Antibiotic-induced dysbiosis alters stress responsiveness and neutrophil dynamics in fish

Immune and stress responses are closely interconnected, with glucocorticoids modulating neutrophil number and activity, and cytokines influencing stress response. Neutrophils, due to their role as primary responders to infection, high sensitivity to glucocorticoid fluctuations, and critical involvement in gut microbiota homeostasis, were selected as the central focus of this study. We examined how antibiotic-induced dysbiosis affects the hypothalamus-pituitary-interrenal (HPI) axis and stress-related neutrophil dynamics in common carp (Cyprinus carpio L.). We analyzed the expression of stress-related genes and characterized neutrophil maturation and function within the hematopoietic niche. Dysbiotic fish exposed to acute stress exhibited significantly elevated cortisol levels compared to stressed fish with intact microbiota. Notably, even non-stressed dysbiotic fish showed increased cortisol level, indicating that microbiota disruption alone impairs HPI axis regulation. Antibiotic-treated and stressed fish displayed upregulation of il1β, gcsfr, cxcl8_l2, and cxcr1, suggesting enhanced granulopoiesis and neutrophil mobilization. However, systemic neutrophilia was attenuated in dysbiotic fish regardless of stress exposure. Transcriptomic profiling of neutrophils from dysbiotic, stressed fish revealed downregulation of mpx and cxcr4, and upregulation of mmp9, mhc1, trb, nlrp12, dhx58, irf3, irf7 and stat1, indicating altered maturation, increased migratory potential, and possible neutrophil-T cell interactions. In contrast, stressed fish with intact microbiota exhibited anti-apoptotic signatures and suppression of antiviral pathways. Across all stressed and dysbiotic groups, neutrophil phagocytic activity was significantly reduced. These findings underscore the pivotal role of gut microbiota in modulating stress responses, neutrophil development and trafficking, and immune function in vertebrates.