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Indolamine 2,3-dioxygenase is expressed in the CNS and down-regulates autoimmune inflammation

Erik Kwidzinski^*,1, Jörg Bunse^*, Orhan Aktas, Daniel Richter^*, Leman Mutlu^*, Frauke Zipp, Robert Nitsch^* and Ingo Bechmann^*

^* Center for Anatomy, Institute of Cell Biology and Neurobiology, Charité University Hospital Berlin; and
Institute of Neuroimmunology, Clinical and Experimental Neuroimmunology, Charité University Hospital Berlin, Germany

¹Correspondence: E-mail: erik.kwidzinski{at}charite.de

SPECIFIC AIMS

The mechanisms of spontaneous recovery from autoimmune central nervous system (CNS) inflammation in multiple sclerosis (MS) and its animal model autoimmune encephalomyelitis (EAE) remain poorly understood. One requirement for recovery from EAE is the control over self-reactive T cells from the CNS and the periphery. The tryptophan (trp)-degrading enzyme IDO is the first and rate-limiting enzyme of the kynurenine pathway. IDO activation reduces T cell proliferation and susceptibility to CD95L-induced apoptosis due to tryptophan depletion and the production of T cell-toxic tryptophan metabolites such as 3-hydrocyanthranilic acid and quinolic acid.

Therefore, the primary aim of our work was to determine whether IDO is expressed and active within the CNS during autoimmune inflammation in EAE-induced SJL mice. To assess time-dependent IDO activity in the relapsing-remitting disease course, we analyzed the relative IDO activity within the CNS and spleen by HPLC. The clinical relevance of IDO expression was analyzed by comparing the clinical disease severity of control mice and mice that were injected daily with the specific IDO inhibitor 1-methyl tryptophan (1-Mt). Finally, we wanted to identify the cells that express IDO within the CNS in vivo and in vitro by immunocytochemistry.

PRINCIPAL FINDINGS

1. IDO activity is increased within the CNS due to autoimmune inflammation
By HPLC measurement of trp and its metabolite kynurenine (kyn), relative IDO activity was determined as the kyn/trp ratio of tissue lysates. The animals were actively immunized by injection of PLP_139-151 in CFA at day 0. At each time point (Fig. 1 ), three animals were transcardially perfused with physiological NaCl solution, and tissue samples were prepared for HPLC analysis. Statistical significance was determined by Student’s t test, setting P<0.05 as significant. Within the spleen, relative IDO activity was significantly increased 4 days after immunization, but not at any later time point (Fig. 1A ). In the CNS, a significant increase in IDO activity was detectable at the acute phase in the three tested areas (Fig. 1B-D ). The kyn/trp ratio within the spinal cord peaked at the time of clinical remission and reached control levels at the chronic phase of the disease (Fig. 1B ), whereas in the rhombencephalon during this phase, the ratio did not completely return to control levels (Fig. 1C ). In general, the increase in IDO activity within the CNS correlated with inflammation (Fig. 1B-D ).

View larger version (22K): [in this window] [in a new window]   Figure 1. HPLC analysis of IDO activity durig EAE. Kyn/trp ratio as determined by HPLC in the spleen and different parts of the CNS after immunizing mice. Probes derived from nonimmunized animals (n=3) and animals killed at the following time points after immunization: 4 dai: 4 days after immunization (n=3), 8 dai: 8 days after immunization (n=3), acute phase: animals exhibiting a clinical score from 3 to 4 (11–13 dai, n=3), remission phase: animals with a clinical score from 0 to 0.5 (14–16 dai, n=3), relapse phase: animals exhibiting a clinical score from 2.5 to 4.0 (18–25 dai, n=3). Statistical significance was determined by t test, *P < 0,05 as significant. A) Within the spleen, IDO activity was increased at the preclinical phase of EAE (4 dai) and returned to control levels after onset of the disease. B–D) At 8 dai, IDO activity was enhanced only in the spinal cord and peaked at the remission phase, while no changes were evident in the rhombencephalon and diencephalon during the preclinical phase. However, in the acute phase of EAE, a several-fold increase in IDO activity was seen in all parts of the CNS. Only within the rhombencephalon was IDO activity still slightly increased during the relapse phase, while it reached almost control levels of untreated animals within the spinal cord and prosencephalon at this time point.

2. IDO inhibition exacerbates the clinical course of EAE
When IDO was inhibited by daily administration of the IDO inhibitor 1-Mt starting at the acute phase of the disease, the mean clinical severity of treated animals (n=30) increased significantly in comparison to control groups (n=30; P=0,038; ANOVA), supporting a role for IDO in controlling T cell-mediated inflammation.

