Purpose: Keratoconus (KC) is characterized by irregular astigmatism along with corneal stromal weakness and is associated with altered immune status. Tissue resident microbiomes are known to influence the immune status in other organs, but such a nexus has not been described in ocular conditions. Therefore, we examined the ocular surface microbiome of patients with KC and correlated it to the immune cell and tear molecular factor profiles.
Methods: Sixty-two patients with KC and 21 healthy controls underwent corneal topography analysis and eye examination followed by a collection of Schirmer's strip, ocular surface wash, and ocular surface swabs. Microbiomes were analyzed by extracting DNA from the swabs followed by 16S rRNA gene V3-V4 amplicon sequencing and analyzed using QIIME. Fifty-two molecular factors from Schirmer's strip tear extracts and 11 immune cells from ocular wash were measured using multiplex ELISA and flow cytometry. Alpha diversity, linear discriminant analysis effect size (LEfSe), relative abundance and receiver operating characteristic - area under the curve (ROC-AUC) analysis were performed. Unsupervised clustering at the genus level with clinical parameters, soluble factors, and immune cells was performed.
Results: Fifty-two phyla/class, 132 order, 283 family, and 718 genera were identified in our cohort. Alpha diversity indices were comparable between patients with KC and the healthy controls. Dominant phyla across groups were Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Alphaproteobacteria increased in KC eyes whereas Actinobacteria, Firmicutes_Bacilli reduced compared with the healthy controls. We found a significant positive correlation of Microbacterium, Cutibacterium, and Brevundimonas genera abundance with keratometry and corneal thickness. Levels of IL-21, IL-9, Fractalkine, and VEGF positively correlated with Tetrasphaera (P < 0.05). β2-microglobulin and CD66bhigh cells correlated with Bacteroides (P < 0.05). CD45+ cells correlated with Escherichia_Shigella (P < 0.02).
Conclusions: We discovered a unique microbiome signature of KC which correlated to disease grades and secreted molecular factors and immune cells. Therefore, the altered microbiome on the ocular surface may drive immune dysregulation in KC and provide scope for potential interventions in the future.