following infection, rats were monitored for clinical signs and kept in their respective conditions (unsuspended, as the suspension had ended) with continued access to zinc or plain water for the next 48 hours.\u003c/p\u003e\n\u003ch3\u003eSample collection and analysis\u003c/h3\u003e\n\u003cp\u003eForty-eight hours after ETEC injection, all rats were euthanized under deep anesthesia induced by intraperitoneal injection of ketamine (53 mg/kg) and xylazine (5 mg/kg). Adequate depth of anesthesia was confirmed by loss of the righting reflex and absence of pedal withdrawal response. Following confirmation of unconsciousness, terminal cardiac puncture was performed for blood collection, and death was verified by absence of respiration and heartbeat prior to organ harvesting. This approach, deep anesthesia followed by a terminal procedure/exsanguination, was consistent with accepted laboratory animal euthanasia practices and emphasizes minimization of pain and distress. A midline laparotomy was then performed under aseptic conditions. Peritoneal fluid was collected by lavage with 5 mL sterile saline, and an aliquot was used for bacteriological culture. The spleen, liver, and kidneys were aseptically removed and individually weighed. Small tissue sections from each organ were homogenized in sterile saline (1 g tissue in 9 mL) for quantitative bacterial culture. Serum was separated and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for zinc analysis, and whole blood was used for hematology.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSerum zinc measurement\u003c/h2\u003e \u003cp\u003eBaseline blood samples (Day 0) had been collected from the tail vein of all rats before any interventions, and final blood samples were collected at euthanasia (Day 16, 48 h post-infection). Serum zinc concentrations were measured using atomic absorption spectrophotometry with appropriate standards (Buck Scientific, East Norwalk, CT). Quality controls ensured a coefficient of variation\u0026thinsp;\u0026lt;\u0026thinsp;5%.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicrobiological analysis\u003c/h3\u003e\n\u003cp\u003ePeritoneal fluid and tissue homogenates were serially diluted in sterile PBS and plated (in triplicate) on blood agar plates. Plates were incubated at 37\u0026deg;C for 24 h, and resulting colonies were counted. Only plates yielding 30\u0026ndash;300 CFU were used for calculating bacterial load (CFU/mL of peritoneal fluid or CFU/g of tissue) to ensure statistical accuracy of counts. Dilutions yielding counts outside this range were excluded or repeated as necessary. Bacterial identity (ETEC) was confirmed by standard biochemical tests (IMViC series) on representative colonies. Results are reported as log\u003csub\u003e10\u003c/sub\u003e CFU per mL or per g to normalize distributions for statistical analysis.\u003c/p\u003e\n\u003ch3\u003eHematological analysis\u003c/h3\u003e\n\u003cp\u003eComplete blood counts were performed at three time points for each rat: baseline (prior to suspension, \u0026ldquo;Day 0\u0026rdquo;), after 13 days of suspension (one day before infection, \u0026ldquo;Penultimate day\u0026rdquo;), and at study end (48 h post-infection, \u0026ldquo;Final day\u0026rdquo;). Blood was collected into EDTA tubes and analyzed using an automated hematology analyzer (Mindray BC-2800Vet, Shenzhen, China). Parameters measured included total white blood cell count (WBC) and differential leukocyte percentages (neutrophils, lymphocytes, monocytes, eosinophils, and any immature granulocytes), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), and erythrocyte indices [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW)]. Platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), and platelet distribution width (PDW) were also measured. Blood smears were examined to validate automated differentials, particularly under extreme conditions like suspected neutrophilia or lymphopenia.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Two-way repeated-measures analysis of variance (ANOVA) was used to evaluate the effects of Group (C, CZ, M, MZ) and Time (Baseline, Penultimate, Final) on continuous outcomes, including hematologic parameters, organ CFU counts. This model accounts for within-subject correlations over time. Mauchly\u0026rsquo;s test was applied to check sphericity; if violated, Greenhouse-Geisser corrections were employed for F-tests. Where ANOVA indicated significant main effects or interactions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Tukey\u0026rsquo;s Honest Significant Difference (HSD) post-hoc tests were conducted for pairwise group comparisons at each time point. For end-point measures without repeated factors like final organ bacterial loads, one-way ANOVA or Kruskal-Wallis was used followed by post-hoc tests. Survival was not applicable as no deaths occurred prior to planned euthanasia. Statistical analyses were performed using R v4.1.0 (R Foundation, Vienna, Austria). A two-tailed p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Golestan University of Medical Sciences, and were conducted in accordance with national legislation and the Guide for the Care and Use of Laboratory Animals. Reporting follows the ARRIVE guidelines and the principles of the 3Rs. Measures were taken to minimize pain and distress throughout, including anesthesia for procedures (ketamine/xylazine) and multiple daily