Introduction
Research into the cause and treatment of myalgic encephalomyelitis (ME), also known as chronic fatigue syndrome (CFS), has involved the use of 20 different case definitions in common use (Brurberg et al.
2014). The widest definition of CFS is favoured in the UK and only mandates the presence of idiopathic fatigue of variable severity (Sharpe et al.
1991), while the narrowest definition favoured by investigating physicians in the USA mandates the presence of severe incapacitating fatigue, pain, compromised sleep, neurocognitive disability symptoms consistent with autonomic dysfunction and a worsening of global symptoms following even trivial increases in activity (Carruthers et al.
2011). This is an important issue as the use of narrow selection criteria identify patients with far higher levels of physical and cognitive disability than the use of wider criteria (Jason et al.
2012,
2015a,
2016) and criteria variance has been identified as the main factor accounting for the lack of replicated data which has impeded progress in this field (Jason et al.
2015a,
b) (reviewed (Morris and Maes
2013a)). Using published criteria, the prevalence of ME/CFS is relatively high, at between 0.2 and 6.4%; together with a low level of employment, reported to be between 27 and 41%, it is clear that this disorder poses a high financial burden on patients and society (Johnston et al.
2013; Rimbaut et al.
2016). It also carries a high cost in terms of symptomology; compared with patients suffering from multiple sclerosis (MS), CFS patients have been reported to suffer from higher levels of symptom severity, higher levels of depression and kinesiophobia, and lower quality of life, lower maximum voluntary muscle contraction and muscle recovery, and lower cognitive performance (Meeus et al.
2016). In spite of this disease burden, research into ME/CFS is relatively low. Indeed, it has been calculated that, in the USA, if the federal research funding for ME/CFS were to take disease burden into account, then, by comparison with the funding pattern for other diseases, the funding for ME/CFS research would need to be increased by at least a factor of 25 (Dimmock et al.
2016).
However, there is a large and accumulating body of evidence reporting the existence of a wide range of biological abnormalities in patients afforded a diagnosis of CFS according to current international consensus criteria (Fukuda et al.
1994), most notably in the neuroendocrine, autonomic, neurological, bioenergetic, redox and immunological domains (Morris and Maes
2013b,
c). It is germane to note that, in the USA, the National Institute of Neurological Disorders and Stroke have developed the common data elements for clinical research in mitochondrial disease project ‘to provide clinical researchers with tools to improve data quality and allow for harmonization of data collected in different research studies’ (Karaa et al.
2017,
2018). Common data elements for ME/CFS are being developed along the lines of the following 11 domains: baseline/covariate; fatigue; post-exertional malaise; sleep; pain; neurologic/cognitive/CNS imaging; autonomic; neuroendocrine; immune; quality of life/functional status/activity; and biomarkers. Eleven corresponding subgroups first met in 2017; a related publication will be written in due course.
Early results indicate that increased production of intracellular nuclear-factor 6B and cyclo-oxygenase-2 (COX-2) may be key phenomena in CFS indicating activation of immune-inflammatory pathways in that illness (Maes et al.
2006). In addition, increased production of inducible nitric oxide (NO) synthase (iNOS) coupled with increased IgM responses to NO-adducts such as NO-tryptophan indicate increased nitrosylation of proteins (Maes et al.
2006).
Numerous research teams have reported the presence of an activated but dysregulated immune system with elevated pro-inflammatory cytokines (PICs), T cell anergy, natural killer (NK) cell dysfunction, and Th1, Th2 and, possibly, Th17 lymphocyte biases being repeatedly reported (Brenu et al.
2011; Hornig et al.
2015,
2017; Maes et al.
2012a,
b; Milrad et al.
2017; Montoya et al.
2017; Peterson et al.
2015; Russell et al.
