Amino acid catabolism has been implicated in immunoregulatory mechanisms present in several diseases, including autoimmune disorders. Our aims were to assess expression and activity of enzymes involved in Trp and Arg catabolism, as well as to investigate amino acid catabolism effects on the immune system of multiple sclerosis (MS) patients. To this end, 40 MS patients, 30 healthy control subjects, and 30 patients with other inflammatory neurological diseases were studied. Expression and activity of enzymes involved in Trp and Arg catabolism (IDO1, IDO2, Trp 2,3-dioxygenase [TDO], arginase [ARG] 1, ARG2, inducible NO synthetase) were evaluated in PBMCs. Expression of general control nonrepressed 2 serine/threonine kinase and mammalian target of rapamycin (both molecules involved in sensing amino acid levels) was assessed in response to different stimuli modulating amino acid catabolism, as were cytokine secretion levels and regulatory T cell numbers. The results demonstrate that expression and activity of IDO1 and ARG1 were significantly reduced in MS patients compared with healthy control subjects and other inflammatory neurological diseases. PBMCs from MS patients stimulated with a TLR-9 agonist showed reduced expression of general control nonrepressed 2 serine/threonine kinase and increased expression of mammalian target of rapamycin, suggesting reduced amino acid catabolism in MS patients. Functionally, this reduction resulted in a decrease in regulatory T cells, with an increase in myelin basic protein–specific T cell proliferation and secretion of proinflammatory cytokines. In contrast, induction of IDO1 using CTLA-4 or a TLR-3 ligand dampened proinflammatory responses. Overall, these results highlight the importance of amino acid catabolism in the modulation of the immunological responses in MS patients. Molecules involved in these pathways warrant further exploration as potential new therapeutic targets in MS.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease that affects the CNS (1). Although MS immune pathogenesis is not yet fully understood, there is considerable evidence to suggest it is an autoimmune disease mediated mainly, but not exclusively, by Th1 and Th17 lymphocytes (2).

Like all other cells, immune cells require continuous access to amino acids to maintain basal metabolism and remain viable. When immune cells are activated by inflammatory and antigenic cues, their demand for amino acids rapidly increases. Although amino acid deprivation per se may attenuate immune response under some conditions, cells also possess amino acid–sensing pathways that trigger profound changes in cell metabolism in response to changes in amino acid levels detected at the cell surface of cytoplasm, particularly of translational activity. Such pathways provide mechanisms to control how immune cells respond under conditions of amino acid depletion (3).

An increasing body of data shows that cells express enzymes that degrade amino acids and modulate APC and lymphocyte function, and reveals amino acid pathways as critical in controlling immune cell gene expression, function, and survival. Basal amino acid catabolism may contribute to immune homeostasis preventing autoimmunity, whereas elevated amino acid catalytic activity may reinforce immune suppression, promoting pathogen persistence and chronic infection. Furthermore, accumulating evidence shows that several downstream amino acid metabolites are important biological mediators of autoimmune response regulation (4, 5).

Although any amino acid can be catabolized into a simpler compound or serve as substrate to build new molecules, not all cell types express the enzymes required to catalyze such changes in response to inflammation. For this reason, we chose to focus mainly on the immunological effects of Trp and Arg metabolism.

Trp is catabolized by Trp 2,3-dioxygenase (TDO) and two isoenzymes of IDO (IDO1, IDO2). Immune regulation by IDO is thought to be mediated either by Trp deprivation, which directly impacts immune cell survival and function, or by downstream Trp immunosuppressive metabolites (57). In contrast, three enzymes catabolize Arg: inducible NO synthetase (iNOS) and two arginase (ARG) isoforms (ARG1, ARG2). Genes encoding IDO, iNOS, and ARG are induced by inflammatory cues such as cytokines, a key feature that distinguishes them from enzymes that catabolize other amino acids (3).

Based on this background, we hypothesized that, in MS patients, amino acid catalytic activity may be decreased. This study evaluated pathways sensing and catabolizing Trp and Arg, and whether these pathways modulated immune cell function in MS patients.

Forty patients with a diagnosis of clinically definite relapsing-remitting MS (RRMS) according to Poser’s (8) and McDonald’s 2010 (9) criteria were studied during remission. No MS patients had received steroids during the 3 mo preceding blood draws. Sixty-two percent of patients were in treatment with disease-modifying drugs (IFN-β1a [64%] and glatiramer acetate [36%]). Patients were subjected to a comprehensive neurological examination every 3 mo, including physical assessment of disease activity and Expanded Disability Status Scale scoring (10). Thirty healthy subjects matched for age and sex served as control subjects. Thorough clinical and neurological examination, as well as standard chemical and hematological laboratory examinations, ruled out other underlying disorders. A second control group consisting of 30 patients suffering from other inflammatory neurological diseases (OIND) (15 cases of viral encephalitis, 10 cases of aseptic meningitis, and 5 patients with bacterial meningitis) was also included.

Demographic and clinical characteristics of all three groups are shown in Table I.

Table I.
Demographic and clinical characteristics of MS patients, patients with OIND, and HCs
CharacteristicsSex Ratio (F/M)Age (y)Expanded Disability Status Scale Score at EntryDuration of Disease (y)Annual Relapse Rate 2 y before Entry
MS (n = 40) 27:13 32 ± 7.9 2.3 ± 0.9 5.2 ± 2.8 0.6 ± 0.2 
OIND (n = 30) 20:10 32 ± 7.8 — — — 
HCs (n = 30) 20:10 32 ± 5.9 — — — 
CharacteristicsSex Ratio (F/M)Age (y)Expanded Disability Status Scale Score at EntryDuration of Disease (y)Annual Relapse Rate 2 y before Entry
MS (n = 40) 27:13 32 ± 7.9 2.3 ± 0.9 5.2 ± 2.8 0.6 ± 0.2 
OIND (n = 30) 20:10 32 ± 7.8 — — — 
HCs (n = 30) 20:10 32 ± 5.9 — — — 

—, not applicable; F, female; M, male.

Study protocol was approved by our Institutional Ethics Committee, and written informed consent was obtained from all participants.

T cell lines (TCLs) were expanded from PBMCs as previously described (11). In brief, PBMCs were isolated from heparinized blood by density-gradient centrifugation over Ficoll-PaquePLUS (Amersham Biosciences, Piscataway, NJ). After isolation, cells were resuspended in complete culture medium (RPMI 1640 medium, containing 100 U/ml penicillin, 100 μg/ml streptomycin; all from Sigma-Aldrich, St. Louis, MO) and 5% heat-inactivated autologous serum to a final concentration of 1 × 106 cells/ml. Five million cells were seeded in 25-cm2 flasks (Sigma-Aldrich) and stimulated with 10 μg/ml of either myelin basic protein (MBP)83–102 or MBP143–168 synthetic peptides. After 5–7 d, cells were recultured in fresh medium containing 50 U/ml recombinant human IL-2 (rhIL-2; R&D Systems, Minneapolis, MN) for an additional week. Weekly restimulation cycles were conducted with autologous adherent irradiated PBMCs (3000 rad) as APC, to which Ag was added, and expansion induced with rhIL-2 rich medium. After four cycles of restimulation and expansion, TCLs were evaluated using standard proliferation assays. Cutoff values for positive response were set at stimulation index >3. TCLs were used in the different assays 10 d after the last addition of Ag and feeder cells. All TCLs exhibited CD3+ CD4+CD8 TCRαβ+ cell-surface phenotype.

CD14+ cells, CD4+ T cells, CD8+ T cells, CD4+CD25+ regulatory T cells (Tregs), and B cells were purified by magnetic cell sorting using specific cell isolation kits (Miltenyi Biotec, Auburn, CA) and following manufacturer’s instructions. Purity of the different cell populations obtained using this method measured by flow cytometry was between 93 and 97%.

For Th cell polarization, human CD4+ T cells were purified from PBMCs as described earlier and cultured for 5 d with PHA (1 μg/ml; Sigma-Aldrich) and rhIL-2 (50 U/ml; R&D Systems) under neutral or polarizing conditions, namely Th1, IL-12 (2 ng/ml; BD Biosciences, San Diego, CA), plus anti–IL-4 mAb (100 ng/ml; BD Biosciences) and Th2, IL-4 (5 ng/ml; Sigma) plus anti–IL-12 (2 μg/ml; BD Biosciences). For Th-17 differentiation, naive CD4+ T cells were stimulated for 5 d with plate-bound anti-CD3 (5 μg/ml; American Type Culture Collection, Manassas, VA) and soluble anti-CD28 (1 μg/ml; BD Biosciences), in the presence of TGF-β (3 ng/ml), IL-6 (20 ng/ml), and IL-23 (20 ng/ml; all from R&D Systems), as well as neutralizing Abs anti–IL-4 (10 μg/ml) and anti–IFN-γ (10 μg/ml; both from BD Biosciences). Cultures were supplemented with rhIL-2 (50 U/ml; R&D Systems) on days 2 and 4. Purity of all three cell populations measured by flow cytometry was >94%.