welfare checks; no tail lesions or adverse events were observed during hindlimb suspension. Animals were euthanized under deep anesthesia at study end, and biosafety procedures appropriate for \u003cem\u003eE. coli\u003c/em\u003e challenge were followed\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSerum Zinc Levels\u003c/h2\u003e \u003cp\u003eAt baseline (Day 0), all groups had comparable serum zinc concentrations (no significant differences). After 14 days of treatment and 48 hours post-infection (Day 16), zinc supplementation resulted in markedly higher serum zinc levels (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Rats given Zn-enriched water (CZ and MZ) showed a\u0026thinsp;~\u0026thinsp;40\u0026ndash;50% increase in serum Zn compared to unsupplemented controls (C and M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, microgravity exposure alone did not significantly affect serum Zn, final levels in the M group were similar to Ground Control C group in the absence of supplementation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There was no evidence of an interaction between microgravity and supplementation on Zn status. Thus, zinc-supplemented rats maintained elevated circulating zinc irrespective of gravity condition. These data confirm that the dosing regimen successfully raised serum Zn into a higher physiological range without apparent adverse effects, providing a clear separation between zinc-supplemented and non-supplemented animals.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum zinc concentrations (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) at baseline and final time points in each group. Day 0\u0026thinsp;=\u0026thinsp;pre-intervention baseline; Day 16\u0026thinsp;=\u0026thinsp;final (48 h post-ETEC infection).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay\u0026nbsp;0 (Baseline)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay\u0026nbsp;16 (Final)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC (Control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.3\u0026nbsp;\u0026plusmn;\u0026nbsp;5.1 \u0026micro;g/dL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.5\u0026nbsp;\u0026plusmn;\u0026nbsp;5.4\u003csup\u003e*\u003c/sup\u003e \u0026micro;g/dL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCZ (Control\u0026thinsp;+\u0026thinsp;Zn)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.1\u0026nbsp;\u0026plusmn;\u0026nbsp;4.8 \u0026micro;g/dL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e118.2\u0026nbsp;\u0026plusmn;\u0026nbsp;7.5\u003csup\u003e**\u003c/sup\u003e \u0026micro;g/dL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM (Microgravity)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81.0\u0026nbsp;\u0026plusmn;\u0026nbsp;5.9 \u0026micro;g/dL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.0\u0026nbsp;\u0026plusmn;\u0026nbsp;6.1\u003csup\u003e*\u003c/sup\u003e \u0026micro;g/dL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMZ (Microgravity\u0026thinsp;+\u0026thinsp;Zn)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.7\u0026nbsp;\u0026plusmn;\u0026nbsp;5.5 \u0026micro;g/dL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e111.0\u0026nbsp;\u0026plusmn;\u0026nbsp;8.2\u003csup\u003e**\u003c/sup\u003e \u0026micro;g/dL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e* \u0026lt; 0.05; ** \u0026lt; 0.01\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHematological Parameters\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eLeukocyte Counts and Differentials\u003c/strong\u003e \u003cp\u003eBaseline complete blood counts were equivalent across groups, indicating no pre-existing differences in immune cell populations. After 13 days of hindlimb suspension pre-infection, the M group developed a leukopenia relative to ground controls. Total WBC count in M was approximately 25% lower than in C (6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 vs 8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 \u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), accompanied by a shift in differential counts. Specifically, suspended rats showed a reduction in lymphocyte percentage and a relative neutrophilia compared to unsuspended controls (neutrophils\u0026thinsp;~\u0026thinsp;40% in M vs\u0026thinsp;~\u0026thinsp;25% in C, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; lymphocytes\u0026thinsp;~\u0026thinsp;55% vs\u0026thinsp;~\u0026thinsp;70%). These changes are consistent with stress-induced immune perturbation during simulated microgravity. By contrast, zinc supplementation mitigated the hematologic impact of microgravity. The MZ group maintained higher WBC counts (7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 \u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L) than M (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), not significantly different from controls at Day 13. Zinc-supplemented rats in normal gravity CZ group had WBC counts similar to C (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Thus, zinc appeared to preserve leukocyte numbers during microgravity exposure, preventing the marked drop observed in unsupplemented suspended rats.