2016). There is also evidence of a longitudinal shift in the immune profiles of patients, with an inflammatory phenotype seen in early disease giving way to an anti-inflammatory or immunosuppressed phenotype, indicating activation of the compensatory anti-inflammatory reflex system (Morris and Maes
2013a,
b,
c) and somewhat reminiscent of the profile seen in endotoxin tolerance in patients who have been ill for many years or even decades (Hornig et al.
2015; Russell et al.
2016). Readers interested in a detailed review of these data are referred to reviews by (Morris and Maes
2013b; Morris et al.
2015a). It should be stressed, however, that there is no evidence of any immune abnormalities in participants afforded a diagnosis of CFS based on any diagnostic schema other than the Fukuda or Canadian criteria (Blundell et al.
2015).
Reported markers of chronic oxidative and nitrosative stress (ONS) include elevated levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), depleted levels of reduced glutathione (GSH), elevated inducible nitric oxide synthase (iNOS) and oxidatively modified proteins and highly reactive metabolites of lipid peroxidation such as 4-hydroxynonenal and malondialdehyde together with the presence of autoantibodies directed at neoepitopes and the presence of damage associated molecular patterns (DAMPs) (Fulle et al.
2000,
2007; Gerwyn and Maes
2017; Maes
2013; Morris et al.
2015a; Morris and Maes
2013b,
c; Rutherford et al.
2016).
Importantly, several research teams have reported that levels of oxidative stress in the muscles of exercising CFS patients are higher than in age- and sex-matched controls and that protective heat shock protein (HSP) responses are impaired (Jammes et al.
2005,
2009,
2011,
2012; Thambirajah et al.
2008). The existence of oxidative stress in individuals afforded a diagnosis of CFS using schemata other than the Canadian or Fukuda criteria is currently uncertain as a literature search failed to uncover any published research investigating this matter.
Chronic ONS is an acknowledged cause of mitochondrial dysfunction (Morris et al.
2017c,
d) and hence the fact that impaired synthesis of adenosine triphosphate (ATP), impaired oxidative phosphorylation and damaged or morphologically abnormal mitochondria in striated muscle and peripheral mononuclear blood cells (PMBCs) of patients have all been extensively reported is unsurprising (Morris and Berk
2015; Morris and Maes
2013b; Naviaux et al.
2016; Tomas et al.
2017).
Inflammation, oxidative stress and mitochondrial dysfunction are also recognised drivers of neuroendocrine and autonomic system dysfunction (Kanjwal et al.
2010; Masson et al.
2015; Schultz
2009; Ulleryd et al.
2017), hence the existence of neuroendocrine abnormalities (reviewed (Morris et al.
2017a; Tomas et al.
2013)) and dysautonomia (Lewis et al.
2013; Naschitz et al.
2004,
2006; Newton et al.
2007; Van Cauwenbergh et al.
2014) is to be expected. This is of importance as some 90% of patients diagnosed via the Fukuda criteria have evidence of autonomic dysfunction characterised by increased sympathetic activity, decreased parasympathetic activity and vagal nerve hypoactivity (Beaumont et al.
2012; Lewis et al.
2013; Robinson et al.
2015). The most common manifestation of dysautonomia reported in trial participants is a suppressed and unresponsive heart rate variability (HRV) both during the day and at night (Boneva et al.
2007; Burton et al.
2010; Kadota et al.
2010; Vollmer-Conna et al.
2006). These findings have been confirmed by a large meta-analysis (Martinez-Martinez et al.
2014). Once again it should be stressed that there is no evidence of any abnormal HRV values in patients afforded a diagnosis of CFS via the application of one of a plethora of alternative criteria (Bozzini
2012; Malfliet et al.
2018).
Disrupted patterns of resting state functional connectivity have been repeatedly reported in patients and appear to correlate with levels of fatigue and pain (Boissoneault et al.
2016; Gay et al.
2016; Kim et al.
2015b; Wortinger et al.
2016). The use of voxel-based morphometric analysis of structural magnetic resonance imaging (MRI) brain scans has revealed abnormalities in brain structure and regional volumes (Barnden et al.