For quantitative assessment of relative mRNA levels, total RNA was prepared using TRIzol LS reagent (Invitrogen, Carlsbad, CA) following manufacturer’s instructions. RNA was reverse transcribed using an M-MLV RT reverse transcription kit with random hexamer primers (Invitrogen). Relative levels of enzymes catabolizing Trp (IDO1, IDO2, and TDO) and Arg (iNOS, ARG1, and ARG2), as well as levels of main molecules involved in amino acid–sensing pathways (general control nonrepressed 2 serine/threonine kinase [GCN2], C/EBP homologous protein [CHOP], and mammalian target of rapamycin [mTOR]) were determined in PBMC mRNAs by real-time PCR using an ABI 7000 sequence detection system (Applied Biosystems, Foster City, CA). Values obtained were normalized to GAPDH amount. Primer sequences used were as follows: GAPDH: sense 5′-GAAGGTGAAGTCGGAGTC-3′, antisense 5′-GAAGATGGTGATGGGATTTC-3′; IDO1: sense 5′-TGCCAAATCCACAGGAAAAT-3′, antisense 5′-GTTTGCCAAGACACAGTCTG-3′; IDO2: sense 5′-CATAGCAAGGAAAGTGGTGAC-3′, antisense 5′-CTAAACACGTGGGTGAAGGATTG-3′; TDO: sense 5′-TGGGAACTAGATTCTGTTCG-3′, antisense 5′-TCGCTGCTGAAGTAAGAGCT-3′; ARG1: sense 5′-CCCTTTGCTGACATCCCTAA-3′, antisense 5′-GACTC CAAGATCAGGGTGGA-3′; ARG2: sense 5′-GACACTGCCCAGACCTTTGT-3′, antisense 5′-CGTTCCATGACCTTCTGGAT-3′; iNOS: sense 5′-CAGGAGGATGCCTCCGCAGCTGG-3′, antisense 5′-ATGATGAGGTAGTCGAGGAGGGTC-3′; GNC2: sense 5′-AAAAAGCTTACACGAAACATCAATTCA-3′, antisense 5′-GATAGATCTTTTGGTGACTGAATTATT-3′; CHOP: sense 5′-CATCACCACACCTGAAAGCA-3′, antisense 5′-TCAGCTGCCATCTCTGCA-3′; mTOR: sense 5′-GGATGTTCCAACGCAAGTTGA-3′, antisense 5′-GCAGAGGTTTTCATGGGATGTC-3′; Raptor: sense 5′-GAGAAGCTCTACAGCCTCCTCTCC-3′, antisense 5′-CCGTCCTCTCTGCAGAGTTGCC-3′; Rictor: sense 5′-CAACTGGGATGCTGTGAGGCATAG-3′, antisense 5′-GTACTAGTAGAGCTGCTGCCAAAC-3′.

Cells collected were centrifuged for 5 min at 1000 × g, 2–8°C. Supernatants were removed and cells resuspended in PBS until cell concentration reached 106−107 cells/ml. Cells were then washed with PBS three times, lysed, and 20 μg of cell extract used for IDO1, ARG1, GCN2, mTOR, Raptor, Rictor, AKT [pS473], and p70-S6K concentration measurement, using commercial ELISA kits according to manufacturer’s instructions. Sample concentrations were determined with standard curves after blank subtraction. ELISA kits for IDO, ARG1, RaptorR, GCN2, and mTOR were purchased from MyBioSource (San Diego, CA), whereas ELISA kits for measuring AKT [pS473], p70-S6K, and Rictor were acquired from R&D Systems, Thermo Fisher (Waltham, MA), and Abnova (Walnut, CA), respectively.

For IDO1 activity evaluation, cells isolated ex vivo and MBP-peptide–specific TCLs were cultured in complete culture medium containing 5% heat-inactivated autologous serum and supplemented with Trp. Seventy-two hours after culture, supernatants were collected and kynurenine concentrations measured with HPLC, using a reversed phase column as previously described (12). Kynurenine was detected using UV detection at 360-nm wavelength, and values referred to a calibration curve previously constructed with defined kynurenine concentrations. ARG1 activity was assessed using a commercially available ELISA kit, according to the manufacturer’s instructions (Abnova).

PBMCs were isolated from heparinized blood by density-gradient centrifugation over Ficoll-PaquePLUS, as previously described, washed in Ca2+ and Mg2+-free HBSS, and red cells were lysed by adding distilled water. After centrifugation, the pellet was suspended in 0.5 ml of 0.16 M potassium chloride, and cell suspension was lysed by applying three freezing and thawing cycles. The suspension was deproteinized by adding sulpho-5-salicylic acid (7 mg/ml suspension), and free amino acids were analyzed in supernatants after sulpho-5-salicylic acid precipitation by reversed phase HPLC (Beckman Instruments, Fullerton, CA), as previously described (13).

We assessed whether free amino acids regulated mTOR by measuring mTOR activation in amino acid–starved cells. For these experiments, PBMCs were initially cultured for 16 h in complete culture medium without serum, washed once with Dulbecco’s PBS containing 0.1 g/l CaCl2, then incubated in modified RPMI 1640 medium lacking amino acids (MyBioSource) and serum, and then supplemented with HEPES 25 mM and glucose 4.5 g/l during the time periods indicated. Refeeding of amino acids involved changing media to modified RPMI 1640 medium containing individualized amino acids as indicated. Both mTORC1 and mTORC2 activity were measured as phosphorylation of p70-S6 protein and Akt protein using ELISA, as previously described.

For peptide Ag-specific stimulation, MBP83–102– or MBP143–168–specific TCLs were cultured in complete culture medium containing 5% heat-inactivated autologous serum at a density of 5 × 104 cells per well, in the presence of 5 × 103 adherent irradiated autologous PBMCs, as APC source, and 10 μg/ml cognate peptide, under different conditions as indicated in each experiment, during 72 h. IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IFN-γ, and TNF-α were measured in the supernatant using commercially available ELISA kits, following the manufacturer’s instructions (R&D Systems). For nonspecific stimulation, peptide-specific T cells were cultured in the presence of 1 μg/ml immobilized anti-CD3 mAb (OKT3; American Type Culture Collection). Meanwhile, ex vivo–isolated CD4+ T cells were plated at a density of 5 × 104 cells per well and stimulated with 1 μg/ml PHA (Sigma-Aldrich).

CD4+CD25+FOXP3+ Treg percentage was evaluated by flow cytometry, using commercially available Treg staining kits and following the manufacturer’s instructions (eBioscience).

Mann–Whitney U test was used to compare clinical and demographic data, as well as immunological variable between patient and control groups, and ANOVA for comparison between multiple groups. The p values <0.05 were considered statistically significant.

As shown in Fig. 1A and 1B, IDO1 expression was significantly reduced in PBMCs from MS patients, compared with healthy control subjects (HCs) and patients with OIND, both at mRNA (0.85 ± 0.11 versus 2.42 ± 0.14 [p < 0.001] and 2.39 ± 0.12 [p < 0.001], respectively) and protein levels (10.3 ± 0.6 versus 41.5 ± 1.7 [p < 0.001] and 42.5 ± 2.6 μM [p < 0.001], respectively). Trp catabolism by IDO1 generates l-kynurenine, among other catabolites, and l-kynurenine levels are considered a surrogate marker of IDO1 activity (3). As illustrated in Fig. 1C, l-kynurenine levels were significantly decreased in MS patients compared with HCs and patients with OIND (4.31 ± 0.33 versus 11.5 ± 0.41 [p < 0.001] and 11.5 ± 0.42 μM [p < 0.001], respectively). ARG1 expression was also dampened in MS patients compared with HCs and patients with OIND both at the mRNA (0.79 ± 0.08 versus 2.08 ± 0.08 [p < 0.001] and 2.06 ± 0.07 [p < 0.001], respectively) and protein levels (8.2 ± 0.6 versus 45.8 ± 2.2 [p < 0.001] and 46.4 ± 1.8 μM [p < 0.001], respectively; Fig. 1D, 1E). Likewise, ARG1 activity was significantly reduced in MS patients compared with HCs and patients with OIND (5.43 ± 0.50 versus 12.4 ± 0.45 [p < 0.001] and 12.5 ± 0.42 U/l [p < 0.001], respectively) (Fig. 1F). In contrast, IDO2, TDO, iNOS, and ARG2 expression were similar in MS patients, patients with OIND, and HCs (data not shown).

FIGURE 1.

Expression and activity of Trp and Arg enzymes. Expression at both mRNA and protein levels, as well as activity of IDO1 (AC) and ARG1 (DF), were significantly reduced in patients with RRMS compared with patients with OIND and HCs. Each circle, triangle, or square represents values from a single individual. Horizontal lines indicate mean group values. (A and D) Data are expressed as IDO1 and ARG1 mRNA levels relative to GAPDH, and are shown as mean ± SEM of mRNA expression in PBMCs from 40 patients with RRMS, 30 HCs, and 30 patients with other inflammatory diseases.

FIGURE 1.

Expression and activity of Trp and Arg enzymes. Expression at both mRNA and protein levels, as well as activity of IDO1 (AC) and ARG1 (DF), were significantly reduced in patients with RRMS compared with patients with OIND and HCs. Each circle, triangle, or square represents values from a single individual. Horizontal lines indicate mean group values. (A and D) Data are expressed as IDO1 and ARG1 mRNA levels relative to GAPDH, and are shown as mean ± SEM of mRNA expression in PBMCs from 40 patients with RRMS, 30 HCs, and 30 patients with other inflammatory diseases.

Close modal

MS patients receiving treatment with disease-modifying drugs and untreated MS patients showed similar levels of expression of both IDO1 and ARG1, both at mRNA and protein levels. Likewise, enzymatic activity was similar in both MS patient groups (p = 0.15–0.93; Supplemental Fig. 1), indicating the decreased amino acid pathway activity observed was not linked to treatment effects. For the remaining studies, both groups were therefore pooled together.