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eForty-eight hours after the ETEC challenge (Day 16, final), all groups exhibited an increase in WBC count due to infection, but the magnitude of this acute leukocyte response differed by treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Ground control rats mounted a robust leukocytosis (WBC 14\u0026ndash;15\u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L), driven largely by neutrophil expansion (neutrophils\u0026thinsp;~\u0026thinsp;60\u0026ndash;65% of WBC in C and CZ). Microgravity without zinc significantly blunted this response. Accordingly, the M group\u0026rsquo;s total WBC on the final day was ~\u0026thinsp;30% lower than controls (10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 vs 14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 \u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and neutrophil counts in M were correspondingly reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs C). In contrast, zinc supplementation in MZ improved the infection-induced leukocyte mobilization. The MZ rats reached WBC counts (13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 \u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L) that were higher than unsupplemented M (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and statistically equivalent to the response in ground controls (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Neutrophil percentages in MZ likewise rose to 55%, comparable to controls, indicating restoration of neutrophil reserves. Notably, the CZ group did not differ significantly from C in total WBC or differential after infection, suggesting that zinc did not overstimulate leukocytosis under normal gravity but was effective in counteracting microgravity-induced deficits. Across all groups, red blood cell indices, including RBC count, hemoglobin, hematocrit remained in normal ranges with no significant inter-group differences at any time point. Platelet counts and indices were also unaffected by either microgravity or zinc.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTotal white blood cell counts (WBC, \u0026times;103/\u0026micro;L) at baseline, after 13 days, and 48 h post-infection for each group (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;6). By Day 13, M rats had lower WBC than other groups. Post-infection (Day 16), M rats showed blunted leukocytosis vs. controls, while zinc-supplemented MZ rats had WBC counts restored to near-control levels.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime Point\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC (Control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCZ (Control\u0026thinsp;+\u0026thinsp;Zn)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM (Microgravity)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMZ (Microgravity\u0026thinsp;+\u0026thinsp;Zn)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaseline (Day\u0026nbsp;0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.6\u0026nbsp;\u0026plusmn;\u0026nbsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.8\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.5\u0026nbsp;\u0026plusmn;\u0026nbsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e7.7\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePost-14\u0026nbsp;days (Day\u0026nbsp;13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.2\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.5\u0026nbsp;\u0026plusmn;\u0026nbsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.0\u0026nbsp;\u0026plusmn;\u0026nbsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e7.8\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePost-infection (Day\u0026nbsp;16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.8\u0026nbsp;\u0026plusmn;\u0026nbsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.3\u0026nbsp;\u0026plusmn;\u0026nbsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.5\u0026nbsp;\u0026plusmn;\u0026nbsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e13.5\u0026nbsp;\u0026plusmn;\u0026nbsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Load and Infection Outcome\u003c/h2\u003e \u003cp\u003e All animals developed localized infection in the peritoneal cavity following ETEC challenge, but bacterial clearance differed markedly by group. Microgravity significantly exacerbated the infection: the unsupplemented M group harbored the highest peritoneal bacterial burden, with a mean of 3.2\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU/mL (log\u003csub\u003e10\u003c/sub\u003e 5.5), which was nearly an order of magnitude higher than in ground control rats (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs C). In contrast, zinc supplementation greatly improved the host\u0026rsquo;s ability to control peritoneal bacteria. The MZ group had a\u0026thinsp;~\u0026thinsp;90% lower peritoneal CFU count than M (\u0026asymp;\u0026thinsp;2.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e vs 3.2\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU/mL), bringing the bacterial load down to levels statistically indistinguishable from unsuspended controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 MZ vs M; MZ vs C n.s.). Notably, zinc-supplemented controls CZ showed the most efficient clearance, with peritoneal fluid counts\u0026thinsp;~\u0026thinsp;3\u0026times;10\u003csup\u003e3\u003c/sup\u003e CFU/mL (log\u003csub\u003e10\u003c/sub\u003e 3.8), significantly lower than in untreated controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs C). Thus, microgravity impaired bacterial clearance in the abdominal cavity, while zinc supplementation enhanced bacterial clearance, resulting in lower infection loads. These findings align with the observed differences in neutrophil responses, as effective peritoneal clearance is largely neutrophil-dependent.