Epstein-Barr Virus dUTPase Induces Neuroinflammatory Mediators: Implications for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract

Purpose: Neuroinflammation is a common feature in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), affecting 85%-90% of all patients, yet the underlying mechanism or mechanisms responsible for the initiation and/or promotion of this process is largely unknown. Multiple reports, however, have suggested a role for Epstein-Barr virus (EBV), in particular, in ME/CFS, but its potential role, if any, in the neuroinflammatory process has not been addressed. In support of this premise, studies by our group have found that the EBV protein deoxyuridine triphosphate nucleotidohydrolase (dUTPase) induces anxiety and sickness behaviors in female mice. We also found that a small subset of patients with ME/CFS exhibited prolonged and significantly elevated neutralizing antibodies against EBV dUTPase protein in serum, which inversely correlated with ME/CFS symptoms. A larger ME/CFS case-control cohort study further confirmed that a significant percentage of patients with ME/CFS (30.91%-52.7%) were simultaneously producing antibodies against multiple human herpesviruses-encoded dUTPases and/or human dUTPase. Altogether, these findings suggest that EBV dUTPase protein may be involved in the neuroinflammatory process observed in ME/CFS. Thus, the aim of the present study was to determine whether the EBV dUTPase protein could contribute to neuroinflammation by altering the expression of genes involved with maintaining blood-brain barrier (BBB) integrity and/or modulating synaptic plasticity.

Methods: With the use of human immortalized astrocytes, microglia, and cerebral microvascular endothelial cells, we conducted time-course (0-24 h) experiments with EBV dUTPase protein (10 μg/mL) to determine what effect(s) it may have on the expression of genes involved with BBB permeability, astrocytes and microglia cell function, tryptophan metabolism, and synaptic plasticity by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In parallel, in vivo studies were conducted in female C57Bl/6 mice. Mice were injected by the intraperitoneal route with EBV dUTPase protein (10 μg) or vehicle daily for 5 days, and the brains were collected and processed for further qRT-PCR analysis of the in vivo effect of the dUTPase on the dopamine/serotonin and γ-aminobutyric acid/glutamate pathways, which are important for brain function, using RT² Profiler PCR Arrays.

Findings: EBV dUTPase protein altered the expression in vitro (12 of 15 genes and 32 of 1000 proteins examined) and in vivo (34 of 84 genes examined) of targets with central roles in BBB integrity/function, fatigue, pain synapse structure, and function, as well as tryptophan, dopamine, and serotonin metabolism.

Implications: The data suggest that in a subset of patients with ME/CFS, the EBV dUTPase could initiate a neuroinflammatory reaction, which contributes to the fatigue, excessive pain, and cognitive impairments observed in these patients.

Keywords: Epstein-Barr virus (EBV); Toll-like receptor 2 (TLR2); deoxyuridine triphosphate nucleotidohydrolase (dUTPase); myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS); synaptic plasticity.