Exosomes derived from IL-4 treated macrophages alleviate steroidogenesis, apoptosis, and inflammatory dysregulation induced by lipopolysaccharide in bovine granulosa cells.

High-yield dairy cows are frequently exposed to bacterial challenges that result in endotoxin release, leading to ovarian granulosa cell dysfunction and reduced fertility. Exosome have emerged as innovative therapeutic agents due to their ability to regulate inflammation and promote tissue repair. The present study aimed to evaluate the impact of M2 macrophage-derived exosomes (M2-Exo) on steroidogenesis, apoptosis, and inflammatory pathways in lipopolysaccharide (LPS) exposed bovine GCs. Primary bovine macrophages were isolated and treated with IL-4 (20 ng/ml) for 24 h to polarize them into the M2 phenotype. The exosomes (M2-Exo) were then isolated, characterized, and applied to treat primary bovine granulosa cells (GCs). GCs were obtained from medium size follicles (5-8 mm) of dairy cows and stimulated with 1 μg/mL LPS for 4 h, followed by treatment with M2-Exo (50 μg/ml) for an additional 20 h. Cell viability was assessed by MTT assay. Cytokines and steroid hormone concentrations were measured using ELISA assay. The expression of steroidogenic, FSHR and NFκB/TLR4 genes was analyzed by qRT-PCR method. Apoptosis was evaluated using Annexin V/PI staining. M2-Exo treatment significantly improved GC viability and reduced apoptosis in LPS-stimulated cells. M2-Exo attenuated the secretion of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), enhanced the expression of steroidogenic (CYP19A1, P450scc) and FSHR, and restored estradiol and progesterone production. In addition, M2-Exo reduced the expression of TLR4 and NFκB, in LPS exposed GCs cells. Overall, M2-Exo significantly mitigated the LPS-induced inflammation, apoptosis, and steroidogenesis impairment in bovine GCs. These findings suggest that M2-Exo may represent a promising candidate for improving ovarian function and fertility in dairy cows affected by inflammatory disorders.