Transcriptional profiling of mouse neutrophils treated ex vivo with a Toll-like receptor 5 agonist

Acute radiation syndrome (ARS) is a major cause of lethality following radiation disasters. A TLR5 agonist, entolimod, is among the most powerful experimental radiation countermeasures and shows efficacy in rodents and non-human primates as a prophylactic (radioprotection) and treatment (radiomitigation) modality. While the prophylactic activity of entolimod has been connected to suppression of radiation-induced apoptosis, the mechanism by which entolimod functions as a radiomitigator remains poorly understood. Here, we demonstrate that, in contrast to radioprotection, neutrophils are essential for the radiomitigative activity of entolimod. We found that neutrophils express functional TLR5 and stimulation by entolimod is a key component to the mechanism of radiomitigation. Given that the underlying effects of TLR5 agonists have been associated with NF-kB-dependent transcriptional events, we hypothesized that changes in the transcriptional profile of neutrophils post-entolimod treatment mitigates radiation damage. However, RNA sequencing data analysis showed that ex vivo stimulation of neutrophils with entolimod for 30 minutes caused changes in transcriptional profiles that were substantially weaker – both in the number of responsive genes and scale of changes – as compared with that of liver cells following in vivo entolimod treatment. These observations suggest that mitigation of radiation damage by entolimod-stimulated neutrophils involves a transcriptional-independent mechanism. Gene expression profiles were generated by deep sequencing of neutrophils 30 minutes after ex vivo treatment with vehicle or entolimod. Neutrophils were magnetically isolated from the bone marrow of BALB/c mice. All experiments were performed in duplicate.