Significance of Neutrophil to Lymphocyte ratio (NLR) and Gut microbiome of the astronauts during space missions and suggestions for beneficial modulations using novel beta-glucans

Nobunao Ikewaki ¹ Koji Ichiyama ² Rajappa Senthilkumar ² Senthilkumar Preethy ³ Samuel JK Abraham ^4*

• ¹ Kyushu University of Medical Science, Nobeoka, Miyazaki, Japan
• ² GN Corporation, Kofu, Yamanashi, Japan
• ³ Nichi-In Centre for Regenerative Medicine (NCRM), Chennai, Tamil Nadu, India
• ⁴ Faculty of Medicine, University of Yamanashi, Yamanashi, Japan

Introduction:Among the multitudes of clinical experiments, human beings subjected to spaceflight travel stand apart because of their complexity owing to a microgravity environment. Before considering any pathological implications due to changes in the gravitational force, the fundamental physiological processes aided by the Earth’s gravity are disrupted and need to be evaluated. Therefore, maintaining physiological stability despite altered gravity is the utmost priority, followed by addressing the pathological implications and their management.Spaceflight, characterised by microgravity, circadian misalignment, isolation, confinement, stress, a semi-closed food system, and increased exposure to space radiation, has been shown to have detrimental effects on the human immune system [1,2]. The immune system dysregulations reported in astronauts include altered leukocyte distribution, changes in plasma cytokines, reduced T-cell function, and reactivation of latent herpesviruses. Persistent low-grade systemic inflammation characterised by increased TNF-α and IL-1RA, which can lead to various diseases, has been documented [3]. The clinical implications of such immune dysfunction include rashes, hypersensitivity, and atopic dermatitis, in addition to accelerated physiological aging, as evidenced by muscle wasting and loss of bone density [1,2].In this context, ongoing research has focused on identifying safe and easily evaluable in-flight biomarkers for monitoring the immune system of astronauts. The neutrophil-to-lymphocyte ratio (NLR) has been identified as a potential biomarker candidate to evaluate the immune status [1,3] because (i) leukocyte counts have been reported to be altered during spaceflight, and (ii) on Earth, elevated NLR is an extremely useful biomarker of chronic persistent subclinical inflammation, which can be a major pre-existing factor for disease development. Furthermore, an elevated NLR has been shown to predict poor prognosis in cancers and chronic conditions such as coronary heart disease, stroke, diabetes, obesity, psychiatric diagnosis, anaemia, and stress. A gradual increase in the NLR, apart from having a positive correlation with age, also serves as a biomarker to predict the overall mortality of a specific population [4]. Significance of NLR:Recent reports [1,3] have documented a gradual increase in the NLR in astronauts, which has been suggested to be an indicator of hastened inflammation. Moreover, compared to other biomarkers of inflammation and immune status, NLR is easy to measure and has been proven to be altered under simulated spaceflight conditions on Earth, as well as in spaceflight experiments, suggesting that it is a critical biomarker for monitoring the immune system and health of astronauts [3].The NLR can be considered a critical biomarker because it acts as a bridge between innate and adaptive immune systems [5]. The NLR in the peripheral blood, serves as a biomarker linking two key components of the immune system: the innate immunity mediated by neutrophils and adaptive immunity mediated by lymphocytes. Neutrophils constitute the frontline defence of the host immune system against pathogens by employing mechanisms such as chemotaxis, phagocytosis, reactive oxygen species (ROS) generation, granular protein release, and cytokine production. Beyond these functions, neutrophils considerably influence adaptive immunity and are pivotal effector cells in systemic inflammatory response syndrome. Acting as regulators of innate immunity, neutrophils recruit, activate, and modulate other immune cells by secreting diverse pro-inflammatory and immunomodulatory cytokines and chemokines, thereby enhancing the activity and recruitment of immune cells such as dendritic, B, natural killer (NK), CD4+, CD8+, γδ T, and mesenchymal stem cells [5].However, although the NLR has been identified as a critical biomarker for monitoring the immune system and prognosis of diseases both in routine clinical settings and in astronauts, safe, easily administrable dietary or nutritional interventions are still needed to beneficially modulate the NLR for maintaining health in astronauts. Even in a head-down tilt bed rest experiment, considered as the best and most integrated Earth-based analogue of the microgravity in spaceflight, the NLR was identified as a critical biomarker for astronauts and was found to increase in the study participants. However, dietary supplementation which was part of the experiment, did not produce any changes in NLR values [3].Interventions to modulate NLR:Some interventions have decreased the NLR in routine clinical settings. One such is Vitamin D, wherein high-dose supplementation of vitamin D reduced NLR distribution in a clinical study in adolescent girls [6]. Vitamin D supplementation has been suggested as a means to beneficially modulate NLR in astronauts, and despite daily vitamin D supplementation, crew members on the Russian space station Mir had serum 25(OH)-D3 concentrations that were 32%–36% less during and after long-duration (3- to 4-mo) missions than before the missions [7]. In another study, an oral food supplement containing Echinacea angustifolia, rosehip, propolis, royal jelly, and