Epstein-Barr Virus Induced Gene-2 Upregulation Identifies a Particular Subtype of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is a chronic multisystem disease characterized by a variety of symptoms, and exhibits various features of an autoimmune-like disease. Subtypes are well recognized but to date are difficult to identify objectively. The disease may be triggered by infection with a variety of micro-organisms, including Epstein-Barr virus (EBV). A subset of CFS/ME patients exhibit up regulation of EBV virus induced gene 2 (EBI2) mRNA in peripheral blood mononuclear cells (PBMC), and these patients appear to have a more severe disease phenotype and lower levels of EBNA1 IgG. EBI2 is induced by EBV infection and has been found to be upregulated in a variety of autoimmune diseases. EBI2 is a critical gene in immunity and central nervous system function; it is a negative regulator of the innate immune response in monocytes. Its heterogeneous expression in CFS/ME could explain the variable occurrence of a variety of immune and neurological abnormalities which are encountered in patients with CFS/ME. The EBI2 subtype occurred in 38–55% CFS/ME patients in our studies. Further work is required to confirm the role of EBV and of EBI2 and its oxysterol ligands in CFS/ME, and to identify the most practical means to identify patients of the EBI subtype. There are two EBI2 antagonists currently in development, and these may hold promise in the treatment of CFS/ME patients of the EBI subtype.


INTRODUCTION
Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is a chronic multisystem disease characterized by at least 6 months of fatigue and a variety of other symptoms, including headache, sore throat, muscle pain, joint pain, muscle weakness, post-exertional malaise, sleep abnormalities, and secondary anxiety and depression (1). It is most likely that heterogeneity in CFS/ME is the reason that although there have been many immune and other abnormalities found in patients, none are universal and so there are currently no biomarkers of CFS/ME per se. This is used as evidence against a biological pathogenesis of the disease, however, the most plausible explanation is that CFS/ME is a heterogeneous autoimmune-like disease with a variety of subtypes, a phenomenon typical of autoimmune disease.

CFS/ME AS A CHRONIC FLU-LIKE ILLNESS TRIGGERED BY VIRUS INFECTION
At its most simple, CFS/ME can be considered to be a chronic flulike illness triggered by virus infection, and that the symptoms of CFS/ME are those of a resulting flu-like illness (fatigue, impairment in short term memory or concentration, sore throat, muscle pain, joint pain without swelling or redness, headaches, unrefreshing sleep, and post-exertional malaise) (7). Psychological symptoms (anxiety and depression) are common during flu-like illnesses, and are secondary in otherwise healthy persons. There is a biological basis for such secondary psychological symptoms in that circulating proinflammatory cytokines result in activation of glial cells in the brain and secretion of proinflammatory cytokines by these cells, causing the symptoms of depression, lethargy and anxiety (8). We are all familiar with such short term illness and its symptoms, including the secondary psychological symptoms. We are familiar with the sickness behavior we exhibit during these short term illnesses. For example, we prefer to go to bed early rather than stay out late (8). The principal difference between a short term flu-like illness secondary to virus infection, and CFS/ME, is the duration, severity and the global effect on the lives of patients. In the case of CFS/ME, generally 6 months of symptoms are required (1), while a short term flu-like illness would typically last <2 weeks.