2011,
2015; Finkelmeyer et al.
2018; Puri et al.
2012; Shan et al.
2016), including grey matter (GM) and white matter (WM) changes (de Lange et al.
2005,
2008; Finkelmeyer et al.
2018; Okada et al.
2004; Puri et al.
2012; Shan et al.
2016). These findings contrast with earlier work with low resolution MRI and manual analysis which revealed abnormalities in some patients but not others (Perrin et al.
2010) (reviewed (Morris et al.
2017b)).
Systematic studies of cerebral chemistry, using magnetic resonance spectroscopy, have shown evidence of increased regional levels of choline-containing compounds (Chaudhuri et al.
2003; Puri et al.
2002).
The existence of regional or global cerebral hypoperfusion indicative of reduced bioenergetic capacity has also been consistently reported by research teams utilising xenon-computed tomography, arterial spin labelling and high-resolution single-photon emission computed tomography (SPECT) (Biswal et al.
2011; Machale et al.
2000; Patrick Neary et al.
2008; Yoshiuchi et al.
2006). These findings have also been reported in large studies utilising older SPECT techniques either globally (Ichise et al.
1992; Schwartz et al.
1994) or regionally (Costa et al.
1995; Goldstein et al.
1995) but the results in studies with far fewer participants have been negative (Fischler et al.
1996; Peterson et al.
1994).
Unsurprisingly, given the evidence regarding neuroimaging abnormalities above, a meta-analysis of 50 studies has confirmed the existence of widespread cognitive dysfunction in patients, which are at their worst in the domains of attention, memory, reaction times, information processing and reasoning (Cockshell and Mathias
2010). These findings are also discussed in two excellent narrative reviews by (Cvejic et al.
2016; Shanks et al.
2013) and global decreases in cognitive and executive functions have recently been reported in adults and adolescents irrespective of sex (Nijhof et al.
2016; Santamarina-Perez et al.
2014). It is interesting that several research teams using functional MRI (fMRI) have noted that cognitive dysfunction worsens in patients with increased effort and/or exercise, as such findings may be of relevance from the perspective of pathogenesis and/or pathophysiology (Cook et al.
2017; DeLuca et al.
2004; Lange et al.
2005; Tanaka et al.
2006). It is also noteworthy that a recent large study primarily containing participants with CFS confirmed the results of earlier research indicating that there was scant evidence of global cognitive dysfunction in patients diagnosed via alternative criteria (Hughes et al.
2018).
Many patients complain of ‘brain fog’, which is often described as slow thinking, difficulty focusing, slow thinking, lack of concentration, confusion, forgetfulness, or, sometimes, hazy thought processes (Ocon
2013). From a more objective perspective, subjective brain fog can be described as a constellation of symptoms that include impaired cognition, loss of long- and short-term memory, and a reduced ability to concentrate and engage in multiple low-level tasks at once (Theoharides et al.
2015). It is important to note that this phenomenon is not confined to CFS patients but also characterises patients with autism spectrum disorders, coeliac disease, postural orthostatic tachycardia syndrome, as well as patients with mild cognitive impairment and a range of neuroprogressive illnesses (reviewed (Theoharides et al.
2015)). The causes of brain fog are not fully delineated but there is accumulating evidence that this symptom complex may relate to autonomic dysfunction and/or decreased regional cerebral blood flow (rCBF) in the context of ongoing neuroinflammation (Ocon
2013; Ross et al.
2013; Theoharides et al.
2015).
Several research teams have reported changes in the DNA sequences or expression of numerous genes involved in the delivery of the immune response and the regulation of metabolic and bioenergetic pathways in patients diagnosed according to the Fukuda criteria (Morris et al.
2016b). Examples of dysfunctional or abnormally expressed genes compared with matched controls include
NFKB1,
IL6,
IL1A,
TNF,
IL17A,
IL7,
CXCL8 (formerly
IL8),
INFG,
IRF3,
TLR4,
CD14,
STAT5A,
HSPA2,
P2RX7,
ATP5J2,
GZMA,
COX5B,
DBI,
PSMA3,
PSMA4,
HINT,
ARHC,
HLA-DQB1-AS1,
RIPK3 and
DEFA1 (Carlo-Stella et al.
