The data indicate that administration of IFN-alpha leads to increased KYN in peripheral blood as well as increased KYN, QUIN and KA in the CNS, likely secondary to activation of IDO. Interestingly, although significant decreases in TRP and increases in the KYN/TRP ratio were found in peripheral blood, no IFN-alpha-induced changes in TRP concentrations were found in the brain as reflected in the CSF. Correlations between CSF markers of inflammation and KYN and its metabolites provide evidence that changes in KYN metabolism are linked to central cytokine responses, which together were associated with IFN-alpha-induced depression.
Previous studies have emphasized the role of TRP depletion in the mechanism by which cytokine-induced activation of IDO leads to behavioral alterations.35
The lack of changes in CSF TRP following IFN-alpha administration, despite decreases in peripheral blood TRP, indicates that the access of TRP to the brain relative to other amino acids which compete with TRP for transport across the blood brain barrier is unaffected. These findings are consistent with previous research demonstrating that the ratio of TRP to competing amino acids does not change during IFN-alpha therapy,30
and may provide an explanation for the lack of a correlation between plasma and CSF TRP concentrations in the current study. Moreover, no relationships were found between TRP and CNS inflammatory markers or CSF KYN and its metabolites or IFN-alpha-induced mood changes. Taken together, these findings indicate that although decreased peripheral blood TRP may reflect inflammation-induced activation of peripheral IDO pathways and their impact on the brain (as indicated by the inverse relationship between peripheral blood TRP and CSF KYN), the data do not support the notion that changes in peripheral blood or CNS availability of TRP are a primary mediator of IFN-alpha-induced mood disturbances.
In contrast to TRP, KYN was found to be significantly elevated in both the periphery and the CSF with a high correlation in KYN concentrations between these two tissue compartments. These data complement previous findings that CNS KYN is derived largely from TRP metabolism in peripheral tissues and is taken up into the brain at a significant rate by the large amino acid transporter.56
In contrast, KYN metabolites including QUIN and KA cross the blood brain barrier at relatively low rates through passive diffusion, suggesting that peripheral stores of QUIN and KA contribute minimally to concentrations in the brain.37,56
Thus, peripheral blood KYN may provide an accessible “window” into the CNS regarding activation of IDO pathways relevant to the brain and behavior in humans.
Additionally compatible with the notion that TRP may not be a primary mediator of behavioral change during IFN-alpha therapy, is that peripheral administration of KYN has been shown to induce depressive-like symptoms in rodents.25,34
Upon entry into the brain, KYN is metabolized into QUIN and KA, both of which are neuromodulators that can influence neurotransmission and ultimately behavior.37
KA inhibits the release of glutamate, which, by extension, may inhibit the release of dopamine (DA), whose release is regulated in part by glutamatergic activity.37,57,58
Indeed, intrastriatal administration of KA dramatically reduces extracellular DA in the rat striatum.58
Of relevance in this regard, decreases in the DA metabolite, homovanillic acid, have been reported in IFN-alpha-treated rhesus monkeys in association with depressive-like huddling behavior,12
and mice treated with IFN-alpha exhibit decreased whole brain DA in association with reduced locomotor activity.59
In contrast to KA, QUIN promotes glutamate release through activation of NMDA receptors.37
QUIN also induces oxidative stress, which in combination with glutamate release may contribute to CNS excitotoxicity.36,37,41,47
As mentioned previously, excess QUIN is associated with several neurodegenerative disorders with behavioral alterations,42-45
and in the current study was found to be correlated with depression both alone and in concert with inflammatory mediators (sTNFR2 and MCP-1) previously found to be elevated in the CSF of IFN-alpha-treated subjects.13
Of note, in mice treated with BCG, the only brain KYN pathway enzyme whose mRNA exhibited a significant increase compared to saline was 3-hydroxyanthranilic acid oxygenase, which converts 3-hydroxyanthranillic acid to QUIN.26
Previous investigators have suggested that the balance between QUIN, which activates glutamate release, and KA which inhibits glutamate release (through inhibition of the alpha 7 subunit of the nicotinic acetylcholine receptor), may determine the relative impact of inflammatory stimuli.30,36,47
Nevertheless, no differences in the ratio between CSF QUIN and KA were found between IFN-alpha-treated and control subjects. These data indicate that both enzymatic pathways leading to the production of these relevant KYN metabolites may be equally activated and can contribute to behavioral changes in the context of inflammatory stimuli such as IFN-alpha.