3. IDO is expressed in leukocytic infiltrates within the spinal cord during EAE
In the acute phase of EAE (clinical score 3.5), IDO-positive cells were identified in the white matter of the spinal cord (Fig. 2 A, B). These IDO-expressing cells exhibited the same morphology as activated macrophages/microglia stained by Iba1 (Fig. 2C, D ). In non-immunized animals, IDO-positive cells were not identified (Fig. 2E, F ) and Iba1 exhibited the typical ramified morphology of non-activated microglia.

View larger version (119K): [in this window] [in a new window]   Figure 2. Expression of IDO within perivascular infiltrates (spinal cord). Spinal cord sections of an animal with a clinical score of 3.5 in the acute phase (A–D) and a nonimmunized control animal (E–H). Region delineated by squares in the left column is shown at higher magnification in the right column. A, B) IDO-positive cells exhibiting typical morphologic features of macrophages/activated microglia (arrows). C, D) HE staining combined with Iba1 staining reveals typical perivascular infiltrates in the white matter of the spinal cord. Besides ramified microglia (open arrows), Iba1-positive microglia/macrophages (arrows) can be detected exhibiting the same amoeboid morphology and size as IDO-positive cells (B). E, F) IDO-positive cells were not present in the spinal cord of healthy animals. G, H) Combined Iba1 and HE staining in these animals did not reveal perivascular infiltrates and amoeboid microglia. Iba1-positive cells within the neuropil exclusively exhibit ramified morphologies (arrows). Original magnification: left column x200; right column x400.

4. Primary microglia express IDO upon stimulation in cell culture
We stimulated primary microglia, astrocytes, and neurons in cell cultures with IFN-, TNF-, or both cytokines together. RT-PCR revealed IDO mRNA in microglia cultures and at a lower quantity in astrocytes after stimulation with IFN- or IFN- and TNF-. Staining of these cell cultures clearly identified IDO-expressing microglia after stimulation with IFN-. At the protein level, IDO was found in microglia, but not in astrocytes. The signal detected in astrocyte cultures at the mRNA level may thus derive from the few microglia which unavoidably contaminate the cultures. Neurons did not express IDO with or without cytokine stimulation.

CONCLUSIONS AND SIGNIFICANCE

Previous studies have shown the involvement of IDO expression in maintaining the immune privilege of the placenta by preventing T cell-mediated inflammation. Macrophages and dendritic cells inhibit T cell proliferation via IDO expression by trp depletion. Moreover, trp starvation sensitizes T cells to CD95L-induced apoptosis in vitro. Some trp degradation products that are produced along the kynurenine pathway also induce CD95L-independent apoptosis, primarily in Th1 cells. SJL EAE is a Th1-mediated autoimmune disease and recovery from CNS inflammation is mediated by central and peripheral deletion of these cells. We demonstrated that IDO expression is induced within the CNS in response to the infiltration by inflammatory leukocytes, and that the detected protein is active. Inhibition of IDO at the acute phase of the disease, the time point of maximal inflammation, reduced the clinical recovery of the animals. Our data support the view that the temporal and local increase in IDO expression in inflamed tissue is involved in reducing autoimmune CNS inflammation. Some trp degradation products can be toxic not only to T cells, but also to neurons. Therefore, time limitation of IDO activity within the CNS appears to be important to prevent the neuronal damage that occurs under long-term expression (e.g., during SIV/HIV encephalitopathy). In light of our results, we hypothesize that infiltrating Th1 cells themselves induce the expression of IDO within the inflamed CNS via IFN- secretion, thereby initializing a self-limiting negative feedback signal triggering their own down-modulation (Fig. 3 ). The dual role of IFN- became apparent in knockout models, where animals lacking the IFN- receptor did not recover from EAE. These and other data showed that proinflammatory cytokines may also exert anti-inflammatory effects in particular phases of a given disease. The protective and destructive effects of IDO expression within the CNS complicate the development of therapeutic strategies. However, a better understanding of the production and effects of trp metabolites may help to unravel mechanisms of the increasingly appreciated neuronal damage during MS/EAE.

View larger version (18K): [in this window] [in a new window]   Figure 3. Hypothesis of the self-limiting negative feedback mechanism that is induced by CNS-infiltrating T cells during the acute and remission phases of EAE. Self-reactive activated Th1 cells infiltrate the CNS and secrete IFN-, thereby inducing IDO expression in microglia/macrophages and CD95L expression in astrocytes. IDO activity decreases extracellular trp levels, which in turn increase CD95L-mediated apoptosis in Th1 cells. Due to IDO activity, T cell-toxic trp metabolites are synthesized along the kynurenine pathway, inducing T cell apoptosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3228fje;

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