Increased intestinal permeability and translocation of commensal antigens into the circulation

Chronically elevated I&ONS in the guise of increased levels of ROS, RNS and PICs induces marked increases in intestinal epithelial barrier permeability (Al-Sadi et al. 2009; Banan et al. 2003; Lee 2015; Tian et al. 2017), ultimately leading to translocation of Gram-negative bacterial LPS and a range of other commensal antigens such as peptidoglycan and flagellin, from the gut lumen into the intestinal mucosa (Lucas et al. 2015; Morris et al. 2016b). Such events lead to the creation of a self-amplifying pattern of localised and then systemic inflammation via several different routes (Delzenne and Cani 2011; Zhang and Zhang 2013).

In the first instance, the presence of LPS in the colon exacerbates inflammation in the intestine and depletes levels of regulatory T cells (Tregs), resulting in increased PIC levels (Im et al. 2012). In addition, increased an concentration of colonic LPS provokes further increases in epithelial tight junction permeability by stimulating the increased synthesis and release of the chemokine IL-8 by intestinal epithelial cells and upregulation of TLR4 on the surface of enterocytes (Angrisano et al. 2010; Guo et al. 2013). Translocated LPS also increases tight junction permeability by inducing increased enterocytic expression of TLR4 and CD14 and changes in the location of their respective proteins (Guo et al. 2013).

The development of gut inflammation interacts with dysbiosis of the microbiome and provokes the recruitment of proinflammatory macrophages into mucosal tissue which exacerbates localised inflammation, further increasing intestinal permeability to the point that enables the translocation of LPS, peptidoglycan and flagellin into the bloodstream (Delzenne and Cani 2011; Zhang and Zhang 2013). This latter phenomenon can have serious consequences in terms of initiating, or in this instance exacerbating, systemic inflammation via the activation of TLR4 and TLR2 on antigen-presenting cells and increasing levels of PICs, ROS and RNS (Morris et al. 2015a, b). The pathogenic consequences of LPS translocation are highlighted by replicated data demonstrating that this phenomenon is the cause of chronic systemic immune activation and I&OS seen in HIV seropositive patients in the absence of viraemia and otherwise well controlled on highly active antiretroviral therapy (Brenchley and Douek 2008; Shan and Siliciano 2014). LPS translocation into the systemic circulation is also held to be the cause of the metabolic endotoxaemia increasingly considered to be a major element in the pathogenesis of a wide range of inflammatory conditions and illnesses such as metabolic syndrome, type 2 diabetes mellitus and MS (Cani et al. 2008, 2009; Puddu et al. 2014; Riccio and Rossano 2015). From the perspective of a model of the pathogenesis and pathophysiology of ME/CFS, the important point is that the advent of LPS translocation in an environment of pre-existing I&ONS would be expected to increase the levels of all these parameters.

Development of autonomic dysfunction

A plethora of human studies have established a causative association between indices of increased systemic inflammation, most notably elevated C-reactive protein and IL-6, and low and unresponsive HRV in a range of inflammatory and infectious illnesses such as MS, type 2 diabetes mellitus and sepsis (de Castilho et al. 2017; Stuckey and Petrella 2013; Studer et al. 2017; Tateishi et al. 2007). More specifically, translocated LPS-mediated systemic inflammation is an acknowledged cause of low and unresponsive HRV in people with metabolic endotoxaemia, which is of relevance given the existence of this phenomenon in many CFS patients (Jan et al. 2010; Lehrer et al. 2010; Morris et al. 2016b). However, the suppressive effect of LPS on HRV is not limited to its effects on systemic inflammation as there is evidence that LPS may also invoke systemic autonomic dysfunction via direct effects on TLR4 receptors on microglia in the paraventricular nucleus of the hypothalamus (Masson et al. 2015; Okun et al. 2014) and the brainstem (Ogawa et al. 2011). The effects of LPS in this instance appear to be mediated via cross-talk with angiotensin II and the ultimate effect is the activation of microglia with resultant increases in I&OS throughout the hypothalamus (Biancardi et al. 2016; Ogawa et al. 2011; Wang et al. 2009).

