Epigenetic impacts of stress priming of the neuroinflammatory response to sarin surrogate in mice: a model of Gulf War illness

Abstract

Background: Gulf War illness (GWI) is an archetypal, medically unexplained, chronic condition characterised by persistent sickness behaviour and neuroimmune and neuroinflammatory components. An estimated 25-32% of the over 900,000 veterans of the 1991 Gulf War fulfil the requirements of a GWI diagnosis. It has been hypothesised that the high physical and psychological stress of combat may have increased vulnerability to irreversible acetylcholinesterase (AChE) inhibitors leading to a priming of the neuroimmune system. A number of studies have linked high levels of psychophysiological stress and toxicant exposures to epigenetic modifications that regulate gene expression. Recent research in a mouse model of GWI has shown that pre-exposure with the stress hormone corticosterone (CORT) causes an increase in expression of specific chemokines and cytokines in response to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor.

Methods: C57BL/6J mice were exposed to CORT for 4 days, and exposed to DFP on day 5, before sacrifice 6 h later. The transcriptome was examined using RNA-seq, and the epigenome was examined using reduced representation bisulfite sequencing and H3K27ac ChIP-seq.

Results: We show transcriptional, histone modification (H3K27ac) and DNA methylation changes in genes related to the immune and neuronal system, potentially relevant to neuroinflammatory and cognitive symptoms of GWI. Further evidence suggests altered proportions of myelinating oligodendrocytes in the frontal cortex, perhaps connected to white matter deficits seen in GWI sufferers.

Conclusions: Our findings may reflect the early changes which occurred in GWI veterans, and we observe alterations in several pathways altered in GWI sufferers. These close links to changes seen in veterans with GWI indicates that this model reflects the environmental exposures related to GWI and may provide a model for biomarker development and testing future treatments.

Keywords: AChE; Acetylcholinesterase inhibitors; CORT; Corticosterone; DFP; Diisopropyl fluorophosphate; Epigenetics; Gulf War illness; Transcriptomics.

Fig. 1

Overview of exposure timeline. CORT + DFP exposed animals were given CORT in the drinking water for 4 days and injected with DFP on the 5th day, before being culled 6 h later

Fig. 2

Frontal cortex RNA-seq significantly enriched gene ontology biological process annotations. Gene ontology biological process annotations significantly enriched in genes which were differentially expressed in the frontal cortex of CORT + DFP exposed mice, with groups of similar annotations highlighted

Fig. 3

Hippocampus RNA-seq significantly enriched gene ontology biological process annotations. Top 50 gene ontology biological process annotations significantly enriched in genes that were differentially expressed in the hippocampus of CORT + DFP exposed mice, with groups of similar annotations highlighted

Fig. 4

Estimated cell proportions from RNA-seq data. a Hippocampus and b frontal cortex cell proportions, estimated from RNA-seq data. Proportion of five cell types of interest in each exposure group, showing significant differences due to exposure. OPC oligodendrocyte precursor cells, MO myelinating oligodendrocytes. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 5

Frontal cortex H3K27ac ChIP-seq significantly enriched gene ontology biological process annotations. Top 50 GO BP annotations significantly enriched for differential enrichment of H3K27ac with CORT + DFP exposure

Fig. 6

Annotated GENCODE genes found in each of our differential analyses. UpSetR diagram [73] of annotated GENCODE genes found in each of our differential analyses: frontal cortex RRBS (FC RRBS), frontal cortex H3K27ac ChIP-seq (FC ChIP), hippocampus RNA-seq (Hipp RNA), frontal cortex RNA-seq (FC RNA) and hippocampus RRBS (Hipp RRBS)