To identify which cell types were involved in IDO1 and ARG1 expression and activity deficiency observed in PBMCs from MS patients, we purified CD14+ cells, CD4+ T cells, CD8+ T cells, CD4+CD25+ Tregs, and B cells from PBMCs of HCs and MS patients, and assessed IDO1 and ARG1 expression/activity in each population, as previously described. IDO1 and ARG1 expression and activity were detected in all cell populations tested (see Table II). When different cell populations were compared, the most significant IDO1 and ARG1 expression and activity deficiency were observed in CD14+ cells (p < 0.0001) from MS patients. No statistically significant differences were observed between CD4+, CD8+ T cells, or B cells isolated from HCs or MS patients. However, when Th1, Th2, Th17, and CD4+CD25+ Tregs were compared, CD4+CD25+ Treg and Th1 cells from MS patients were found to exhibit a significant decrease in IDO1 and ARG1 expression and activity compared with HCs (p = 0.002–0.005), whereas no significant differences between Th2 and Th17 cells were observed.

Table II.
IDO1 and ARG1 enzyme expression and activity in different cell populations
IDO1 mRNA ExpressionaIDO1 Protein Expression (μM)l-Kynurenine (μM)ARG1 mRNA ExpressionaARG1 Protein Expression (μM)ARG1 Activity (U/l)
HCs       
 CD14+ cells 7.74 ± 0.36 41.13 ± 0.83 18.19 ± 1.02 7.01 ± 0.44 41.66 + 1.93 21.22 ± 1.25 
 CD4+ T cells 1.58 ± 0.11 37.00 ± 2.57 10.80 ± 0.33 1.41 ± 0.12 38.33 + 2.85 10.70 ± 0.43 
 CD8+ T cells 1.02 ± 0.10 41.93 ± 1.70 10.31 ± 0.41 1.38 ± 0.16 32.06 + 3.24 10.93 ± 0.32 
 B cells 0.54 ± 0.05 40.40 ± 2.84 7.9 ± 0.38 0.52 ± 0.06 36.73 + 2.68 6.60 ± 0.39 
 Tregs 1.88 ± 0.15 37.93 ± 2.48 11.92 ± 0.36 1.91 ± 0.17 32.72 + 2.43 12.35 ± 0.41 
 Th1 1.60 ± 0.14 37.66 ± 3.00 11.06 ± 0.30 1.94 ± 0.20 38.33 + 3.17 13.40 ± 0.38 
 Th2 0.81 ± 0.07 42.73 ± 1.47 9.3 ± 0.70 0.91 ± 0.07 43.06 + 2.76 10.10 ± 0.77 
 Th17  0.90 ± 0.09 41.2 ± 1.50 8.91 ± 0.66 0.93 ± 0.06 49.08 + 2.70 9.89 ± 0.89 
MS patients       
 CD14+ cells 1.50 ± 0.16* 9.23 ± 0.84* 2.72 ± 0.60* 1.21 ± 0.18* 10.40 ± 1.12* 2.9 ± 0.70* 
 CD4+ T cells 1.72 ± 0.14 32.93 ± 2.70 10.16 ± 0.28 1.32 ± 0.15 35.40 ± 3.26 10.66 ± 0.40 
 CD8+ T cells 1.1 ± 0.09 35.06 ± 2.45 10.31 ± 0.32 1.35 ± 0.16 30.33 ± 3.07 10.89 ± 0.55 
 B cells 0.55 ± 0.05 37.40 ± 2.39 7.50 ± 0.20 0.50 ± 0.06 35.00 ± 3.37 6.65 ± 0.42 
 Tregs 1.06 ± 0.12** 21.86 ± 3.20** 10.41 ± 0.38*** 1.04 ± 0.11** 19.73 ± 1.35** 10.20 ± 0.44*** 
 Th1 1.08 ± 0.14** 20.33 ± 2.64** 9.28 ± 0.30*** 1.01 ± 0.10** 20.46 ± 1.54** 10.33 ± 0.48*** 
 Th2 0.77 ± 0.09 40.80 ± 2.17 8.90 ± 0.51 0.88 ± 0.08 39.93 ± 2.73 10.02 ± 0.66 
 Th17 0.84 ± 0.08 37.8 ± 1.46 8.62 ± 0.55 0.89 ± 0.10 47.00 ± 3.04 9.35 ± 0.69 
IDO1 mRNA ExpressionaIDO1 Protein Expression (μM)l-Kynurenine (μM)ARG1 mRNA ExpressionaARG1 Protein Expression (μM)ARG1 Activity (U/l)
HCs       
 CD14+ cells 7.74 ± 0.36 41.13 ± 0.83 18.19 ± 1.02 7.01 ± 0.44 41.66 + 1.93 21.22 ± 1.25 
 CD4+ T cells 1.58 ± 0.11 37.00 ± 2.57 10.80 ± 0.33 1.41 ± 0.12 38.33 + 2.85 10.70 ± 0.43 
 CD8+ T cells 1.02 ± 0.10 41.93 ± 1.70 10.31 ± 0.41 1.38 ± 0.16 32.06 + 3.24 10.93 ± 0.32 
 B cells 0.54 ± 0.05 40.40 ± 2.84 7.9 ± 0.38 0.52 ± 0.06 36.73 + 2.68 6.60 ± 0.39 
 Tregs 1.88 ± 0.15 37.93 ± 2.48 11.92 ± 0.36 1.91 ± 0.17 32.72 + 2.43 12.35 ± 0.41 
 Th1 1.60 ± 0.14 37.66 ± 3.00 11.06 ± 0.30 1.94 ± 0.20 38.33 + 3.17 13.40 ± 0.38 
 Th2 0.81 ± 0.07 42.73 ± 1.47 9.3 ± 0.70 0.91 ± 0.07 43.06 + 2.76 10.10 ± 0.77 
 Th17  0.90 ± 0.09 41.2 ± 1.50 8.91 ± 0.66 0.93 ± 0.06 49.08 + 2.70 9.89 ± 0.89 
MS patients       
 CD14+ cells 1.50 ± 0.16* 9.23 ± 0.84* 2.72 ± 0.60* 1.21 ± 0.18* 10.40 ± 1.12* 2.9 ± 0.70* 
 CD4+ T cells 1.72 ± 0.14 32.93 ± 2.70 10.16 ± 0.28 1.32 ± 0.15 35.40 ± 3.26 10.66 ± 0.40 
 CD8+ T cells 1.1 ± 0.09 35.06 ± 2.45 10.31 ± 0.32 1.35 ± 0.16 30.33 ± 3.07 10.89 ± 0.55 
 B cells 0.55 ± 0.05 37.40 ± 2.39 7.50 ± 0.20 0.50 ± 0.06 35.00 ± 3.37 6.65 ± 0.42 
 Tregs 1.06 ± 0.12** 21.86 ± 3.20** 10.41 ± 0.38*** 1.04 ± 0.11** 19.73 ± 1.35** 10.20 ± 0.44*** 
 Th1 1.08 ± 0.14** 20.33 ± 2.64** 9.28 ± 0.30*** 1.01 ± 0.10** 20.46 ± 1.54** 10.33 ± 0.48*** 
 Th2 0.77 ± 0.09 40.80 ± 2.17 8.90 ± 0.51 0.88 ± 0.08 39.93 ± 2.73 10.02 ± 0.66 
 Th17 0.84 ± 0.08 37.8 ± 1.46 8.62 ± 0.55 0.89 ± 0.10 47.00 ± 3.04 9.35 ± 0.69 

Values are expressed as mean values ± SEM from 15 control subjects and 15 MS patients.

a

Data are expressed as IDO1 and ARG1 mRNA levels relative to GAPDH.

*

p < 0.001, **p = 0.002, ***p = 0.005.

Cells sense amino acids through at least two distinct pathways involving serine/threonine kinases: the GCN2 pathway and the mTOR pathway. Both are interconnected and cooperate to assess nutrient deficiency (GCN2) and sufficiency (mTOR). This means that an increase in amino acid catabolism (e.g., through an activation of IDO1 by inflammatory cytokines) results in a depletion of amino acids, which subsequently activates GCN2 and inactivates mTOR (3). PBMCs from RRMS patients stimulated with the TLR-9 agonist CpG resulted in low expression of GCN2 at both mRNA and protein levels compared with levels observed in HCs and patients with OIND, as well as of CHOP, a well-established marker of GCN2 activation, (p = 0.001 to <0.0001; Fig. 2A–C). In contrast, both mTOR mRNA and protein levels were significantly increased in RRMS patients compared with HCs and patients with OIND using a similar stimulus (p < 0.001; Fig. 2D, 2E). Overall, these results indicate the presence of high levels of Trp and Arg, and low amino acid catabolism, in activated PBMCs from RRMS patients.

FIGURE 2.

Expression of GCN2, CHOP, and mTOR. Stimulation of PBMCs from MS patients using the TLR9 agonist CpG showed lower expression of GCN2 at both mRNA (A) and protein (B) levels, as well as lower CHOP mRNA expression (C), compared with levels observed in patients with OIND and HCs. By contrast, mTOR levels at both mRNA (D) and protein (E) levels were significantly increased in RRMS patients compared with both other cohorts. Raptor mRNA (F) and protein (G) expression, as well as mTORC1 activity measured according to p70-S6 protein phosphorylation (H), were significantly increased in RRMS patients compared with patients with OIND and HCs. By contrast, Rictor mRNA (I) and protein (J) expression, as well as mTORC2 activity represented as Akt protein phosphorylation (K), were similar in all three groups. (A, C, D, F, and I) Data are expressed as specific mRNA levels relative to GAPDH. Values are reported as mean values ± SEM from 30 RRMS patients, 30 HCs, and 30 patients with other inflammatory diseases.

