Tryptophan Metabolites and Their Impact on Multiple

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Tryptophan Metabolites and Their Impact on Multiple Sclerosis Progression Jens O. Watzlawik, Bharath Wootla and Moses Rodriguez* Departments of Neurology and Immunology, Mayo Clinic College of Medicine, 200 1st Street SW, Rochester, Minnesota 55905, USA Abstract: Accumulating evidence demonstrates involvement of tryptophan metabolites and in particular activation of the kynurenine pathway (KP) in neurocognitive disorders under CNS inflammatory conditions. The KP is involved in several brain-associated disorders including Parkinson’s disease, AIDS dementia, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, schizophrenia, and brain tumors. Our review is an attempt to address any relevant association between dysregulation of KP and multiple sclerosis (MS), an inflammatory CNS disorder that ultimately leads to demyelinated brain areas and severe neurological deficits. Modulation of KP is a new topic for the field of MS and warrants further research. The availability of potential KP modulators approved for MS may shed some light into the therapeutic potential of KP antagonists for the treatment of MS patients.

Jens O. Watzlawik

Keywords: Demyelination, microglia, macrophages, kynurenine pathway, quinolinic acid, kynurenic acid. 1. INTRODUCTION The kynurenine pathway (KP) is the main route for non-protein metabolism of the essential amino acid tryptophan. This pathway ultimately leads to the production of nicotinamide adenine dinucleotide (NAD+). Complete disruption of KP is associated with the genetic disorder hydroxykynurenuria, while enzyme deficiencies in kynurenine-3-monooxygenase (KMO) are associated with brain disorders (schizophrenia, tic disorders) and liver diseases resulting in accumulation of kynurenine and an increase in kynurenic acid (KYNA) and anthranilic acid [1-5]. Dysfunctional states of distinct steps of KP (e.g. quinolinic acid (QUIN), 3hydroxykynurenine, KYNA, kynurenine, anthranilic acid) are involved in a number of disorders including tic disorders, tourette syndrome, HIV-dementia, psychiatric disorders (e.g. anxiety disorders, schizophrenia, major depression), multiple sclerosis (MS), eosinophilia-myalgia syndrome, lipid metabolism, Huntington's disease, encephalopathies, systemic lupus erythematosus, vitamin B6 deficiency [6]. Surprisingly, in CNS disorders KP metabolites have been suggested to either act as neuroprotective or neurotoxic agents [7, 8]. In this review, we discuss known relationships between KP and MS. We will also emphasize disease conditions adequate for activation of KP. 1.1. Multiple Sclerosis, an Inflammatory CNS Disease MS is a relapsing, and often progressive white matter disorder of the CNS with unclear pathogenesis. So far, there is no treatment available to stop disease progression or reverse existing disabilities in MS patients. March 2015 statistics from the MS Foundation estimated more than 400,000 people in the United States and about 2.5 million people worldwide with MS (http://j.mp/MS_Statistics). The most common MS subtype, relapsing–remitting MS (RRMS) is present in 80 percent of patients and typically begins in the second or third decade of life with a female predominance of 2:1. Patients typically improve spontaneously or respond to corticosteroids administered. Unfortunately, the patient's responsiveness to corticosteroids typically fades over time. A certain level of CNS dysfunction may persist between relapses or progresses over time (secondary progressive MS). Approximately 20 percent of MS patients are *Address correspondence to this author at the Departments of Neurology and Immunology, Mayo Clinic College of Medicine, 200 1st Street SW, Rochester, Minnesota 55905, USA, Tel: (507) 284-3734; Fax: (507) 2841086; E-mail: [email protected] 1381-6128/16 $58.00+.00

diagnosed with primary progressive MS, which progresses gradually in the absence of obvious relapses and remissions. Primary progressive MS has a similar incidence among men and women [9]. 1.2. Pathological Hallmarks in MS CNS demyelination is a primary inflammatory process and the pathological hallmark in MS leading to brain lesion formation and neurological deficits [9, 10]. Demyelination disables saltatory nerve conduction and increases axonal vulnerability to environmental stressors, a key aspect in MS pathogenesis, which results in loss of neural function and ultimately neuronal death. Despite extensive investigations in MS research during the last decades the initial trigger(s) causing the disease have not been identified yet. Given the complexity of the disease with potentially different demyelinating disorders covered under the umbrella of MS, different mechanisms may contribute to tissue injury and MS lesion formation. Infiltrating T cells and peripheral macrophages attacking/engulfing myelinating oligodendrocytes are believed to initiate acute MS lesions [11]. This hypothesis is strongly supported by studies using an inflammatory rodent model of MS, termed experimental autoimmune encephalomyelitis (EAE). Myelin proteins in combination with complete Freunds adjuvants (CFA) (typically mycobacteria tuberculosis plus mineral oil) induce EAE. Based on EAE studies in rodents, it was hypothesized that invading T-cells reactive to myelin components are the major disease initiators in MS [12-14]. Similar to EAE, demyelination of the spinal cord in the Theiler's murine encephalomyelitis (TMEV) - model of MS was shown to be T-cell mediated. Different from EAE, immunization with spinal cord homogenates containing substantial myelin components in TMEVinfected chronically demyelinated mice induced substantial remyelination of demyelinated spinal cord areas. IgM antibodies targeting myelin components were responsible for the beneficial outcome in this model, which questioned a deleterious effect of anti-myelin antibodies in EAE. An alternative to the immune-mediated hypothesis is that oligodendrocyte death represents the first and earliest lesion stage, which in turn results in primary demyelination and secondary autoimmune inflammation [15]. Levels of subsequent lesion extension and oligodendrocyte loss may follow the extent of secondary inflammation. Recent studies also emphasize mitochondrial damage with subsequent energy failure as a major contributor to MS pathogenesis [16-20], which may in part explain demyelination and oligodendrocyte apoptosis [21], death of small diameter axons [22, © 2016 Bentham Science Publishers