The relationship between systemic inflammation and HRV is important in a wider context as it is a measure of suppressed vagal nerve activity and a dysfunctional cholinergic anti-inflammatory reflex (reviewed (Huston and Tracey 2011)) and hence is accepted as a surrogate marker of global autonomic dysfunction as the vagus nerve acts on both the cardiac sinoatrial node and the reticulo-endothelial system (Borovikova et al. 2000) (reviewed (Herlitz et al. 2015)). It is also of interest that given the existence of data demonstrating that the extent of HRV suppression correlates with the levels of systemic inflammation (Durosier et al. 2015; Herry et al. 2016) (review (Cooper et al. 2015)), that HRV parameters could act as surrogate markers of systemic inflammation, which could potentially prove to be objective biomarkers for CFS patients in the early stages of their illness (Durosier et al. 2015). However, low HRV values are also seen in patients suffering from major depressive disorder (Brunoni et al. 2013), possibly as a result of associated high levels of peripheral PICs (reviewed (Berk et al. 2013)) and hence HRV values are unlikely to be able to differentiate patients with CFS from those with a similar symptom presentation as a result of major depressive disorder.

Development of neuroinflammation, neurocognitive and neuroimaging abnormalities

Development of neuroinflammation

There is accumulating data demonstrating a causative association between the development of chronic systemic inflammation and disruptions in resting state connectivity, the development of GM and WM atrophy and lesions, and reduced cerebral perfusion (Adam et al. 2013; Felger et al. 2015; Labrenz et al. 2016; Lekander et al. 2016; Marsland et al. 2015; Riverol et al. 2012; Sankowski et al. 2015; Sonneville et al. 2013). There is also a large and increasing body of evidence indicating a causative relationship between the presence of chronic systemic inflammation and the existence of cognitive disability in patients diagnosed with a range of neuroinflammatory, neurodegenerative and neuroprogressive conditions (Gorelick 2010; Marsland et al. 2015; Sartori et al. 2012).

Inflammatory signals can reach the brain via humoral and neural routes to activate the HPA axis (Morris and Berk 2015). The humoral route involves direct or indirect cytokine signalling, either through direct access to the brain via regions where the integrity of the blood-brain barrier (BBB) is compromised or absent, such as the choroid plexus or other circumventricular organs (CVOs) (Morris et al. 2013), or by direct entry via saturable BBB transport systems or an indirect induction of cytokines and other inflammatory mediators, such as prostaglandins, and their subsequent release into the CNS parenchyma or via provocation of an increase in BBB permeability (Morris et al. 2015b; Seruga et al. 2008). The neural route involves direct stimulatory action of PICs on peripheral afferent neurons of the vagus nerve (Goehler et al. 2000; Johnston and Webster 2009).

Entry of PICs into the brain can have profound pathological consequences either directly or indirectly. There is now overwhelming evidence that transduced inflammatory signals provoke the development of chronic neuroinflammation secondary to the sequential activation of microglia and astrocytes. Activated microglia secrete a range of neurotoxic molecules such as tumour necrosis factor (TNF)-α, IL-6, IL-1β, ROS, RNS, COX-2, prostaglandin E 2 (PGE 2 ), glutamate and, in some cases, quinolinic acid (Morris et al. 2015b; Morris and Maes 2013b). Moreover, the release of PICs can in themselves act as independent sources of RNS, primarily NO, and other neurotoxins via their capacity to upregulate iNOS, COX-2 and PGE 2 (Morris et al. 2015a; Sofroniew and Vinters 2010). Unsurprisingly, the production of these neurotoxins can exert profound and detrimental effects on neurotransmitter systems, and neural integrity and function (Morris et al. 2015b).

Activated microglia and subsequent release of PICs and glutamate, combined with reduced glutamate reuptake by activated astrocytes, can lead to the development of glutamate neurotoxicity with resulting damage to glutamatergic neurones and disruption to glutamatergic neurotransmission (Noda 2016; Robel et al. 2015; Takeuchi et al. 2006), while the I&OS generated by such activation damage A9 dopamine and A6 noradrenaline neurones thereby respectively disrupting dopaminergic and noradrenergic neurotransmission (Nagatsu and Sawada 2006; Tripathy et al. 2015). There is also an accumulating body of evidence indicating that elevated ROS and RNS in the CNS can inhibit dopaminergic neurotransmission by inhibiting dopamine receptors (Morris et al. 2017d). Elevated CNS PIC levels, and subsequent activation of the p38 mitogen-activated protein kinase (MAPK) signalling system, can also adversely affect the synthesis, reuptake and release of serotonin (Miller et al. 2013). Elevated PICs in the CNS also provoke the activation of the tryptophan catabolite pathway, depleting levels of tryptophan, which is the precursor of 5-HT, as well as creating another dimension of neuropathology via the synthesis of several neurotoxic tryptophan catabolites including the potentially neurotoxic quinolinic acid (Miller et al. 2013) (reviewed (Morris et al. 2016e)).

