Chronic fatigue syndrome, the immune system and viral infection

Brain, Behavior, and Immunity 26 (2012) 24–31

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Named Series: Fatigue, Brain, Behavior, and Immunity

Chronic fatigue syndrome, the immune system and viral infection A.S. Bansal a,⇑, A.S. Bradley a, K.N. Bishop b, S. Kiani-Alikhan c, B. Ford a a Dept. of Immunology, Epsom and St. Helier University Hospitals NHS Trust, Carshalton, Surrey, SM5 1AA and Chronic Illness Research Team, Stratford Campus, University of East London, London E15 4LZ, UK b Division of Virology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK c Dept. of Immunology, Kings College Hospital, Denmark Hill London, UK

a r t i c l e

i n f o

Article history: Received 8 April 2011 Received in revised form 14 June 2011 Accepted 28 June 2011 Available online 2 July 2011 Keywords: Chronic fatigue syndrome Cytokines T cells NK cells Immune memory Viruses

a b s t r a c t The chronic fatigue syndrome (CFS), as defined by recent criteria, is a heterogeneous disorder with a common set of symptoms that often either follows a viral infection or a period of stress. Despite many years of intense investigation there is little consensus on the presence, nature and degree of immune dysfunction in this condition. However, slightly increased parameters of inflammation and pro-inflammatory cytokines such as interleukin (IL) 1, IL6 and tumour necrosis factor (TNF) a are likely present. Additionally, impaired natural killer cell function appears evident. Alterations in T cell numbers have been described by some and not others. While the prevalence of positive serology for the common herpes viruses appears no different from healthy controls, there is some evidence of viral persistence and inadequate containment of viral replication. The ability of certain herpes viruses to impair the development of T cell memory may explain this viral persistence and the continuation of symptoms. New therapies based on this understanding are more likely to produce benefit than current methods. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction The chronic fatigue syndrome (CFS) is characterised by severe and disabling fatigue (Afari and Buchwald, 2003) but without a patho-physiologic explanation. In addition to fatigue, individuals with CFS also report a variety of other symptoms including musculoskeletal pain, sleep disturbance, impairment in short term memory and concentration, sore throat, and headaches of new type, pattern and severity (Reid et al., 2000; Afari and Buchwald, 2003). In nearly all cases there is an exacerbation of these symptoms, but particularly the fatigue by any form of physical, mental and sometimes emotional exertion. Symptom severity may also fluctuate on a daily or weekly basis without obvious cause. Studies of the general population suggest a prevalence rate for CFS of between 0.2% and 2.6% depending on the criteria used (Reid et al., 2000; Afari and Buchwald, 2003). Most of the research on prognosis and treatment outcome has focussed on people attending specialist centres, who may be assumed to have more severe and complex difficulties. Nevertheless, studies suggest that a significant proportion of people with CFS will continue to experience symptoms for some time (Afari and Buchwald, 2003). Indeed, as few as 6% of people with CFS return to pre-morbid levels of functioning in the medium to long term (Reid et al., 2000).

⇑ Corresponding author. Fax: +44 208 641 9193. E-mail address: [email protected] (A.S. Bansal). 0889-1591/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2011.06.016

The diagnosis of CFS presently rests on the exclusion of any medical or psychiatric causes of fatigue in someone with new onset persistent tiredness for over six months. In 1988 Holmes et al. from the US Centre for Disease Control (CDC) drafted the working case definition for CFS to help standardise the patient population for research purposes and to avoid the connection with viral infection after investigations failed to confirm past or current infections. A 1994 revision of the CDC case definition constitutes the current criteria for chronic fatigue syndrome and is the most widely used definition internationally (Fukuda et al., 1994). Amendments have been proposed since and the Canadian criteria that highlight the importance of a post-exertional malaise have gained some favour (Carruthers et al., 2003). Nevertheless, problems remain in case definition and the clear influence that differing criteria may have on research results (Christley et al., 2010). CFS has long been thought as having a significant immunological component. This is because of the nature of the symptoms and the finding of abnormalities in the immune system. However, it is still not clear whether these defects are the cause or the result of CFS. What is clear though is that therapies that modulate the immune system can result in a clinical improvement. This review will focus on the role of impaired immunological memory in CFS, with particular reference to viral infections and possible therapeutic interventions. It should, however, be noted that the immune system is significantly influenced by stress, mood and by disturbance of sleep. Varying degrees dysfunction in these areas may initiate or perpetuate immune changes that contribute to a susceptibility to

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severe or prolonged viral infection and the development of CFS (Fig. 1).