FIGURE 2.

Expression of GCN2, CHOP, and mTOR. Stimulation of PBMCs from MS patients using the TLR9 agonist CpG showed lower expression of GCN2 at both mRNA (A) and protein (B) levels, as well as lower CHOP mRNA expression (C), compared with levels observed in patients with OIND and HCs. By contrast, mTOR levels at both mRNA (D) and protein (E) levels were significantly increased in RRMS patients compared with both other cohorts. Raptor mRNA (F) and protein (G) expression, as well as mTORC1 activity measured according to p70-S6 protein phosphorylation (H), were significantly increased in RRMS patients compared with patients with OIND and HCs. By contrast, Rictor mRNA (I) and protein (J) expression, as well as mTORC2 activity represented as Akt protein phosphorylation (K), were similar in all three groups. (A, C, D, F, and I) Data are expressed as specific mRNA levels relative to GAPDH. Values are reported as mean values ± SEM from 30 RRMS patients, 30 HCs, and 30 patients with other inflammatory diseases.

Close modal

The kinase mTOR is an evolutionarily conserved member of the phosphatidylinositol-3-OH kinase–related family. It forms the core of two distinct signaling complexes whose activation is regulated differentially (14). The first complex, mTORC1, contains the scaffolding protein Raptor and subunits mLST8, PRAS40, and Deptor (15). It promotes phosphorylation of the translational regulators p70-S6K, which controls protein translation via ribosome biosynthesis, and 4E-BP1 kinase, which regulates mRNA translation (16). The second mTOR signaling complex, mTORC2, contains the scaffolding protein Rictor and subunits mSINI, mLST8, and Protor (15). mTORC2 activation leads to phosphorylation of Akt at Ser473, linked to cytoskeletal rearrangement control and spatial aspects of cell growth, in particular, cell polarity (17). Therefore, we next investigated the differential role of mTORC1 and mTORC2 on amino acid catabolism regulation in PBMCs from RRMS patients, by measuring the expression of Raptor and Rictor. As shown in Fig. 2F and 2G, Raptor mRNA and protein expression were significantly higher in PBMCs from RRMS patients compared with HCs and patients with OIND (p = 0.002 and p < 0.0001). These findings were reflected in a significant increase in mTORC1 activity, measured as phosphorylation of the p70-S6K protein (Fig. 2H). In contrast, Rictor mRNA and protein expression, as well as mTORC2 activity assessed as phosphorylation at Ser473 of Akt protein, were similar in RRMS patients, patients with OIND, and HCs (Fig. 2I–K).

Intact GCN2 genes in CD4+ T cells are essential for naive CD4+ T cells to undergo conversion into CD4+CD25+FOXP3+ Tregs, whereas mTOR is a crucial negative regulator of FOXP3 induction inhibiting Treg de novo differentiation. In addition, CD4+CD25+FOXP3+ Tregs lacking GCN2 genes not only fail to acquire regulatory phenotypes, but actually undergo functional reprogramming to acquire helper/effector functions, as indicated by the expression of proinflammatory cytokines (3). In these experiments, high levels of Trp and Arg were associated with significant decrease in both GCN2 mRNA and protein expression (Fig. 3A, 3B), as well as a robust increase in both mTOR mRNA and protein expression (Fig. 3C, 3D). This in turn resulted in: 1) significant decrease in CD4+CD25+FOXP3+ Treg numbers (Fig. 3E); 2) increased proliferation of MBP-peptide–specific TCLs (Fig. 3F); and 3) robust increase in IL-2, IL-6, IL-17 IFN-γ, and TNF-α production (Fig. 3G). T cell cytokine production was assessed using both ex vivo–isolated CD4+ T cells and MBP-peptide–specific TCLs, stimulated with either PHA, the specific Ag, or immobilized anti-CD3 mAb. Similar patterns of cytokine secretion were seen for both MBP-peptide–specific TCLs and when ex vivo–isolated CD4+ T cells were stimulated either with PHA or immobilized anti-CD3 mAb.

FIGURE 3.

GCN2 and mTOR pathways regulate immune cell behavior. High levels of Trp (0.5 g/l) resulted in decreased GCN2 mRNA (A) and protein (B) expression, and increased mTOR mRNA (C) and protein (D) expression, which, in turn, were associated with robust decrease in CD4+CD25+FOXP3+ Treg percentage (E), significant increase in MBP-peptide–specific TCL proliferation (F), and significant increase in IL-2, IL-6, IL-17, IFN-γ, and TNF-α production by PBMCs (G). Overexpression of GCN2 and inhibition of mTOR using siRNA or culture media containing low levels of Trp (0.0005 g/l) countered these effects (E–G). Data represent mean values ± SEM from 15 individual experiments, using TCLs specific for MBP83–102 and MBP143–168 isolated from 14 different RRMS patients. Similar results were observed regardless of which Ag was used. In all cases, cell viability was normal when assessed by trypan blue dye exclusion.

FIGURE 3.

GCN2 and mTOR pathways regulate immune cell behavior. High levels of Trp (0.5 g/l) resulted in decreased GCN2 mRNA (A) and protein (B) expression, and increased mTOR mRNA (C) and protein (D) expression, which, in turn, were associated with robust decrease in CD4+CD25+FOXP3+ Treg percentage (E), significant increase in MBP-peptide–specific TCL proliferation (F), and significant increase in IL-2, IL-6, IL-17, IFN-γ, and TNF-α production by PBMCs (G). Overexpression of GCN2 and inhibition of mTOR using siRNA or culture media containing low levels of Trp (0.0005 g/l) countered these effects (E–G). Data represent mean values ± SEM from 15 individual experiments, using TCLs specific for MBP83–102 and MBP143–168 isolated from 14 different RRMS patients. Similar results were observed regardless of which Ag was used. In all cases, cell viability was normal when assessed by trypan blue dye exclusion.

Close modal

In contrast, low levels of Trp in culture media induced a significant increase in GCN2 mRNA expression, as well as decreased mTOR mRNA expression (Fig. 3A, 3C). These changes were associated with a significant increase in CD4+CD25+FOXP3+ Treg percentages, a decrease in proliferation of MBP-peptide–specific TCLs, and a robust decrease in production of IL-2, IL-6, IL-17 IFN-γ, and TNF-α (Fig. 3E–G). Similar results were observed when T cells overexpressed GCN2 or when mTOR was blocked using siRNA (Fig. 3E–G).

It has long been appreciated that amino acid levels are crucial for mTOR activation and represent one of the most preserved growth signals in this pathway (18). However, whether all amino acids, one in particular, or products thereof are being sensed remains unknown. Therefore, we next investigated intracellular concentrations of free amino acids in PBMCs from MS patients and HCs, and the relationship between these concentrations and mTOR activation. As shown in Table III, intracellular concentrations of Trp, Arg, Leu, Ile, and glutamine (Glm) were significantly higher in MS patients than in HCs, indicating that in addition to Trp and Arg, catabolism of additional amino acids was also impaired. To investigate the role of these amino acids in mTOR activation, cells were incubated under amino acid starvation conditions. Withdrawal of amino acids from the medium (1 h) significantly reduced phosphorylation of the mTORC1 substrate p70-S6K protein. This inhibition of mTOR activity is reversible. PBMCs refed with optimal concentrations of Trp, Arg, Leu, Ile, or Glm restored p70-S6K protein phosphorylation within 30 min. However, no significant changes were observed in AKT phosphorylation, suggesting that, under these conditions, there is little or no effect on mTORC2 activity (Fig. 4A–F). Importantly, amino acids that showed normal intracellular levels (e.g., valine and methionine) were unable to stimulate mTORC1 activity (Fig. 4G, 4H), indicating that effects of individual amino acids are not equal.