There are also some data to suggest that activated microglia inhibit GABAergic neurotransmission, but perhaps counterintuitively this action would appear to have a neuroprotective effect (Chen et al. 2014). It is also noteworthy that this would appear to be contrary to the effects of systemic inflammation on the GABA system, leading to its activation, which is considered to be one source of apparently idiopathic chronic pain (Jang et al. 2017). This may be a mechanism underpinning the presence of the chronic intractable pain reported by many CFS patients (Nijs et al. 2012). In this context it is also worth noting that microglial activation, most notably in the basal ganglia, is also a well-documented cause of ‘unexplained’ chronic pain (Jeon et al. 2017).

Systemic LPS can enter the CNS via regions of BBB permeability such as the circumventricular organs and area postrema, in much the same way as PICs, and engage TLR4 receptors on microglia leading to their activation (Hines et al. 2013; Konsman et al. 2002; Sandiego et al. 2015). The resulting neurocognitive dysfunction has much the same origins as microglial activation induced by PICs but there is some evidence to suggest that the adverse effects on adult neurogenesis, memory deposition and recall, synaptic plasticity and long-term potentiation following elevated systemic LPS is primarily mediated by elevated IL-1β in the hippocampus (Abareshi et al. 2016; Li et al. 2017; Nolan et al. 2005).

Chronic neuroinflammation as the cause of neurocognitive and neuroimaging abnormalities

It should also be stressed at this junction that microglia and astrocytes play indispensable roles in maintaining CNS homeostasis in areas such as synaptic plasticity and long-term potentiation, which are essential for the deposition and retrieval of memory representations, together with oxygen and nutrient delivery to neurones; thus dysregulated activity of these glial cells is detrimental to cognitive function (Sofroniew and Vinters 2010; Xavier et al. 2014). Hence the development of functional gliopathology in combination with disturbances to neurotransmission following the activation of microglia and astrocytes discussed above could explain, at least in part, the multiple lines of evidence demonstrating cognitive dysfunction in patients with CFS.

This would also seem to be true of neuroimaging abnormalities seen in CFS patients as there is direct evidence that neuroinflammation resulting from microglial activation disrupts resting state functional connectivity (Colasanti et al. 2016) and there is copious evidence that neuroinflammation is a cause of structural damage and of GM and WM atrophy in a range of neurological and medical conditions (Calabrese et al. 2015; Chen et al. 2015; Cheriyan et al. 2012; Chiang et al. 2017; Raj et al. 2017; Tóth et al. 2017; Zhang et al. 2016).

The existence of chronic ONS in the CNS following the activation of microglia and astrocytes subsequent to the existence of chronic peripheral inflammation may also explain the development of cerebral hypoperfusion in CFS patients. Briefly, high levels of NO and ROS result in oxidative damage to lipids, proteins and DNA in the endothelial cells of the BBB resulting in a pattern of escalating damage to such cells and a concomitant loss of the cytoprotective effects of NO normally derived from endothelial nitric oxide synthase (eNOS) (Lucas et al. 2015; Morris and Maes 2014). This is the result of oxidative inactivation of tetrahydrobiopterin (BH 4 ), which is one of the enzyme’s essential cofactors, and changes in levels of arginine and calcium ions (Burghardt et al. 2013; Mitchell et al. 2007; Montezano and Touyz 2012). It should also be noted at this juncture that chronic peripheral inflammation can also impair endothelial eNOS function (Burghardt et al. 2013).

The mechanism underpinning depleted BH 4 levels under such conditions involves ROS-induced oxidation of BH 4 to dihydrobiopterin (BH 2 ), subsequently reducing levels of the former molecule in the endothelium of the BBB (Najjar et al. 2013). The subsequent decrease in the BH 4 to BH 2 ratio results in the inhibition of eNOS while simultaneously uncoupling arginine as its substrate thereby enabling engagement with environmental oxygen and increased production of superoxide ions (Bouloumie et al. 1999; Moens and Kass 2006; Najjar et al. 2013). The resultant combination of superoxide ions with NO results in further increases in levels of ONOO−, thereby inducing increased oxidation of BH 4 to BH 2 , further decreasing the activity of eNOS in an escalating positive feedback loop (Chen et al. 2010; Szabó et al. 2007).