2. Heterogeneity of CFS in relationship to immune dysfunction Persistent fatigue lasting more than 6 months may be observed in several viral and bacterial infections and in numerous rheumatological conditions. Previous investigations of the biochemical, microbiological and immunological abnormalities in subjects with CFS have often considered CFS to be a single disorder. The problem is compounded by difficulties in diagnosing this condition which is primarily one of exclusion. This may explain the absence of any consistent set of abnormalities in this group of people. Even comparing subjects with acute onset CFS with those whose symptoms had a gradual onset confirms significant differences in premorbid personality, prognosis and response to treatment (Masuda et al., 2002a,b). There also appears to be differences in the levels of certain immune parameters depending on the mode of onset of the CFS symptoms (Masuda et al., 2002a,b). Thus dividing patients on the basis of their Natural Killer (NK) cell function appears to select a subgroup of individuals who may respond favourably to immune based therapy using interferon alpha (See and Tilles, 1996). Therefore it is important that subjects with CFS are not grouped into a single entity based simply on a common set of symptoms. At the very least they should be divided into those with an acute versus gradual onset symptoms and those with and without abnormality of immune function. In the absence of such a division inconsistent results may be evident in regard to precipitating factors, prognosis and response to specific therapies. (Natelson et al., 2002) drew attention to this problem several years ago in relationship to the immune system and summarised the inconsistency in the results obtained by several groups.

3. Cytokine dysregulation Despite the heterogeneity in CFS, there is growing evidence suggesting immune dysfunction plays an important role in CFS (Patarca-Montero et al., 2001; Stewart et al., 2003). However, the past two decades has seen a confusing array of reports on the levels of different cytokines with sometimes conflicting results. In equal measure this is likely due to patient related variables and those arising from methodological differences. The former include the precise patient selection criteria used, the different stages of the relapse/remission cycle when patients were assessed, their precise levels of stress, physical activity and sleep disturbance and the time of day that blood sampling occurred. Methodological issues are particularly important in the reports on cytokine measurements in those with CFS. Thus these can be studied by direct immunoassay of serum/plasma, by immunoassay of in vitro culture supernatants of stimulated or unstimulated cultures of whole blood or separated mononuclear cells, by gene expression in mononuclear cells or by quantitative flow cytometry of intracellular protein. Unfortunately the results obtained by one method are difficult to compare to those obtained by another. Early reports looking particularly at the inflammatory cytokines showed a possible increase in several of these proteins. Indeed (Moss et al., 1999) reported significantly elevated levels of tumour necrosis factor (TNF) a in patients with CFS compared to healthy controls. It is possible that such elevations may have accounted for the elevated levels of the inflammatory markers C-reactive protein (CRP), beta 2-microglobulin, and neopterin in the patients with CFS reported by (Buchwald et al., 1997). However, patients with CFS also have significantly higher levels of bioactive transforming growth factor (TGF) b compared to healthy controls, sub-

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jects with major depression, lupus and multiple sclerosis (Bennett et al., 1997). In women with CFS, abnormalities of interleukin (IL) 1b and IL1Ra release by peripheral blood mononuclear cells (PBMC) were demonstrated particularly in the premenstrual phase (Cannon et al., 1997). However, no difference in IL1b was evident in the peripheral blood of CFS patients undergoing sub-maximal and self paced exercise (Nijs et al., 2010). While IL1b and IL6 assessed by multiplex technology were raised in the CFS patients reported by (Fletcher et al., 2009), the level of TNFa was no different from the healthy controls. Confusingly, there was a mixed elevation of Th1 and Th2 cytokines and no alteration in the Th17 and T regulatory cell associated cytokines. In a large study of well characterised patients with CFS, (Raison et al., 2009) found highly sensitive CRP to be no different from controls when adjusted for age, sex, race, location of residence, body mass index (BMI), depressive status and immune-modulating medications. Interestingly, depressive symptoms were associated with increased log hs-CRP. IL6 was also found to be similar in CFS versus controls when BMI was taken into account by (Nater et al., 2008). In our own unpublished work we have observed no significant or consistent change in the level of plasma IL1b, IL6 and TNFa when measured at 3 month intervals in patients with CFS and when correlated with degree of fatigue. A similar lack of variation within individuals in mitogen stimulated cytokine production has also been seen in girls with severe fatigue, amongst whom those with CFS had an increased profile of anti-inflammatory cytokines and reduced inflammatory cytokines (ter Wolbeek et al., 2007). Regarding the immune stimulatory and regulatory cytokines, lipopolysaccharide (LPS)-induced IL10 secretion in whole blood cultures was significantly increased in patients with CFS compared with controls and with a trend to decreased IL-12. Importantly, this IL10 secretion appeared to be resistant to suppression by dexamethasone in the CFS patients only (Visser et al., 2001) although in a later study this group found IL10 secretion to be no different from the control group. A slightly increased level of IL10 has also been observed in CFS patients with and without fibromyalgia compared to healthy controls during sleep by (Nakamura et al., 2010). However, there was no difference in the pro-inflammatory cytokines in the serum, peripheral blood lymphocytes (PBL) mRNA or resting and stimulated PBL between these groups. There was also no difference in serum levels of IL4, IFNc and soluble CD23 measured by ELISA in 79 monozygotic (MZ) and 45 dizygotic (DZ) twins discordant for prolonged fatigue (Hickie et al., 1995). However, this work did suggest the importance of genetic factors in encouraging fatigue. Thus persistent fatigue was more frequently concordant in the MZ versus the DZ twins. Additionally, this work also confirmed the major influence of a shared early environment in affecting current immune function and unique environmental influences in encouraging fatigue. More recent work has looked at cytokine groups mediating differing patterns of immune activity as quite often individual cytokine levels may not have differed significantly between patients with CFS and age and sex matched healthy controls. Using a network analysis Broderick et al. (2010) using the same data set used earlier by Fletcher et al. (2009) assessed the co-expression of IL-1a, 1b, 2, 4, 5, 6, 8, 10, 12, 13, 15, 17 and 23, interferon (IFN) c, lymphotoxin-a (LT-a) and TNF-a in the plasma of 40 female CFS and 59 case-matched controls. Cytokine co-expression networks were constructed from the pair-wise mutual information (MI) patterns found within each subject group and showed diminution of T helper (Th) 1 and Th17 function with an increase in Th2 type immunity. There was also evidence of an attenuation of those networks that contribute to NK cell activation and IL12 and LT-a in particular. While the evidence for significant alterations in the levels of the