PSYCHOLOGICAL STRESS IS KEY TO VIRUS TRANSMISSION, INFECTION, AND CFS/ME
For any given virus infection, outcomes vary according to many factors. However, for the purposes of CFS/ME, the key factor is psychological stress. It has been shown for a variety of viruses that psychological stress is necessary for successful virus transmission from one person to another (9). Furthermore, it has been shown that psychological stress is necessary for symptoms to develop after successful transmission, as opposed to asymptomatic infection (9). It is well known that psychological stress is key in the reactivation of herpes viruses, and this precedes the recurrence of cold sores (herpes simplex virus) (10), shingles (varicella-zoster virus) (11), and Epstein-Barr virus (EBV) (12,13). Psychological stress has been shown to be important in triggering a large proportion of cases of CFS/ME, and this fits perfectly with a viral pathogenesis. Psychological stress is universal and is expected under various circumstances, for example, student examinations, loss of a parent or partner, etc.  (34). It is recognized that for each microbial trigger of CFS/ME, that there are a variety of possible clinical outcomes of acute infection, including CFS/ME. It is also recognized, that of those patients who suffered an acute microbial infection which led to development of CFS/ME, there are a number of possible resulting CFS/ME phenotypes, and that this varies depending on other, as yet unknown factors. Therefore, of those patients who developed CFS/ME following parvovirus B19 infection, for example, some will have a CFS/ME phenotype with predominant musculoskeletal pain, while others will have less predominant pain, and more problems with sleep, memory, and concentration, for example. Therefore, there is a lack of correlation between the particular microbial or other trigger and the resulting CFS/ME phenotype.

CFS/ME EXHIBITS FEATURES OF AN AUTOIMMUNE DISEASE
A variety of features suggest that CFS/ME may be an autoimmune-like disease. CFS/ME may be triggered by virus infection, and its course characterized by a typical "viral" flu-like illness (7). These observations have led to the recognition that the immune response plays a large and significant role in the pathogenesis of the disease. There are striking similarities between CFS/ME and various autoimmune diseases, for example, Multiple Sclerosis (MS). And, the existence of subtypes of CFS/ME is a further parallel to autoimmune diseases, in which subtypes are well recognized.  (53). There is considerable co-morbidity of CFS/ME with other immune or autoimmune diseases, including fibromyalgia (30-77%), postural orthostatic tachycardia syndrome (POTS) (11-40%), Hashimoto's thyroiditis (17-20%), and a family history of an autoimmune disease (18-41%) (53). We have also found upregulated EBI2 mRNA expression in a subset of CFS/ME patients which also occurs in autoimmune diseases (see below). Two clinical trials of monoclonal anti-CD20 antibody, rituximab, in CFS/ME patients demonstrate partial or complete benefit in 60%, and in some of these the remission was sustained. This treatment, in both trials, exhibited a delayed onset of response of ∼4 months, suggesting that benefit was not directly mediated by CD20 depletion, but by plasma cell depletion followed by washout of short-lived autoantibodies (54,55).