Crucially, reduced eNOS activity can deplete endothelial NO levels, ultimately resulting in significantly impaired CBF (Najjar et al. 2013; Toda and Okamura 2012). The development of this phenomenon also appears to be associated with impaired vasodilation which also stems from impaired neurovascular eNOS-dependent synthesis of NO (Li et al. 2016a; Liu et al. 2016; Najjar et al. 2013). Furthermore, persistent cerebral hypoperfusion can compromise endothelial mitochondrial respiration further increasing the formation of ROS in BBB endothelial cells (Aliev et al. 2010, 2014; Liu and Zhang 2012), which in turn promotes increased eNOS uncoupling, further lowering endothelial NO levels, and a pattern of incrementally decreasing cerebral perfusion in a positive feedback loop (Antoniades 2006; Chen et al. 2010; Lavoie et al. 2010).

Importantly, the existence of positive feedback loops in the brain and periphery, such as those discussed above, can lead to a pattern of increasing I&ONS, which can trigger a state of metabolic and immune downregulation potentially accounting for a range of apparently conflicting data reported by researchers investigating these domains even in participants afforded a diagnosis of CFS according to the international consensus criteria. These processes form the focus of the remainder of this paper and are discussed below.

Increasing I&ONS and the switch to immune and metabolic downregulation

Advent of hypernitrosylation

Reversible protein S-nitrosylation, denitrosylation and transnitosylation of protein cysteine thiols effects the vast bulk of NO cellular signalling and enables the homeostatic regulation of virtually every dimension of redox-dependent protein signalling, largely determining protein function, stability and trafficking (Banerjee 2012; Hill and Bhatnagar 2012; Paulsen and Carroll 2010; Winterbourn and Hampton 2008).

From the perspective of this paper, the key point to stress is that this NO-induced post-translational modification plays an indispensable role in maintaining cellular homeostasis in the face of increasing levels of oxidative stress (Gorelenkova Miller and Mieyal 2015; Okamoto and Lipton 2015). In such an environment, moderate increases in levels of ROS and NO lead to a defensive pattern of increased S-nitrosylation of crucial structural and functional proteins as a shield against irreversible oxidation of cysteine thiols and subsequent prolonged or even permanent changes in their secondary and tertiary conformation leading to inactivity and/or immunogenicity (Kohr et al. 2014; Penna et al. 2014; Sun and Murphy 2010; Sun et al. 2006). However, further increases in O&NS lead to impaired activity of denitrosylases such as the thioredoxin system, S-nitrosogluthathione reductase, protein disulphide isomerase, superoxide dismutase and glutathione peroxidase, which maintain the reversibility of S-nitrosylation leading to a state of protracted or even irreversible nitrosylation which has been described as hypernitrosylation (Wu et al. 2010; Wu et al. 2011) (reviewed (Morris et al. 2017c)).

A state of increased nitrosylation in CFS is indicated by findings that IgM responses to NO-tryptophan, NO-tyrosine, NO-albumin and NO-cysteinyl are increased in CFS (Maes et al. 2006). Interestingly, in CFS, increased bacterial translocation is associated with indicants of increased nitrosylation (Maes and Leunis 2014). A wider discussion of the potential role of NO and ONOO− in the pathogenesis and pathophysiology of CFS may be found in a recent review by Monro and Puri (2018). This is of importance as the development of ‘hypernitrosylation’ can provoke a switch from an inflammatory environment with excessive activation of immune pathways to an environment of hypo-inflammation, impaired energy production and metabolic downregulation, which may be exacerbated in individuals with high levels of translocated LPS as high systemic concentrations of this antigen may result in the same ultimate endpoints (Morris et al. 2016b, c). This will form the theme of the remainder of this paper with initial impairment in oxidative phosphorylation and mitochondrial dynamics considered immediately below.