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proinflammatory cytokines remains unclear the possibility that unchecked Th17 function with reduced suppression of the wild type IL17F allele by the His161Arg variant (C allele) has recently been presented by (Metzger et al., 2008). Thus the frequency of the C allele was significantly reduced in patients with CFS. 4. Cellular dysfunction Although different patterns of raised circulating and stimulated cytokines have been reported by different investigators, the only abnormality consistently demonstrated by the majority of reports on CFS is the reduction in the number (Masuda et al., 1994) or function (Barker et al., 1994) of NK cells. (Tirelli et al., 1994) also found the reduced NK cell population to express an increased number of adhesion (CD11b, CD11c and CD54) and activation (CD38) markers. (Klimas et al., 1990), however, found NK cells numbers to be increased in subjects with CFS but for the NK cell cytotoxicity to be reduced compared to healthy controls. (Levine et al., 1998) found NK cell function assessed in a 51Cr release assay to be lower in a family with CFS compared to family members without CFS. Interestingly the latter in turn had 51Cr release results that were intermediate between those with CFS and healthy controls. (Stewart et al., 2003) have stressed the importance of ensuring comparable geographic controls in the comparison of subjects with and without CFS. (Ogawa et al., 1998) have shown the L-Arg-induced activation of NK activity by Nitrous Oxide to be impaired in CFS patients. More recently (Brenu et al., 2010) have reported a decrease in the CD56 (bright) CD16 population of NK cells with a significantly reduced neutrophil oxidative burst both assessed flow cytometrically in 10 patients with CFS. Thus several aspects of immune function appear to be affected in patients with CFS. NK cell proliferation, maturation and activation are increased by several cytokines but particularly interleukin (IL) 21 and IFNc and especially in the presence of IL2, IL12, IL15 and IL18 (Strengell et al., 2002, 2003). NK cells recognize their targets by the absence of classical HLA class I proteins and NK cell receptors of the KIR superfamily. NK cell inhibitory receptors are also recognized and important in regulating cytolytic activity. CD69 is one of the earliest specific markers of NK cell activation (Craston et al., 1997; Marzio et al., 1999; Llera et al., 2001). Activated NK cells release cytokines that activate other NK cells and the cellular immune system generally (Marzio et al., 1999). Elevated NK cell CD69 expression is associated with increased cytotoxicity and target cell lysis (Lanier et al., 1988; De Maria et al., 1994). The latter is achieved by NK cell release of perforin and granzymes that induce target cell apoptosis and cell membrane destruction. As NK cells are important in the elimination of virally infected/altered host cells it is possible that impaired NK cell function may allow the persistence of chronic viral infection in subjects with CFS. Interestingly, (Maes et al., 2005) found reduced levels of CD69 T cells and (Mihaylova et al., 2007) reduced levels of CD69 T and NK cells in patients with CFS. It is therefore possible that CFS may be associated with the impaired T and NK cell activation as reduced secretion of those cytokine important in regulating NK cell function. This in turn may be caused by specific polymorphisms in the promoter regions of IL21, IFNc, IL2, IL12, IL15 and IL18. However, NK cell activity has been shown to be adversely affected by depression and sleep (Irwin et al., 1992). 5. CFS and viral infection Several investigators have reported increased 20 50 oligoadenylate synthetase (OAS) activity by mononuclear cells of patients with CFS and the levels correlating with disease severity (Vojdani and Lapp, 1999; Ikuta et al., 2003; Nijs and Fremont, 2008). This