EBV REACTIVATION IS A MODEL FOR PSYCHOLOGICAL STRESS AND TRIGGERING OF CFS/ME
Acute EBV infection has been shown to be an important virus trigger of CFS/ME (20)(21)(22). Study of acute EBV infection in medical students shows that EBV preferentially reactivates during the psychologically stressful time of examinations as compared with other less stressful periods of the academic year (56). This has also been shown for military recruits in training at examination times (57). This reactivation is also paralleled by changes in a large variety of immune markers of cellular immunity which are important in the long-term control and suppression of replication of persistent and asymptomatic EBV in the normal person (12,13). These studies elegantly document the importance of the balance between persistent EBV and cellular immune system competency which can be disrupted by psychological stress, leading to reactivation and replication of EBV, and subsequent manifestation of symptoms of EBV infection, which if the stress is maintained, can become prolonged and lead to CFS/ME and other diseases, such as nasopharyngeal cancer and post-transplant lymphoproliferative disorder (PTLD) (58). The various types of stress that can adversely affect the efficacy of the cellular immune response include marital stress (59), student examination stress (56), attachment anxiety or fear of abandonment and rejection (60), NK cell dysfunction (40) Reduction in CD3-/CD16+ and CD57+/CD56+ NK cells with an expansion of the CD8+/CD56+ and CD16-/CD56+ NK subsets and total circulating B cells (36) Deficiency in NKH.1+ T3 cell numbers and decreased NK cell function in patients with CFS who had evidence of EBV reactivation (41) Deficiency in cellular immunity with reduced cytotoxicity of NK cells with increased NK numbers (35) Total NK numbers normal, with decreased NK cell activity as compared to normal (CFS family) (42,43) Decreased antibody-mediated cellular cytotoxicity (ADCC) Th2 profile of CD4 helper T cell responsiveness (44)(45)(46) Reduced TGF1 mRNA expression (47) Increased neutrophil apoptosis (48) Deficiency of IgG1 in 2 CFS patients (49) Deficiency of IgG1 and IgG3 in CFS compared with healthy sedentary controls. IgG1 and IgG3 were even lower in CFS with concurrent axis-I depression as compared with CFS itself (50) Deficiency of IgG1, IgG3, and IgG4 (51) Deficiency of IgG3 (52) loneliness (61), etc. But physical stress by itself does not have this effect (62). Psychological stress triggers release of glucocorticoids which activate EBV lytic infection through the upregulation of the immediate early BZLF1 gene expression (63). The cause of the pro-inflammatory state with EBV reactivation is the EBV-encoded deoxyuridine triphosphate nucleotidohydrolase (dUTPase) which modulates innate immunity in human primary monocyte-derived macrophages through toll-like receptor (TLR)-2 signaling leading to NF-κB activation and the production of pro-inflammatory cytokines. EBV dUTPase induces sickness responses in mice (64). Restraint stress (unavoidable stress which causes autonomic and behavioral changes) results in impairment of learning and memory which is due to expression of EBV dUTPase (65).
CFS/ME patients exhibit prolonged raised antibody titers against EBV dUTPase and EBV DNApol which are neutralizing, and may be used to identify CFS patients in which their disease pathogenesis is due to ongoing EBV reactivation (66). However, global screening of serum antibody responses to an EBV peptide array in serum of CFS/ME patients compared with controls revealed strikingly similar patterns (67).
EBI2 also plays an important role in the central nervous system (69). Astrocytes are the macrophages of the brain and protect it against invading pathogens and astrocyte abnormalities are implicated in multiple sclerosis (MS), Parkinsons Disease (PD), Amyotrophic Lateral Sclerosis (ALS), and Alzheimer Disease (AD). Oligodendrocytes are the main cells involved in myelination of nerve fibers of the brain and are implicated in leukodystrophies and leukoencephalopathies. Cholesterol is a crucial component of myelin and cholesterol deficiency results in motor symptoms (81). Abnormal levels of oxysterols have been found in AD, MS and Experimental Allergic Encephalomyelitis (EAE). Mutations in CYP7B1 gene have been demonstrated in Spastic Paraplegia Gene 5 (SPG5) and lead to lesions of upper motor neurones, periventricular areas and subcortical white matter (82,83). Brain cholesterol is synthesized in the brain as it can't traverse the blood brain barrier (BBB) (84,85). The cholesterol metabolite, 24(S)hydroxycholesterol (24(S)HC) is a brain-specific oxysterol which is thought to be synthesized exclusively in the brain and secreted into the circulation via the BBB to maintain steady levels of brain cholesterol (85). Circulating 24(S)HC is believed to be a biomarker for brain cholesterol homeostasis and neurodegenerative disease (MS, PD, AD) (85)(86)(87). Oxysterols also have detrimental effects on myelin and oligodendrocyte viability (88,89).
Dysregulation of EBI2 expression has been demonstrated in EBV infection (68,69,73,74,76), melanoma metastasis, lymphoblastic leukemia, glioblastoma, bone cancer metastasis, systemic lupus erythematous, chronic rhino sinusitis with nasal polyps, Type 1 Diabetes (69), and CFS/ME (90) (see below). Aberrant oxysterol signaling has been demonstrated in Multiple Sclerosis, Experimental Allergic Encephalomyelitis (EAE), Alzheimer's disease, Parkinsons Disease, Motor Neurone Disease, Cerebrotendinous Xanthomatosis, Hereditary spastic paraplegia type 5 (SPG5), Huntingdon Disease, Age related macular degeneration, atherosclerosis, Inflammatory bowel disease, and osteoporosis (69). EBI2 regulates several genes, important in monocyte function, which are important in the pathogenesis of glioblastoma multiforme and Type 1 Diabetes Mellitus. Knock down of the EBI2 gene in rat monocytes, results in upregulated IRF7 expression. As IRF7 is a critical regulator of the type 1 interferon response, this suggests that EBI2 is a negative regulator of the innate immune response in macrophages (91). However, the precise role played by EBI2 in EBV infection remains to be clarified (68,69).