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23], differentiation arrest of oligodendrocyte progenitor cells (OPCs) [24] and astrocytic dysfunction [25]. 2. THE KP Researchers studied the KP over the last decades and identified pathological pathway changes in several neurological diseases. Some key metabolites and enzymes in KP are highlighted below (Fig. 1). 2.1. Indoleamine 2,3 Dioxygenase Indoleamine 2,3 Dioxygenase also termed Indoleamine-pyrrole 2,3-dioxygenase (IDO), encoded by the IDO1 gene, is one of the first and rate-limiting enzymes of tryptophan catabolism in the KP that catalyzes the synthesis of L-kynurenine from L-tryptophan [26, 27]. Stimulation of IDO-1 expression in macrophages/microglia and allografted tumor cells was observed through administration of interferons and (IFN-, IFN-), tumor necrosis factor (TNF), platelet activating factor (PAF), the HIV proteins Nef and Tat and the A peptide 1-42 (A1-42) [28-31]. Munn et al., (1999) reported a mechanism by which macrophages and other antigen-presenting cells (APCs) are able to regulate activation of T-cells through tryptophan degradation. IDO expression in antigen presenting cells (APCs) in vivo may enable them to overcome deleterious T-cell responses [32, 33]. A more recent study in mice demonstrated a synergistic effect between TNF together with IFN- to stimulate IDO expression in vivo as well as in primary microglia [34]. Both cytokines were required to induce a depressive-like behavior in rodents after Mycobacterium bovis infection [34]. 2.2. Kynurenine-3-Monooxigenase (KMO) KMO is a -nicotinamide adenine dinucleotide 2-phosphate (NADPH)-dependent flavin monooxygenase, that converts LKynurenine to 3-hydroxykynurenine. Within the CNS KMO is localized in the outer mitochondrial membrane and predominantly

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expressed in microglia [35, 36]. KMO specifically catalyzes the incorporation of one oxygen atom into L-Kynurenine in the presence of NADPH as an electron donor. The cytokine-mediated regulation of kynurenine enzymes other than IDO by proinflammatory cytokines has not been studied extensively. However, emerging studies in rodents indicate increased KMO expression through systemic LPS administration [37, 38]. Similarly, IFN--treated murine macrophages (MT2) and microglia (N11) upregulate KMO expression, while kynureninase expression was increased only in MT2 macrophages, and 3-hydroxyanthranilic acid oxygenase (3-HAO) was not effected [39]. IL-1 treatment of human hippocampal progenitor cells stimulates expression of KMO and kynureninase [40]. 2.3. L-Kynurenic acid (KYNA) (IUPAC Nomenclature: 4Hydroxyquinoline-2-Carboxylic Acid) In the CNS, formation of KYNA is catalyzed by the enzyme kynurenine aminotransferase (KAT) I and II through an intramolecular cyclization of kynurenine to form the quinoline backbone present in KYNA. KYNA acts as an anti-excitotoxic and anticonvulsing agent that blocks/attenuates glutamate-mediated as well as quinolinic acid-mediated excitotoxicity on ionotrophic glutamate receptors including -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate receptors. In millimolar concentrations, KYNA blocks the AMPA and kainate receptors [41], but in lower, micromolar concentrations, it blocks the glycine site of the NMDAR as well as the cholinergic a7 nicotinic receptor (a7nAChR) [42]. It is still under debate whether KYNA is an antagonist for the 7 nicotinic acetylcholine receptor [42]. Two independent studies failed to identify an antagonistic effect of KYNA on the 7 nicotinic acetylcholine receptor at various concentrations. Results suggested that KYNA has no blocking effect on 7 nicotinic acetylcholine receptor currents in adult hippocampal neurons [43, 44].

Fig. (1). Molecular pathways of tryptophan metabolism. Of the dietary tryptophan that is not used in protein synthesis, 99% is metabolized along the KP (red arrows). Alternative pathways are the conversion of tryptophan to 5-hydroxykynurenine and then 4-hydroxyquinoline (or kynuramines). 3-HAO, 3hydroxyanthranilic acid oxidase; IDO, indoleamine 2,3-dioxygenase; KAT, kynurenine aminotransferase; MAO, monoamineoxidase; QPRT, quinolinic-acid phosphoribosyl transferase; TDO, tryptophan 2,3-dioxygenase.

