Effects of psychosocial stress on laryngeal microbiology and epithelial barrier integrity

Psychosocial stress and laryngeal physiology are linked. However, the biological mechanisms of psychosocial stress on voice have not been studied. This study delineated the effects of psychosocial stress on laryngeal microbiota composition and vocal fold epithelial integrity. We hypothesized that stress would result in reduced microbial diversity and abundance in laryngeal microbiota, and reduced vocal fold epithelial barrier integrity, with more pronounced differences in females and with increased duration of stress. One hundred and eight, conventionally-raised, C56BL/7 mice (8-10 weeks of age, 54 males, 54 females) were allocated to short stress, prolonged stress or control groups. Psychosocial stress involved restraint stress for 7 days (short stress) and 14 days (prolonged stress). Laryngeal microbiota profiles were compared across stress groups using 16S rRNA sequencing (N = 66). Outcome measures of alpha and beta diversity, differentially abundant taxa were obtained. Independently, stress-altered epithelial targets were delineated using RT-qPCR (N = 24) and immunofluorescence (N = 18).We found that prolonged stress, but not short stress, altered measures of alpha, beta diversity, indicating distinct laryngeal microbiota composition compared to control samples. Prolonged stress samples were dominated by Firmicutes phyla, whereas, short stress and control groups by Actinobacteria, and Proteobacteria phyla. Within genera, prolonged psychosocial stress decreased relative abundance of Corynebacterium and increased Streptococcus. Laryngeal microbial differences were more pronounced in females following psychosocial stress, as hypothesized. In addition, short and prolonged psychosocial stress downregulated gene and/or protein expression of inflammatory cytokines, sensory receptors, adherens and tight junction (E cadherin, Zo-1), TLRs and mucins (MUC2) within the larynx, with more severe effects in the prolonged stress group. Short and prolonged psychosocial stress alters laryngeal microbiota composition and vocal fold epithelial barrier integrity. Future studies should delineate causal host epithelial-microbiome interactions in the larynx in response to stress.