2006; Gow et al.
2009; Kerr et al.
2008a,
b; Light et al.
2009,
2012,
2013; Nguyen et al.
2017; Saiki et al.
2008; Shimosako and Kerr
2014; White et al.
2012; Zhang et al.
2010). There is also evidence of abnormalities in the mitochondrial genome of patients as a research team has recently reported the presence of polymorphisms in the mitochondrial DNA (mtDNA) of their trial participants which were associated with increased symptom severity (Billing-Ross et al.
2016). These findings were reiterated in (Hanson et al.
2016). These are interesting observations as this study contained 196 patients who were recruited via criteria which mandated the existence of what many researchers view as the defining characteristic of CFS, namely an exacerbation of symptoms following even trivial increases in activity (Morris and Maes
2013a).
The observation that mtDNA polymorphisms appear to influence the severity of CFS is consistent with observations in other disease areas where such polymorphisms increase the susceptibility to the development of metabolic and neurodegenerative diseases and susceptibility to microbial infection (review (Hendrickson et al.
2008)). Polymorphisms in mtDNA also play a role in structuring the composition of the microbiota and determining the levels of IgG and IgM autoantibody production (Ma et al.
2014; Zhou et al.
2017). This may be of pathophysiological relevance in the light of data demonstrating elevated IgA and IgM responses to lipopolysaccharide (LPS)/antigens of
Gram-negative gut commensal bacteria and gut dysbiosis in patients afforded a diagnosis of CFS via the Fukuda criteria (Maes et al.
2006; Morris et al.
2016b; Morris and Maes
2013b). Mutations in mtDNA can increase levels of inflammation and oxidative stress (I&OS) via direct effects on the innate immune system involving PIC production and NF-κB activity and hence can influence the intensity of the immune response (Imanishi et al.
2013; Ishikawa et al.
2010; Novak and Mollen
2015).
There is also evidence of abnormalities in the epigenetic regulation of gene expression in CFS patients diagnosed via narrow criteria, most notably in gene promoter methylation patterns and elevation of microRNAs (miRNAs) involved in the regulation of the immune system (Brenu et al.
2014a; de Vega et al.
2014,
2017; Petty et al.
2016; Vangeel et al.
2015,
2018). The work of de Vega and others is of particular interest as these authors also selected patients according to criteria mandating the presence of post-exertional malaise and examined global patterns of gene methylation rather than a single gene as was the case for Vangeel and colleagues (de Vega et al.
2014,
2017; Vangeel et al.
2015,
2018).
Importantly, de Vega and others reported a global hypomethylation of cytosine residues in the promoter regions of immune system-related genes consistent with a chronically activated but dysregulated immune system, and abnormal patterns of DNA methylation in genes regulating metabolic pathways and various aspects of cellular homeostasis (de Vega et al.
2014,
2017). The work of (Vangeel et al.
2015,
2018) is also of interest as the pattern of hypomethylation of the glucocorticoid receptor gene
NR3C1 1F region suggests an activated hypothalamic-pituitary-adrenal (HPA) axis in an attempt to counter peripheral inflammation rather than a blunted HPA response reported in people diagnosed with CFS according to wider criteria (reviewed (Morris et al.
2017a)). The lack of association between childhood trauma and levels of methylation reported by these authors in studies where participants universally reported this phenomenon is also of interest as these patients were diagnosed according to the Fukuda criteria whereas studies which have reported a significant but slight association between childhood trauma and CFS involved participants recruited via alternative criteria (reviewed (Morris et al.
2017a)).
Abnormalities in miRNA levels in CFS patients diagnosed according to the Fukuda criteria may also suggest dysregulation of immune and metabolic pathways. For example, upregulated
hsa-miR-127-3p,
hsa-miR-142-5p and
hsa-miR-143-3p was reported by (Brenu et al.