Tryptophan Metabolites and Their Impact on Multiple Sclerosis Progression

The clinical relevance of KYNA for disease states remains unclear due to low physiological KYNA concentrations in the brain and the fact that at least three times higher concentrations of KYNA are required to effectively block the excitotoxic effects of QUIN when injected into the hippocampus [45]. Interestingly, KYNA concentrations are decreased in neuroinflammatory and neurodegenerative diseases including MS, amyotrophic lateral sclerosis and HIV-dementia. The data suggests a potential neuroprotective effect of KYNA in some neurobiological disorders. In contrast, patients with schizophrenia show increased brain and cerebrospinal fluid (CSF) concentrations of KYNA [46-49]. Clinical observations show that systemic administration of Nmethyl-D-aspartate (NMDA) receptor antagonists evokes schizophrenia-like symptoms in healthy individuals and provokes symptoms in patients with schizophrenia [50]. In addition, increased CSF KYNA levels were associated with an increased suicide risk in schizophrenia [51]. Based on the glutamate deficiency theory to explain pathophysiological findings in schizophrenia (e.g. disturbances in dopamine transmission), a hypoglutamatergic state of the brain can be achieved by elevation of the endogenous NMDA receptor antagonist kynurenic acid [52]. Elevated levels of KYNA in the rat brain are associated with increased midbrain dopamine firing [53-55] and disrupted prepulse inhibition [56], a deficit that has also been observed in patients with schizophrenia [57]. Support for a detrimental role of KYNA for cognitive function and plasticity comes from a transgenic mouse study in animals lacking the astrocytic kynurenine aminotransferase II (KAT II). Kat II-deficient mice show reduced KYNA levels and increased glutamate levels which were accompanied with enhanced cognitive abilities and improved synaptic plasticity [58]. In WT rats KYNA exposure during adolescence resulted in long-term behavioral consequences. Rats previously treated with KYNA exhibited no longterm potentiation (LTP) after a burst of high-frequency stimulation that was sufficient to induce robust LTP in vehicle-treated rats [59]. In summary, KYNA appears to be a double-edged sword for several brain disorders. In at least some neurodegenerative disorders including MS, KYNA may have neuroprotective effects when present at physiological concentrations. However, unusual high KYNA concentrations appear to be associated with psychotic symptoms in patients with schizophrenia and may substantially be involved (together with dopamine) in disease pathogenesis. 2.4. 3-Hydroxykynurenine (IUPAC Nomenclature: (2S)-2Amino-4-(2-Amino-3-Hydroxyphenyl)-4-Oxobutanoic Acid) The formation of 3-Hydroxykynurenine (3-HK) from kynurenine is catalyzed by the enzyme KMO. 3-Hydroxykynurenine is a potential endogenous neurotoxin and increased 3Hydroxykynurenine concentrations were reported in neurodegenerative disorders including HIV dementia [60], Parkinson's disease [61] and Huntington disease [62, 63]. In patients with schizophrenia, 3-hydroxykynureneine may predict the severity of early clinical symptoms before exposure to antipsychotic drugs [64]. 3Hydroxykynurenine is able to induce hydrogen peroxide-mediated neuronal death [65] under pathological conditions and acts synergistically together with QUIN to mediate CNS excitotoxicity [66]. 2.5. Quinolinic Acid (QUIN) (IUPAC Nomenclature: Pyridine2, 3-Dicarboxylic Acid) In the KP the enzyme kynureninase catalyzes the conversion of 3-hydroxykynurenine into 3-hydroxyanthranilic acid, which in turn is converted by 3-HAO into QUIN. QUIN is assumed to play a critical role in the pathogenesis of a variety of human neurological diseases. QUIN is detectable in the cerebrospinal fluid (CSF) and the CNS within a nanomolar concentration range. QUIN mediates its neurotoxic effects as a NMDA receptor agonist and by induction of mitochondrial dysfunction [67, 68]. In addition, QUIN dysregulates the cellular ability to reduce/neutralize reactive oxy-