Table III.
Intracellular free amino acids in PBMCs from MS patients and HCs (μmol/l)
Amino acidsMS PatientsHCsp Value
Histidine 225.8 ± 101.3 230.2 ± 120.1 NS 
Ile 216.7 ± 20.8 160.3 ± 16.7 0.03 
Leu 472.0 ± 41.2 280.1 ± 27.8 0.001 
Lysine 205.4 ± 65.4 177.2 ± 86.2 NS 
Methionine 88.2 ± 23.3 73.4 ± 21.1 NS 
Phenylalanine 99.9 ± 29.4 108.6 ± 31.1 NS 
Threonine 220.3 ± 36.8 187.2 ± 42.2 NS 
Tyrosine 121.3 ± 38.5 109.3 ± 31.4 NS 
Valine 206.5 ± 66.3 201.1 ± 54.3 NS 
Arg 329.9 ± 38.6 156.9 ± 38.6 0.0005 
Asparagine 93.6 ± 32.1 73.3 ± 18.4 NS 
Aspartic acid 295.4 ± 69.3 288.5 ± 59.6 NS 
Citrulline 28.4 ± 10.1 21.2 ± 6.1 NS 
Glutamic acid 1908.4 ± 320.7 1730.7 ± 238.2 NS 
Glm 873.5 ± 107.0 500.6 ± 55.9 0.01 
Glycine 825.4 ± 155.3 765.3 ± 128.8 NS 
Ornithine 449 ± 59.6 418.9 ± 55.3 NS 
Serine 438.7 ± 78.9 399.3 ± 53.2 NS 
Trp 89.4 ± 8.5 52.3 ± 5.9 0.0007 
Alanine 188.1 ± 59.6 158.4 ± 45.6 NS 
Amino acidsMS PatientsHCsp Value
Histidine 225.8 ± 101.3 230.2 ± 120.1 NS 
Ile 216.7 ± 20.8 160.3 ± 16.7 0.03 
Leu 472.0 ± 41.2 280.1 ± 27.8 0.001 
Lysine 205.4 ± 65.4 177.2 ± 86.2 NS 
Methionine 88.2 ± 23.3 73.4 ± 21.1 NS 
Phenylalanine 99.9 ± 29.4 108.6 ± 31.1 NS 
Threonine 220.3 ± 36.8 187.2 ± 42.2 NS 
Tyrosine 121.3 ± 38.5 109.3 ± 31.4 NS 
Valine 206.5 ± 66.3 201.1 ± 54.3 NS 
Arg 329.9 ± 38.6 156.9 ± 38.6 0.0005 
Asparagine 93.6 ± 32.1 73.3 ± 18.4 NS 
Aspartic acid 295.4 ± 69.3 288.5 ± 59.6 NS 
Citrulline 28.4 ± 10.1 21.2 ± 6.1 NS 
Glutamic acid 1908.4 ± 320.7 1730.7 ± 238.2 NS 
Glm 873.5 ± 107.0 500.6 ± 55.9 0.01 
Glycine 825.4 ± 155.3 765.3 ± 128.8 NS 
Ornithine 449 ± 59.6 418.9 ± 55.3 NS 
Serine 438.7 ± 78.9 399.3 ± 53.2 NS 
Trp 89.4 ± 8.5 52.3 ± 5.9 0.0007 
Alanine 188.1 ± 59.6 158.4 ± 45.6 NS 

Values are expressed as mean values ± SEM from 15 control subjects and 15 MS patients.

FIGURE 4.

Effects of amino acid withdrawal on p70-S6 and AKT [pS473] activity. PBMCs from MS patients were starved for all amino acids during 1 h and then refed for 30 min with culture medium (A), l-Trp (B), l-Arg (C), l-Leu (D), l-Ile (E), l-Glm (F), l-Val (G), and l-Met (H). Final concentrations of each amino acid were as follows: l-Arg: 300 μM, l-Glm: 850 μM, l-Leu: 450 μM, l-Ile: 200 μM, l-Met: 90 μM, l-Trp: 90 μM, and l-Val: 200 μM. Data represent mean values ± SEM from five individual experiments.

FIGURE 4.

Effects of amino acid withdrawal on p70-S6 and AKT [pS473] activity. PBMCs from MS patients were starved for all amino acids during 1 h and then refed for 30 min with culture medium (A), l-Trp (B), l-Arg (C), l-Leu (D), l-Ile (E), l-Glm (F), l-Val (G), and l-Met (H). Final concentrations of each amino acid were as follows: l-Arg: 300 μM, l-Glm: 850 μM, l-Leu: 450 μM, l-Ile: 200 μM, l-Met: 90 μM, l-Trp: 90 μM, and l-Val: 200 μM. Data represent mean values ± SEM from five individual experiments.

Close modal

It has been shown that stimulation of costimulatory molecules (CTLA-4 and CD80/86) or TLR upregulates IDO1 in inflammatory sites, through IFN-α–dependent pathways (19). Treatment of MBP-peptide–specific T cells with TLR3 agonist polyinosinic-polycytidylic acid significantly increased IDO expression (Fig. 5A). The effect was associated with a significant reduction in IL-2, IL-6, IL-17, TNF-α, and IFN-γ and a robust increase in TGF-β levels (Fig. 5B). Likewise, proliferation of MBP83–102 and MBP143–168 peptide-specific T cells was significantly inhibited (Fig. 5C). Similar results were observed during CTLA-4 treatment (data not shown).

FIGURE 5.

Induction of IDO1 dampens proinflammatory responses. PBMCs from 30 RRMS patients stimulated with the TLR-3 agonist polyinosinic-polycytidylic acid induced a robust expression of IDO mRNA (A). IDO1 induction was associated with decreased IL-2, IL-6, IL-17, TNF-α, and IFN-γ production and a robust increase in TGF-β (B). Likewise, treatment with TLR3 agonist was associated with a significant decrease in MBP-peptide–specific TCL proliferation (C). (B and C) Data represent mean values ± SEM from 14 individual experiments using TCLs specific for both MBP83–102 and MBP143–168, isolated from 12 RRMS patients. Similar results were observed regardless of the Ag used.

FIGURE 5.

Induction of IDO1 dampens proinflammatory responses. PBMCs from 30 RRMS patients stimulated with the TLR-3 agonist polyinosinic-polycytidylic acid induced a robust expression of IDO mRNA (A). IDO1 induction was associated with decreased IL-2, IL-6, IL-17, TNF-α, and IFN-γ production and a robust increase in TGF-β (B). Likewise, treatment with TLR3 agonist was associated with a significant decrease in MBP-peptide–specific TCL proliferation (C). (B and C) Data represent mean values ± SEM from 14 individual experiments using TCLs specific for both MBP83–102 and MBP143–168, isolated from 12 RRMS patients. Similar results were observed regardless of the Ag used.

Close modal

As mentioned previously, one mechanism of immune suppression elicited by IDO results from a direct effect of Trp metabolites (kynurenine and 3-hydroxyantranilic [3-HAA]) on target cells. Treatment of PBMCs with 3-HAA significantly increased CD4+CD25+FOXP3+ Treg percentage (Fig. 6A), reduced IL-6, IL-17, and IFN-γ levels, and increased TGF-β production (Fig. 6B), as well as inhibited MBP-peptide–specific TCL proliferation (Fig. 6C, 6D). To dissect potential mechanisms underlying 3-HAA–mediated upregulation of CD4+CD25+FOXP3+ Tregs, we stimulated PBMCs with anti-CD3/anti-CD28 mAb in the presence of IL-6 or neutralizing anti–TGF-β mAb. Both IL-6 and TGF-β are critical in controlling the differentiation of CD4+ T cells into Tregs or Th17 lineage. Addition of IL-6 (10–50 ng/ml) to anti-CD3/anti-CD28–activated PBMCs without 3-HAA treatment did not affect CD4+CD25+FOXP3+ Treg numbers, whereas addition of neutralizing anti–TGF-β mAb to culture during 3-HAA priming significantly reduced CD4+CD25+FOXP3+ Treg numbers (Fig. 6E). These results suggest 3-HAA–induced TGF-β plays a critical role in CD4+CD25+FOXP3+ Treg induction.

FIGURE 6.

Downstream Trp metabolite 3-HAA inhibits proinflammatory responses. PBMCs were stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml), in the presence of 3-HAA (200 μM) or vehicle control. 3-HAA promoted a significant increase in CD4+CD25+FOXP3+ Treg percentages (A), significantly reduced IL-6, IFN-γ, and IL-17 production, and increased TGF-β levels (B), as well as markedly suppressed MBP83–102- and MBP143–168-peptide–specific TCL proliferation, in a dose-dependent manner (C and D). Addition of IL-6 (20 ng/ml) did not affect CD4+CD25+FOXP3+ Treg numbers. In contrast, addition of neutralizing anti–TGF-β mAb (30 μg/ml) significantly decreased 3-HAA–induced CD4+CD25+FOXP3+ Treg numbers (E). Values are reported as mean values ± SEM from 12 individual experiments.

FIGURE 6.

Downstream Trp metabolite 3-HAA inhibits proinflammatory responses. PBMCs were stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml), in the presence of 3-HAA (200 μM) or vehicle control. 3-HAA promoted a significant increase in CD4+CD25+FOXP3+ Treg percentages (A), significantly reduced IL-6, IFN-γ, and IL-17 production, and increased TGF-β levels (B), as well as markedly suppressed MBP83–102- and MBP143–168-peptide–specific TCL proliferation, in a dose-dependent manner (C and D). Addition of IL-6 (20 ng/ml) did not affect CD4+CD25+FOXP3+ Treg numbers. In contrast, addition of neutralizing anti–TGF-β mAb (30 μg/ml) significantly decreased 3-HAA–induced CD4+CD25+FOXP3+ Treg numbers (E). Values are reported as mean values ± SEM from 12 individual experiments.