2014a) in an analysis of whole blood profiles and elevated expression of
hsa-miR-99b,
hsa-miR-330,
hsa-miR-126 and
hsa-miR-30c was reported by (Petty et al.
2016) in an analysis involving NK cells and monocytes.
miR-99b upregulation is involved in immune downregulation in macrophages and DCs by reducing levels of IL-6, IL-12 and IL-1β upregulated in response to an infection (Singh et al.
2013; Zheng et al.
2015).
miR-127-5p upregulation exerts an immunosuppressive effect by inhibiting the phosphorylation and subsequent translocation of p65 into the nucleus leading to the inhibition of NF-κB signalling and the downregulation of c-Jun N-terminal kinase (JNK)/p38 and reduced levels of PICs (Huan et al.
2016; Park et al.
2013).
miR-30 also acts to suppress the immune response by inhibiting the toll-like receptor (TLR)/myeloid differentiation primary response 88 (MyD88) signalling pathway (Wu et al.
2017). Upregulation of
miR-143 and
miR-142-5 also signals a downregulated immune response as the production both molecules is increased following transforming growth factor beta 1 (TGF-β1) activation and plays positive and inhibitory roles in signalling pathways instigated by this cytokine (Cheng et al.
2014b; Long and Miano
2011; Ma et al.
2016; Yu et al.
2017). miR-126 is an effector of TGF-β1 signalling and regulates the activity of the PI3K/Akt signalling pathway (Guo et al.
2008,
2016). This molecule also regulates multiple aspects of the immune response in macrophages and monocytes, plays a major role in the preconditioning of the immune system against future infections with the same pathogen and activates mechanistic (or mammalian) target of rapamycin kinase (mTOR) and glycogen synthase kinase 3 (GSK-3) (Ye et al.
2013) (reviewed (Ferretti and La Cava
2014; Bai et al.
2014)). In addition, miR-126 regulates cytosolic TLR signalling and modulates the duration and intensity of such signalling in DCs (Rogers and Herzog
2014). Finally, miR-330 also regulates the activity of the PI3K/Akt signalling pathway and also exerts an inhibitory effect on T cell proliferation and NK cell activation (Grigoryev et al.
2011; Kim et al.
2015a; Petty et al.
2016).
Several pathogens have been associated with the development of CFS and evidence of chronic virus infections have been repeatedly reported in the gastrointestinal (GI) tract (Chia et al.
2010; Chia and Chia
2008), serum (Clements et al.
1995) and muscle (Cunningham et al.
1991; Gow and Behan
1991; Lane et al.
2003). Pertinently, these latter findings were not replicated in patients diagnosed according to alternative criteria (Swanink et al.
1994). Several authors have reported the presence of activated Epstein-Barr virus, human herpesvirus 6, cytomegalovirus and parvovirus B19, but these findings are not universal even in patients diagnosed according to narrow criteria (Morris and Maes
2013b) reviewed (Morris et al.
2016d)). Hence it is difficult to conclude that persistent or chronic infections are at the root of CFS/ME/SEID, but of course they could be in some patients. However, many patients have a history of a severe infection before the development of their symptoms (Gow et al.
2009; Hickie et al.
2006; Stormorken et al.
2015; White
2007; Zhang et al.
2010).
In this context it is noteworthy that the intensity of the immune response is not determined solely by the virulence or otherwise of an invading pathogen but by genetic and epigenetic variation in immune response genes (Bronkhorst et al.
2013; Morandini et al.
2016; Rautanen et al.
2015; Smelaya et al.
2016). Epigenetic variation in immune response genes also plays a major role in determining the development of DAMPs in an individual post-infection (reviewed (Morandini et al.
