Brucella suis vaccine strain 2 promotes intracellular proliferation by inducing M2 macrophage polarization and autophagy through PI3K/Akt/mTOR signalling

Introduction. The persistent intracellular survival of Brucella is a key factor contributing to immune evasion and chronic infection as evidenced by clinical recurrence. This ability of establishing long-term intracellular residency is a major driver of brucellosis relapse. Pathogens such as Brucella often manipulate host cell polarization and exploit autophagy to sustain their intracellular survival. Hypothesis/Gap Statement. Real-world understanding of how macrophage polarization and autophagy jointly influence Brucella intracellular replication remains limited, and the specific signalling pathways mediating this interaction have not been fully characterized. Aim. This study aimed to investigate the interplay between macrophage polarization, autophagy and intracellular proliferation of Brucella suis strain 2 and to characterize the involvement of the PI3K/AKT/mTOR signalling pathway in this process. Methodology. We examined infection-induced alterations in BV2 cell polarization and autophagic activity following Brucella S2 infection. Autophagy-related protein expression (LC3B-I, LC3B-II, ULK1 and p62) was assessed by Western blot, and autophagosome-to-autolysosome conversion was visualized by laser confocal microscopy. Macrophage polarization markers (iNOS and Arg1) and cytokine secretion (TNF- α and IL-10) were measured by ELISA. Intracellular bacterial replication was quantified by c.f.u. assays, and pharmacological inhibitors were used to modulate PI3K/Akt/mTOR signalling and autophagic activity. Results. Following Brucella S2 infection in BV2 cells, we observed elevated expression of iNOS, Arg1, p-Akt, mTOR and autophagy-related proteins (LC3B-I, LC3B-II and ULK1), accompanied by decreased p62 levels. Laser confocal microscopy revealed efficient autophagosome-to-autolysosome conversion. Time-dependent increases in TNF- α and IL-10 secretion indicated macrophage plasticity and the establishment of a mixed M1/M2 phenotype. c.f.u. assays demonstrated that PI3K/Akt/mTOR inhibition and autophagy induction promoted Brucella S2 intracellular proliferation. Notably, pharmacological inhibition of the PI3K/Akt/mTOR pathway reversed this effect by promoting M1 polarization while suppressing autophagic activity. Conclusion. Collectively, our findings demonstrate that Brucella S2 infection induces both M1/M2 mixed polarization and autophagic activation in BV2 cells. Importantly, we identified that the M2-polarized state and autophagy induction synergistically promote intracellular bacterial replication, a process mediated through the PI3K/Akt/mTOR signalling pathway. These results suggest that targeted modulation of PI3K/Akt/mTOR signalling could represent a promising therapeutic approach to control Brucella S2 intracellular proliferation.