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gen/nitrogen species and free radicals [69, 70] by affecting the glutathione redox potential [71], depleting the activity of copper- and zinc-dependent superoxide dismutase activity (Cu, Zn-SOD) [72, 73] and contributing to lipid peroxidation [74, 75], which may lead to neuronal and astrocytic apoptosis [76]. In response to bacterial infections, CNS QUIN synthesis can be higher 100-fold upregulated. Under cell culture conditions, micromolar QUIN concentrations were toxic within hours [77, 78]. It is of note that 3hydroxykynurenine, 6-hydroxydopamine and reactive oxygen species potentiate quinolinic acid-mediated neuronal damage in vivo through NMDA receptor overactivation [66, 79, 80]. 2.6. 5-Hydroxytryptamine (5-HT) (Serotonin) Serotonin or 5-hydroxytryptamine (5-HT) is a tryptophanderived monoamine neurotransmitter that is primarily located in the mammalian gastrointestinal tract. The cellular localization of 5-HT or serotonin receptors is at the level of the neuronal plasma membrane where it mediates serotonin effects as well as several pharmaceutical and hallucinogenic drugs. With the exception of the ligand-gated ion channel 5-HT3 receptor, all other 5-HT receptors are G-protein-coupled receptors (also called seven-transmembrane) that activate an intracellular second messenger cascade. Termination of serotonergic effects are primarily mediated via serotonin uptake through the monoamine transporter SERT located at the presynaptic neuron. Different molecule classes were identified with the ability to inhibit serotonin uptake including selective serotonin reuptake inhibitors (SSRIs), cocaine, tricyclic antidepressants and dextromethorphan. In addition to SERT, the plasma membrane monoamine transporter (PMAT) was identified to translocate monoamine neurotransmitter like serotonin across the neuronal presynaptic membrane [81]. In contrast to SERT, PMAT monoamine transporters are relatively insensitive to SSRIs. At present, there are no pharmaceuticals available to inhibit PMAT at therapeutic doses. 3. CNS CELL TYPE SPECIFICITY OF KP METABOLITE EXPRESSION 3.1. Glial Cells and Infiltrating Macrophages Lipopolysaccharide (LPS)- or IFN--stimulated human microglia and macrophages significantly increased QUIN synthesis [29, 82]. In contrast to macrophages, levels of QUIN synthesis were very heterogeneous in individual microglial cells and approximately 32-fold lower compared to macrophages [82]. The difference in QUIN production between human microglia and macrophages may be due to different KP enzymes expression levels in both cell types (Fig. 2) [83]. Under pathological conditions with blood–brain barrier (BBB) breakdown and substantial leukocyte infiltration, QUIN levels in the CNS may substantially be derived from peripheral macrophages (Fig. 2). QUIN production by macrophages and at the same time release of cytotoxic, pro-inflammatory cytokines by macrophages and microglia results in an amplifying feedback mechanism that further stimulates QUIN synthesis and likely contributes to MS lesion pathology (see section 4). Astrocytes represent the most abundant cell type in the CNS with important roles in cellular homeostasis, trophic and metabolic support and glutamate recycling. Guillemin et al. (2001) demonstrated absence of kynurenine 3-hydroxylase in astrocytes and therefore lack of all downstream KP metabolites including QUIN [84]. Astrocytes are capable of generating large amounts of the potentially neuroprotective agent KYNA under cytokine stimulation, which may imply an astrocyte-mediated protection under inflammatory conditions (Fig. 2). In support of this hypothesis, expression of glial specific S100 in astrocytes correlated with increased KYNA levels in the CSF of MS patients [85].


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Fig. (2). Glial cells and KP activation. Tryptophan degradation and kynurenine synthesis is regulated by steroid hormones, cytokines and growth factors in the periphery, which stimulate indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and kynurenine 3-monooxygenase (KMO). Circulating tryptophan, kynurenine and 3-hydroxykynurenine (3-HK) are able to cross the blood–brain barrier. Inflammatory conditions stimulate the KP both in the periphery and in the brain. Increased influx of the brain-permeable metabolites, in some cases aided by a leaky blood–brain barrier, as well as infiltration of macrophages, then leads to an excess of QUIN in the brain parenchyma. Furthermore, infiltrating cytokines stimulate the KP in activated microglial cells and macrophages. KYNA, kynurenic acid; QUIN, quinolinic acid.

It is of note that human astrocytes catabolize only small amounts of QUIN [86], whereas pathological QUIN concentrations (0.5-1.2 M) are sufficient to induce astrocytic apoptosis [87]. Human fetal oligodendrocytes lack IDO expression, TDO expression and KATII expression, a finding that still needs to be confirmed by other groups not only in human tissue. In addition, it is very unclear whether these findings have any relevance for the adult situation [88]. It is of note that oligodendrocytes express NMDA receptors [89], which in theory makes them vulnerable towards QUINmediated excitotoxicity. However, to our best knowledge pathophysiological relevant QUIN concentrations have not been tested in vivo or in vitro to show deleterious effects on oligodendrocytes [90]. 3.2. Neurons Axonopathy and substantial neuronal loss are important pathological features during the chronic demyelinating phase of MS and in the TMEV model [91-93]. Accumulating evidence suggests a detrimental effect of excess calcium ions potentially due to ischemia and mitochondrial dam-

age/dysfunction responsible for axonopathy. Mitochondrial dysfunction results in limited energy supplies required for ATPdependent ion (Na+) pumps to guarantee stable ion gradients across neuronal membranes, which is the basal requirement for proper nerve conductivity [94]. Demyelinated "naked" axons are more vulnerable towards stressors including monocyte- and macrophagederived pro-inflammatory cytokines [19, 95, 96]. Glutamate- and QUIN-mediated excitotoxicity through monocytic cells is another possible cause of axonal damage [97]. Guillemin et al., (2007) characterized the KP in human neurons and demonstrated that cytokine stimulation can trigger the production of neuronal neuroprotective agents KYNA and picolinic acid [98]. However, levels of detectable neuronal KYNA were in the low micromolar range, which is unlikely sufficient to antagonize the excitotoxic effects of QUIN under pathological conditions. Schwarcz and co-workers demonstrated the neurotoxic potential of QUIN under pathophysiological concentrations in rat brain slice cultures [99, 100]. In a similar study, chronic exposure of QUIN to the rat striatum led to cognitive deficits [101].


