Iron oxide nanozymes modulate autophagy involving the JNK pathway and inhibit Salmonella Enteritidis infection in chickens

Salmonella Enteritidis ( S . Enteritidis) is a significant zoonotic foodborne pathogen primarily hosted by poultry, causing gastroenteritis and substantial economic losses on the poultry industry while threatening human food safety. Macrophages play a dual role in combating Salmonella infections but can also serve as bacterial carriers, allowing bacteria to evade antibiotic treatment. Therefore, developing effective antibacterial strategies targeting intracellular Salmonella is of critical importance. Iron oxide nanozymes (IONzymes), possessing peroxidase-like activity and magnetic properties, have shown potential in inhibiting bacterial proliferation. Autophagy, a conserved cellular degradation process, is vital for defending against pathogenic bacterial infections, and c-Jun NH 2 -terminal kinases (JNKs) are key regulators of autophagy. In this study, chicken macrophages (HD11) and specific pathogen-free (SPF) chicks were used to establish a S . Enteritidis infection model to evaluate the ability of IONzymes to mitigate infection both in vitro and in vivo , with a focus on their effects on the autophagic pathway, especially the JNK-mediated autophagy pathway, and to elucidate the mechanism by which IONzymes inhibit bacterial proliferation. The results demonstrated that IONzymes promoted autophagy in infected cells, as evidenced by increased formation of autophagosomes and elevated expression of autophagy-related proteins ATG16L1 and LC3-II. Inhibition of autophagy using LY294002 reversed the antibacterial effect of IONzymes, indicating that autophagy is essential for their action. Further investigation showed that the JNK pathway mediates IONzyme-induced autophagy; inhibition of JNK with SP600125 attenuated both autophagy and the antibacterial effect, while JNK activation alone (by anisomycin) was insufficient to eliminate intracellular bacteria. In vivo , 150-nm IONzymes significantly reduced S . Enteritidis loads in the spleen, thymus, and liver of infected chicks, improved spleen pathology, and increased the expression of autophagy proteins and phosphorylated JNK. Moreover, IONzymes restored Lactobacillus abundance, which negatively correlated with bacterial load. These findings provide insights into the potential use of IONzymes as novel antibacterial agents against S. Enteritidis infections in poultry and humans.