Study suggests gut phages may be involved in the development of asthma

In a recent study published in Nature Medicine, a group of researchers investigated the relationship between early-life gut virome, specifically temperate phage taxa, and the development of asthma in childhood while considering the interaction with host genetics and the bacteriome.

Background 

Asthma, a prevalent chronic inflammatory disease of the airways, often begins in early childhood. Its pathophysiology is complex and influenced by genetic, environmental, and immunological factors. The gut microbiota, crucial in immune development, has been linked to asthma, allergies, and other immune-mediated chronic diseases. Despite this, research has primarily focused on gut bacteria, leaving the role of gut viruses, particularly phages, less explored.

The gut harbors numerous viruses, with phages predominantly targeting bacteria. Phages, either virulent or temperate, can impact host immunity by influencing bacterial colonization and interacting with the mucosal immune system. Further research is essential to unravel the complex interactions between gut virome, bacteriome, and host immunity. This could lead to novel biomarkers and therapeutic strategies for asthma and other immune-mediated diseases.

About the study 

In compliance with the Declaration of Helsinki, the present study on asthma in children adhered to stringent ethical standards. Parental consent was secured, and strict adherence to research integrity, patient safety, and data protection regulations, including Good Clinical Practice (GCP) and General Data Protection Regulation (GDPR), was maintained.

The study was a part of the Danish COPSAC2010 mother–child cohort, involving 700 children from pregnancy to five years of age. Asthma diagnoses were carefully recorded based on comprehensive criteria, including symptom severity, duration, and response to treatment. For this study, any diagnosis of asthma by age five was considered.

Fecal samples from 647 children were analyzed for their virome at one year of age. Sample collection, storage, and processing followed specific protocols, ensuring the preservation of the samples until analysis. The bioinformatic processing involved detailed procedures for virome extraction, library preparation, and sequencing. For bacterial deoxyribonucleic acid (DNA), specific methods and tools were used, including the MoBio PowerSoil kits and the MiSeq viz MiSeq Sequencing System platform for sequencing.

Statistical analysis was conducted using R, employing various packages for data treatment and visualization. The study used two-sided P values, with a significance threshold set at P ≤ 0.05, and employed the Benjamini–Hochberg correction for multiple testing. Asthma associations with the virome were investigated using logistic regression and other statistical methods while controlling for potential confounders and batch effects.

The study also assessed the correlation between the virome and bacteriome, employing Spearman correlations and Procrustes analysis. The impact of the virome on asthma development was analyzed using logistic regression and quasi-poisson regression analyses. Additionally, mediation analysis was performed to test the significance of indirect bacterial effects.

The trajectories of asthma development were examined using generalized estimating equations, which allowed for a longitudinal assessment of asthma persistence over time. The study further investigated the timing of preschool asthma onset in relation to virome and bacteriome signature scores. Four groups were compared using Kaplan–Meier curve analysis and a log-rank test, providing insights into the impact of these microbial communities on asthma development.

Environmental factors influencing the virome signature score were also examined through linear regression analysis. Lastly, the study delved into the genetic aspect by focusing on the Toll-Like Receptor 9 (TLR9) genotype and its interaction with the virome in relation to asthma. Logistic regression analyses were used to explore the effects of different genotypes on asthma, with a specific emphasis on the TLR9 genotype rs187084. This genetic aspect was crucial in understanding the complex interplay between genetics, the gut virome, and asthma development in children.

The interaction between the TLR9 genotype and virome signature scores was evaluated, providing valuable insights into how genetic predispositions might influence the risk of asthma in early childhood. 

Study results 

The present study focused on analyzing the gut virome of 647 one-year-old children, and these children were deeply phenotyped from birth, with longitudinally assessed asthma diagnoses. It was found that specific temperate gut phage taxa were associated with the later development of asthma. Notably, the joint abundances of 19 caudoviral families significantly contributed to this association.

The research revealed that combining asthma-associated bacteriome and virome signatures had increased effects on asthma risk, suggesting an independent virome-asthma association. Interestingly, the virome-associated asthma risk was modulated by the host TLR9 rs187084 gene variant, indicating a direct interaction between phages and the host immune system. This finding opens up new avenues for further studies to explore whether phages, alongside bacteria and host genetics, can be used as preclinical biomarkers for asthma.

A key finding of the study was the variation in the relative abundances of both caudoviruses and microviruses between children with and without asthma by age five. The relative abundance of temperate phages was particularly associated with asthma. Adjustments for potential confounders like siblings, birth weight, urbanicity, and age did not alter these results. This association was primarily driven by caudoviruses, and differences in the relative abundance of phages with an unknown lifestyle were driven by microviruses.

The study also noted the high degree of uniqueness of gut viromes among children, with a median richness across samples of 1306 Viral Operational Taxonomic Units (vOTUs) and an overall sparsity of 86% across the samples. However, overall observed richness and evenness were not associated with the development of asthma before the age of five.

The compositional differences in the temperate virome between children with and without asthma were most notable, leading to the identification of 19 temperate viral family-level clades (VFCs) that were jointly associated with later asthma.

The research further explored the independence of the asthmatic virome from the bacteriome. Despite some correlation in species richness and composition between the temperate virome and bacteriome, the results suggested only a minor indirect effect of the virome association with preschool asthma was mediated through bacteria. This was further supported by the finding that both virome and bacteriome signature scores captured the relationship with asthma better than either alone, indicating independent and additive effects.

Finally, the study examined the impact of early life exposures on the asthmatic virome and the genetic link between the gut virome and the host immune system in preschool asthma. It was found that the risk of asthma derived from the virome signature score seemed dependent on the TLR9 genotype. This suggests a direct interaction between phages and the host immune system, contributing to the development of asthma in early childhood.