Integrative analysis of the meibum microbiome in dry eye disease: from dysbiosis and diagnostic biomarkers to immunomodulation by Bradyrhizobium-derived outer membrane vesicles

BACKGROUND: Dry eye disease (DED) is a multifactorial disorder involving tear film instability and inflammation. The ocular surface microbiome (OSM) modulates local immunity, but its functional role in DED remains elusive. We characterized OSM alterations in DED and investigated the immunomodulatory effects of bacterial outer membrane vesicles (OMVs). METHODS: We enrolled 1,262 participants (520 DED, 742 controls). Clinical DED phenotypes were systematically identified using univariate, multivariate, and least absolute shrinkage and selection operator (LASSO) regression analyses. Meibum samples were subjected to 16S rRNA gene sequencing; amplicon sequence variants were generated using the divisive amplicon denoising algorithm 2 (DADA2) and taxonomically annotated against the SILVA database. Microbial diversity, community composition, and diagnostic biomarkers were evaluated, complemented by functional prediction (PICRUSt2) and co-occurrence network analyses. In vitro, OMVs isolated from Bradyrhizobium were applied to M1 macrophages to assess immunomodulatory effects through cytokine quantification, flow cytometric analysis of surface markers, and polarization-related gene expression profiling. RESULTS: Clinical profiling identified eight independent risk factors associated with DED, including ocular surface disease index scores, tear break-up time, and meibomian gland dysfunction. DED patients exhibited markedly elevated alpha-diversity (P < 0.001) and distinct microbial communities. Taxonomic shifts included reduced Alphaproteobacteria and Bradyrhizobium but enriched Gammaproteobacteria and Ralstonia. A six-genus panel distinguished DED from controls (AUC = 0.793). Functional prediction highlighted upregulated pathways related to inflammation, tight junctions, and PI3K-Akt-mTOR signaling. In vitro, Bradyrhizobium OMVs attenuated pro-inflammatory cytokines (TNF-α, IL-6, IL-1β; P < 0.001) while enhancing anti-inflammatory mediators (IL-10, TGF-β1). OMVs treatment decreased CD86⁺ and increased CD206⁺ macrophages (P < 0.001), consistent with M2 polarization, supported by downregulated inducible nitric oxide synthase (iNOS) and upregulated arginase-1 (Arg-1) expression (P < 0.001). CONCLUSION: This study establishes DED as a multifactorial condition associated with sleep, systemic disease, meibomian gland blockage, and radiation. These clinical features coexist with ocular surface dysbiosis characterized by depletion of beneficial Bradyrhizobium and expansion of Ralstonia taxa. Notably, Bradyrhizobium-derived OMVs confer protective effects by driving M2 macrophage polarization and resolving inflammation. Collectively, these findings underscore the microbiome-immune axis as a critical node in DED pathogenesis and provide a mechanistic rationale for microbiome-targeted diagnostics and therapeutics. Given the low-biomass nature of meibum and the absence of sequenced negative controls, a contribution of reagent-associated taxa to the observed signal cannot be fully excluded and warrants confirmation in future studies with comprehensive contamination controls.