zinc, was shown to decrease NLR inpatients with COVID [8]. Other nutritional supplements reported to influence NLR include omega-3 fatty acids [9] and synbiotic supplements [10]. However, the search continues for a dietary intervention that is safe, easy to administer, and can work on immunity as well as other aspects contributing to optimal health, including the gut microbiome.Gut microbiome and NLR:Regarding the relevance of gut microbiome to NLR and health, it is well established that 70% of the immune cells in our body are found in the gastrointestinal (GI) tract, where their development and maturation are influenced by their interactions with the gut microbiota. When gut dysbiosis occurs, a clinically “maladaptive” immune response can arise [11]. Changes in NLR have already been found to be directly correlated with gut dysbiosis, and gut microbiome abundance has been reported to differ considerably between patients with normal NLR and those with increased NLR [12]. Thus, NLR can be considered a critical indicator along with the correlation of the gut microbiome for monitoring health status and disease prognosis [12-14]. On spaceflight and in the gut microbiome, notable changes in 44 microbiome species, including relative reductions in bile acid and butyrate-metabolising bacteria such as Extibacter muris and Dysosmobacter welbionis, have been reported [15]. Increases in the genera Clostridium, Romboutsia, Ruminiclostridium, and Shuttleworthia, along with the decreases in Hungatella and significant enrichment of Dorea sp. and Lactobacillus murinus have also been reported [16].Beta-glucans beneficially modulate NLR:Given the above background of several known and unknown factors that affect the health of astronauts and the critical nature of maintaining NLR and gut homeostasis, a major solace is the result of our work on the safe and beneficial modulation of NLR in pre-clinical and clinical studies using the biological response modifier (BRM) Beta-1,3-1,6-glucans produced as an exo-polysaccharide by the AFO-202 and N-163 strains of a black polyextremotolerant yeast, Aureobasidium pullulans. Beta-glucans are naturally occurring polysaccharides found in the cell walls of yeast, fungi (including mushrooms), certain bacteria, seaweed, and cereals such as oats and barley. These bioactive compounds possess multiple functional properties including hypocholesterolaemic, hypoglycaemic, immunomodulatory, antitumour, antioxidant, and anti-inflammatory activities. The structural and functional properties of Beta-glucans vary depending on their source. Among the various types, yeast-derived Beta-1,3-1,6-glucans have demonstrated superior BRM effects compared to those derived from cereal sources such as oats or barley. Clinical applications of Beta-glucanshave gained prominence worldwide, with Japan being a leader in their therapeutic utilisation [17]. Since 1983, Beta-glucans derived from Lentinula edodes (lentinan) and Coriolus versicolor (polysaccharide-K) have been approved as pharmaceutical agents. As of 2019, more than 177 clinical trials have been registered in the United States, evaluating beta-glucans for their potential in cancer therapy, cholesterol regulation, and immune modulation [18]. The immunomodulatory properties of beta-glucans stem from their ability to interact with a range of immune receptors, including Dectin-1, complement receptor 3, lactosylceramide, natural cytotoxicity receptor p30, and scavenger receptors. These receptors are expressed on key immune cells such as macrophages, neutrophils, and NK cells, enabling Beta-glucansto modulate immune responses effectively. Owing to their capacity to either enhance or regulate immune functions, Beta-glucans have been extensively explored as potential therapeutic adjuvants, particularly in immunotherapy. Extensive research has established that beta-glucans produced as an exo-polysaccharide by two novel strains of black yeast (A. pullulans), AFO-202 and N-163, exhibit unique immunomodulatory and metabolic-immune enhancing benefits [17].AFO-202 beta-glucan has been shown to reduce NLR in Sprague–Dawley rats [19]. In a 30-day study involving patients with COVID-19, a 70% reduction was observed from baseline in the group administered AFO-202 beta-glucan. In the group receiving a combination of AFO-202 and N-163 beta-glucan, a 66% reduction was observed from baseline [20]. Apart from NLR reduction, a decrease in other inflammatory markers such as IL-6 and D-dimer and an increase in the lymphocyte-to-C-reactive and leukocyte-to-C-reactive protein ratios were observed [20]. In patients with pancreatic cancer undergoing surgery, perioperative administration of AFO-202 led to a 40% reduction in the NLR [21]. The clinical outcomes were decreased Serum Amyloid A, sCD44, and CA19-9 levels and increased mean survival time [21]. The AFO-202 beta-glucan has been shown to have anti-infective properties against Leishmania amazonensis and malaria through the increase of NK cell activity and cellular immunity [22] and has also shown potential as a vaccine adjuvant, enhancing the immune response to the avian influenza A H5N1 and H5N2 vaccines [23]. N-163 produced beta-glucan has been reported to attenuate lipotoxicity, as evident by a decrease in non-esterified fatty acids [24], with anti-inflammatory and anti-fibrotic effects in animal and human clinical studies of metabolic dysfunction-associated diseases such as non-alcoholic steatohepatosis (NASH) [25] and Duchenne muscular dystrophy (DMD) [26,27].In the gut microbiome, AFO-202 beta-glucan in children with autism spectrum disorder has been shown to decrease the abundance of harmful Enterobacteriaceae, including Escherichia coli, Akkermansia muciniphila CAG:154, Blautia