Close modal

Amino acid catabolism is crucial in immunoregulatory mechanisms responding to inflammatory stimuli, participating in the defense against infectious agents, in maternal–fetal tolerance, in oncologic processes as well as in autoimmune diseases (7). In this study, we observed decreased Trp, Arg, Leu, Ile, and Glm catabolism in immune cells from MS patients. All amino acids can be catabolized into a simpler compound or serve as a substrate for other molecules. However, not all cell types express catabolic enzymes, especially in response to inflammation. IDO, iNOS, and ARG are induced by inflammatory cues, a key feature that distinguishes them from enzymes catabolizing other amino acids (3). For this reason, we focused our study mainly on the immunological effects of Trp and Arg metabolism, to prove the hypothesis that impaired amino acid catabolism in immune cells from MS patients could regulate immune response. Decreased Trp and Arg catabolism was mediated by decreased expression and activity of IDO1 and ARG1. Trp is an essential amino acid, obtained only through dietary intake or protein catabolism. Emerging evidence suggests the kynurenine pathway of Trp metabolism is responsible for a broad spectrum of effects, including endogenous regulation of neuronal excitability and initiation of immune tolerance (20). The broad immunological effects of this pathway are principally attributable to increased activity of IDO, the rate-limiting enzyme involved in Trp catabolism to kynurenines. IDO-dependent suppression of T cell responses has been proposed as an immunoregulatory pathway, implicated in maternal tolerance to allogeneic fetuses during mammalian gestation, tumor tolerance, as well as protective regulation in autoimmune disease (3, 7, 21). Different mechanisms have been proposed to explain the underlying immunosuppression after IDO activation. First, Trp deprivation results in cell-cycle arrest and apoptosis of reactive T cells (22, 23). Second, kynurenines promote Treg differentiation by direct activation of aryl hydrocarbon receptors on CD4+ T cells, an effect potentiated by TGF-β, which increases expression of aryl hydrocarbon receptors (7, 24, 25). Third, Trp metabolites and Trp depletion act synergistically on CD8+ effector T cells, downregulating the expression of TCR ζ chain and subsequently impairing its cytotoxic activity (26). Finally, IDO modifies dendritic cell (DC) function by decreasing Ag-presenting action, increasing secretion of anti-inflammatory cytokines and expression of suppressive ligands, turning them into tolerogenic cells, and subsequently shifting the T cell immune response toward development and activation of Tregs (21, 27).

The importance of IDO in the prevention of rejection of allogeneic fetuses was first shown by Munn et al. (28) in a pregnant mouse animal model, in which systemic inhibition of IDO by 1-methyl-Trp lead to placental immune-tolerance breakdown and subsequent rejection of allogeneic embryos, but not syngeneic ones. Similar to observations resulting from Trp metabolism, Arg catabolism was proposed as a mechanism involved in fetal tolerance, after ARG1 activity was shown to be increased in normal term pregnancy (29). Increased Arg catabolism induced T cell hyporesponsiveness through a mechanism involving reduced half-life of mRNA encoding the TCR CD3ξ chain (30).

Several lines of evidence indicate that IDO acts as a negative endogenous regulator of CNS inflammation. In the experimental autoimmune encephalomyelitis (EAE) model, IDO activity is increased in the spleen during preclinical phases, and IDO-positive microglia and macrophages have been detected in brain and spinal cord at symptom onset, correlating with disease severity, later tapering off during remission phases. Furthermore, inhibition of IDO using 1-methyl-Trp exacerbates disease scores, revealing IDO expression influence over immune regulation. These findings suggest IDO is induced by IFN-γ from encephalitogenic Th1 cells, thereby participating in a negative-feedback loop contributing to autolimitation of inflammation (31). Likewise, IDO-deficient mice showed clinical EAE exacerbation with an increase in Th1 and Th17 cells, as well as reduction of Tregs (5). In contrast, administration of downstream Trp metabolite 3-HAA inhibited effector Th1 and Th17 cells and alleviated EAE by promoting Treg responses through upregulation of TGF-β production by DCs (5).

Studies evaluating kynurenine levels in CSF and plasma from MS patients have shown inconsistent results (3234). Discrepancies observed may be the result of evaluation of different patient populations (i.e., individuals undergoing acute exacerbations versus patients in chronic inactive phases of disease). The role of kynurenine in the CNS during the course of MS would need to be evaluated in longitudinal follow-up studies before conclusions can be drawn.

In contrast with our findings, a recent comprehensive metabolome analysis demonstrated increased kynurenine levels in lymphocytes from systemic erythematosus lupus (SLE) patients and found the amino acids stimulated mTORC1 in T cells. The effects were most prominent in CD4 CD8 double-negative (DN) T cells and metabolically linked to oxidative stress (35). DN T cells are expanded in SLE patients and have been implicated in different mechanisms mediating lupus pathogenesis, including elevated IL-4 and IL-17 levels (36, 37), as well as B cell activation and autoantibody production (38) leading to tissue damage (39). These results confirmed previous observations showing that DN T cells from MRL/lpr mice produced high levels of IL-17 during disease progression (40). In addition, in vitro treatment of MRL/lpr lymph node cells with IL-23, a cytokine required for sustained Th17 cell differentiation, induced generation of highly pathogenic DN T cells causing nephritis after adoptive transfer in lymphocyte-deficient Rag1−/− mice (41). But DN T cells can also exert a homeostatic role by suppressing excessive immune responses deleterious to the host. Indeed, rat DN T cell clones do not elicit disease, but rather inhibit EAE development (42). In our study, the kynurenine pathway was not studied in DN T cells from MS patients; further studies are therefore warranted to address what might be the role in MS of the pathway demonstrated in DN T cells from SLE patients.

Although IDO activation plays an important role in immunological tolerance development, the effect can be countered by neurotoxic metabolites of the kynurenine pathway generated within the CNS, such as quinolinic acid and 3-hydrokynurenine (20, 32, 43). Concentrations of these metabolites increased to neurotoxic levels in the spinal cord and lower brainstem of rats with EAE, particularly in cells expressing MHC class II and iNOS, but not glial fibrillary acidic protein, localized in perivascular, subependymal, or subpial regions. These observations would indicate that most quinolinic acid and 3-hydrokynurenine synthesis occurs in macrophages (43). Quinolinic acid is a weak but specific agonist of the N-methyl-d-aspartate receptor; however, its neurotoxic potential may be attributed to different additional mechanisms, including inhibition of glutamate uptake by astrocytes, generation of reactive oxygen intermediates, depletion of endogenous antioxidants, and peroxidation of lipid molecules (20, 32, 43). Toxic effects of 3-hydrokynurenine are mediated through the production of free radicals (44). By contrast, kynurenic acid synthesis in neurons and astrocytes exerts neuroprotective effects by acting as an endogenous glutamate receptor antagonist and free radical scavenger (45). Overall, IDO activation triggers complex immune mechanisms producing immune tolerance, most probably as a self-protective mechanism. However, some metabolites of the kynurenine pathway are potentially toxic for neurons and oligodendrocytes. Therefore, overactivation of IDO in the CNS should be considered as an event that can have both favorable and unfavorable consequences, requiring further investigation.

We found IDO1 and ARG1 activity and expression were reduced in MS patients, primarily in monocytes and, to a lesser extent, in CD4+CD25+ Tregs and Th1 cells. In line with these observations, IDO+ monocyte-derived DCs suppress potent T cell responses in vivo, promoting systemic tolerance (46) by, at least in part, increasing CD4+CD25+ Tregs numbers and activity (47). In contrast, it has been suggested that CD4+CD25+ Tregs expressing CTLA-4 induce functional IDO expression in monocyte-derived DCs (48). Therefore, CD4+CD25+ Tregs and IDO+ monocyte-derived DCs might interact to form a self-reinforcing network capable of suppressing local T cell responses and promoting systemic tolerance.

GCN2 and mTOR are involved in sensing amino acid deficiency or sufficiency. These pathways play important roles as downstream links between amino acid catabolism, regulation of T cell proliferation, and generation of Tregs. When assessing the functional consequences of decreased amino acid catabolism, we observed that high levels of Trp and Arg were associated with significant downregulation of GCN2 and upregulation of mTOR expression and activity, which in turn resulted in decreased Treg numbers, increased proliferation of MBP-peptide–specific TCLs, and increased proinflammatory cytokine production.

The GCN2-dependent pathway is essential for naive CD4+ T cells to undergo conversion to FOXP3+ Tregs, through the combined effect of Trp withdrawal and presence of Trp catabolites (26). GCN2 has also been shown to have a pivotal role in activating resting FOXP3+ Tregs, which exhibit little suppressor activity and must be activated by mitogenic signals to acquire adequate suppressor function (49). Indeed, regulatory responses of FOXP3+ Tregs were absent when these cells were obtained from GCN2 knockout mice (50). Furthermore, GCN2-dependent activation of the amino acid starvation response was found to inhibit mouse and human Th17 differentiation (51). Supporting these findings, GNC2-deficient mice develop a form of EAE characterized by absence of remission (52). In contrast, the mTORC1 pathway regulates metabolic programs to facilitate the switch from quiescent to activated T cell and plays a critical role in Th17 cell differentiation (53, 54). Meanwhile, mTORC2 through its substrate SGK1 regulates the ability of Th cells to differentiate into Th2 cells (55, 56). Interestingly, availability of significant levels of amino acids is critical to mTORC1 function, and amino acid depletion rapidly inactivates mTORC1 signaling, inhibiting Th17 cell differentiation (57). Both mTORC1 and mTORC2 seem to interfere with the differentiation and function of Tregs. Overactivation of the mTORC1 pathway has been associated with decreased expression of the 44- and 47-kDa forms of FOXP3 in ex vivo–isolated Tregs from RRMS patients compared with HCs, together with increased expression of the cell-cycle inhibitor p27kip1. These findings have been linked to the Treg hyporesponsiveness observed in RRMS patients both in vivo and in vitro (58). Interestingly, impaired proliferation capacity of Tregs in RRMS patients correlates with patient clinical condition, such that increasing disease severity is associated with a decline in Treg expansion (58). mTORC2 activation may also influence FOXP3 ability to maintain suppressive Treg function, because Rictor deficiency restores Treg function in vitro and partially reverses fatal autoimmune disease observed in mice bearing Raptor-deficiency Tregs (59). Although mTORC1 drives proinflammatory responses in the adaptive immune system, it may also exert anti-inflammatory effects on the innate immune system, favoring M2 over M1 polarization of macrophages (60). Overall, these findings suggest GCN2 and mTOR pathways are interconnected to fine-tune control of cell growth and effector function (3).