EXPRESSION OF EBI2 IN CFS/ME
We have previously found that CFS/ME patients exhibit significantly upregulated expression of EBI2 in PBMC as compared with normal controls, in gene expression arrays and reverse-transcriptase polymerase chain reaction (RT-PCR) confirmation assays (90). EBI2 mRNA (NM_004951) expression was found to be significantly upregulated by a factor of 1.3 in microarray experiments using PBMC from 25 CFS patients vs. 50 normal human controls matched 2:1. This upregulation was confirmed using RT-PCR in 55 CFS/ME patients vs. 75 normal human controls at a fold difference of 3.44 (P = 0.0012) using ABI assay number Hs00270639_s1 (90). In this study, EBI2 was found to be unregulated in 55% CFS/ME patients, one of whom was a 26 year old woman whose CFS/ME had been triggered by laboratory documented EBV infection 10 years prior (90). All those with raised expression of EBI2 were also positive for serum anti-VCA IgG.
Microarray experiments identified 88 human genes which were upregulated in CFS/ME, and the RT-PCR expression data on these 88 human genes in 52 CFS patients was then clustered and this identified seven gene expression subtypes. These gene expression derived subtypes differed significantly in measures of clinical symptomatology and neurocognitive functioning (90). Of these 88 human genes which were differentially expressed in CFS patients, they could be divided into two groups each with 44 genes, one group of which showed quite predictable up regulation across most CFS/ME patients and in the other group, the expression was much more variable. EBI2 was one of those which was more variably upregulated in PBMC of CFS/ME patients. This is illustrated in Figure 1, in which 12 of 31 (38%) patients exhibited EBI2 up regulation as compared with none of 40 normal controls.
Although it was known in 2008 that EBI2 was upregulated in PBMC of CFS/ME patients (90), the significance was not understood at the time as little was known about the gene and its function. However, with the recent identification of EBI2 as a critical regulator of the immune response with importance for a variety of autoimmune diseases and cancer, its significance in the pathogenesis of CFS/ME has been recognized.