In summary, QUIN can act as a harmful neurotoxin under pathophysiological concentrations in vivo and in vitro. However, the role of QUIN in the pathogenesis of MS is still undetermined. 4. INFLAMMATORY CYTOKINES REGULATE THE KP IDO and Tryptophan 2,3-dioxygenase (TDO) initiate tryptophan catabolism and are regulated by different mechanisms. Corticosteroids and glucagon induce TDO synthesis, while LPS- and endotoxin B-mediated cellular production of IFN- induce IDO synthesis [102]. IDO is expressed in a variety of immune cells including monocytes, dendritic cells, microglia and macrophages [34, 39, 103-105]. IDO expression is predominantly induced by IFN- and interferoninducers LPS, polyinosinic:polycytidylic acid (Poly(I:C)), viruses and endotoxin B [34, 102, 105-108]. Cytokines IFN- and IFN- also induce IDO and QUIN production in human macrophages, however to a lesser degree compared to IFN- [109, 110]. With IFN- as the major physiological agent in stimulation of IDO expression, other cytokines were identified that enhance effects of IFN- on IDO expression. Both IL-1 and TNF- enhance IFN- receptor expression in an NF-B-dependent manner, which in turn lowers the threshold for IFN--mediated IDO induction [111, 112]. Moreover, IFN- and TNF- synergistically stimulate IDO expression through STAT-1 activation and induction of IRF-1 expression in an NFB dependent manner [111-115]. In contrast to proinflammatory cytokines IFN- and TNF-, studies involving cytokines interleukin-4 (IL-4), interleukin-10 (IL10), -carboline and indole derivatives demonstrated inhibition of IDO in human monocytes and bone marrow derived dendritic cells [116, 117] and in mice [118]. Controversies exist regarding the outcome of IL-4 and IL-10 on IDO expression and IDO activation levels particularly when used in combination with other cytokines/LPS [116, 117, 119, 120]. This may reflect the complexity of molecular interactions, differences in cell types/tissues used or differences in experimental conditions. For example, IL-4 enhances IDO expression in mouse microglia [119] but not peripheral myeloid cells (monocytes) [117]. Intracerebral co-administration of IL4 together with LPS in rats potentiated the behavioral effects of LPS to induce an IDO-dependent depressive-like phenotype with respect to social exploration. However, IL-4 injection preceding LPS administration by 12 hours blocked the depressive-like behavioral phenotype, which emphasizes the complex interactions between IL-4 and LPS on IDO expression and activation in the CNS [121]. In addition to IFN--dependent IDO expression several studies performed in vivo and in vitro demonstrated an IFN--independent mechanism of LPS-mediated IDO expression leading to upregulation of IL-6 and TNF- levels combined with basically unchanged IFN- levels [37, 122]. Fujigaki et al., (2001) demonstrated in IFN antibody-treated WT mice and in IFN-- and TNF- genedisrupted mice that LPS-mediated IDO expression is TNF- but not IFN- dependent [122]. TNF-, IL-1 and IL-6 were able to synergistically induce IDO expression and activation after LPS stimulation in an IFN--independent mechanism [103]. In LPSstimulated primary microglia IDO expression was associated with undetectable IFN- mRNA levels [37, 123]. Wang et al., (2010) showed inhibition of IDO expression in LPS-stimulated mouse microglia using a c-Jun-N-terminal kinase (JNK) inhibitor [123]. Similarly, LPS-stimulated L-kynurenine synthesis was independent of IRF-1 or STAT-1, but blocked using NF-B- and p38 inhibitors [103, 122]. In summary, studies indicate IFN--dependent and IFN- independent mechanisms of LPS induced IDO expression. The IFN- independent mechanism present in monocytes and macrophage-like cells involves mitogen-activated protein (MAP) kinases like JNK and p38 as well as NF-B [103, 122, 123]. The toll-like-receptor 3 (TLR3) agonist Poly(I:C) stimulates astrocytic IDO transcription in