protein is induced by IFNa and IFNc and is an important defence against viral proliferation leading to proposals that chronic viral infection could be a possible cause of CFS. However, the detection of several herpes viruses, enteroviruses and Borna viruses in patients with CFS by serology and PCR has provided conflicting results. Thus (Ablashi et al., 2000) found evidence of HHV-6 reactivation in their patients with CFS by detecting a raised frequency of anti-HHV-6 IgM and the HHV-6 antigen in short term PBMC cultures. In contrast, (Koelle et al., 2002) in a study of a cohort of 22 monozygotic twins in which one sibling from each twin was diagnosed with CFS, suggested no serological evidence for a significant difference in past or current infections with HHV-8, cytomegalovirus, herpes simplex virus 1 and 2 or hepatitis C virus. Importantly the raw serological data such as antibody class or specificity were not reported in this study. Additionally, the frequency of DNA detection by PCR for HHV-6, HHV-7, HHV-8, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, varicella zoster virus, JC virus, BK virus, and parvovirus B19 was not different between the patients who fulfilled the criteria for CFS diagnosis and their siblings who did not. However, the overall frequency of EBV detection in this study was considerably lower than the general population (20% versus 80–90%) casting a doubt over the methodology. In an earlier study, (Buchwald et al., 1996) were also unable to find serological evidence to support a role for viruses in 548 chronically fatigued patients. In their analysis they included herpes simplex virus 1 and 2, rubella, adenovirus, human herpesvirus 6, EpsteinBarr virus, cytomegalovirus, and Cox-sackie B virus, types 1–6. In contrast, (Manian, 1994) found serological evidence of an increased frequency of previous EBV and Cox-sackie viruses B1 and B4 in their investigation of 20 patients with CFS using standard well tested methodology. IgM antibodies to non structural genes in human CMV have also been detected in a subset 16 out of 34 CFS patients with positive IgG against CMV envelope glycoproteins and in none of the 59 controls, 44 of whom were CMV IgG positive (Lerner et al., 2002). This group also found IgM antibodies to EBV in a subset of CFS patients suggesting that a defect in the immune system could be permitting reactivation of the virus (Lerner et al., 2004). The current literature is therefore mixed in relation to the seroprevalence of the common viruses in CFS patients and this, at least to some degree, may be attributed to the different viral antigens used in different serological studies. Furthermore, the criteria used in diagnosing CFS have been different in the early pre-2000 period compared to subsequent studies. In a study by (Kerr et al., 2000), although no difference in seroprevalence for parvovirus B19 was found, in contrast to healthy blood donor controls those fulfilling the Fukada criteria for CFS had significantly raised frequency of IgG antibodies to the parvovirus B19 NS1 protein (41.5% versus 7%). Additionally, viral DNA detected by real time PCR was evident in 11 out of the 200 CFS patients and in none of the 200 healthy controls. The results were suggested to indicate deficient control of parvovirus B19 perhaps in relation to impaired cellular immunity. On the other hand detection of antibodies with unusual specificities may suggest an altered immune response to viruses or altered viral replication in patients diagnosed with CFS. Further dysregulation of viral immunity is also suggested by the finding of antibodies to mitochondrial components and also to serotonin, microtubule-associated protein 2 and muscarinic cholinergic receptor 1 (Bassi et al., 2008). Fatigue is a known consequence of several viral infections and in the case of EBV this has been reported to last a median of eight weeks and with an interquartile range of four to sixteen weeks (White et al., 1998). Stress has also been reported to reactivate EBV (Glaser et al., 2005) and it is possible that the increased stress suffered by patients with CFS may contribute to recurrent relapses in CFS. With this is in mind it is interesting that valacyclovir has