In addition to decreased catabolism of Trp and Arg, measurement of intracellular free amino acids in PBMCs from MS patients showed a significant increase of Leu, Ile, and Glm compared with HCs (Table III), suggesting impaired catabolism of these amino acids as well. Furthermore, in starvation experiments, addition of Trp, Arg, Leu, Ile, and Glm resulted in marked increase of p70-S6 protein, reflecting mTOR activity. It seems likely that specific cell types differ in their sensitivity to the omission or addition of particular amino acids (61). Interestingly, Glm in combination with Leu activates mammalian mTORC1 by enhancing Glm metabolism, promoting cell growth, and inhibiting autophagy (62). Moreover, blocking Leu and glucose metabolism inhibits mTORC1 activation, which, in turn, inhibits T cell effector functions and promotes the induction of anergy in Th1 cells (63). Collectively, these observations indicate that controlling intracellular amino acid availability can regulate immune responses through different mechanisms.

Although evidence supporting the importance of amino acid catabolism in immune regulation is increasing, the relevance of this pathway in MS is mostly inferred from animal models; clinical studies in MS patients are still lacking. IDO appears to be a critical feedback inductor of immune response termination, which can be detrimental in some settings such as chronic infections and cancer. IDO inhibitors are currently being explored as therapeutic targets, because high IDO expression in tumors has been linked to poor prognosis and chemoresistance (64). Conversely, persistent IDO expression may be an optimal target to elicit long-term tolerogenicity, as recently demonstrated for adjuvant autoantigens vaccines (65). Our results suggest amino acid catabolism is decreased in MS patients, and that this decrease has functional consequences, increasing proinflammatory cytokines and decreasing Treg numbers. Further exploration of these pathways in MS patients may allow potential future manipulation for therapeutic benefit.

This work was supported by an internal grant from the Raúl Carrea Institute for Neurological Research, FLENI (to J.C.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

ARG

arginase

Arg

arginine

CHOP

C/EBP homologous protein

DC

dendritic cell

DN

double-negative

EAE

experimental autoimmune encephalomyelitis

GCN2

general control nonrepressed 2 serine/threonine kinase

Glm

glutamine

3-HAA

3-hydroxyantranilic

HC

healthy control subject

Ile

isoleucine

iNOS

inducible NO synthetase

Leu

leucine

MBP

myelin basic protein

MS

multiple sclerosis

mTOR

mammalian target of rapamycin

OIND

other inflammatory neurological diseases

rhIL-2

recombinant human IL-2

RRMS

relapsing-remitting MS

SLE

systemic erythematosus lupus

TDO

Trp 2,3-dioxygenase

Treg

regulatory T cell

Trp

tryptophan.