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an IFN--dependent manner that requires both NF-B and IRF-3 [124]. The regulation of enzymes in the KP other than IDO has not been studies extensively. However, some evidence suggests that LPS is able to induce expression of CNS resident KMO similarly to IDO [37, 38]. A study by Alberati-Giani et al., (1996) demonstrated IFN--mediated induction of KMO and kynureninase (KYNU) but not 3-HAO in a macrophage-derived cell line and in immortalized microglial cells [39]. A similar outcome on KMO and KYNU expression was shown using IL-1 as a stimulus in human hippocampal progenitor cells [40] Interestingly, proinflammatory cytokines IFN- and IL-1 or LPS do not stimulate induction of KATs involved in synthesis of the potential neuroprotective agent KYNA [37-40]. Instead, IL-1 down regulated levels of KAT I+II in human hippocampal progenitor cells [40]. Surprisingly, even though a majority of CNS derived KATs is expressed in astrocytes, this has not been the focus of many studies. Surprisingly, even though CNS derived KATs are mainly expressed in astrocytes, this has not been the focus of many studies. 5. THE KP AND MULTIPLE SCLEROSIS 5.1. Lessons from the EAE Model Activation of resident microglia and CNS infiltration of peripheral macrophages are common features of EAE and early stages of the human disease. A potential involvement of KP metabolites in EAE pathogenesis was demonstrated in IFN--treated human macrophages that produced neurotoxic levels of QUIN [125, 126]. Lewis rats intracerebrally inoculated using equal amounts of mycobacterium tuberculosis and myelin basic protein (MBP) developed EAE with increased QUIN levels in the spinal cord [127]. Increased spinal cord QUIN and 3-hydroxy-kynureninase levels were attributed to higher IDO and KMO expression levels and activity [128]. Administration of the KMO inhibitor Ro 61-8048 in EAE rats reduced QUIN and 3-hydroxykynurenine levels and enhanced Lkynurenine and KYNA levels in the spinal cord, but did not change the severity of symptoms in rodents [128]. This outcome argues against a QUIN-mediated neurotoxic effect or a KYNA-mediated neuroprotective effect in EAE. Different from their potential involvement in acute EAE pathogenesis, IDO and specific KP metabolites were implicated in limiting autoimmunity and promoting immune tolerance, which was suggested to partially account for the periodic remissions in MS and EAE. In EAE mice immunized with MBP or proteolipid protein (PLP), brain and spinal cord kynurenine/tryptophan ratios and microglial/macrophage-derived IDO levels increase simultaneously with EAE symptoms, while IFN- mRNA levels decrease [129, 130]. This data may suggest an IDO-mediated down-regulation of pathogenic IFN--producing T helper type1 (Th1) cells. In support of this hypothesis, inhibition of IDO activity in EAE mice using 1methyl-tryptophan resulted in earlier disease onset, significantly higher clinical scores, and more spinal cord inflammation [129, 130]. In EAE, IDO KO mice exhibit more severe clinical scores and enhanced Th1/Th17- like cytokine profiles compared to WT mice [131]. Thus, a model of IDO-mediated negative feedback in EAE is emerging. T-cell derived IFN- leads to IDO induction in microglia or infiltrating macrophages and dendritic cells, which in turn, limits the survival of pathogenic Th1 and Th17 T-cells and promotes the expansion of immunoregulatory T-cell phenotypes (i.e., Th2 and regulatory T-cells (Tregs)). Proliferation rates of pathogenic T-cells are suppressed by IDO induction and therefore consumption of free tryptophan levels in human macrophages and dendritic cells [32, 132, 133]. Thus, IFN-mediated IDO induction in macrophages and microglia may provide a hostile environment for pathogenic T-cells during inflammation. In addition, tryptophan metabolites 3-hydroxykynurenic acid,


N-(3,4-dimethoxycinnamoyl) anthranilic acid and 3-hydroxyanthranilic acid inhibit proliferation of myelin-specific Th1 and Th17 T-cells and improve symptoms in EAE mice [131, 134]. 3hydroxyanthranilic acid also enhances the expression of TGF- in dendritic cells (DCs), which stimulates the differentiation of naive T-cells into Tregs [131]. Thus, KP metabolism may suppress autoimmunity in EAE not only through local tryptophan depletion, but also through the influence of KP metabolites on DC-mediated Tcell differentiation. In brief, IFN--mediated IDO induction in macrophages and microglia as well as specific tryptophan metabolites suppress proliferation of pathogenic T-cells and improve symptoms in EAE mice either via tryptophan depletion or by directly targeting pathogenic T-cell populations. In addition, 3-hydroxyanthranilic acid stimulates DC-mediated TGF- production, which causes differentiation of naive T-cells into Tregs. A major disadvantage of the EAE model for the investigation of kynurenine pathway activity is the use of mycobacterium tuberculosis as the most important component of the Freund's adjuvant. Increased QUIN and 3-hydroxy-kynureninase levels in the spinal cord of EAE rats [127] were attributed to higher IDO and KMO expression levels and activity [128], which are likely due to the presence of bacterial antigens but not myelin protein MBP. This point of view is supported by the fact that mycobacterium tuberculosis but not MBP is a potent activator of IDO-1 in mice [135]. Moreover, increased IDO activity levels assayed via serum Trp/Kyn ratios are associated with poor prognosis in bacteremia and cancers. Pulmonary tuberculosis patients have significantly higher kynurenine levels associated with increased IDO activity and at the same time significantly lower tryptophan concentrations compared to control patients [136]. It is of note that EAE is not a rodent variant of MS but a chemically induced autoimmune-mediated disease model with different pathological features compared to the human disease. Thousands of drugs were tested in EAE during the last decades and it was possible to stop disease progression at any timepoint during the relapsing disease process [137]. Unfortunately, all tested drugs lacked efficacy to stop disease progression or prevent long-term disability in patients. In summary, the KP is activated by the sole presence of mycobacterium tuberculosis in the absence of myelin components in humans and rodents. Despite the unknown etiology for MS with potentially many different disease-causing triggers, bacterial toxins are not particularly prevalent in the human disease in high enough concentrations to be responsible for KP modulations. It is therefore essential to study MS-associated KP modulations in animal models lacking bacterial antigens unless proven otherwise. 5.2. Kynurenine Metabolites and MS Accumulating evidence suggests dysregulation of the KP in different psychiatric and neurodegenerative disorders including MS. Given the (proinflammatory) factors known to modulate KP activity (see above, section 3) MS patients with acute lesions but not during chronic stages of the disease with little or no CNS inflammation are likely to show involvement of tryptophan metabolites in disease progression. It was first reported in 1979 that plasma and CSF tryptophan levels are decreased in MS patients [138]. Confirming a possible involvement of the KP in MS pathology a number of subsequent studies showed changes in tryptophan metabolites in patients with RRMS. When compared to healthy controls KYNA levels in the CSF were decreased during the patients remission phase but elevated during acute clinical exacerbation [85, 139, 140]. A recent study by Mancuso et al., (2015) analyzed potential changes in IDO expression and activity in peripheral blood mononuclear cells (PBMCs) from RRMS patients [141]. Different from KYNA levels, IDO expression and activity remained unchanged between healthy controls (n = 15) and acute phase RRMS patients (n = 21) and between healthy controls (n = 15) and stable RRMS