Hypernitrosylation and impaired mitochondrial performance

There is a considerable and accumulating body of data demonstrating that S-nitrosylation of key mitochondrial enzymes and structural proteins plays an indispensable role in the redox-based regulation of mitochondrial respiration and other aspects of energy production (Doulias et al. 2013; Mailloux et al. 2014). In sum, readily reversible S-nitrosylation negatively regulates the function of a myriad of proteins involved in oxidative phosphorylation, the tricarboxylic cycle, gluconeogenesis, glycolysis, generation of mitochondrial ROS, mitochondrial permeability transition, as well as apoptosis (Doulias et al. 2013; Mailloux et al. 2014). Hence hypernitrosylation has the capacity potentially to impair cellular energy generation and, in particular, to lead to inhibitory nitrosylation of crucial cysteine thiols of enzymes in the electron transport chain (ETC) such as complex I (Drose et al. 2014; Murray et al. 2012; Piantadosi 2012), cytochrome c oxidase (complex IV) and, to a lesser extent, complex II (Sarti et al. 2012a; Zhang et al. 2005). Unsurprisingly, such inhibition impairs oxidative phosphorylation and the production of ATP, and depletes levels of mitochondrial glutathione owing to increased production of ROS by a dysfunctional ETC (Sarti et al. 2000, 2003a, b, 2012b; Zhang et al. 2005). There are also data demonstrating that prolonged inhibition of cytochrome c oxidase increases ATP production by glycolysis at least in some cell types as an attempt to mitigate against cell death via apoptosis or necrosis (Almeida et al. 2001; Bolanos et al. 2010; Burwell et al. 2006; Shiva et al. 2007; Sun et al. 2007).

In addition, prolonged nitrosylation of the mitochondrial regulatory proteins PINK-1, PARKIN and DRP-1 compromises mitochondrial dynamics as a result of impaired mitophagy, increased degeneration and increased fission (Nakamura et al. 2010; Oh et al. 2017; Ozawa et al. 2013; Reddy et al. 2011). This may be relevant from the perspective of activity intolerance reported by many CFS patients as intact mitochondrial dynamics enables the adaptation of an individual to continuous or incremental exercise regimes (Trewin et al. 2018; Yan et al. 2012).

Hypernitrosylation and the development of immune suppression and cellular hibernation

Prolonged or intractable S-nitrosylation characteristic of hypernitrosylation may lead to changes in the activity of proteins and signalling pathways regulating inflammation and energy production, leading to a state of hypo-inflammation and metabolic downregulation. For example, hypernitrosylation of a crucial cysteine residue of p50 silences NF-κB-dependent gene transcription via several different mechanisms (Bogdan 2001; DelaTorre et al. 1997, 1998, 1999; Kelleher et al. 2007). In addition, hypernitrosylation of cytosolic enzyme inhibitory kappaB kinase beta impairs its levels of phosphorylation (Reynaert et al. 2004) leading to diminished proteasomal degradation of inhibitory kappaB which maintains NF-κB in an inactive state (Hess et al. 2005; Marshall et al. 2004; Reynaert et al. 2004). In addition, hypernitrosylation can further contribute to the development of immune suppression or an hypo-inflammatory state by the downregulation of TLR signalling via inhibition of MyD88 thereby inhibiting the immune response to acute pathogen invasion (Into et al. 2008). Increased NO levels may also upregulate IRAK-M mRNA and protein expression of interleukin receptor associated kinase-M (IRAK-M), which is a key negative regulator of TLR signalling although there is no compelling data indicating that this process is enabled by S-nitrosylation either directly or indirectly (del Fresno et al. 2004; Gonzalez-Leon et al. 2006). These pathways are illustrated in Fig. 1.

Fig. 1 Acute stimulation of TLR4 leads to the recruitment of MyD88, MyD88-adaptor-like (MAL), IL-1 receptor-associated kinase-4 (IRAK-4), toll/IL-1 receptor (TIR)-domain-containing adaptor-inducing IFNβ (TRIF) and TRIF-related adaptor molecule (TRAM), ultimately provoking the initiation of signalling cascades that converge to activate NFκB, MAPKs and IFN response factors (IRFs) with the subsequent production of inflammatory mediators such as type 1 interferons and PICs. However, in a state of endotoxin tolerance the TLR4 response is reprogrammed via the upregulation of the inhibitory proteins SHIP1, suppressor of cytokine signalling 1 (SOCS1) and the pseudokinase IRAK-M leading to the production of IL-10 and TGF-β1 leading to a downregulated and anti-inflammatory immune response Full size image

