In vitro and in vivo Generation of Heterophil Extracellular Traps after Salmonella Exposure

The release of extracellular traps (ETs) by granulocytes is a unique strategy to stop the dissemination of microbial pathogens. This study was undertaken to elucidate the potential of avian granulocytes (heterophils) to form ETs that can arrest and kill Salmonella organisms. After in vitro exposure of isolated heterophils and in vivo infection of day-old chicks with Salmonella enterica subsp. enterica serovars Infantis (SI) or Enteritidis (SE), the generation of ETs as well as the trapping and survivability of Salmonella organisms in the ET meshwork were determined by means of microscopy and spectrophotometry. In vitro, heterophils were able to form ETs within 15 minutes after SE and SI inoculation. At 120 minutes and with a multiplicity of infection of 1 and 5, SI induced significantly more ETs and DNA release than SE. Both SE and SI were found to be associated with the ET structures. Live-dead staining showed most of the microorganisms within the extracellular scaffold alive. In vivo, heterophils were detected in cecal lumen of SE-, but not SI-infected chicks. In cecum of the SE-exposed chicks, ET formations were scarcely detected whereas intact heterophils with phagocytosed bacteria were frequently found. The results evidence the capability of heterophils to generate ETs after SE and SI exposure in vitro. However, an infection of chicks with Salmonella did not significantly induce the formation of ET structures in cecum. Thus, the process to form ETs (ETosis) seems not to be of special relevance for Salmonella defense within the cecal lumen of young chicks.