Exploring the Role of Chemokine Receptor 6 (Ccr6) in the BXD Mouse Model of Gulf War Illness

Abstract

Gulf War illness (GWI) is a chronic and multi-symptomatic disorder with persistent neuroimmune symptomatology. Chemokine receptor 6 (CCR6) has been shown to be involved in several inflammation disorders in humans. However, the causative relationship between CCR6 and neuroinflammation in GWI has not yet been investigated. By using RNA-seq data of prefrontal cortex (PFC) from 31 C57BL/6J X DBA/2J (BXD) recombinant inbred (RI) mouse strains and their parental strains under three chemical treatment groups - saline control (CTL), diisopropylfluorophosphate (DFP), and corticosterone combined with diisopropylfluorophosphate (CORT+DFP), we identified Ccr6 as a candidate gene underlying individual differences in susceptibility to GWI. The Ccr6 gene is cis-regulated and its expression is significantly correlated with CORT+DFP treatment. Its mean transcript abundance in PFC of BXD mice decreased 1.6-fold (p < 0.0001) in the CORT+DFP group. The response of Ccr6 to CORT+DFP is also significantly different (p < 0.0001) between the parental strains, suggesting Ccr6 is affected by both host genetic background and chemical treatments. Pearson product-moment correlation analysis revealed 1473 Ccr6-correlated genes (p < 0.05). Enrichment of these genes was seen in the immune, inflammation, cytokine, and neurological related categories. In addition, we also found five central nervous system-related phenotypes and fecal corticosterone concentration have significant correlation (p < 0.05) with expression of Ccr6 in the PFC. We further established a protein-protein interaction subnetwork for the Ccr6-correlated genes, which provides an insight on the interaction of G protein-coupled receptors, kallikrein-kinin system and neuroactive ligand-receptors. This analysis likely defines the heterogeneity and complexity of GWI. Therefore, our results suggest that Ccr6 is one of promising GWI biomarkers.

Keywords: BXD strain; CORT; Ccr6; DFP; GWI; RNA-seq; neuroinflammation.

FIGURE 1

The expression of Ccr6 across the BXD strains. (A) The relative fold change of Ccr6 expression in the DFP and CORT+DFP groups compared to the CTL across the BXD RI strains. (B) Comparison of the expression (Mean ± SEM) of Ccr6 between the treatments (CTL, DFP, and CORT+DFP) by unpaired t-test. *p < 0.05, ***p < 0.0001. The Ccr6 expression is a normalized value by the DEseq2.

FIGURE 2

eQTLs mapping of Ccr6. eQTLs mapping demonstrates Ccr6 is cis regulated in the prefrontal cortex mRNA. (A) CTL (Max likelihood ratio statistic score (LRS) = 53.5), (B) DFP (Max LRS = 34.6), (C) CORT+DFP (Max LRS = 26.0). Chromosome number can be found across the top of the plot with megabases (Mb) on the x-axis. The y-axis contains the LRS. The location of Ccr6 is marked by an arrowhead found on the x-axis is at Chr 17 at 8.236 Mb. (D–F) The expression (Mean ± SEM) of Ccr6 is significantly different between B and D allele by unpaired t-test, ***p < 0.0001. The Ccr6 expression is a normalized value by the DEseq2.

FIGURE 3

Phenotype Pearson correlation analysis with the expression of Ccr6. Five BXD central nervous system (CNS) related phenotypes including brain to body weight ratio (A), novel open field behavior (B), anxiety assay (C), acoustic startle response (D), and learning and memory (E) were significantly correlated with the expression of Ccr6 in PFC. The fecal CORT content was positively correlated with the expression of Ccr6 (F). GeneNetwork BXD phenotype identifiers (e.g., ID “BXD_17494”) are at the top of each plot. n, Number of strains, p, p-value.

FIGURE 4

The gene set enrichment analysis of Ccr6 correlated genes. The bubble plot shows the top 20 GO (A) and KEGG pathways (B) of Ccr6 correlated genes in the CORT+DFP group.

FIGURE 5

PPI subnetwork of Ccr6 correlated genes. (A) Ccr6 PPI subnetwork includes 38 genes with the interaction score ≥0.9. The nodes represent genes while edges represent PPIs between two genes. Bubble plot of the Top 10 GO (B) and 3 KEGG (C) enrichment categories for the 38 PPI subnetwork genes.