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patients (n = 15). Glucocorticoid-induced disease remission resulted in significantly reduced IDO and IFN- expression levels and reduced IDO activity [141]. A more comprehensive study with 71 MS patients, 20 non-inflammatory neurological disease control patients and 13 patients with inflammatory neurological disease showed no absolute differences in CSF levels of tryptophan, kynurenine, KYNA or QUIN between MS patients and non-inflammatory neurological disease control patients [142]. However, stratification of patients into MS subtypes and different phases of the disease revealed increased QUIN concentrations and quinolinic acid/kynurenine ratios in RRMS patients during the relapsing phase, whereas patients with secondary progressive MS (SPMS) had lower tryptophan and KYNA levels [142]. Patients with primary progressive MS (PPMS) displayed increased levels of all investigated tryptophan metabolites, which was similar to control patients with inflammatory neurological disease. The results demonstrate that clinical disease activity and differences in disease courses are reflected by changes in KP metabolites [142]. 5.3. Potential KP Modulation Using Current and Future Therapeutics for MS Teriflunomide (Aubagio) is a pyrimidine synthesis inhibitor that inhibits T-cell and B-cell proliferation and prevents T-cell mediated cytokine production. Teriflunomide is an immunosuppressive drug approved for the treatment of rheumatoid arthritis. Teriflunomide was approved by the U.S. Food and Drug Administration (FDA) in September, 2012 for patients with RRMS. The efficacy of teriflunomide in MS was shown in a placebo-controlled phase III study (Teriflunomide Multiple Sclerosis Oral (TEMSO)), in which the drug reduced both the annualized relapse rate (by about 30% versus placebo) and disability progression [143, 144]. Teriflunomide inhibits the enzyme dihydroorotate dehydrogenase (DHODH) that converts dihydroorotate into orotate during pyrimidine de novo synthesis [145, 146]. Two of the most important intracellular signaling pathways, the mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-B) pathways, are inhibited by teriflunomide in cultured cells. Finally, teriflunomide inhibits type-2 cyclooxygenase (COX-2) activity, with an IC50 of 0.5–20 μM, and it has been suggested that teriflunomide acts as an inhibitor of the KP [147]. Laquinimod is a potential novel immunomodulatory treatment for RRMS. The quinoline-3-carboxamide analog laquinimod may combine neuroprotective and anti-inflammatory effects. The efficacy of laquinimod in MS was evaluated in a placebo-controlled phase III study (ALLEGRO (Placebo-controlled Trial of Oral Laquinimod for Multiple Sclerosis)), where it reduced disability progression with modest effect on patients relapse rates [148]. The mechanism of action for laquinimod is only partially known and includes modulation of antigen presentation in DCs and other APCs. Interestingly, laquinimod is structurally similar to KYNA and xanthurenic acid (Fig. 1). Laquinimod might influence the KP, thus driving kynurenine metabolism towards the formation of molecules that promote immune tolerance at the immunological synapse. This interesting hypothesis warrants in-depth investigation [147]. The most important questions to be addressed are 1. The enzyme specificity of laquinimod in the KP and 2. Whether and to what extent laquinimod-mediated effects on the KP ameliorate the human disease. 5.4. KP Activation and Depression in MS Patients Psychiatric disorders, especially depression, are frequent in patients with MS. Depression is commonly associated with decreased serotonin levels, suggesting low melatonin and N-acetylserotonin (NAS) levels which, in turn, may contribute to increased immuno-inflammatory pathways in depressed patients [149]. Recent data by Hesse et al., show changes in serotonin transporter levels in MS patients [150], suggesting that differences in serotonin


