Fig. 3

Blood neutrophil transcriptional profiles and spleen neutrophil functions are chronically dysregulated during the chronic stages of TBI. Bulk RNA-seq was performed on blood neutrophils at 120 dpi. (A) PLSDA of all normalized gene counts revealed a clear separation of aged sham and aged TBI samples into individual groups across the first two principal components. n = 3 mice/group. (B) Pathways involved in biosynthetic processes and translation were down-regulated after TBI, whereas those involved in stress and viral defense responses were up-regulated. (C) Heatmap of unsupervised clustering of the top 30 differentially expressed genes. Color coding was based on z-score scaling. (D-E) Sample-level enrichment analysis (SLEA) for chromatin remodeling pathway (D) and epigenetic regulation (E) of gene expression. (F) Venn Diagram of chromatin remodeling pathway, epigenetic regulation of gene expression pathway, and differentially expressed (DE) epigenetic pathway genes genes between AT vs. AS. (G) Six out of 268 genes from chromatin remodeling pathway were differentially expressed after TBI, including Smarcad1, Tspyl4 and Hgfl3 up-regulated after TBI and Bcl7c, Chd3 and Wdhd1 down-regulated after TBI. (H-I) Neutrophil function including phagocytosis (MFI of pHrodo E. coli., H) and oxidative stress levels [mean fluorescence intensity (MFI) of DCF, I] from the spleen was evaluated by flow cytometry. n = 4–6 mice/group. For all histograms, light gray = fluorescence minus one (FMO) control. Data were analyzed using Mann-Whitney for two group comparisons. **p < 0.01, *p < 0.05. AS: aged sham; AT: aged TBI