\u003c/p\u003e \u003cp\u003eConsistent with the above, microgravity also facilitated higher spread of bacteria to distant organs, whereas zinc mitigated this spread (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;D; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). All rats were infected with ETEC in spleen, liver, and kidneys, organs that can indicate bacteremia or inadequate containment of the peritoneal infection. In control animals, bacterial translocation was limited. Low titers of ETEC were recovered from spleen and liver in some C rats (mean\u0026thinsp;~\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e CFU/g in spleen, ~10\u003csup\u003e2.5\u003c/sup\u003e in liver), and none or minimal from kidneys (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, M group rats showed high bacterial loads in all organs, reflecting systemic dissemination. Spleens of M rats contained on average\u0026thinsp;~\u0026thinsp;1\u0026times;10\u003csup\u003e4\u003c/sup\u003e CFU/g, a level significantly higher than in C (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and ETEC was consistently recovered from liver and kidneys of M rats (~\u0026thinsp;10\u003csup\u003e3.5\u003c/sup\u003e CFU/g, vs. near or below detection in C). Importantly, zinc supplementation significantly reduced organ invasion. The MZ group had much lower bacterial counts than M in the spleen (~\u0026thinsp;8\u0026times;10\u003csup\u003e3\u003c/sup\u003e vs 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e CFU/g, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and liver (~\u0026thinsp;4\u0026times;10\u003csup\u003e2\u003c/sup\u003e vs 3\u0026times;10\u003csup\u003e3\u003c/sup\u003e CFU/g, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In kidneys, 4 of 6 MZ rats had no detectable bacteria whereas all M rats had positive cultures, and the overall mean in MZ was 50% of that in M (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, the CZ group again showed the greatest resistance to spread, and only sporadic colonies were found in CZ livers or kidneys, and splenic counts in CZ were approximately 10-fold lower than in unsupplemented C (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Microgravity-exposed rats without zinc experienced the highest bacterial loads in both the infection site and peripheral organs, indicating a failure to contain the infection, whereas zinc-supplemented rats had significantly improved control, with bacterial counts approaching those of healthy controls.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBacterial loads in peritoneal fluid and organs 48 h after infection (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Values represent log10 CFU/mL of peritoneal lavage fluid or per g of tissue. Within each column, the M group had significantly higher CFU than C (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Zinc supplementation lowered bacterial counts: MZ vs M was significant for peritoneal fluid and spleen (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CZ vs C was also significant in peritoneal fluid and spleen (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating enhanced clearance with zinc even under normal gravity.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC (Control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCZ (Control\u0026thinsp;+\u0026thinsp;Zn)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM (Microgravity)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMZ (Microgravity\u0026thinsp;+\u0026thinsp;Zn)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeritoneal fluid (CFU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.54\u0026nbsp;\u0026plusmn;\u0026nbsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.80\u0026nbsp;\u0026plusmn;\u0026nbsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.50\u0026nbsp;\u0026plusmn;\u0026nbsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.20\u0026nbsp;\u0026plusmn;\u0026nbsp;0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpleen (CFU/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.10\u0026nbsp;\u0026plusmn;\u0026nbsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.40\u0026nbsp;\u0026plusmn;\u0026nbsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.00\u0026nbsp;\u0026plusmn;\u0026nbsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.30\u0026nbsp;\u0026plusmn;\u0026nbsp;0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver (CFU/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.75\u0026nbsp;\u0026plusmn;\u0026nbsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.00\u0026nbsp;\u0026plusmn;\u0026nbsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.54\u0026nbsp;\u0026plusmn;\u0026nbsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.62\u0026nbsp;\u0026plusmn;\u0026nbsp;0.