2016)). This may be pertinent from the perspective of the aetiology of CFS, as the production or presence of these molecules can at least in some circumstances ‘convert’ an acute pathogenic infection into a state of escalating chronic systemic inflammation, and hence this mechanism could conceivably underpin the development of chronic symptoms in CFS patients diagnosed according to the Fukuda criteria (Lucas and Maes
2013; Lucas et al.
2015). However, could this relatively simple concept explain all the observations relating to CFS reviewed above? Accordingly, this paper aims to answer this question in the context of developing an explanatory model of illness development and progression, commencing with a proposed mechanism explaining the development of chronic systemic inflammation, oxidative and nitrosative stress (I&ONS) following a pathogen invasion in genetically predisposed individuals.
Acute infection and the development of chronic I&ONS: The role of DAMPs
The relationship between polymorphisms in immunity-related genes and the intensity of the immune response, irrespective of pathogen virulence, has been repeatedly demonstrated (Cvejic et al.
2014; Helbig et al.
2005; Piraino et al.
2012; Vollmer-Conna et al.
2008). Equally, the relationship between an exaggerated immune response and increased production of DAMPs stemming from cellular stress and tissue damage is well documented (Fichna et al.
2016; Kakihana et al.
2016; Rittirsch et al.
2008; Wiersinga et al.
2014).
Mechanistically, the genesis of DAMPs following TLR or nucleotide-binding oligomerisation (NOD)-like receptor activation by a range of pathogens in the context of a hyper-responsive immune system involves prolonged or excessive activation of NFκB and other transcription factors such as nuclear factor of activated T cells and activated protein-1. This leads to upregulation of macrophage and monocyte PICs and activation of T and B lymphocytes (Lucas et al.
2015; Morris et al.
2015a). Abnormally elevated PIC production in turn leads to elevated upregulation of iNOS and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, leading to the production of superoxide, nitric oxide and peroxynitrite, leading to further upregulation of NF-κB and hence further increases in PICs, ROS and RNS levels (Morris et al.
2015a; Morris and Maes
2014). This bidirectional self-amplifying association between the development of chronic systemic inflammation and chronic ONS is sometimes described as an ‘autotoxic loop’ (Ortiz et al.
2013; Reuter et al.
2010). Excessive levels of ROS and RNS can lead to damage to proteins, lipids and DNA and the formation of oxidative specific epitopes and products of lipid peroxidation, which function as DAMPs capable of activating TLRs on cell membranes and cytosolic pathogen recognition receptors (PRRs) (Bowie
2013; Leibundgut et al.
2013; Miller et al.
2011; Uchida
2013).
Importantly, several research teams have reported the presence of such DAMPs in CFS patients (Brkic et al.
2010; Maes et al.
2011; Maes and Leunis
2014; Richards et al.
2007; Tomic et al.
2012; Wang et al.
2014). In addition, there is accumulating evidence indicating that an environment of chronic ONS leads to release of mitochondrial components into the cytosol which also have the capacity to activate cytosolic PRRs and logically are categorised as mitochondrial DAMPs (reviewed (Nakahira et al.
2015)). There are a range of molecular entities which fall into this category other than mtDNA and one such species is cardiolipin (Wenceslau et al.
2014). This is of importance from the perspective of this paper as immunogenic cardiolipin has been repeatedly detected in CFS patients diagnosed according to internationally agreed criteria (Hokama et al.
2008,
2009). The work of Hokama and others would appear to be especially noteworthy as the study contained 320 participants satisfying the requirements of the Fukuda criteria (Hokama et al.
2008).
Research investigating the role of DAMPs in human diseases is advancing apace and the categorisation of these molecules (Jammes et al.
2009) as uniquely proinflammatory entities is changing; one such change which may be relevant to the pathogenesis of CFS is the realisation that HSPs, once thought to be exclusively proinflammatory, have a key anti-inflammatory function and play a crucial role in restraining the intensity and/or duration of the immune response (van Eden et al.
2012) (reviewed (van Eden et al.