availability and subsequent NAS and melatonin production likely occur in both depressed and MS patients. Based on disease modulating effects of melatonin in MS with respect to myelination and remyelination, it was suggested that depression is not a sole psychiatric comorbidity of MS, but an integral part of the human disease [151, 152]. In support of this hypothesis, a metaanalysis by Chong et al., (1997) demonstrated a specific and frequent association between MS and depression, which was not just due to physical and cognitive disabilities present in any chronic disease [153]. Cytokines IL-1, IL-6, IL-18, TNF-, particularly IFN-, increase IDO expression and therefore depletion of serotonin, NAS and melatonin [2]. Kynurenine is able to cross the BBB and increases CNS tryptophan catabolite levels that contribute to depression, somatization and fatigue [154]. Aeinehband et al., (2015) evaluated the correlation between KP metabolite levels and neuropsychiatric symptoms in MS patients with active disease and short disease duration [142]. Depressed MS patients displayed higher KYNA/tryptophan and kynurenine/tryptophan ratios, which was mainly due to low tryptophan levels. The predictive value of this model was low however, and did not completely separate clinically depressed patients from non-depressed MS patients. Levels/pattern of KP metabolites in MS patients were not predictive for neurocognitive symptoms [142]. 5.5. Therapeutic Intervention in MS may Lead to KP Activation IFN- is a standard first line treatment in MS. Three clinical trials BENEFIT, BEYOND and a 16-year Long-Term Follow-up (LTF) trial provided safety data on IFN--1b for MS patients. Depression was among the most common adverse events observed in those three trials [155-157]. This raised the question whether IFN-treatment was at least in part responsible for depression in MS patients. In vitro studies in human monocyte-derived macrophages supported the hypothesis by showing that therapeutically relevant concentrations of IFN- are able to induce QUIN production and IDO expression [109]. In MS patients IFN--treatment resulted in significantly elevated plasma and serum concentrations of Lkynurenine and increased kynurenine/tryptophan ratios compared to baseline measurements, which is in line with IFN--mediated IDO induction [158, 159]. It is, however, still unclear whether IFN-mediated changes in KP metabolite levels are causative involved in the development of depressive symptoms in treated MS patients. It is also undetermined whether IFN--mediated IDO induction causes the low efficacy of IFN- treatment to improve MS symptoms [160]. The hypothesis originates from cell culture experiments in human macrophages where IFN--treatment resulted in increased levels of QUIN [109], combined with the fact that QUIN acts as a (weak) NMDAR agonist [160]. In brief, one of the adverse effects of IFN- treatment in MS patients is the development of depression. So far, there is no direct evidence demonstrating that therapeutic IFN- concentrations result in increased CNS QUIN levels in concentrations high enough to cause depressive symptoms in patients. CONCLUSION The KP is important in several fundamental biological processes including neuronal excitability, cell growth and division and the cellular antioxidant status. Pathological changes in the KP as the dominant non-protein route of the tryptophan metabolism likely have substantial effects on all tryptophan pathways that generate kynuramines, serotonin or melatonin. Results from clinical MS studies demonstrate a correlation between disease activity and changes in KP metabolites. The significance of these findings is however unclear. KP modulation is unlikely sufficient to trigger MS, to substantially exacerbate (quinolinic acid) or to ameliorate (KYNA) the disease course. Increased IDO activity/expression and QUIN concentrations in CSF may rather be a bystander phenomenon reflecting levels of inflammation or more precisely levels of activated macrophages and to a lower degree activated microglia in


7

the CNS. The authors are hesitant to believe in a beneficial outcome of KP antagonists for MS patients given the complexity of the nonprotein tryptophan metabolism with subsequent changes in nonblocked arms of the pathway. Immunomodulatory, antiinflammatory and immunosuppressive agents are central to the treatment of MS and effective during early stages of the disease. Unfortunately, all current treatments including those targeting the immune system are so far unable to stop disease progression from early to chronic disease stages or reverse disability in chronic MS patients. KP antagonists are expected to act as immunomodulatory/anti-inflammatory agents and may therefore benefit MS patients at early stages but not during chronic phases of the disease. It is likely that at least some immunomodulatory and/or immunosuppressive agents available for the treatment of MS already affect the KP without stopping or reversing long-term disability in patients. The authors would also like to caution the choice of the disease model used to identify potential interactions between KP changes and MS. While EAE is by far the most investigated and established MS model that led to the development of immunomodulatory and immunosuppressive agents, the involvement of bacterial toxins required to initiate EAE in studies mentioned (see above) is not acceptable to provide a proper foundation to investigate KP involvement in the human disease. Results obtained from these EAE studies may have misled the field into unsubstantiated clinical studies. Different from therapeutic approaches, CSF-isolated KP metabolites may be useful to determine levels of CNS inflammation and could broaden the spectrum of criteria for the subclassifications of MS. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS This work was supported by grants from the National Institutes of Health (R01 GM092993, R01 NS048357 and R21 NS073684), the National Science Foundation (CAREER Award), the Minnesota Partnership Award for Biotechnology and Medical Genomics, the National Multiple Sclerosis Society (CA1060A), and the Mayo Clinic Center for Clinical and Translational Science (CCaTS). We acknowledge support from the Applebaum, Hilton, Peterson and Sanford Foundations, the Moon and Marilyn Park Directorship Fund and the McNeilus family. REFERENCES [1] [2] [3] [4] [5]

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Received: November 1, 2015

Accepted: December 14, 2015


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