Respiratory microbiota transplantation: optimized framework and its impact on metabolic and immune characteristics

The respiratory microbiota is critical to maintaining local immune homeostasis and respiratory health. Microbiota transplantation has proven transformative in gut microbiome studies. However, a standardized approach for the respiratory tract remains lacking, hindered by technical difficulty of establishing a recipient airway niche conducive to stable donor microbial engraftment, and limited systematic evaluation of key parameters that influence transplantation efficacy. This study aims to establish an optimized respiratory microbiota transplantation (RMT) framework and determine whether RMT reshapes metabolic and immunological characteristics.

We developed and optimized a method for RMT in murine models. Key parameters, including sample storage, delivery route, and treatment regimen, were systematically evaluated for their effects on the microbiome using 16S ribosomal RNA gene sequencing-based profiling. The influence of microbiota transplantation on host metabolism and immunity was also assessed through metabolomic and transcriptomic characterization. Wilcoxon rank-sum test was used to compare Bray-Curtis dissimilarity between groups. Additionally, a one-sample Wilcoxon signed-rank test was used to determine whether the relative abundance changes within each recipient-donor pair significantly deviated from zero. Differential metabolomic and transcriptomic features were identified using trend analysis.

We established mouse-to-mouse RMT by transferring bronchoalveolar lavage fluid (BALF)-derived microbial communities from specific pathogen-free (SPF) donors to germ-free (GF) recipients. To model lung dysbiosis, we induced sepsis via cecal ligation and puncture in SPF mice and transplanted their BALF microbiota into normal SPF recipients. The highest compatibility of donor-recipient microbiota was observed using glycerol-preserved samples, delivered either intratracheally or intranasally every other day, for a duration of 14 days (Wilcoxon rank-sum test, SPF-GF mice: intratracheal delivery 7 days vs. 14 days, W = 12.0, P = 0.057, intranasal delivery 7 days vs. 14 days, W = 12.0, P = 0.057; CLP-SPF mice: intratracheal delivery 7 days vs. 14 days, W = 35.0, P = 0.003, intranasal delivery 7 days vs. 14 days: W = 36.0, P = 0.035). This microbiota transplantation partially shifted the metabolomic and immunological characteristics of GF recipients toward those of SPF donors, reversing 188 metabolites and 2721 host genes that were altered in GF mice compared with SPF mice. We then adapted this protocol for human-to-mouse RMT, transplanting microbiota from human BALF and sputum into SPF mice. Intratracheal and intranasal delivery of human BALF yielded comparable donor-recipient microbiota similarity. However, intratracheal administration significantly increased donor-recipient similarity when sputum-derived microbiota were transplanted (Wilcoxon rank-sum test, W = 53.5, P = 0.004).

Our study establishes an optimized protocol for RMT using glycerol-preserved samples, delivered either intratracheally or intranasally every other day over 14 days. This approach should empower preclinical investigation of respiratory microbiota and pave the way for clinical translation.