A transcriptomic reporter assay employing neutrophils to measure immunogenic activity of septic patients' plasma

Background: There are diverse molecules present in blood plasma that regulate immune functions and also present a potential source of disease biomarkers and therapeutic targets. Genome-wide profiling has become a powerful method for assessing immune responses on a systems scale, but technologies that can measure the plasma proteome still face considerable challenges. An alternative approach to direct proteome assessment is to measure transcriptome responses in reporter cells exposed in vitro to plasma. In this report we describe such a transcriptomic reporter assay to assess plasma from patients with sepsis, which is a common and severe systemic infectious process for which physicians lack efficient diagnostic or prognostic markers. Methods: Plasma samples collected from patients with culture-confirmed bacterial sepsis and uninfected healthy controls were used to stimulate three separate cell types - neutrophils, peripheral blood mononuclear cells, and monocyte-derived dendritic cells. Whole genome microarrays were generated from stimulated cells to assess transcriptional responses. Unsupervised analysis and enriched functional networks were evaluated for each cell type. Principal component analyses were used to assess variability in responses. A random K-nearest neighbor - feature selection algorithm was used to identify markers predictive of sepsis severity, which were then validated in an independent data set. Results: Neutrophils demonstrated the most distinct response to plasma from septic patients with 709 genes showing altered expression profiles, many of which are involved in established immunologic pathways. The amplitude of the neutrophil transcriptomic response was shown to be correlated with sepsis severity in two independent sets of patients comprised of 64 total septic patients. A subset of 30 transcripts selected using one set of patients was demonstrated to have a high degree of accuracy (82-90%) in predicting sepsis severity and outcomes in the other independent set. This subset included several genes previously established in sepsis pathogenesis as well as novel genes. Conclusions: These results demonstrate both the suitability and potential clinical relevance of a neutrophil reporter assay for studying plasma, in this case from septic patients. The distinctive transcriptional signature we found could potentially help predict severity of disease and guide treatment. Our findings also shed new light on mechanisms of immune dysregulation in sepsis.