Divergent age-dependent peripheral immune transcriptomic profile following traumatic brain injury

Abstract

The peripheral immune system is a major regulator of the pathophysiology associated with traumatic brain injury (TBI). While age-at-injury influences recovery from TBI, the differential effects on the peripheral immune response remain unknown. Here, we investigated the effects of TBI on gene expression changes in murine whole blood using RNAseq analysis, gene ontology and network topology-based key driver analysis. Genome-wide comparison of CCI-injured peripheral whole blood showed a significant increase in genes involved in proteolysis and oxidative-reduction processes in juvenile compared to adult. Conversely, a greater number of genes, involved in migration, cytokine-mediated signaling and adhesion, were found reduced in CCI-injured juvenile compared to CCI-injured adult immune cells. Key driver analysis also identified G-protein coupled and novel pattern recognition receptor (PRR), P2RY10, as a central regulator of these genes. Lastly, we found Dectin-1, a c-type lectin PRR to be reduced at the protein level in both naïve neutrophils and on infiltrating immune cells in the CCI-injured juvenile cortex. These findings demonstrate a distinct peripheral inflammatory profile in juvenile mice, which may impact the injury and repair response to brain trauma.

Figure 1

Age-dependent comparative transcriptomic analysis of the peripheral whole blood. (a) Heat map of top 30 genes significantly upregulated in the juvenile whole blood at 4d post-CCI injury compared to juvenile sham. (b) Identification of Genes upregulated in the adult CCI-injured whole blood compared to sham injury. (c) Comparison of genes upregulated following CCI injury in juvenile vs adult whole blood. No upregulated genes were found to overlap. (d) Downregulated genes in juvenile CCI-injured and in (e) adult whole blood compared to respective shams. (f) No downregulated genes were found to overlap. (g–i) Comparison of genes found to be significantly increased or reduced in sham juvenile compared to sham adult. (j) Genes that were increased in juvenile sham vs adult sham that were also increased in juvenile CCI vs adult CCI samples. (k) Genes that were decreased in juvenile sham vs adult sham that were also increased in juvenile CCI vs adult CCI samples.

Figure 2

Age-dependent Gene Ontology analysis of CCI-injured peripheral immune response. (a,b) GO biological process groupings of changing genes show differentially regulated pathways in juvenile vs adult CCI-injured mice. Increased genes were identified in the categories of transport (p = 0.0002), metabolic process (p = 0.0033), oxidation-reduction (p = 1.85E-05) and proteolysis (p = 0.0005). Decreased genes were identified in the categories of cell cycle (p = 8.18E-07), RNA polymerase (p = 9.02E-06), apoptotic processes (p = 0.0004), cell adhesion (p = 0.0274), inflammatory response (p = 0.0011), cytokine-mediated signaling (p = 0.0018) and cell migration (p = 0.0063). (c,d) Enrichment analysis of GO biological process categories for significantly changing genes in juvenile vs adult CCI-injured mice. (e) Ingenuity Pathway Analysis (IPA) shows that MAP3K8, JAK1, ETS1 and RHO (red highlight), among others, represent predicted signaling pathways identified as central to the functions of the down-regulated genes confirmed from above ontology analysis. Interaction of downregulated genes in CCI-injured juvenile vs adult. Networks were generated through the use of IPA: https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis . (f) Enrichment analysis of KEGG categories for significantly changing genes in juvenile vs adult CCI-injured mice. P values were calculated using the chi-square method.

Figure 3

P2ry10 is a key driver of the juvenile gene profile following CCI injury. Mergeomics analysis of differentially reduced genes in juvenile vs adult CCI-injured mouse peripheral blood identified significant interactions between genes and identified P2ry10 as a key driver implicated in the genes found to be reduced in the juvenile peripheral immune response to injury. Red diamond, key driver; Blue circles, reduced juvenile genes; red dots, other known neighboring genes within the network.

Figure 4

Age-dependent Dectin-1 protein expression and Gene Ontology analysis of sham whole blood. (a–d) Flow cytometry analysis showed reduced dectin-1 protein expression on Ly6G+ neutrophils in juvenile peripheral blood and spleen compared to adult. (e–g) Infiltrating CD45+ cells in the lesion of the adult brain also express dectin-1. (h–j) Dectin-1 expression is decreased in infiltrating CD45+ cells in the lesion of the juvenile brain. (k,l) Enrichr GO Biological Process analysis of significantly downregulated genes in juvenile vs adult sham peripheral blood. (m,n) KEGG pathway analysis of significantly downregulated genes in juvenile vs adult sham peripheral blood. P values were calculated using the hypergeometric method.