Bacterial Extracellular Vesicles Derived from Weissella paramesenteroides and Lactobacillus paracasei Modulate Trained Immunity-Associated Responses in THP-1 Cells

Trained innate immunity refers to the capacity of innate immune cells, such as macrophages, to undergo long-term functional reprogramming following primary stimulation, leading to altered responses upon secondary challenges. In this context, bacteria-derived extracellular vesicles have emerged as promising mediators of this process. To investigate whether extracellular vesicles (EVs) derived from the lactic-acid bacteria (LAB) Weissella paramesenteroides and Lactobacillus paracasei can modulate trained immunity-associated responses in human macrophage-like cells. EVs were isolated by centrifugation, filtration, and ultracentrifugation, followed by characterization by dynamic light scattering (Zetasizer) and protein quantification using the BCA assay. THP-1 cells were maintained in RPMI-1640 supplemented with 10% FBS and 1% antibiotics. Then, cells were differentiated into macrophage-like cells by treatment with PMA (phorbol 12-myristate 13-acetate) for 48 h. Subsequently, cells were stimulated with EVs or β-glucan (positive control), followed by a 3-day resting period. Afterward, the cells were challenged with LPS for 24 h. Cell culture supernatants were collected for ELISA and nitric oxide (NO) assays, while gene expression analysis was performed by polymerase chain reaction (PCR) in THP-1 cells.  EVs showed protein concentrations of 1.2–1.4 mg/mL and sizes of 146.2 nm and 269.9 nm for Lactobacillus paracasei and Weissella paramesenteroides derived vesicles, respectively. EV treatment did not significantly affect cell viability or NO production. However, in LPS-stimulated cells, probiotic-derived EVs reduced IL-6 mRNA expression compared with the LPS-only group. These findings suggest that LAB-EVs may modulate macrophage responses and influence innate immune memory, promoting a balanced response upon secondary stimulation. Further studies are needed to elucidate the molecular and metabolic mechanisms underlying this process.