Enduring and Sex-specific Changes in Hippocampal Gene Expression after a Subchronic Immune Challenge

Abstract

Major illnesses, including heart attack and sepsis, can cause cognitive impairments, depression, and progressive memory decline that persist long after recovery from the original illness. In rodent models of sepsis or subchronic immune challenge, memory deficits also persist for weeks or months, even in the absence of ongoing neuroimmune activation. This raises the question of what mechanisms in the brain mediate such persistent changes in neural function. Here, we used RNA-sequencing as a large-scale, unbiased approach to identify changes in hippocampal gene expression long after a subchronic immune challenge previously established to cause persistent memory impairments in both males and females. We observed enduring dysregulation of gene expression three months after the end of a subchronic immune challenge. Surprisingly, there were striking sex differences in both the magnitude of changes and the specific genes and pathways altered, where males showed persistent changes in both immune- and plasticity-related genes three months after immune challenge, whereas females showed few such changes. In contrast, females showed striking differential gene expression in response to a subsequent immune challenge. Thus, immune activation has enduring and sex-specific consequences for hippocampal gene expression and the transcriptional response to subsequent stimuli. Together with findings of long-lasting memory impairments after immune challenge, these data suggest that illnesses can cause enduring vulnerability to, cognitive decline, affective disorders, and memory impairments via dysregulation of transcriptional processes in the brain.

Keywords: RNA sequencing; hippocampus; inflammation; learning and memory; neuroimmune; sex differences.

Figure 1.. Subchronic, peripheral LPS challenge induces changes in hippocampal gene expression 12 weeks after last injection.

(A) Males (blue) show a greater number of DEGs than females (yellow). Males showed 183 upregulated and 47 downregulated genes, whereas females showed 7 and 18 up- and down-regulated genes, respectively. (B) Top upregulated and downregulated genes in males and females. (C, D) Volcano plots showing differentially expressed genes in males (C) and females (D) obtained from Advaita iPathway Analysis. Differentially expressed genes (DEG) are represented in terms of their measured expression change (x-axis) and the significance of the change (y-axis), with upregulated genes shown in red and downregulated genes shown in blue. (E) Genes differentially expressed in the hippocampus of both males and females are all dysregulated in opposite directions.

Figure 2.. Sex-specific functions of differentially expressed genes 12 weeks after subchronic, peripheral LPS challenge.

(A,B) Biological pathways and processes enriched in gene set (A) in males and (B) in females were generated through Metascape. Distinct biological pathways are observed in males and females, with greater plasticity and immune-related targets in males and greater monoaminergic signaling in females. (C, D) Protein-protein interaction (PPI) networks of targets were generated using STRING 10.5 and clustered by biological function (C) in males and (D) in females. Edges between nodes are color coded for relationship type. Blue: known interactions; Pink experimentally determined interactions; Black: Co-expression of targets; Purple: protein homology; Green, yellow, and dark blue: predicted interactions.

Figures 3.. Prior subchronic, peripheral LPS challenge alters hippocampal gene expression in response to a subsequent, acute challenge in a sex-specific manner.

(A) Females (yellow) show a greater number of differentially expressed genes than males (blue). Females show 192 upregulated and 240 downregulated genes, whereas males show 67 up- and 1 downregulated gene compared with the response to acute immune challenge in previously naïve animals. (B) Top upregulated and downregulated genes in males and females. (C, D) Volcano plots showing differentially expressed genes in males (C) and females (D) obtained from Advaita iPathway Analysis. Differentially expressed (DE) genes are represented in terms of their measured expression change (x-axis) and the significance of the change (y-axis), with upregulated genes shown in red and downregulated genes shown in blue.

Figures 4.. Subsequent acute immune challenge leads to dysregulation of sex-specific targets in the hippocampus.

(A,B) Distinct biological pathways and processes are enriched in DEGs of (A) males and (B) females who showed greater numbers of pathways and changes in immune-related pathways. (C,D) Protein interaction (PPI) networks of targets, clustered by biological function (C) in males and (D) in females. Edges between nodes are color coded for relationship type. Blue: known interactions; Pink experimentally determined interactions; Black: Co-expression of targets; Purple: protein homology; Green, yellow, and dark blue: predicted interactions.

Figures 5.. Differential gene expression in hippocampus of males vs females prior to immune challenge.

(A,B) Top 10 differentially expressed (DEGs) in immune-naïve male versus female mice. 95 genes were more highly expressed in male hippocampi whereas 125 genes were more highly expressed in females. (C) Volcano plots showing genes that are higher in males (blue) and higher in females (red) at baseline obtained from Advaita iPathway Analysis. Differentially expressed (DE) genes are represented in terms of their measured expression change (x-axis) and the significance of the change (y-axis), with upregulated genes shown in red and downregulated genes shown in blue.

Figures 6.. Functions of differential gene expressed genes in hippocampus of males vs females prior to immune challenge.

(A,B) Biological pathways and processes enriched in (A) males compared with females and (B) in females compared with males. (C,D) Protein interaction (PPI) networks of targets, clustered by biological function (C) in males and (D) in females. Edges between nodes are color coded for relationship type. Blue: known interactions; Pink experimentally determined interactions; Black: Co-expression of targets; Purple: protein homology; Green, yellow, and dark blue: predicted interactions.

Figure 7.. Meta-analysis comparing differentially expressed genes and pathways amongst long-term and acute conditions in males and females.

(A) Males. Circos plot of differentially expressed genes amongst Long-term (3 months after subchronic LPS injections) and Long-term+Acute (LPS injection 3 months after subchronic challenge) conditions. Genes dysregulated in both experimental conditions are depicted by pink lines; different genes that share similar biological pathways are depicted in blue. (B) Males. Heatmap of selected enriched gene ontology (GO) terms compared between Long-term and Long-term + Acute conditions in males. (C) Females. Circos plot of differentially expressed genes amongst Long-term and Long-term+Acute conditions. Genes dysregulated in both experimental conditions are depicted by pink lines; different genes that share similar biological pathways are depicted in blue. (D) Heatmap of selected enriched gene ontology (GO) terms compared between Long-term and Long-term + Acute conditions in females.