spp., Coprobacillus sp., and Clostridium bolteae CAG:59, with an increase in butyrate producers, such as Faecalibacterium prausnitzii and Roseburia [28]. In the NASH model [29], the gut microbial diversity increased greatly in the AFO-202+N-163 group. In the AFO-202+N-163 group, the abundance of Firmicutes decreased, whereas those of Bacteroides and Lactobacillus increased. In NASH mice fed AFO-202 beta-glucan alone, there was a decrease in the abundance of Enterobacteriaceae and other Firmicutes, whereas in the N-163 group, there was a decrease in the abundance of harmful bacteria such as Turicibacter and Bilophila. In the same study, an increase in the abundance of butyrate precursors and amino acids such as tryptophan was also reported [29,30]. The administration of N-163 produced beta-glucan, resulting in an increase in butyrate-producing species, such as Roseburia and F. prausnitzii, and a decrease in harmful bacteria associated with inflammation, such as Enterobacteria and Alistipes in patients with DMD. In patients with multiple sclerosis, there was an increase in the abundance of beneficial genera such as Bifidobacterium, Collinsela, Prevotella, and Lactobacillus and species such as Prevotella copri, Bifidobacterium longum, F. prausnitzii, and Siphoviridae, whereas there was a decrease in inflammation-associated genera such as Blautia, Ruminococcus and Dorea [31].Discussion:These findings on the novel beta-1,3-1,6--glucan-based BRMs produced from AFO-202 and N-163 strain of A. pullulans align with the requirements for astronauts. As mentioned earlier in this manuscript, studies have documented increases in NLR and imbalances in microbial species during space missions [1,3,15,16], which have been reported to be modulated in a beneficial manner in the studies using these beta-glucans [17-31]. The beneficial effects of lowering the NLR are primarily mediated through the suppression of excessive neutrophil activity and the restoration of balanced lymphocyte function [32]. The mechanisms underlying the improvement in disease outcomes include: (1) Neutrophilia reduction because elevated neutrophil counts, which are common in chronic inflammatory conditions, are linked to excessive ROS production, prolonged inflammation, and tissue damage. Lowering NLR reduces neutrophil-driven inflammation and oxidative stress. (2) NETosis regulation, where excessive neutrophil extracellular trap (NET) formation, which is implicated in microvascular complications in diabetes mellitus, atherosclerosis in coronary artery disease, and airway damage in chronic obstructive pulmonary disease (COPD), is attenuated with decreased NLR, reducing endothelial and tissue injury [32]. (3) Decreased pro-inflammatory cytokine secretion: A decreased neutrophil burden limits the release of TNF-α, IL-6, and other pro-inflammatory mediators, thereby curbing chronic systemic inflammation. (4) Enhanced lymphocyte recruitment to inflamed sites: A balanced NLR ensures optimal lymphocyte trafficking, aiding in effective immune surveillance and resolution of inflammation [32]. (5) Treg and Th17 balance restoration: Decreased NLR is associated with increased regulatory T cell (Treg) activity, which suppresses excessive inflammation, and reduced Th17-driven autoimmunity, which contributes to chronic inflammatory pathology. (6) Improved adaptive immune response: Lymphocytopenia, which is linked to immune dysfunction is mitigated by lowering NLR, enhancing antigen-specific immune responses, and reducing infection susceptibility. Thus, lowering the NLR contributes to immune homeostasis by regulating neutrophil overactivation and enhancing lymphocyte function [32]. This balance is critical for mitigating chronic inflammation, improving disease prognosis, and reducing complications of various inflammatory conditions. Thus, A. pullulans AFO-202 and N-163 produced beta-glucans, with a long safety track record as a food supplement, which are water soluble and produced with any ingredient in the commonly notified list of allergens, are promising candidates for consideration in nutrition studies for astronauts and in space flight experiments due to their beneficial NLR modulating effects in a safe manner [17-31]. Figure 1 summarizes the potential of NLR-modifying beta-glucans in astronaut diets during space missions. It also illustrates the possible mechanisms through which NLR modulation occurs, highlighting the effects of these beta-glucans on the immune system and gut microbiome.These novel NLR modifying beta-glucans’ ability to exert beneficial immune modulation, as observed in studies involving healthy human volunteers as well [33], further supports their potential, after necessary validation for inclusion in astronaut diets as a routine intervention. Despite experiencing physiological stressors such as microgravity, circadian misalignment, and space radiation exposure, astronauts differ considerably from patients with chronic diseases and healthy terrestrial subjects. Unlike individuals with preexisting inflammatory or metabolic conditions, astronauts are highly trained and undergo rigorous physical and psychological preparation, which may influence their immune adaptability and inflammatory responses. Therefore, although the observed effects of A. pullulans AFO-202 and N-163 beta-glucans on immune regulation, particularly in modulating the NLR and systemic inflammation, align with the physiological challenges encountered during spaceflight, further research is warranted to validate these findings, specifically in the astronaut population. Controlled studies on spaceflight-relevant models and actual space missions are required to establish the extent of their benefits in the unique physiological environment of space.