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney (CFU/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.40\u0026nbsp;\u0026plusmn;\u0026nbsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.70\u0026nbsp;\u0026plusmn;\u0026nbsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.50\u0026nbsp;\u0026plusmn;\u0026nbsp;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.36\u0026nbsp;\u0026plusmn;\u0026nbsp;0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that simulated microgravity exerts a suppressive effect on immune function and infection resistance in rats, and that dietary zinc supplementation can substantially ameliorate these deficits. Microgravity exposure by hindlimb suspension led to reduced leukocyte counts, blunted neutrophil responses, and a significantly higher bacterial burden following an \u003cem\u003eE. coli\u003c/em\u003e challenge. These findings are consistent with the well-documented immune dysregulation observed in true spaceflight [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous research has shown that more than half of astronauts on long-duration missions experience some form of immune impairment, including altered leukocyte distributions, diminished function of immune cells, and increased susceptibility to infections [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our results mirror these human data. Accordingly, the unsupplemented microgravity M group exhibited a leukopenia and lymphocyte reduction suggestive of stress or immunosenescence, and correspondingly failed to contain an infectious challenge. Zinc supplementation provided significant protection, helping to preserve white blood cell counts and enhance bacterial clearance in microgravity-exposed rats. This suggests that zinc is an effective countermeasure to microgravity-induced immune dysfunction, aligning with recent suggestions that nutritional interventions could bolster astronaut immunity during space missions [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe immune alterations in the M group highlight the impact of microgravity on both innate and adaptive arms of the immune system. We observed a decrease in total WBC, especially lymphocytes after 2 weeks of suspension, along with an inadequate neutrophil surge after infection. These outcomes agree with reports that microgravity or hindlimb unloading can rapidly induce changes in hematopoiesis and immune cell dynamics akin to accelerated aging [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It was reported that just 21 days of hindlimb unloading in mice caused a significant reduction in bone marrow B-cell lymphopoiesis and a shift in the IgM antibody repertoire, mirroring changes seen in much older animals [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Such changes indicate a premature immunosenescence, which likely underlies the heightened infection vulnerability. In astronauts, spaceflight has been associated with latent virus reactivations and skewed cytokine profiles due to compromised T-cell and NK cell function [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our rodent model reinforces that microgravity impairs the immune system\u0026rsquo;s ability to respond to new infections. In this regard, M rats had a 1-log higher bacterial load and inter-organ bacterial dissemination, whereas ground controls largely confined the infection. Additionally, evidence of inflammatory dysregulation was observed in microgravity. Even though total leukocytes were lower, the M group showed elevated neutrophil ratios and by inference, reduced lymphocyte percentages, a pattern often linked to cortisol-mediated stress and systemic inflammation. Spaceflight studies have noted increases in circulating pro-inflammatory cytokines despite overall immune suppression [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This paradoxical combination of heightened inflammation ,inflammaging, with weakened immune defense is a known consequence of microgravity and chronic stress [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The excessive bacterial growth in M rats can be seen as a direct result of such dysfunction. Neutrophils and monocytes are fewer in number and functionally impaired in microgravity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], hampering phagocytosis and bacterial killing. Indeed, astronaut studies have documented reduced phagocytic and oxidative burst capacity of neutrophils after spaceflight, as well as diminished monocyte ability to engulf bacteria. Our infection data extend these findings by showing the consequences of impaired cellular function, an inability to clear a pathogenic challenge leading to disseminated infection.\u003c/p\u003e \u003cp\u003eCompounding matters, microgravity may also enhance pathogen virulence, further tipping the scales against the host. Experiments have shown that bacteria cultured in real or simulated microgravity become more aggressive: for instance, \u003cem\u003eSalmonella\u003c/em\u003e grown during Space Shuttle flight returned with triple the virulence measured as a threefold lower LD\u003csub\u003e50\u003c/sub\u003e in mice compared to Earth-grown controls [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Similarly, \u003cem\u003eE. coli\u003c/em\u003e has exhibited increased growth, biofilm formation, and antibiotic resistance under microgravity conditions [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Although our study maintained identical inoculum across groups (the bacteria were not grown in microgravity \u003cem\u003ein vivo\u003c/em\u003e), the \u003cem\u003ein situ\u003c/em\u003e replication of ETEC was clearly much greater in microgravity-exposed hosts. This could reflect a synergy between a compromised immune system and the microgravity-induced stress responses in bacteria. The