POSSIBLE LINK BETWEEN EBI2 EXPRESSION, EBV LATENCY, AND CLINICAL SEVERITY IN CFS/ME
As part of a separate study of microbial infections in CFS/ME, we analyzed EBV antibody markers in 117 CFS/ME patients which had been grouped into eight subtypes (A-H) based on clustering of RT-PCR expression data for 88 CFS/ME-associated genes (34). These 117 CFS/ME patients included 55 CFS/ME patients who had been included in the previous study (90), 56 CFS/ME patients who had not previously been studied and six whose disease had been triggered by acute Q fever (QFS) (34). The CFS/ME patients exhibited 90% EBV seropositivity which is to be expected (34).
Subtype D was the most interesting in terms of EBV infection markers and clinical phenotype (34). Subtype D consisted only of females and had the most severe clinical phenotype, with the lowest functional level on SF-36 scoring (physical role, vitality, general health, bodily pain, and total score) and a high frequency of muscle pain and sleep problems (Figures 2A,B)   There were no discernible differences in EBV antibody markers [viral capsid antigen (VCA) IgM and IgG, early antigen (EA) IgG, and Epstein-Barr nuclear antigen (EBNA) IgG] between subtypes, except that in Subtype D, CFS/ME patients had a markedly reduced titer of EBNA IgG (Figure 2D).
Epstein-Barr virus nuclear antigen 1 gene (EBNA1) is important in establishing and maintaining the altered state that cells undergo during EBV infection, and is the only EBV protein found in all EBV-associated malignancies. EBNA1 has a glycine-alanine repeat which stabilizes the protein, prevents its breakdown, impairs antigen processing, and MHC class Irestricted antigen presentation, resulting in inhibition of the CD8-restricted cytotoxic T lymphocyte response against EBV infected cells, thus favoring latency (92). The finding that CFS/ME patients of subtype D with EBI2 mRNA upregulation had lower titers of EBNA IgG than the other subtypes (Figure 2D), supports the idea that subtype D is associated with a higher prevalence of EBV latency, as lytic infection is required to expose this antigen to circulating lymphocytes, a necessary step in developing serum EBNA IgG positivity.
Within the CFS/ME-associated gene signature of 88 human genes, 12 had recognized associations with EBV infection. One of these was EBI2, as discussed, and the others were NFKB1, EGR1, ETS1, GABPA, CREBBP, CXCR4, HIF1A, JAK1, IL6R, IL7R, and PIK3R1. Striking associations were found for these 12 genes across subtypes, and subtype D had the highest levels of all of them in PBMC ( Figure 2F).

HETEROGENEOUS EBI2 UPREGULATION MAY CONTRIBUTE TO THE VARIABILITY OF IMMUNE AND NEUROLOGICAL ABNORMALITIES IN CFS/ME
The following have been variably been found in CFS/ME patients; increase in the number of circulating B lymphocytes, increase in activated T lymphocytes, reduction in NK cell numbers and/or function, deficiency in antibody-mediated cellular cytotoxicity (ADCC), Th2 profile of helper T cell responsiveness, reduced TGF1 expression, increased neutrophil apoptosis, and deficiencies in particular IgG subsets ( Table 2). During the normal humoral immune response, activated B cells upregulate EBI2 which mediates their journey to the outer follicle where they interact with T helper cells. After CD40 engagement, EBI2 expression results in cells moving away from the B-T boundary toward the outer and inner areas of the follicle (73), differentiation into plasmablasts, and mounting of a rapid antibody response. Some B cells move to the central follicle, differentiate into germinal center B cells, to later exhibit antibody affinity maturation (71)(72)(73)(74)(75). A higher than normal expression of EBI2 could result in both increased numbers of B cells in the circulation, and in reduced T cell help and therefore deficiencies in particular IgG subsets, and reduced antibody affinity maturation. EBV reactivation is associated with expansion of differentiated and activated CD4 and CD8 T lymphocytes and later with decline in these cells as exhaustion takes over (94), and so the timing of EBV infection in CFS/ME will affect research studies on immune abnormalities. NK cells are important in defense during the early stages of primary EBV infection (94). Innate immune control of lytic EBV infection by early differentiated NK cells was found to attenuate infectious mononucleosis (IM) (95). It has been proposed that NK cells are important in the long-term control of EBV (96), which may account for the variable findings related to NK numbers and function in CFS/ME, as not all CFS/ME patients will have EBV reactivation at the time of sampling.
Abnormalities in white matter, gray matter and in cerebral perfusion have been found in CFS/ME patients, and these occur in a similar presentation to those of MS patients. Both CFS/ME and MS patients have reduced cerebral perfusion, gray matter reduction and white matter hyper intensities, although individual patients are variably affected (97). As EBI2 expression and oxysterol dysregulation have been linked with the pathogenesis of MS (69), and as MS is characterized by a relapsing and remitting course, in which subtypes exist and in which EBI2 is variably upregulated in some of these, it is logical to suggest that heterogeneous EBI2 expression may similarly play a role in the neurological abnormalities found in CFS/ME.
Although we know that there are a variety of immune abnormalities which occur with regularity in CFS/ME patients, these do not occur invariably, and none can be used as a marker for the presence of the disease. However, the upregulation of EBI2 in a subset of CFS/ME patients may contribute to this phenomenon.