Prolonged S-nitrosylation (see Fig. 2) can also lead to profound metabolic changes via upregulation of the protein subunit hypoxia-inducible factor-1α (HIF-1α), through upregulation of HIF1A and/or stabilisation of this subunit, under non-hypoxic conditions (Kasuno et al. 2004; Li et al. 2007; Yasinska and Sumbayev 2003) and activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR signalling (Gupta et al. 2017; Kwak et al. 2010; Lopez-Rivera et al. 2014; Numajiri et al. 2011). There is also evidence that mTOR is directly activated as a result of inhibitory S-nitrosylation of tuberous sclerosis complex 2 (TSC2), an inhibitor of mTOR (Lopez-Rivera et al. 2014) and nitrosylation-mediated activation of Ras, a small GTPase which is a positive regulator of mTOR (Lee and Choy 2013). Prolonged nitrosylation may also provoke changes in metabolic pathways via the upregulation of GSK-3 (Morris et al. 2017c). Furthermore, activated GSK-3 and PI3K/Akt signalling in tandem or separately can provoke metabolic and bioenergetic dysregulation by inhibiting the enzyme 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) (Park et al. 2014; Suzuki et al. 2013).

Fig. 2 Proteins undergo reversible S-nitrosylation and denitrosylation initiated by the covalent addition and release of NO probably derived from N 2 O 3 in a hydrophobic environment. Denitrosylation may be mediated by several molecular players such as GSNOR (S-nitrosoglutathione (GSNO) reductase) and the thioredoxin system. Transnitrosylation is another major route mediating protein nitrosylation. Transnitrosylation of GSH leading to the formation of native protein and GSNO is a major element enabling this process. Reformation of GSH by NADPH involves NADH-dependent reduction by GSNOR to generate GSH. The GSH and thioredoxin systems are progressively inactivated in an environment of increasing chronic ONS leading to the inhibition of denitrosylation and transnitrosylation and a state of protein ‘hypernitrosylation’ Full size image

These observations may be directly relevant as far as the pathogenesis of CFS is concerned as activation of HIF-1α, PI3K/Akt/mTOR and GSK-3 signalling pathways and inhibition of AMPK, in the context of decreased canonical NF-κB activation, are major elements in the development of a phenomenon described as endotoxin tolerance which involves profound systemic immune and metabolic downregulation, with the latter phenomenon involving sequential shifts in energy production via glycolysis and oxidation of fatty acids, following prolonged TLR upregulation in macrophages and monocytes (Liu et al. 2011a, 2012). This phenomenon is mediated, at least in part, by epigenetic reprogramming ‘masterminded’ by sirtuin 1 (SIRT1) (Liu et al. 2011a, 2012) and in this context decreased levels of canonical NF-κB signalling induced by persistent nitrosylation would be expected to activate SIRT1 (Kauppinen et al. 2013; Vaziri et al. 2001; Yang et al. 2012). This is important as SIRT1 nitrosylation in the context of high levels of classical NF-κB signalling secondary to chronically elevated I&ONS or the response to acute pathogen invasion leads to its inactivation (Kalous et al. 2016; Nakazawa et al. 2017; Shinozaki et al. 2014).

Before moving on to consider the mechanisms underpinning the development of endotoxin tolerance, it is important to note that this phenomenon resolves within days and sometimes weeks in the context of DAMP- or pathogen-associated molecular pattern-mediated engagement of PRRs, but in the context of the same result mediated by chronically elevated I&ONS and hypernitrosylation a state similar to endotoxin tolerance could be protracted or even permanent in the absence of ameliorative interventions.

Mechanisms underpinning metabolic downregulation in endotoxin tolerance

During the initial inflammatory phase following TLR activation, NF-κB upregulates the Akt-mTOR-HIF-1α pathway, leading to a surge in ATP production via aerobic glycolysis (Cheng et al. 2014a; Srivastava and Mannam 2015; van Uden et al. 2008). Such upregulation leads to inhibition of the tricarboxylic acid (TCA) cycle and increased production of mitochondrial ROS, leading to a fall in mitochondrial ATP production, mitochondrial structural damage, increased mitophagy and a switch in cytochrome oxidase subunits designed to increase the efficiency of the ETC (Cheng et al. 2014a; Semenza 2011; Zhong et al. 2010). Inhibition of the TCA cycle and oxidative phosphorylation is mediated by increase in the transcription of pyruvate dehydrogenase kinase with the subsequent inhibition of pyruvate dehydrogenase and conversion of pyruvate into acetyl coenzyme A (acetyl-CoA). This inhibition is accompanied by a concomitant HIF-1α-mediated increase in the conversion of pyruvate to lactate with the resultant production of ATP and NAD+ by aerobic glycolysis (Kim et al. 2006).