overal result is a perfect storm for infection. The host is less equipped to fight microbes, while the microbes may be proliferating more readily. This dual threat has been noted in reviews as a significant risk for deep-space missions [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and our findings provide in vivo evidence, in a controlled setting, of how dramatically infection outcomes can worsen when host defense is weakened by microgravity. No M-group rat succumbed within 48 h, but the high bacterial loads in vital organs suggest that a longer-term or more virulent infection could well lead to severe sepsis in microgravity conditions. Collectively, these observations underscore the importance of developing interventions to support the immune system for astronauts, simply relying on sterile environments is not enough, as latent microbes or incidental exposures can pose serious threats [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA central finding of this study is that zinc supplementation strengthened the rats\u0026rsquo; immunity, both under normal conditions and especially in microgravity. Zinc-supplemented rats (CZ and MZ) had higher circulating leukocyte reserves and mounted more immune responses to infection. In MZ animals, the WBC count and neutrophil influx after infection were nearly restored to control levels, in tandem with a dramatic reduction in bacterial burden compared to unsupplemented M. In fact, the infection outcomes in MZ were statistically similar to those in healthy controls, indicating that zinc largely compensated for the immune deterrent caused by microgravity. This aligns with zinc\u0026rsquo;s well-established role as an essential immunomodulator. Zinc is required for the development and function of immune cells, affecting neutrophils, NK cells, macrophages, and lymphocytes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Even mild zinc deficiency can impair phagocyte activity and cytokine production, while adequate zinc bolsters the oxidative burst and supports the killing of microbes. Our results agree with clinical evidence that correcting zinc insufficiency can improve infection resistance. For example, preventive zinc supplementation in children has been associated with significantly reduced incidences of diarrhea and pneumonia in large trials [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and with enhanced antibody-mediated responses to \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In older adults, who often exhibit immunosenescence similar to what we observed in microgravity, zinc supplementation is shown to rejuvenate aspects of immune function, reducing inflammation and infections [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The parallel between aging and microgravity immune changes has been noted, and zinc\u0026rsquo;s effectiveness in our model reinforces the idea that nutritional strategies targeting immunosenescent features can be translated to the spaceflight context.\u003c/p\u003e \u003cp\u003eMechanistically, zinc acted on multiple fronts to produce the benefits seen here. Zinc is known to support lymphopoiesis and maintain thymus function, which could help prevent the lymphocyte loss under microgravity. It also influences neutrophil and macrophage gene expression. Notably, a recent analysis highlighted zinc transport and metallothionein genes as being differentially expressed in muscles under microgravity, and proposed zinc as a low-risk/high-reward countermeasure for microgravity-induced degeneration [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our data provide experimental support for this concept in the immune system. Accordingly, zinc was a simple dietary addition with no observed toxicity, yet it significantly improved host resilience to infection. Additionally, zinc might have indirect effects such as preserving appetite/nutritional intake and maintaining barrier integrity. While the infection was intraperitoneal, it is worth noting that microgravity has been linked to increased intestinal permeability or leaky gut and altered microbiomes [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], which can promote systemic infection. Zinc has well-documented benefits in strengthening gut epithelial barriers and reducing translocation of bacteria. Therefore, zinc\u0026rsquo;s protective role could extend to limiting bacterial spread from mucosal surfaces during spaceflight conditions. In the present study, the CZ group\u0026rsquo;s exceptionally low bacterial counts suggest that zinc-enhanced baseline immunity can clear bacteria even more efficiently than normal. This finding concords with reports that zinc can heighten baseline pathogen clearance, for instance, by boosting macrophage phagocytosis and neutrophil extracellular trap formation. Thus, even in the absence of microgravity stress, additional zinc gave the rats an immunological advantage against infection.