2017)). This is pertinent given that HSP production appears to be deficient in CFS patients, and the HSPs produced appear to be dysfunctional, which could potentially provide another mechanism underpinning a prolonged and/or exaggerated immune response to pathogen invasion and other sources of inflammation such as stress, medical comorbidity and lifestyle factors in such patients (Elfaitouri et al.
2013; Jammes et al.
2005,
2009,
2011,
2012).
Chronic engagement of TLRs by DAMPs leads to the development of a positive feedback loop, whereby increasing tissue damage caused by elevated PICs, ROS and RNS perpetuates and escalates pro-inflammatory responses, leading to a state of chronic inflammation, ONS, mitochondrial dysfunction and glial cell activation (Drexler and Foxwell
2010; Goh and Midwood
2012; Morris and Berk
2015; Piccinini and Midwood
2010). Unsurprisingly, chronic engagement of TLRs, NOD-like receptors and retinoid acid-inducible gene I (RIG-I)-like receptors is implicated in the pathogenesis and pathophysiology of systemic lupus erythematosus (SLE), rheumatoid arthritis and MS (review (Drexler and Foxwell
2010; Goh and Midwood
2012; Piccinini and Midwood
2010)). Pertinently, the presence of DAMPs can also lead to chronic activation of the inflammasome (Anders and Schaefer
2014), which is also implicated in the development of neuro-inflammation and abnormal central nervous system (CNS) signalling characteristic of neurodegenerative and neurodevelopmental disorders (Singhal et al.
2014; Tan et al.
2013).
I&ONS and the development of endotoxin tolerance via IDO upregulation
Chronic I&ONS can also provoke the development of endotoxin tolerance by inducing the transcriptional activation of IDO (Kim et al.
2015c; Wichers and Maes
2004) leading to upregulation of the kynurenine pathway, aryl hydrocarbon receptor (AhR) activity and increased levels of TGF-β1 (Bessede et al.
2014; Wirthgen and Hoeflich
2015) and IL-10 (Alexeev et al.
2016; Lanis et al.
2017) via well documented mechanisms (reviewed (Wirthgen and Hoeflich
2015)).
The upregulation of AhR activity is of interest given data presented in the previous section as increased activity of this cytosolic transcription factor leads to upregulation of RelB and non-canonical NF-κB signalling (Salazar et al.
2017; Vogel et al.
2013). Mechanistically, these effects appear to be mediated by transcriptional upregulation of RelB (de Souza et al.
2014; Thatcher et al.
2007) and subsequent physical engagement between RelB and AhR to produce dimers capable of modulating the expression of NF-κB-sensitive genes (Vogel et al.
2008). AhR-upregulated RelB also stimulates and maintains the transcription of miR-146a (Zago et al.
2014,
2017). This is of importance as miR-146a is a dominant player in the development and maintenance of the hypo-inflammatory environment characteristic of endotoxin tolerance (Banerjee et al.
2013; Nahid et al.
2009). Mechanistically, this inhibitory effect is enabled by suppressing TLR signalling pathways by reducing the translation of
TNF receptor associated factor 6 (
TRAF6),
interleukin-1 receptor-associated kinase 1 (
IRAK1),
IRAK2 and
interferon regulatory factor 3 (
IRF3), which are positive adaptor kinases of MyD88-mediated signalling and hence their inactivation results in reduced activity of both NF-κB and IRF3 (Nahid et al.
2011) (reviewed (Testa et al.
2017)).
The upregulation of TGF-β1 also results in upregulated non-canonical NF-κB signalling (Pallotta et al.
2011; Shi and Massague
2003). Increased activation of this cytokine also upregulates pseudokinase IRAK-M (Pan et al.
2010; Srivastav et al.
2015; Standiford et al.
2011). This is significant because IRAK-M would appear to be the ‘master regulator’ of the TLR pathway suppression characteristic of the state of endotoxin tolerance in PMBCs (del Fresno et al.
2007; Escoll et al.
2003; Stiehm et al.
2013; van’t Veer et al.