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been shown beneficial in subset of patients with CFS with previous EBV infection and particularly in regard to cardiac function (Lerner et al., 2007). Mechanistically purified EBV deoxyuridine triphosphate nucleotidohydrolase (dUTPase) has been shown to inhibit the replication of human PBMCs in vitro and to increase the production of several cytokines (Glaser et al., 2005). These included TNFa, IL1b, IL6, IL8, and the immune regulatory IL10. Additionally, it increased NK cell lysis of target cells. In mice this group also found EBV dUTPase to significantly inhibit the replication of mitogen-stimulated lymphocytes and the synthesis of IFNc by lymph node and splenic cells (Glaser et al., 2005). Inoculation was associated with an increase in body temperature, decrease in body mass and physical activity known to be induced by pro-inflammatory cytokine secretion. Subsequently this same group has shown that depletion of CD14+ monocytes attenuated cytokine secretion (Glaser et al., 2006). Furthermore, the pathway of proinflammatory cytokine secretion by EBV dUTPase involved the initial binding of Toll like receptor 2 and subsequent activation of NF-kappaB through the recruitment of the MyD88 adaptor molecule (Ariza et al., 2009). Of relevance to patients with CFS, glucocorticoids that are secreted as part of the stress response have been shown to induce lytic replication of latent EBV through the induction of the immediate early gene BZLF1 (Yang et al., 2010). Importantly, however, dexamethasone also induced the early BLLF3 gene that encodes EBV dUTPase as well as BALF5 that encodes the EBV DNA polymerase. Thus stress induced EBV reactivation may represent the initial problem that leads to a disturbance of immune memory which in turn leads to a prolongation and accentuation of viral symptoms. Regarding other viruses, (Lane et al., 2003) have reported enterovirus sequences in the quadriceps of their patients with confirmed CFS and evidence of muscle weakness. (Chia et al., 2010) detected enteroviral RNA in peripheral blood of two and gastric antral biopsy of one patient after an acute illness that had progressed to chronic fatigue. These findings support previous work suggesting a persistence of enteroviral infection in patients with CFS (Galbraith et al., 1997). In addition to viruses several other organisms have also been considered to be associated with chronic fatigue. These include several types of bacteria including mycoplasma species in particular (Vojdani et al., 1998) but also borrelia. In 2006, researchers investigating a link between the OAS pathway and familial prostate cancer identified a novel retrovirus in samples from patients with a deficiency in RNase L function. This virus was similar to known murine leukaemia viruses (MLV) and

Severe/prolonge d viral and/or other infections

Stress associated with anxiety, depression, insomnia, inactivity

was thus called xenotropic MLV-related virus (XMRV). Although there is no evidence to suggest an increase in prostate cancer among CFS patients, the link with RNase L function led a team from the Whittemore Peterson Institute to look for the virus in their CFS cohort. In late 2009, they reported that they had found XMRV nucleic acid in white blood cells from 68/101 CFS patients compared to only 8/218 controls (Lombardi et al., 2009), although they did not find a link to RNase L deficiency. The extremely high prevalence of the virus in CFS patients caused great excitement, especially as the authors claimed to have cultured virus from these patient samples. However, the story was soon mired in confusion as three groups quickly published contrary reports that they could find little evidence of the virus in their patient cohorts (Erlwein et al., 2010; Groom et al., 2010; van Kuppeveld et al., 2010). Two of the reports were criticised for only using PCR based assays, however the third also used serological tests (Groom et al., 2010). Additional negative reports soon followed, and, to date, no other group has published similar findings of XMRV in CFS patients. The story has been complicated further by reports of other MLV-like viruses in CFS patients (Lo et al., 2010) and sample contamination (Smith, 2010). Despite a high profile, the association between XMRV and CFS is still very uncertain and requires further investigation. However, there is increasing evidence that this has little to do with CFS and current evidence is far more in favour of one or more herpes viruses and/or possibly an enterovirus being involved.

6. CFS, immunodeficiency and disturbed immunological memory We have recently observed the frequency of fatigue and other symptoms compatible with CFS, diagnosed on the basis of the Canadian and Fukada criteria, to be increased at least twenty fold in patients with primary antibody deficiency (unpublished). Additionally, we have also observed impaired specific antibody production in a number of patients with CFS whose serum antibody levels are otherwise within normal levels. These patients had recurrent sore throats accompanied by bronchitis but not pneumonia or invasive disease. Together these findings suggest that immune dysfunction predisposes to CFS type symptoms and that at least some patients with CFS have a defect of immune memory. Indeed our early work confirms a defect of both B and T cell memory in patients fulfilling the Fukada and Canadian criteria for CFS. Fig. 1 in a simplified way summarises the interaction between viral

Cellular Immune dysfunction/exhaustion, impaired T and B cell memory and altered NK cell activity

Viral proteins induced pro-inflammatory cytokine release with elevated parameters of inflammation

Reactivation of preexisting chronic viral infection or new viral infections

FATIGUE

Fig. 1. The inter-relationship between psychosocial, immune and viral factors in the initiation and perpetuation of chronic fatigue.

