Longitudinal multi-omics analysis of host microbiome architecture and immune responses during short-term spaceflight

The impact of spaceflight on the human microbiome is a critical area of research, especially given the rise of commercial space travel. Microbes significantly influence human physiology, and understanding their interplay with the space environment is crucial for ensuring astronaut health. Previous studies have been limited by small sample sizes, lack of longitudinal data, and reliance on single sequencing modalities. Spaceflight has been associated with various health issues, including gastrointestinal discomfort, skin barrier disruption, and immune system suppression, all potentially linked to microbiome alterations. The space environment itself, including microgravity and radiation, also affects microbial physiology, genetics, and community composition. Early studies revealed shifts in human and environmental microbiomes during spaceflight, but crucial questions regarding microbiome architecture (the totality of compositional and expression shifts), the contribution of crewmember-to-crewmember versus environment-to-crewmember transmission, and the persistence of post-flight changes remain unanswered. This study addresses these gaps by employing a longitudinal, multi-omics approach.

Existing literature highlights the complex relationship between spaceflight and the human microbiome. Studies have shown a correlation between spaceflight and gastrointestinal issues, often linked to changes in gut microbiome composition. Skin barrier disruption and inflammation are also commonly observed, potentially facilitating the invasion of harmful microorganisms. Immune system suppression during spaceflight can lead to inflammation and the reactivation of latent infections. However, these studies often lack the depth and breadth of data needed to fully understand the intricacies of these relationships. Moreover, the impact of the space environment itself on microbial communities, including the selection for antibiotic resistance, has also been documented, but further investigation is needed to understand the mechanisms and implications. The lack of high-resolution, longitudinal studies and the absence of metatranscriptomic data have hindered a complete understanding of spaceflight's impact on the human microbiome.

This study utilized data from the SpaceX Inspiration4 mission, the first all-civilian commercial spaceflight. Over a six-month period, the four-person crew collected environmental and multiple body site (skin, nasal, oral, stool) samples at eight time points: three before flight, two during flight, and three after flight. Peripheral blood mononuclear cells (PBMCs) were also collected. Samples underwent metagenomic and metatranscriptomic sequencing, along with single-cell immunome profiling. A diverse set of short-read alignment and de novo assembly approaches were used to characterize microbial community taxonomic and functional composition. Linear mixed-effect (LME) models and generalized linear models (GLMs) were used in a microbiome-association study (MAS) to identify associations between flight and microbial features (bacterial species, viral genera, microbial genes). Beta diversity metrics (Bray-Curtis) were calculated to assess microbiome similarity. Strain-level analysis was performed to determine potential microbial exchange. Finally, host immune single-nuclei transcriptome data from PBMCs were integrated with MAS results using lasso regression to investigate links between microbiome changes and host immune responses. Stringent quality control measures, including the use of negative controls, were implemented throughout the process.

The study revealed predominantly transient restructuring of the oral, nasal, and skin microbiomes during spaceflight. Over 821,337 statistically significant associations were identified, comprising 314,701 distinct microbial features. The majority (73.5%) of significant features were transiently increased in abundance during flight. The oral microbiome showed the most persistent changes, including increases in plaque-associated bacteria (*Fusobacteriota*) such as *Fusobacterium hwasookii*, *Fusobacterium nucleatum*, and *Leptotrichia hofstadii*, which correlated with immune cell gene expression. Skin microbiome changes were largely transient, with increases in various bacterial taxa. Functional analysis revealed enrichment of microbial genes associated with antibiotic and heavy metal resistance, heme binding/export, lantibiotic-associated genes, phage-associated genes, and toxin-antitoxin systems, particularly in metagenomic data. Analysis of microbial strain sharing revealed that while in flight, marker genes were mostly shared between skin microbiome samples from the same individual, with increasing sharing between crew members and the capsule observed over time. Integration of microbiome data with host immune data revealed numerous associations between oral microbiome changes and host immune cell gene expression. Notably, skin bacteria showed limited association with immune response, suggesting that acquired microbes during flight may have limited immediate impact on the host immune system.

This study, the largest of its kind to date, provides a comprehensive view of microbiome changes during short-term spaceflight and their association with host immune responses. The findings highlight the dynamic nature of the microbiome in response to spaceflight, with transient changes predominating. However, significant long-term shifts in the oral microbiome, potentially contributing to oral health issues, warrant further investigation. The increased prevalence of genes related to stress response and antibiotic resistance raises concerns about the potential for microbial adaptation and pathogen emergence in the space environment. While the observed microbiome convergence may be partially attributed to close quarters and environmental exposure, the strong correlation between oral microbiome shifts and host immune responses suggests a complex interplay between these factors. The lack of strong immune-skin microbiome associations may imply that the immediate impact of environmentally acquired microbes on the host immune system is limited.

This multi-omics study offers the most detailed characterization yet of microbiome and immune system responses to spaceflight. The findings highlight the need for continuous monitoring and mitigation strategies for astronaut health. Future studies should focus on larger crew sizes, longer mission durations, and more rigorous experimental designs to further elucidate the causality of microbe-immune interactions in the space environment. Investigating these hypotheses using animal or organoid models and comparisons to large ground-control cohorts would be valuable.

The study's observational nature and relatively small sample size (four astronauts) limit the generalizability of the findings. Confounding factors such as diet and cleaning habits may have influenced the results. Challenges in analyzing low-biomass samples and the limitations of current viral taxonomic classification methods warrant consideration. The opportunistic study design did not allow for a truly controlled experiment and therefore causality between specific changes and spaceflight is inferential. Further, the lasso-based approach for immune-microbe interaction modeling does not allow statistical inference or account for inter-individual variations.