EBI2 MODULATORS
EBI2 is a key receptor in B, T and dendritic cells, modulating the T and B cell response to blood borne antigens (76). As EBI2 and/or its oxysterol ligands are upregulated in B cell malignancies and autoimmune diseases (Type 1 Diabetes, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis), two EBI2 modulators are being developed. GSK682753A is a small molecule, potent EBI2 antagonist which blocks 7a25HC stimulation of the EBI2 receptor in a recombinant system (98). NIBR189 is a potent selective antagonist of EBI2, which has been developed in paradigms relevant to cardiovascular disease (99).

LIMITATIONS OF THE HYPOTHESIS THAT EBI2 UPREGULATION IS IMPORTANT IN A SUBSET OF CFS/ME PATIENTS
The raw data underpinning the present review were generated by only one research group. Therefore, it would be important that these findings are replicated in additional CFS/ME patients and normal controls by independent research groups.
Elevated levels of antibodies to EBV VCA, EA, and DNase have been reported to occur in CFS/ME patients (20,21,66,(100)(101)(102)(103)(104)(105) albeit inconsistently (106)(107)(108). However, it is important to understand that EBV antibody markers may associate with CFS/ME, but this does not prove that EBV has triggered the disease in those particular cases. The seroprevalence of EBV in the general population and in CFS/ME patients is ∼90%. And the proportion of CFS/ME patients with EBI2 upregulation was found to be between 38 and 55% CFS/ME patients, all of whom had IgG to EBV VCA. For a disease in which a variety of microbial triggers are recognized, our findings are consistent with the hypothesis that upregulation of EBI2 is important in the pathogenesis of disease in a subset of CFS/ME patients. However, this hypothesis remains wholly unproven and it is not understood what factors, in addition to EBV infection, are required for upregulation of EBI2 in an individual patient.
Although EBI2 was found to be the most upregulated gene in EBV-infected Burkitt lymphoma cells (68) and has been shown to be important in a variety of autoimmune diseases and cancers, the particular role of EBI2 in the pathogenesis of EBV infection still remains to be elucidated. Therefore, the possible pathogenetic role of EBI2 upregulation in CFS/ME patients remains speculative at present.

CONCLUSION
CFS/ME is a heterogeneous disease which is frequently triggered by virus infection, including EBV. Subtypes are well recognized but to date are difficult to identify objectively. Evidence is presented to document that a subset of CFS/ME patients exhibit up regulation of EBI2 mRNA in PBMC. EBI2 is a gene which is induced by EBV infection and which has been found to be upregulated in a variety of autoimmune diseases. EBI2 is a critical gene in immunity and central nervous system function; it is a negative regulator of the innate immune response in monocytes. Its heterogeneous expression in CFS/ME may indicate an ongoing host response to EBV reactivation and on this basis, could explain the heterogeneous occurrence of many of the immune and neurological abnormalities reported in CFS/ME patients. The EBI subtype may account for 38-55% CFS/ME patients. EBI2 antagonists may hold promise for the treatment of CFS/ME patients of the EBI subtype. Further work is required to confirm the role of EBV and of EBI2 and its oxysterol ligands in CFS/ME, and to identify the most practical means to identify patients of the EBI subtype.

DATA AVAILABILITY
All relevant data is contained within the manuscript.

AUTHOR CONTRIBUTIONS
JK conceived the idea for this review, collated the data, and wrote the paper, without assistance from any other person.

FUNDING
The author acknowledges the kind support of the New Jersey Myalgic Encephalomyelitis / Chronic Fatigue Syndrome Association (NJME/CFSA) with the production costs of this article.