Impact of Prolonged Continuous Stress Exposure on Immune Function and the Gut Microbiome in a Perpetual Avoidance of Water on a Wheel Mouse Model

Abstract

Both the immune system and gut microbiome play important roles in the physiological response to stress, yet their interplay under conditions of long-term continuous stress remains poorly understood due to the lack of suitable experimental models. We employed our previously developed Perpetual Avoidance of Water on a Wheel (PAWW) model to investigate the effects of sustained stress on immune-related cytokine expression and gut microbiome composition in mice. We demonstrated that PAWW stress can be sustained for six weeks without signs of behavioral or physiological adaptation. Circadian locomotor rhythms remained disrupted throughout the exposure period, accompanied by elevated levels of norepinephrine and epinephrine in plasma. Quantitative real-time PCR revealed significant upregulation of inflammation-related cytokine genes, including TNF-α in the brain and IL-6 and IL-1β in intestinal Peyer’s patches (PPs). Microbiome profiling by 16S rRNA gene amplicon sequencing showed that stressed mice underwent more pronounced compositional changes than controls, particularly in members of the Lachnospiraceae family. These findings indicate that the PAWW model provides a valuable approach for investigating the effects of chronic stress on the brain–immune–gut axis. Chronic PAWW stress was associated with both inflammatory immune responses and microbiome alterations, highlighting a potential role for PPs in mediating intestinal immune regulation under stress conditions.