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infections, stress/sleep disturbance and impaired immune memory function. Normally, B cell memory appears to be maintained by a combination of long lived memory cells and constant antigen stimulation of B cells within lymph nodes by antigen retained by follicular dendritic cells. In patients with established immunodeficiency recent work has confirmed reductions in the various memory B cell populations in those with common variable immunodeficiency (CVID). Interestingly, reduced numbers of switched memory B cells (CD19, CD27+ IgD) have been found in CVID patients with splenomegaly and a tendency to granulomatous organ infiltration (Mouillot et al., 2010). In those patients with autoimmune manifestations the number of unswitched memory B cells is increased. In patients with CFS a reduction in the numbers of CD19/IgM+ B cells has been observed (Lundell et al., 2006) although the exact significance of this is unclear as CFS has never been linked to a deficiency of antibody immunity or recurrent bacterial infections. However, in a recent paper, a monoclonal antibody that depletes B cells was found to markedly improve the clinical symptoms in three patients with CFS (Fluge and Mella, 2009) suggesting that B cells have a role in the pathogenesis of CFS. Whether B cells harbouring EBV were removed by this process was not formally assessed but clearly likely. T cell memory appears to be more complex and is based partly on the strength of the initial Tcell receptor – antigen MHC interaction (Kim and Williams, 2010). While acute viral infections often stimulate marked expansion of the naïve T cell pool, chronic viral infections with continued immune stimulation may lead to immune exhaustion particularly of the CD8 T cells (Angelosanto and Wherry, 2010). The extent of clonal expansion is important and CD8 T cell memory requires a combination of IL15 and to a lesser degree IL7 while CD4 memory requires both T cell receptor stimulation and IL7. Downstream to this, the balance of pro-apoptotic factors such as TNFR-6 (Fas) and Bcl2-like protein 11 (BIM) and anti-apoptotic factors Bcl2 determine the fate of T cells (Beverley, 2008). Survival genes such as Tbet and eomesodermin gain importance because of their ability to maintain expression of the IL15a receptor (CD122). This appears to be particularly important in CD8 T cell memory while the HIV type I enhancer protein 2 (HIVEP2 or Schnurri-2) appears more important for CD4 T cells. For all T cells long lived memory is maintained most significantly by continued antigen stimulation or cross reactive antigen stimulation. This is certainly evident in persistent viral infections such as those caused by EBV and HIV. During an initial immune response CD8 T cells appear to show massive expansion and then contraction with subsequent long lived stable memory populations. These phases are much less intense in CD4 T cells which also show very slow loss of memory cells (Beverley, 2008). Recent work has also confirmed the importance of IL15 in the generation and maintenance of CD8/CD44hi memory T cells. Thus, transfer experiments have shown IL15 dependent dendritic cells (DC) in optimising the survival and proliferation of NK cells and CD8/CD44hi memory T cells (Koka et al., 2004; Burkett et al., 2004). IL15 and its receptor were induced by IFNc and NFjB relA inducers and conferred an autocrine loop resistance to apoptosis that accompanied DC maturation (Dubois et al., 2005). More recent work has shown stable complexes of IL15 with its receptor on cell surfaces. The IL15a receptor here presented IL15 in trans configuration that allowed stimulation of neighbouring T and NK cells (Burkett, Koka et al., 2004; Sato et al., 2007). While these complexes underwent endosomal internalisation they appeared to be resistant to lysosomal degradation and were re-circulated to the cell surface as a reservoir of IL15 that maintained memory function by CD8/CD44hi memory T cells (Sato et al., 2007). Such complexes would explain the absence of any significant circulating levels of IL15 and the need for cell to cell proximity in ensuring strong

costimulatory function. Interestingly recent work shows reduced CD8 T cell and NK cell cytotoxicity in 95 patients with CFS compared to 50 healthy controls (Brenu et al., 2011). Our own work has also shown significantly reduced activated CD8+ CD27+ CD28+ cytotoxic T memory cells in 14 patients with CFS as well as a trend to reduced IL15Ra expression after mitogen activation (unpublished). There is now good evidence that EBV can cause major alterations in T cell memory function. Thus in acute EBV induced mononucleosis, the expression of IL-7Ra was lost by all CD8+ T cells, including EBV epitope-specific populations (Sauce et al., 2006). While expression was rapidly regained on total CD8+ cells it was only slowly and incompletely regained on EBV-specific memory cells. In contrast, although the expression of IL-15a was also lost in acute EBV mononucleosis it remained undetectable not just on EBV-specific CD8+ populations but on the whole peripheral Tand natural killer (NK)-cell pool. Of importance this defect in IL15Ra expression and defective IL-15 responsiveness in vitro, was consistently observed in patients up to 14 years after infectious mononucleosis (IM). However, it was absent in patients after cytomegalovirus (CMV)-associated mononucleosis, in healthy EBV carriers with no history of IM and in EBV-naive individuals. It is possible that a similar situation is likely evident in at least a proportion of patients with CFS of acute onset and following a viral illness. Thus EBV may be responsible for not only causing a defect in its own control but may be also a reduction in immune activity to other infectious agents. Interestingly, EBV infection can be associated with the production of a variety of auto-antibodies of varying avidity and clinical significance. It is presently unclear whether anti-cytokine antibodies are present in patients with CFS as has been uncovered in several seemingly unrelated conditions. These include chronic mucocutaneous candidiasis (IL17), pulmonary alveolar proteinosis (GM-CSF), certain types of disseminated nontuberculous mycobacterosis (IFNc) and some people with severe staphylococcal skin infection (IL6) (Browne and Holland, 2010).