The continuation of ATP production and NAD+ by this route is enabled by HIF-1α-induced upregulation of lactate dehydrogenase (Semenza et al. 1996). The switch from ATP generation from oxidative phosphorylation instigated by the upregulation of the transcription factor is further enabled by increased expression of glucose transporters and glycolytic enzymes (Hanahan and Weinberg 2011; Semenza 2003). The importance of Akt-mTOR-HIF-1α signalling in the development of endotoxin tolerance is emphasised by data demonstrating that inhibition of this pathway prevents the development of immune and metabolic downregulation in macrophages and monocytes (Cheng et al. 2014a).

However, this rapid burst of ATP production and concomitant increases in NADH production enabled by activation of the of Akt-mTOR-HIF-1α signalling system is a short-lived phenomenon and the subsequent fall in ATP and NADPH generation leads to a relative rise in AMP and NAD+ (Adriouch et al. 2007; Haag et al. 2007), leading to the activation of AMPK (Gómez et al. 2015) and the sequential activation of the NAD+-sensitive SIRT family members sirtuins 1, 6 and 3 (Liu et al. 2011a, 2012, 2015a). Once activated, these SIRTs play the dominant role in the development of a hypo-inflammatory state and an environment of metabolic downregulation and reductions in glycolysis and TCA-induced oxidative phosphorylation characterised by mitochondrial ATP production via fatty acid oxidation (Gómez et al. 2017; Vachharajani et al. 2016). This adaptive low-energy state is often described as cellular ‘hibernation’ (Levy et al. 2005; Liu et al. 2011a; Singer 2008) (reviewed (Singer 2017)) and it is important to stress that this phenomenon is not limited to macrophages and monocytes but also takes place in hepatocytes and striated muscle cells (Carré and Singer 2008; McCall et al. 2011; Singer 2007, 2008).

Once activated, SIRT1 upregulates the transcription of peroxisome proliferator-activated receptor γ coactivator-1 alpha with a subsequent and concomitant increase in mitochondrial biogenesis and respiration mediated by fatty acid oxidation. The mechanisms underpinning this process include increased cellular uptake of fatty acids via the CD36 membrane transporter and increased transfer of these molecules into mitochondria via upregulation of several enzymes including palmitoyltransferase I, which governs the rate of fatty acid oxidation (Liu et al. 2012; Vachharajani et al. 2014; Wanders et al. 2010) (reviewed (Qu et al. 2016)). SIRT1 also exerts a range of protective effects on mitochondria aimed at maximising energy generation and fostering cellular and organelle survival. These effects include: upregulating cellular anti-oxidant defences, via activation of nuclear factor (erythroid-derived 2)-like 2; increasing mitophagy to remove damaged mitochondria; and maintaining mitochondrial membrane potential, thereby inhibiting the development of mitochondrial permeability transition pore opening (Price et al. 2012; Song et al. 2017).

SIRT1-induced upregulation of SIRT6 also encourages the switch from ATP generation via aerobic glycolysis to ATP generation via fatty acid oxidation, via direct and indirect inhibition of glycolysis, increased fatty acid oxidation and a range of effects broadly encouraging mitochondrial survival in a low-energy state (Cheng et al. 2016; Sebastian et al. 2012). Direct inhibitory effects of SIRT6 on glycolysis include downregulation of glucose transporter 1 and inhibition of lactate production (Elhanati et al. 2016; Long et al. 2017), while indirect effects stem from inhibition of HIF-1α (Sebastian et al. 2012). SIRT6 elevation also preserves mitochondrial membrane potential, preventing mitochondrial permeability transition pore (mPTP) opening in a similar manner to SIRT1 (Cheng et al. 2016).

Upregulation of SIRT3, an NAD+-dependent mitochondrial protein, also deacetylates and activates mitochondrial enzymes involved in fatty acid β-oxidation, amino acid metabolism, the TCA cycle, the ETC and antioxidant defences leading to increased mitochondrial ATP production (Ahn et al. 2008; Ansari et al. 2017). In addition, increased SIRT3 activity inhibits mitochondrial ROS production and the activity of several components of the mPTP, thereby encouraging mitochondrial survival (Kincaid and Bossy-Wetzel 2013; Tseng et al. 2013) (see Fig. 3).

