Immunomodulation represents a key mechanism through which the microbiota-gut-brain axis exerts its effects. While probiotics can promote central nervous system (CNS) development via this axis, current in vitro models hard to recapitulate the process whereby probiotics activate local immunity and subsequently deliver immune mediators to the CNS through systemic circulation. To address this gap, we established a probiotic-RAW264.7 cell-primary hippocampal neuron system for investigation of how live and heat-inactivated Bifidobacterium bifidum TMC3115 promotes hippocampal neuron development via macrophage-conditioned media. We found that the peptidoglycan (PGN) induced relatively low levels of IL-6, IL-1β, IL-10, and TNF-α secretion, whereas lipopolysaccharide (LPS) elicited markedly higher levels of IL-6, IL-1β, and IL-10, as measured by Enzyme-Linked Immunosorbent Assay (ELISA). In contrast, both live and heat-inactivated TMC3115 induced only moderate cytokine secretion. Conditioned mediums (CMs) derived from PGN and LPS treated macrophages inhibited synaptic and morphological development of primary hippocampal neurons, whereas CMs from both live and heat-inactivated TMC3115-RAW264.7 significantly enhanced neuronal survival and upregulated the mRNA expression of Cellular Fos (c-Fos), Postsynaptic Density Protein 95 (PSD95), and Synaptophysin (SYP). Notably, CMs from live TMC3115 elicited stronger pro-neurodevelopmental effects than that from the heat-inactivated counterpart. Importantly, IL-6 depletion in both live and heat-inactivated TMC3115-RAW264.7 CMs suppressed the expression of neurodevelopmental genes and proteins and impaired neuronal morphological maturation. Mechanistically, CMs of both live and heat-inactivated TMC3115-RAW264.7 activated the IL-6/JAK2/STAT3 signaling pathway in neurons, as indicated by increased pJAK2/JAK2 and pSTAT3/STAT3 ratios. Consistently, pharmacological inhibition of JAK2 or STAT3 markedly impaired synaptic development in primary hippocampal neurons. In summary, our results demonstrate that both live and heat-inactivated TMC3115 strain-specifically induce RAW264.7 cells to secrete IL-6 at physiologically relevant concentration, which contributes to the synaptic and morphological development of primary hippocampal neurons and is partially mediated through activation of the IL-6/JAK2/STAT3 signaling pathway.
