In a recent study published in Nature Medicinea team of researchers investigated the relationship between early-life enterovirus, specifically temperate phage taxa, and the development of childhood asthma, while examining the interaction with host genetics and the bacterium.
Record
Asthma, a widespread chronic inflammatory disease of the airways, often begins in early childhood. Its pathophysiology is complex and influenced by genetic, environmental and immunological factors. Gut microbiota, vital for immune development, has been linked to asthma, allergies and other immune-mediated chronic diseases. However, research has focused mainly on gut bacteria, leaving the role of gut viruses, particularly phages, less explored.
The gut is home to many viruses, with phages primarily targeting bacteria. Phages, whether virulent or temperate, can influence host immunity by affecting bacterial colonization and interacting with the mucosal immune system. Further research is necessary to unravel the complex interactions between the enteric virus, the bacterium and host immunity. This could lead to new biomarkers and therapeutic strategies for asthma and other immune-mediated diseases.
About the study
In compliance with the Declaration of Helsinki, the present study of asthma in children adhered to strict 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 part of the Danish COPSAC2010 mother-child cohort, which involved 700 children from pregnancy to age five. 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 the age of five was considered.
Stool samples from 647 children were analyzed for their virus at one year of age. Sample collection, storage, and processing followed specific protocols, ensuring sample preservation until analysis. Bioinformatic processing included detailed procedures for virus extraction, library preparation, and sequencing. For bacterial deoxyribonucleic acid (DNA), specific methods and tools were used, including MoBio PowerSoil kits and the MiSeq viz MiSeq Sequencing System platform for sequencing.
Statistical analysis was performed using R, using various packages for data processing and visualization. The study used two-sided P values, with a significance threshold set at P ≤ 0.05, and used the Benjamini-Hochberg correction for multiple testing. Associations of asthma with viremia were explored using logistic regression and other statistical methods, while controlling for potential confounders and cohort.
The study also assessed the association between virus and bacteria, using Spearman correlations and Procrustes analysis. The impact of virus on the development of asthma was analyzed using logistic regression and quasi-poisonous regression analyses. In addition, mediation analysis was conducted to test the significance of indirect bacterial effects.
Asthma development trajectories 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 viral and bacterial signature scores. Four groups were compared using Kaplan-Meier curve analysis and a log-rank test, providing information on the impact of these microbial communities on the development of asthma.
Environmental factors influencing the virus signature score were also examined through linear regression analysis. Finally, the study delves into the genetic aspect by focusing on Toll-Like receptor 9 (TLR9) genotype and its interaction with the virus in relation to asthma. Logistic regression analyzes were used to investigate the effects of different genotypes on asthma, with particular emphasis on the TLR9 rs187084 genotype. This genetic aspect was crucial to understanding the complex interplay between genetics, the gut virus and the development of asthma in children.
The interaction between TLR9 genotype and viral signature scores was evaluated, providing valuable insights into how genetic predispositions may influence early childhood asthma risk.
Study results
The present study focused on gut virus analysis of 647 one-year-old children, and these children were profoundly phenotyped from birth, with longitudinally assessed asthma diagnoses. It was found that specific species of temperate gut phages were associated with the later development of asthma. Notably, the common abundances of 19 caudovirus families contributed significantly to this association.
The research revealed that the combination of asthma-associated bacteria and virus signatures had increased effects on asthma risk, suggesting an independent virus-asthma association. Interestingly, virus-associated asthma risk was modulated by the host TLR9 gene variant rs187084, indicating a direct interaction between phages and the host immune system. This finding opens new avenues for further studies to investigate whether phages, along with bacteria and host genetics, can be used as preclinical biomarkers for asthma.
A key finding of the study was the variation in the relative abundance of both caudoviruses and microviruses between children with and without asthma by the age of five. The relative abundance of temperate phages was highly associated with asthma. Adjustments for potential confounders such as siblings, birth weight, urbanicity, and age did not change these results. This association was mainly caused by caudoviruses, and differences in the relative abundance of phages with an unknown lifestyle were due to microviruses.
The study also noted the high degree of uniqueness of gut viruses among children, with a median richness across samples of 1306 Viral Operational Taxonomic Units (vOTUs) and an overall sparseness of 86% across samples. However, overall observed richness and smoothness were not associated with the development of asthma before age five.
Differences in temperate virus composition between children with and without asthma were more marked, leading to the identification of 19 family-level temperate virus clades (VFCs) that were jointly associated with later asthma.
The research further explored the independence of the asthma virus from the bacterium. Despite the association of species richness and composition between the temperate virus and the bacterium, the results indicated that only a small indirect effect of the association of the virus with preschool asthma was mediated by bacteria. This was further supported by the finding that both the viral and bacterial signature scores captured the association with asthma better than either alone, indicating independent and additive effects.
Finally, the study examined the impact of early life exposure to the asthma virus and the genetic relationship between the gut virus and the host immune system in preschool asthma. It was found that the asthma risk derived from the viral signature score appeared to depend on the TLR9 genotype. This suggests a direct interaction between phages and the host’s immune system, contributing to the development of asthma in early childhood.