Fig. 3 S-Nitrosylation leads to the inhibition of Complex 1, Complex IV, the F1F0ATPase (Complex V) and possibly Complex II leading to the reduction of ATP production and a decrease in ROS production with a subsequent increase in production of ATP via aerobic glycolysis. S-Nitrosylation can also compromise mitochondrial function while increasing the survival of the organelle by inhibiting uptake of calcium ions and reduction of cytosolic calcium ions via inhibition of SERCA, further reducing mitochondrial ATP and ROS production. S-Nitrosylation also inhibits key enzymes of the TCA cycle, such as aconitase and α-ketoglutarate dehydrogenase, and regulates those involved in fatty acid metabolism, thus further inhibiting oxidative phosphorylation and stimulating aerobic glycolysis and mitochondrial fatty acid oxidation Full size image

It is also noteworthy that the sequential activation of these SIRTs corresponds with the inactivation of AMPK (Jiang et al. 2014; Liu et al. 2015b). Unsurprisingly, the causes of this phenomenon have been the subject of intensive research, and while the process may be multifactorial in origin, the weight of data implicates inhibition by activated GSK-3 (Park et al. 2014; Suzuki et al. 2013). The upregulation of GSK-3 in turn appears to result from the termination of the inhibitory influence of Akt/mTOR signalling by the upregulation of SIRT1 and SIRT6 and the termination of ATP production by aerobic glycolysis (Frost and Lang 2011; Ghosh et al. 2010; Hermida et al. 2017; Pillai et al. 2014). Another contributing factor underpinning the inactivation of AMPK might be the relative increase in mitochondrial ATP production and a reduction in mitochondrial ROS production as a result of a switch to fatty acid β-oxidation from aerobic glycolysis (reviewed (Nsiah-Sefaa and McKenzie 2016)) would be below the cellular AMP and ROS thresholds expected to trigger the activation of the enzyme (Morris et al. 2017d).

SIRT1 activation and subsequent immune downregulation

Once activated, SIRT1 also plays a pivotal role in the development of a hypo-inflammatory immune response. In essence, this is achieved by inhibiting the transcription of IL-1β, TNF-α and other proinflammatory genes and suppressing inflammatory responses by deacetylating and inhibiting the incumbent p65 component of the NF-κB complex while inducing the expression of RelB (Kauppinen et al. 2013; Liu et al. 2011a; Yang et al. 2012) as well as causing gene-specific regulation as histone modifiers (Foster et al. 2007; Gazzar et al. 2008; Liu et al. 2015a; McCall et al. 2010; Yoza et al. 2006).

From a more mechanistic perspective, SIRT1 deacetylates histone protein H1K27 and lysine 310 (Lys310) of RelA/p65 and its continued binding recruits RelB to promoters of target anti-inflammatory genes (Liu et al. 2011a; Millet et al. 2013). In this instance RelB acts as a dual-function transcription factor; it increases silent facultative chromatin at promoters of pro-inflammatory genes via direct interaction with histone H3 lysine 9 methyltransferase G9a, heterochromatin protein 1, assembly of high mobility group box 1 and DNA CpG methylation; (Chen et al. 2009; Gazzar et al. 2009; Millet et al. 2013; Yoza and McCall 2011) and active euchromatin at anti-inflammatory genes via association with the NF-κB subunit p50 and the resultant transcription of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (McCall et al. 2010). In addition, SIRT6 also deacetylates RelA/p65 and histone H3K9Ac to exert additional anti-inflammatory activity (Gil et al. 2013; Kawahara et al. 2009).

However, the activation of SIRT 1 and SIRT 6 are not the only elements involved in the immune downregulation characteristic of endotoxin tolerance as miRNA activity, soluble factors such as IL-10 and TGF-β1 and the concomitant downregulation of proteins enabling TLR signalling and upregulation of proteins such as SH2 (Src Homology 2)-containing inositol phosphatase (SHIP) and IRAK-M, known to inhibit TLR signalling, all play an indispensable role (reviewed (Wisnik et al. 2017; Fu et al. 2012)). Crucially, these processes may begin with the activation of indoleamine 2,3-dioxygenase (IDO) which is activated in an environment of excessive I&OS (Morris et al. 2016a), as discussed below.