Cronobacter sakazakii induces necrotizing enterocolitis by regulating NLRP3 inflammasome expression via TLR4

Abstract

<b>Introduction.</b> Neonatal infection with <i>Cronobacter sakazakii</i> can cause severe intestinal damage and necrotizing enterocolitis (NEC). The inflammasome and Toll-like receptors mediate intestinal damage caused by other intestinal pathogens causing NEC, but the exact mechanism is unclear.<b>Aim.</b> We evaluated the molecular mechanisms underlying <i>C. sakazakii</i>-induced NEC.<b>Methodology.</b> The effects of <i>C. sakazakii</i> treatment on two cell lines and a Sprague-Dawley rat model of NEC were evaluated by a cell death assay, western blot and real-time PCR analyses of the NLRP3 inflammasome and downstream factors, and observation of cell and intestinal damage.<b>Results.</b> <i>C. sakazakii</i> caused cellular damage <i>in vitro</i>, as well as intestinal damage in an animal model. NLRP3, caspase-1, TLR4 and MyD88, as well as the downstream factor IL-1β, were upregulated in <i>C. sakazakii</i>-infected J774A.1 and HT-29 cells. Western blotting showed that <i>C. sakazakii-</i>infected J774A.1 and HT-29 cells and the NEC rat model had higher expression levels of N-terminal gasdermin D (GSDMD) compared with those in the control groups. <i>C. sakazakii</i> and its components promote NF-κB expression via the TLR4/MyD88 signalling pathway, thereby regulating the NLRP3 inflammasome and mediating GSDMD cleavage, resulting in pyroptosis-induced intestinal damage.<b>Conclusion.</b> We found that <i>C. sakazakii</i> upregulates NF-κB via TLR4/MyD88 to promote activation of the NLRP3 inflammasome, leading to the up-regulation of downstream caspase-1, release of IL-1β, GSDMD-mediated pyroptosis and development of NEC. These findings clarify the mechanisms by which <i>C. sakazakii</i> contributes to NEC.