Sex and stress paradigm influence behavioural, neuroimmune, and microbiome responses in pubertal CD-1 mice

Puberty is a critical developmental period marked by heightened vulnerability to stress. Exposure to pubertal stress can have lasting effects on behaviour, immune function, and the gut-brain axis. The NLRP3 inflammasome and gut microbiome are key regulators of neuroinflammation, yet their roles across distinct pubertal stress paradigms remain unclear. This study compared behavioural, immune, and microbial outcomes in 100 CD-1 mice following chronic unpredictable stress (CUS; 14 or 28 days) or 7-day sleep disruption (SD) in pubertal males and females. Behavioural testing revealed stressor- and sex-specific effects: females spent more time in the open arms of the elevated plus maze, SD reduced open-arm exploration, 14-day CUS reduced centre activity in the open field, and SD increased immobility in the forced swim test. Plasma cytokines showed divergent patterns, with IL-22 elevated across stress conditions and IL-17A highest following 28-day CUS and in females overall. In the prefrontal cortex, SD males exhibited increased NLRP3 and CASP1 mRNA expression, whereas 28-day CUS females showed reduced NFκB1, and NFκB2 expression was elevated in stress-exposed males. In the hippocampus, 28-day CUS females demonstrated coordinated reductions in NFκB1, NLRP3, and CASP1 expression. Microbiome analyses indicated that SD and 14-day CUS produced the most substantial alterations, characterized by reductions in short-chain fatty acid-producing taxa (Ligilactobacillus, Acetatifactor, Christensenella) and enrichment of stress-associated genera (Flintibacter, Oscillospiraceae). Together, these findings indicate that stressor type and sex differentially shape behavioural, neuroimmune, and microbial profiles during puberty, with sleep disruption exerting particularly strong effects on behavioural and gut-immune regulation.