7. Treatment options for CFS by immune modulation and antiviral therapy It is clear that current treatment strategies have only a limited ability to ‘cure’ CFS and restore premorbid physical and mental stamina. Routine anti-inflammatory agents while helpful in a small proportion of individuals do little to arrest continued viral proliferation and restore immune memory function that prevents further viral infections. This is also true of glucocorticoid steroids that can reduce the synthesis of pro-inflammatory cytokines but are unable to stimulate anti-viral cellular immunity and immune memory. Indeed they often worsen anti-viral immunity. Intravenous immunoglobulin therapy likewise does not address the areas of immune dysfunction and like steroids reduce further NK cell activity (Thum et al., 2008). Unsurprisingly it provided no benefit in 99 patients with CFS treated with three different doses of IVIg at monthly intervals for 3 months in a double blind placebo controlled trial (Vollmer-Conan et al., 1997). Regarding interferon therapy this is able to stimulate cellular immune function. In seven CFS patients with initially impaired NK cell function treated with 12 weeks interferon a 2a therapy a significantly improved quality of life was evident (See and Tilles, 1996). It was unclear why there was no improvement in patients with impaired lymphocyte proliferation or CFS patients generally. Also inexplicable is why NK cell function was impaired in these patients when other investigators have reported increased levels of interferon inducible proteins in patients with viral and chemical induced CFS (Vojdani and Lapp, 1999). Nonetheless, further work is required to see if interferon therapy may help that subset of CFS patients with demonstrably

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impaired cellular immunity. Clearly the possibility of inducing new autoimmunity particularly to thyroid tissue would need to be balanced against any benefit. Based on impaired immune memory function in patients with CFS, and particularly affecting the CD8 T cell population which is especially important in controlling EBV infected B cells, four methods of treatment may prove beneficial. The first would involve restoring CD8 T cell memory function using complexes of IL15Ra and IL15. In mice this has markedly increased IL-15 half-life and bioavailability leading to a significant proliferation of memory CD8 T cells, NK cells, and NK T cells (Stoklasek et al., 2006). As yet there are no studies of IL15 on memory T cells in CFS. The second method would involve using agents active against EBV. In this case the use of valacycolvir has been shown in a double blind placebo controlled trial lasting 36 months to improve cardiac dysfunction and resume normal life in patients with confirmed CFS (Lerner et al., 2007). Additionally, valgancyclovir, in an open labeled study, has also been shown to be extremely beneficial in 12 patients with symptoms highly suggestive of CFS and who had high titre antibodies to HHV6 and EBV (Kogelnik et al., 2006). The results of the randomized double blind study while not formally published appeared not to have shown significant benefit suggesting that continued EBV/viral suppression requires at least partial restoration of global T cell memory to be effective. Lastly, and most recently, (Lerner et al., 2010) in a retrospective analysis have reported significant benefit of unblinded valacyclovir or valgancyclovir in 142 patients with CFS treated between 2001 and 2007 who had active EBV, CMV or HHV6 infection. Improvement in CFS was measured using an energy index point score. Active infection was diagnosed if IgM serology was positive to viral capsid antigen p18 and/or early antigen-D (EBV), highly raised antibodies to HCMV strain AD69 lysate and IgM to HCMV p52 (CMV) and IgM and IgG tires >1/160 for HHV6 infection. The third method centres on recent work suggesting that rapamycin which inhibits mTOR may also encourage memory CD8 T cell responses by viral infections and by vaccination (Araki et al., 2010). It may therefore be of possible benefit in those with CFS. The fourth method invokes the adoptive transfer of ex-vivo expanded CD8 T cells stimulated by EBV in particular but perhaps other chronic viruses also. This type of therapy has been suggested to be helpful in EBV related malignancy (Merlo et al., 2010) and has also been used in post-transplant lymphoproliferative disease. Regardless, further studies should also look at altered Treg cell and Th17 cell function as well as checking the significance of increased Th2 cells (Skowera et al., 2004) and impaired Th1 cells in CFS. These various lymphocyte subsets have central roles in immunity to viruses and the immune response and are likely dysregulated in CFS.

8. Summary There are major challenges in the analysis of immune function in a condition as heterogenous as CFS where the cause is unknown. Add to this the marked variability in symptom severity from day to day and from hour to hour and there is bound to be variation in the levels of proteins such as cytokines that have short half lives. Further complications are evident in the analysis of immune cells which can vary in number depending on time of day and even mild exertion. Moreover, it is now established that stress and sleep disturbance, which are common in CFS, can alter immune function. Couple this with the fragile nature of most cytokines demanding immediate blood separation and the marked variation in assay sensitivity and reproducibility and it is easy to understand why there is little consensus in the literature on the exact immune dysfunction in CFS. Nevertheless the CFS literature taken as a whole