A high correlation was found between QUIN in the plasma and QUIN in the CSF. However, as noted above, direct access of QUIN to the brain has been shown to occur through passive diffusion at a relatively low rate.56
These data indicate that QUIN may be produced directly in the brain by cells that are represented both in the periphery and the CNS. One cell type that has been shown to actively produce QUIN is macrophages.40,60
In the context of peripheral immune stimulation, activated macrophages can be recruited to the brain by MCP-1 secreted by microglia following activation by peripherally released TNF-alpha.15
Previous studies have shown that IFN-alpha administration is associated with increased MCP-1 both in the peripheral blood and CSF, and IFN-alpha-induced TNF-alpha has been associated with IFN-alpha-induced depression.13,53
Upon activation, macrophages produce QUIN in amounts 20-fold greater than microglia.40
Thus, consistent with previous studies using pokeweed mitogen as a peripheral immune stimulus in gerbils,38
correlations between QUIN in the periphery and the brain may be related to activated macrophages, which are apparent in both peripheral and central compartments and can convert KYN to QUIN.40,60
Interestingly, QUIN has been found to induce the production of MCP-1,61
potentially creating a feed forward cascade in which QUIN leads to increased MCP-1 which in turn recruits more peripheral blood macrophages to the brain that can produce high quantities of QUIN. Blockade of macrophage access to the brain through inhibition of MCP-1 or its receptor CCL2R may be one strategy to prevent this cascade. Indeed, CCLR2 knock out mice were shown to exhibit marked decreases in infiltrating macrophages in a model of peripherally-induced liver inflammation.15
Finally, increased CSF KA suggests activation of astrocytes, which express the appropriate enzymatic machinery to produce KA,37
and like microglia can be stimulated to produce MCP-1.62
Of note, cells at the blood brain barrier can also synthesize KA as well as KYN upon activation.63
Although activation of IDO pathways has been implicated in depression and depressive-like behavior in the context of medical illnesses as well as the administration of inflammatory stimuli including IFN-alpha, LPS and BCG in laboratory animals and humans, data supporting a role for IDO activation in depression in otherwise medically healthy individuals is somewhat limited. Nevertheless, several studies have implicated a role for activation of IDO pathways in pre- and postpartum mood alterations,64-66
where inflammation may also be involved.66
In addition, at least one study found a correlation between KYN and symptoms of anxiety in patients with a variety of affective disorders including major depression, bipolar disorder and schizoaffective disorders.67
However, there have been several reports where no differences in KYN were found between depressed and non-depressed subjects.68-70
These data indicate that activation of IDO pathways and the generation of KYN and its metabolites may be relatively unique to inflammation-induced depression, and further investigation into IDO pathway activation in patients with depression should focus on depressed individuals with increased inflammatory markers.
Several limitations of the current study warrant consideration. Although treatment with IFN-alpha has been widely used as a model system for examining mechanisms by which chronic innate immune activation produces behavioral disturbances in humans, it is important to recognize that exogenously administered IFN-alpha may not be equivalent to endogenously produced IFN-alpha (or cytokines induced by endogenous IFN-alpha) in terms of either physiological or behavioral effects. Similarly, although the analysis of CSF provides a unique “window” into the CNS effects of chronic IFN-alpha exposure, findings in CSF cannot be directly compared to results from animal studies of IFN-alpha or other cytokines and cytokine-inducers where specific brain regions can be examined directly. As a result of practical and ethical issues related to randomizing patients who qualify for IFN-alpha/ribavirin therapy to “no treatment”, assignment to treatment and control conditions was dictated by clinical decisions occurring between patients and treating physicians. Although treatment and control groups were well matched on key variables at baseline, it is possible that differences between the groups in variables, such as motivation or expectation bias, which we did not measure, may have influenced outcomes. Additional limitations include the fact that the small sample size in the current study provided sufficient statistical power to detect only relatively large effect sizes. Moreover, correlations between CSF, blood and behavioral variables may have been compromised by the separation in time (of one week) between blood and behavioral variables and measures in the CSF. However, this time separation allowed blood and behavioral assessments to be uncontaminated by the stress of knowing one was about to receive a LP. IFN-alpha-treated subjects were also on two different preparations of pegylated IFN-alpha, although statistical analyses revealed significant differences between IFN-alpha treatment preparations only for CSF KYN, and multivariate analyses examining the association of CSF TRP, KYN, QUIN, KA and the immune variables with depression were conducted using IFN-alpha preparation as a covariate. Finally, all subjects in the current study were infected with HCV, which may have amplified observed effects through the interaction of viral infection with IFN-alpha administration. Consistent with current standards of care for the treatment of HCV, all subjects who received IFN-alpha also received the antiviral agent ribavirin, which may have contributed to study findings, especially in light of evidence that ribavirin may contribute to the depressive burden of HCV treatment.49
Of note, however, is the consistency between behavioral findings from the current study and studies in patients receiving IFN-alpha monotherapy for other therapeutic indications.71
Current findings are also consistent with those seen across a range of inflammatory stimuli (including IFN-alpha, LPS and BCG), indicating the relevance of IDO and KYN metabolic pathways in contributing to behavioral change during immune activation in both humans and laboratory animals.