2007; Wiersinga et al.
2009). Indeed, the weight of evidence suggests that increased activity of this enzyme alone is sufficient to maintain an LPS-induced hypo-inflammatory state in human macrophages and monocytes (van’t Veer et al.
2007). This is unsurprising given that this molecule can inhibit TLR signalling at multiple levels. TGF-β1 has been established as an indispensable element in the development of endotoxin tolerance-associated SHIP upregulation (Sly et al.
2004; Yang et al.
2015). This may be of particular relevance from the perspective of a putative explanatory model of CFS aetiology as elevated levels of this cytokine in PMBCs and whole blood are a common finding in patients diagnosed according to narrow international consensus criteria and correlate with the severity of a range of symptoms (Blundell et al.
2015; Wyller et al.
2017). Once again, it is noteworthy that this phenomenon is not observed in patients diagnosed according to broader schema which are not internationally recognised such as the ‘alternative CDC criteria’ (Clark et al.
2017).
Upregulated IL-10 also exerts negative effects on TLR signalling by increasing the ubiquination and proteasome-mediated degradation of a range of MyD88-dependent signalling effector molecules such as IRAK-4 and TRAF6 ultimately resulting in reduced phosphorylation and activity of inhibitor of kappa B kinase (IKK), p38 and JNK (Chang et al.
2009). IL-10 is produced by monocytes, macrophages, Tregs and Th2-polarised T cells in a state of endotoxin tolerance, and suppresses the CD8 T and CD4 Th1 type cell response making an indispensable contribution to the development of an anti-inflammatory environment (Jiang and Chess
2006; Littman and Rudensky
2010). The indispensable contribution of IL-10 to the development of endotoxin tolerance (Liu et al.
2011b; Quinn et al.
2012) is of importance from the perspective of this paper as the upregulation of this cytokine is a common observation in CFS patients (Roerink et al.
2017; Wong et al.
2015).
It should be noted that once activated, IDO activity can be maintained by two positive feedback mechanisms. First, TGF-β can target its cellular receptor leading to the upregulation of NF-κB-RelB signalling leading to further transcription of IDO (Pallotta et al.
2011; Shi and Massague
2003). Second, IDO-activated AhRs can in turn upregulate the transcription of
IDO1 (the gene that encodes IDO) via genomic and non-genomic routes (Li et al.
2016b; Litzenburger et al.
2014). Hence once activated, IDO upregulation could be protracted or even chronic.
In addition, there is evidence obtained from human studies that chronic or intermittent translocation of LPS into the systemic circulation can induce a state of tolerance and alternative activation in macrophages and monocytes characteristic of endotoxin tolerance via the activation of IDO, kynurenine and the AhR (Banerjee et al.
2013; del Campo et al.
2011; del Fresno et al.
2008; Pena et al.
2011; Wisnik et al.
2017). Given the existence of LPS translocation in CFS, this mechanism could also contribute to the development of a chronic state resembling endotoxin tolerance.
The importance of IDO activation in the development of endotoxin tolerance is further emphasised by data confirming that interactions between the AhR, kynurenine and TGF-β1 are responsible for the polarisation of activated naïve T cells into the Treg phenotype by the presentation of antigen by tolerogenic antigen-presenting cells (Gandhi et al.
2010; Mezrich et al.
2010). Such phenotypic presentations are considered below.
IDO2 is a homologue of IDO (also known as IDO1), being an immunomodulatory enzyme which catalyses L-trytophan; like
IDO1,
IDO2 is also located on chromosome 8 in humans but
IDO2 is not as widely expressed as
IDO1 and IDO2 has a distinct signalling role (Metz et al.
2007; Cha et al.
2018). B cell
IDO2 expression has recently been identified as being an essential mediator of autoreactive B and T cells in autoimmune responses (Merlo and Mandik-Nayak
2016; Merlo et al.
2016,
2017). It seems likely, therefore, that IDO2 may be found to play an important role in ME/CFS.