\u003c/p\u003e \u003cp\u003eOverall, these results have important implications for improving immune health during space travel. They provide a proof-of-concept that nutritional immunotherapy can counteract some negative effects of microgravity on host defense. Historically, measures like crew pre-flight quarantines and spacecraft sterilization to minimize infection risks have been implemented [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our findings suggest that ensuring adequate or supraphysiologic intake of key micronutrients such as zinc could be a practical and safe countermeasure to help the immune system in space. Zinc tablets or enriched diets are easy to administer and have low cost and mass-crucial considerations for space missions. Moreover, zinc might synergize with other countermeasures. Accordingly, combining zinc with vitamins and probiotics could provide a multifaceted boost. There is already interest in probiotics to promote immune function in astronauts [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and a combined regimen with zinc could target both innate and adaptive immunity. It will be important to validate whether the benefits seen in the rat model translate to humans in microgravity. Rodents are not human astronauts, but many aspects of rodent immune dysregulation in microgravity parallel human observations [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A recent review on spaceflight immune dysregulation explicitly recommends exploring nutritional supplements, including zinc, to boost immunity in astronauts [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our study provides experimental support for this recommendation.\u003c/p\u003e \u003cp\u003eWhile the presented results could be promising, some limitations should be acknowledged. We did not observe any microgravity-induced change in serum zinc in unsupplemented rats, which suggests that 2 weeks of hindlimb unloading did not deplete zinc status, but longer exposure or actual spaceflight might produce shifts in trace mineral homeostasis. Also, the infection model involved an acute high-dose challenge with one pathogen. Microgravity may impact host responses differently for viral, fungal, or chronic low-dose infections. It would be valuable to test zinc\u0026rsquo;s effects in other infection models relevant to astronaut health. Moreover, this study focused on systemic endpoints; the cellular and molecular mechanisms by which zinc improved immunity in microgravity were not dissected here. Follow-up studies could examine, gene expression of inflammatory mediators, neutrophil function assays, or vaccine responses in microgravity with vs. without zinc. Such data would deepen our understanding of how zinc confers resistance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe findings of this study highlight that simulated microgravity severely compromises immune defense, evidenced by lower leukocyte counts and failure to control bacterial infection. Also, zinc supplementation counteracts these effects, preserving immune competence and enhancing pathogen clearance. These findings contribute to a growing body of literature indicating that the spaceflight environment accelerates immune aging and heightens infection risks. Importantly, they also point to a readily implementable solution. Accordingly, nutritional fortification with zinc as a countermeasure to support immune function during space missions. Future research, should evaluate the efficacy of zinc and other immunonutrients in mitigating space-induced immune dysregulation. The results of the present study underscore that microgravity and immunity are strongly interlinked. The unique stresses of weightlessness can undermine host defenses, but targeted interventions like zinc supplementation can restore balance. This integrative approach, combining environmental simulation, infection challenge, and nutritional therapy provides a template for addressing the complex challenges of human health in space.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehindlimb unloading\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHU\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003enatural killer\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNK\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eEnterotoxigenic Escherichia coli\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eETEC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWhite blood cell count\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWBC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRed blood cell count\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRBC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHemoglobin\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHGB\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHematocrit\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHCT\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMean corpuscular volume\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMCV\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMean corpuscular hemoglobin\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMCH\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMean corpuscular hemoglobin concentration\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMCHC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRed cell distribution width\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRDW\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePlatelet count\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePLT\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePlateletcrit\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePCT\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMean platelet volume\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMPV\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePlatelet distribution width\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePDW\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eStandard error of the mean\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHonest Significant Difference\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHSD\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrinciples of laboratory animal care (NIH publication No. 86\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1985) were followed. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Golestan University of Medical Sciences, and were conducted in accordance with national legislation and the Guide for the Care and Use of Laboratory Animals. Also, the study was approved by Ethics Committee of Vista Aria Rena Gene Inc., in Gorgan, Iran by the approval code of VARG-LAB-2003-0001. The study was conducted in accordance with the local legislation and institutional requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eClinical trial number\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAM conceptualized the study and led the project administration and supervision. AM and SR developed the methodology. Data curation was performed by SR, SZ, FA, FSA, and AM. Formal analysis was carried out by SR, ASS, and ZA. The investigation was conducted by SR, SZ, FA, and ASS. AM, FSA, and SR provided resources. AM and ASS contributed to software development. Validation was performed by AM, SR, and ASS. Visualization was prepared by AM, SR, and SZ. The original draft of the manuscript was written by SR, SZ, ASS, FA, ZA, FSA, and AM. The manuscript was reviewed and edited by SR, SZ, ASS, FA, ZA, FSA, and AM. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHicks J, Olson M, Mitchell C, Juran CM, Paul AM. The Impact of Microgravity on Immunological States. Immunohorizons. 2023;7:670.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, et al. 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Front Nutr. 2024;11:1385591.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Zinc Supplementation, Microgravity, Enterotoxigenic Escherichia coli, Immunoprotective, Immunity","lastPublishedDoi":"10.21203/rs.3.rs-8301013/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8301013/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSpaceflight and simulated microgravity impair immune function, increasing infection risk. Zinc, was investigated as a nutritional countermeasure. This study examined whether dietary zinc supplementation mitigates \u0026micro;G-induced immune alterations and improves host response to bacterial infection in a rodent model. Accordingly, Twenty-four male Wistar rats were randomized into Control (C), Control\u0026thinsp;+\u0026thinsp;Zinc (CZ), Microgravity (M), and Microgravity\u0026thinsp;+\u0026thinsp;Zinc (MZ) groups. Simulated \u0026micro;G was induced by 14-day hindlimb suspension, with zinc provided in drinking water. All rats were challenged with \u003cem\u003eE. coli\u003c/em\u003e. Serum zinc, blood counts, and bacterial loads in multiple organs were measured.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAt 48 h post-challenge, total leukocytes were reduced versus ground controls (WBC 4.0\u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L vs 9.8\u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L; \u0026minus;59%), and peritoneal bacterial burden was highest (\u0026asymp;\u0026thinsp;981 CFU units) with greater dissemination to spleen, liver, and kidney (+\u0026thinsp;46% to +\u0026thinsp;197% vs control). Zinc supplementation substantially rescued these deficits. In MZ, WBC rebounded to control levels (\u0026asymp;\u0026thinsp;10.0\u0026times;10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;L; +150% vs microgravity alone) and infection loads fell sharply at the peritoneum (\u0026minus;\u0026thinsp;97%) and across organs (\u0026minus;\u0026thinsp;94% to \u0026minus;\u0026thinsp;98% vs microgravity). Under normal gravity, zinc also improved bacterial clearance (peritoneum \u0026minus;\u0026thinsp;87% vs control) without materially altering final WBC.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eZinc nutrition restored leukocyte responsiveness and dramatically reduced pathogen burden, effectively counteracting microgravity-induced vulnerability. These findings demonstrate that simulated \u0026micro;G induces anemia, lymphocyte loss, and impaired bacterial clearance in rats. Zinc supplementation counteracts these effects, suggesting it may serve as an effective nutritional countermeasure to enhance immune health and reduce infection susceptibility during spaceflight.\u003c/p\u003e","manuscriptTitle":"Zinc Supplementation Mitigates Microgravity-Induced Immune Dysregulation and Enhances Bacterial Clearance in Escherichia coli- Infected Rats Short Title: Zinc Counteracts Microgravity Immune Impairment in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-05 05:37:04","doi":"10.21203/rs.3.rs-8301013/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9892644e-6e6c-4f67-ae34-aeed4ad7e706","owner":[],"postedDate":"January 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-11T18:40:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-05 05:37:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8301013","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8301013","identity":"rs-8301013","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}