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suggests mildly raised circulating pro-inflammatory cytokines and a skewing towards impaired cellular immunity. More work is needed to take into account the level of stress and sleep disturbance amongst the study population and to correlate the immune function with the patient’s perception of how severe their symptoms were at the time of immune analysis. Most importantly of all more longitudinal studies investigating immune function with changes in the severity of CFS symptoms are urgently needed. It is likely that viral infection(s) and immune dysfunction in CFS interact in a manner which perpetuates the conditions necessary for maintaining symptoms. Fig. 1 summarises the interplay between the important variables. It is likely that an initial viral infection or stress acting singly or in combination leads to a state of impaired cellular immunity, immune memory dysfunction and disturbed NK cell activity. This promotes reactivation of previously acquired EBV or related virus infection and wide dissemination of the original viral infection. EBV and other viral proteins stimulate the release of pro-inflammatory cytokines which contribute to fatigue, low grade fever, aching, disturbance of sleep and inactivity. The severity and prolonged nature of these symptoms encourages further stress leading to continued immune paresis and production of immune dysregulating viral proteins. The latter then perpetuate the immune dysfunction with continuation of symptoms. In view of the significant interaction between each of these areas, treatments targeting several areas simultaneously are more likely to be successful than those used selectively in one area. References Ablashi, D.V., Eastman, H.B., et al., 2000. Frequent HHV-6 reactivation in multiple sclerosis (MS) and chronic fatigue syndrome (CFS) patients. J. Clin. Virol. 16 (3), 179–191. Afari, N., Buchwald, D., 2003. Chronic fatigue syndrome: a review. Am. J. Psychiatry 160 (2), 221–236. Angelosanto, J.M., Wherry, E.J., 2010. Transcription factor regulation of CD8+ T-cell memory and exhaustion. Immunol. Rev. 236, 167–175. Araki, K., Youngblood, B., et al., 2010. The role of mTOR in memory CD8 T-cell differentiation. Immunol. Rev. 235 (1), 234–243. Ariza, M.E., Glaser, S.F., Kaumaya, P.T., Jones, C., Williams, M.V., et al., 2009. The EBVencoded dUTPase activates NF-kappa B through the TLR2 and MyD88dependent signaling pathway. J. Immunol. 182 (2), 851–859. Barker, E., Fujimura, S.F., et al., 1994. Immunologic abnormalities associated with chronic fatigue syndrome. Clin. Infect. Dis. 18 (Suppl. 1), S136–41. Bassi, N., Amital, D., et al., 2008. Chronic fatigue syndrome: characteristics and possible causes for its pathogenesis. Isr. Med. Assoc. J. 10 (1), 79–82. Bennett, A.L., Chao, C.C., et al., 1997. Elevation of bioactive transforming growth factor-beta in serum from patients with chronic fatigue syndrome. J. Clin. Immunol. 17 (2), 160–166. Beverley, P.C., 2008. Primer: making sense of T-cell memory. Nat. Clin. Pract. Rheumatol. 4 (1), 43–49. Brenu, E.W., Staines, D.R., et al., 2010. Immune and hemorheological changes in chronic fatigue syndrome. J. Transl. Med. 8, 1. Brenu, E.W., van Driel, M.L., et al., 2011. Immunological abnormalities as potential biomarkers in chronic fatigue syndrome/myalgic encephalomyelitis. J. Transl. Med. 9 (1), 81. Broderick, G., Fuite, J., et al., 2010. A formal analysis of cytokine networks in chronic fatigue syndrome. Brain Behav. Immun. 24 (7), 1209–1217. Browne, S.K., Holland, S.M., 2010. Anticytokine autoantibodies in infectious diseases: pathogenesis and mechanisms. Lancet Infect. Dis. 10 (12), 875–885. Buchwald, D., Ashley, R.L., et al., 1996. Viral serologies in patients with chronic fatigue and chronic fatigue syndrome. J. Med. Virol. 50 (1), 25–30. Buchwald, D., Wener, M.H., et al., 1997. Markers of inflammation and immune activation in chronic fatigue and chronic fatigue syndrome. J. Rheumatol. 24 (2), 372–376. Burkett, P.R., Koka, R., et al., 2004. Coordinate expression and trans presentation of interleukin (IL)-15Ralpha and IL-15 supports natural killer cell and memory CD8+ T cell homeostasis. J. Exp. Med. 200 (7), 825–834. Cannon, J.G., Angel, J.B., et al., 1997. Interleukin-1 beta, interleukin-1 receptor antagonist, and soluble interleukin-1 receptor type II secretion in chronic fatigue syndrome. J. Clin. Immunol. 17 (3), 253–261. Carruthers, B.M., Jain, A.K., et al., 2003. Myalgic encephalomyelitis/chronic fatigue syndrome: clinical working case definition, diagnostic and treatment protocols. J. Chronic Fatigue Syndr. 2003 (11), 7–115. Chia, J., Chia, A., et al., 2010. Acute enterovirus infection followed by myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and viral persistence. J. Clin. Pathol. 63 (2), 165–168.

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