1
Compston
A.
,
Coles
A.
.
2008
.
Multiple sclerosis.
Lancet
372
:
1502
1517
.
2
Hemmer
B.
,
Kerschensteiner
M.
,
Korn
T.
.
2015
.
Role of the innate and adaptive immune responses in the course of multiple sclerosis.
Lancet Neurol.
14
:
406
419
.
3
McGaha
T. L.
,
Huang
L.
,
Lemos
H.
,
Metz
R.
,
Mautino
M.
,
Prendergast
G. C.
,
Mellor
A. L.
.
2012
.
Amino acid catabolism: a pivotal regulator of innate and adaptive immunity.
Immunol. Rev.
249
:
135
157
.
4
Fallarino
F.
,
Grohmann
U.
,
Vacca
C.
,
Orabona
C.
,
Spreca
A.
,
Fioretti
M. C.
,
Puccetti
P.
.
2003
.
T cell apoptosis by kynurenines.
Adv. Exp. Med. Biol.
527
:
183
190
.
5
Yan
Y.
,
Zhang
G. X.
,
Gran
B.
,
Fallarino
F.
,
Yu
S.
,
Li
H.
,
Cullimore
M. L.
,
Rostami
A.
,
Xu
H.
.
2010
.
IDO upregulates regulatory T cells via tryptophan catabolite and suppresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis.
J. Immunol.
185
:
5953
5961
.
6
Platten
M.
,
Ho
P. P.
,
Youssef
S.
,
Fontoura
P.
,
Garren
H.
,
Hur
E. M.
,
Gupta
R.
,
Lee
L. Y.
,
Kidd
B. A.
,
Robinson
W. H.
, et al
.
2005
.
Treatment of autoimmune neuroinflammation with a synthetic tryptophan metabolite.
Science
310
:
850
855
.
7
Mbongue
J. C.
,
Nicholas
D. A.
,
Torrez
T. W.
,
Kim
N. S.
,
Firek
A. F.
,
Langridge
W. H.
.
2015
.
The role of indoleamine 2, 3-dioxygenase in immune suppression and autoimmunity.
Vaccines (Basel)
3
:
703
729
.
8
Poser
C. M.
1965
.
Clinical diagnostic criteria in epidemiological studies of multiple sclerosis.
Ann. N. Y. Acad. Sci.
122
:
506
519
.
9
Polman
C. H.
,
Reingold
S. C.
,
Banwell
B.
,
Clanet
M.
,
Cohen
J. A.
,
Filippi
M.
,
Fujihara
K.
,
Havrdova
E.
,
Hutchinson
M.
,
Kappos
L.
, et al
.
2011
.
Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria.
Ann. Neurol.
69
:
292
302
.
10
Kurtzke
J. F.
1983
.
Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS).
Neurology
33
:
1444
1452
.
11
Correale
J.
,
McMillan
M.
,
McCarthy
K.
,
Le
T.
,
Weiner
L. P.
.
1995
.
Isolation and characterization of autoreactive proteolipid protein-peptide specific T-cell clones from multiple sclerosis patients.
Neurology
45
:
1370
1378
.
12
Widner
B.
,
Werner
E. R.
,
Schennach
H.
,
Wachter
H.
,
Fuchs
D.
.
1997
.
Simultaneous measurement of serum tryptophan and kynurenine by HPLC.
Clin. Chem.
43
:
2424
2426
.
13
Canepa
A.
,
Perfumo
F.
,
Carrea
A.
,
Sanguineti
A.
,
Piccardo
M. T.
,
Gusmano
R.
.
1989
.
Measurement of free amino acids in polymorphonuclear leukocytes by high-performance liquid chromatography.
J. Chromatogr. A
491
:
200
208
.
14
Kim
D. H.
,
Sarbassov
D. D.
,
Ali
S. M.
,
King
J. E.
,
Latek
R. R.
,
Erdjument-Bromage
H.
,
Tempst
P.
,
Sabatini
D. M.
.
2002
.
mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.
Cell
110
:
163
175
.
15
Laplante
M.
,
Sabatini
D. M.
.
2009
.
mTOR signaling at a glance.
J. Cell Sci.
122
:
3589
3594
.
16
Holz
M. K.
,
Blenis
J.
.
2005
.
Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase.
J. Biol. Chem.
280
:
26089
26093
.
17
Sarbassov
D. D.
,
Guertin
D. A.
,
Ali
S. M.
,
Sabatini
D. M.
.
2005
.
Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.
Science
307
:
1098
1101
.
18
Bar-Peled
L.
,
Sabatini
D. M.
.
2014
.
Regulation of mTORC1 by amino acids.
Trends Cell Biol.
24
:
400
406
.
19
Penberthy
W. T.
2007
.
Pharmacological targeting of IDO-mediated tolerance for treating autoimmune disease.
Curr. Drug Metab.
8
:
245
266
.
20
Vécsei
L.
,
Szalárdy
L.
,
Fülöp
F.
,
Toldi
J.
.
2013
.
Kynurenines in the CNS: recent advances and new questions.
Nat. Rev. Drug Discov.
12
:
64
82
.
21
Mellor
A. L.
,
Munn
D. H.
.
2004
.
IDO expression by dendritic cells: tolerance and tryptophan catabolism.
Nat. Rev. Immunol.
4
:
762
774
.
22
Munn
D. H.
,
Shafizadeh
E.
,
Attwood
J. T.
,
Bondarev
I.
,
Pashine
A.
,
Mellor
A. L.
.
1999
.
Inhibition of T cell proliferation by macrophage tryptophan catabolism.
J. Exp. Med.
189
:
1363
1372
.
23
Lee
G. K.
,
Park
H. J.
,
Macleod
M.
,
Chandler
P.
,
Munn
D. H.
,
Mellor
A. L.
.
2002
.
Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division.
Immunology
107
:
452
460
.
24
Harden
J. L.
,
Egilmez
N. K.
.
2012
.
Indoleamine 2,3-dioxygenase and dendritic cell tolerogenicity.
Immunol. Invest.
41
:
738
764
.
25
Mezrich
J. D.
,
Fechner
J. H.
,
Zhang
X.
,
Johnson
B. P.
,
Burlingham
W. J.
,
Bradfield
C. A.
.
2010
.
An interaction between kynurenine and the aryl hydrocarbon receptor can generate regulatory T cells.
J. Immunol.
185
:
3190
3198
.
26
Fallarino
F.
,
Grohmann
U.
,
You
S.
,
McGrath
B. C.
,
Cavener
D. R.
,
Vacca
C.
,
Orabona
C.
,
Bianchi
R.
,
Belladonna
M. L.
,
Volpi
C.
, et al
.
2006
.
The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor ζ-chain and induce a regulatory phenotype in naive T cells.
J. Immunol.
176
:
6752
6761
.
27
Pallotta
M. T.
,
Orabona
C.
,
Volpi
C.
,
Vacca
C.
,
Belladonna
M. L.
,
Bianchi
R.
,
Servillo
G.
,
Brunacci
C.
,
Calvitti
M.
,
Bicciato
S.
, et al
.
2011
.
Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells.
Nat. Immunol.
12
:
870
878
.
28
Munn
D. H. M.
,
Zhou
M.
,
Attwood
J. T.
,
Bondarev
I.
,
Conway
S. J.
,
Marshall
B.
,
Brown
C.
,
Mellor
A. L.
.
1998
.
Prevention of allogeneic fetal rejection by tryptophan catabolism.
Science
281
:
1191
1193
.
29
Kropf
P.
,
Baud
D.
,
Marshall
S. E.
,
Munder
M.
,
Mosley
A.
,
Fuentes
J. M.
,
Bangham
C. R.
,
Taylor
G. P.
,
Herath
S.
,
Choi
B. S.
, et al
.
2007
.
Arginase activity mediates reversible T cell hyporesponsiveness in human pregnancy.
Eur. J. Immunol.
37
:
935
945
.
30
Zea
A. H.
,
Rodriguez
P. C.
,
Culotta
K. S.
,
Hernandez
C. P.
,
DeSalvo
J.
,
Ochoa
J. B.
,
Park
H. J.
,
Zabaleta
J.
,
Ochoa
A. C.
.
2004
.
L-Arginine modulates CD3zeta expression and T cell function in activated human T lymphocytes.
Cell. Immunol.
232
:
21
31
.
31
Kwidzinski
E.
,
Bunse
J.
,
Aktas
O.
,
Richter
D.
,
Mutlu
L.
,
Zipp
F.
,
Nitsch
R.
,
Bechmann
I.
.
2005
.
Indolamine 2,3-dioxygenase is expressed in the CNS and down-regulates autoimmune inflammation.
FASEB J.
19
:
1347
1349
.
32
Hartai
Z.
,
Klivenyi
P.
,
Janaky
T.
,
Penke
B.
,
Dux
L.
,
Vecsei
L.
.
2005
.
Kynurenine metabolism in multiple sclerosis.
Acta Neurol. Scand.
112
:
93
96
.
33
Rejdak
K.
,
Petzold
A.
,
Kocki
T.
,
Kurzepa
J.
,
Grieb
P.
,
Turski
W. A.
,
Stelmasiak
Z.
.
2007
.
Astrocytic activation in relation to inflammatory markers during clinical exacerbation of relapsing-remitting multiple sclerosis.
J Neural Transm. (Vienna)
114
:
1011
1015
.
34
Rejdak
K.
,
Bartosik-Psujek
H.
,
Dobosz
B.
,
Kocki
T.
,
Grieb
P.
,
Giovannoni
G.
,
Turski
W. A.
,
Stelmasiak
Z.
.
2002
.
Decreased level of kynurenic acid in cerebrospinal fluid of relapsing-onset multiple sclerosis patients.
Neurosci. Lett.
331
:
63
65
.
35
Perl
A.
,
Hanczko
R.
,
Lai
Z. W.
,
Oaks
Z.
,
Kelly
R.
,
Borsuk
R.
,
Asara
J. M.
,
Phillips
P. E.
.
2015
.
Comprehensive metabolome analyses reveal N-acetylcysteine-responsive accumulation of kynurenine in systemic lupus erythematosus: implications for activation of the mechanistic target of rapamycin.
Metabolomics
11
:
1157
1174
.
36
Chan
R. W. Y.
,
Lai
F. M. M.
,
Li
E. K. M.
,
Tam
L. S.
,
Chow
K. M.
,
Li
P. K. T.
,
Szeto
C. C.
.
2006
.
Imbalance of Th1/Th2 transcription factors in patients with lupus nephritis.
Rheumatology (Oxford)
45
:
951
957
.
37
Kato
H.
,
Perl
A.
.
2014
.
MTORC1 expands Th17 and Il-4+ DN T cells and contracts Tregs in SLE.
J. Immunol.
192
:
4134
4144
.
38
Shivakumar
S.
,
Tsokos
G. C.
,
Datta
S. K.
.
1989
.
T cell receptor alpha/beta expressing double-negative (CD4-/CD8-) and CD4+ T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis.
J. Immunol.
143
:
103
112
.
39
Crispín
J. C.
,
Oukka
M.
,
Bayliss
G.
,
Cohen
R. A.
,
Van Beek
C. A.
,
Stillman
I. E.
,
Kyttaris
V. C.
,
Juang
Y. T.
,
Tsokos
G. C.
.
2008
.
Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys.
J. Immunol.
181
:
8761
8766
.
40
Nalbandian
A.
,
Crispín
J. C.
,
Tsokos
G. C.
.
2009
.
Interleukin-17 and systemic lupus erythematosus: current concepts.
Clin. Exp. Immunol.
157
:
209
215
.
41
Kyttaris
V. C.
,
Zhang
Z.
,
Kuchroo
V. K.
,
Oukka
M.
,
Tsokos
G. C.
.
2010
.
Cutting edge: IL-23 receptor deficiency prevents the development of lupus nephritis in C57BL/6-lpr/lpr mice.
J. Immunol.
184
:
4605
4609
.
42
Lider
O.
,
Miller
A.
,
Miron
S.
,
Hershkoviz
R.
,
Weiner
H. L.
,
Zhang
X. M.
,
Heber-Katz
E.
.
1991
.
Nonencephalitogenic CD4-CD8- V alpha 2V beta 8.2+ anti-myelin basic protein rat T lymphocytes inhibit disease induction.
J. Immunol.
147
:
1208
1213
.
43
Chiarugi
A.
,
Cozzi
A.
,
Ballerini
C.
,
Massacesi
L.
,
Moroni
F.
.
2001
.
Kynurenine 3-mono-oxygenase activity and neurotoxic kynurenine metabolites increase in the spinal cord of rats with experimental allergic encephalomyelitis.
Neuroscience
102
:
687
695
.
44
Szalardy
L.
,
Klivenyi
P.
,
Zadori
D.
,
Fulop
F.
,
Toldi
J.
,
Vecsei
L.
.
2012
.
Mitochondrial disturbances, tryptophan metabolites and neurodegeneration: medicinal chemistry aspects.
Curr. Med. Chem.
19
:
1899
1920
.
45
Szalardy
L.
,
Zadori
D.
,
Toldi
J.
,
Fulop
F.
,
Klivenyi
P.
,
Vecsei
L.
.
2012
.
Manipulating kynurenic acid levels in the brain - on the edge between neuroprotection and cognitive dysfunction.
Curr. Top. Med. Chem.
12
:
1797
1806
.
46
Grohmann
U.
,
Bianchi
R.
,
Belladonna
M. L.
,
Silla
S.
,
Fallarino
F.
,
Fioretti
M. C.
,
Puccetti
P.
.
2000
.
IFN-γ inhibits presentation of a tumor/self peptide by CD8 alpha- dendritic cells via potentiation of the CD8 alpha+ subset.
J. Immunol.
165
:
1357
1363
.
47
Wakkach
A.
,
Fournier
N.
,
Brun
V.
,
Breittmayer
J. P.
,
Cottrez
F.
,
Groux
H.
.
2003
.
Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo.
Immunity
18
:
605
617
.
48
Fallarino
F.
,
Grohmann
U.
,
Hwang
K. W.
,
Orabona
C.
,
Vacca
C.
,
Bianchi
R.
,
Belladonna
M. L.
,
Fioretti
M. C.
,
Alegre
M. L.
,
Puccetti
P.
.
2003
.
Modulation of tryptophan catabolism by regulatory T cells.
Nat. Immunol.
4
:
1206
1212
.
49
Thornton
A. M.
,
Piccirillo
C. A.
,
Shevach
E. M.
.
2004
.
Activation requirements for the induction of CD4+CD25+ T cell suppressor function.
Eur. J. Immunol.
34
:
366
376
.
50
Sharma
M. D.
,
Baban
B.
,
Chandler
P.
,
Hou
D. Y.
,
Singh
N.
,
Yagita
H.
,
Azuma
M.
,
Blazar
B. R.
,
Mellor
A. L.
,
Munn
D. H.
.
2007
.
Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase.
J. Clin. Invest.
117
:
2570
2582
.
51
Sundrud
M. S.
,
Koralov
S. B.
,
Feuerer
M.
,
Calado
D. P.
,
Kozhaya
A. E.
,
Rhule-Smith
A.
,
Lefebvre
R. E.
,
Unutmaz
D.
,
Mazitschek
R.
,
Waldner
H.
, et al
.
2009
.
Halofuginone inhibits TH17 cell differentiation by activating the amino acid starvation response.
Science
324
:
1334
1338
.
52
Orsini
H.
,
Araujo
L. P.
,
Maricato
J. T.
,
Guereschi
M. G.
,
Mariano
M.
,
Castilho
B. A.
,
Basso
A. S.
.
2014
.
GCN2 kinase plays an important role triggering the remission phase of experimental autoimmune encephalomyelitis (EAE) in mice.
Brain Behav. Immun.
37
:
177
186
.
53
Ren
W.
,
Yin
J.
,
Duan
J.
,
Liu
G.
,
Tan
B.
,
Yang
G.
,
Wu
G.
,
Bazer
F.W.
,
Peng
Y.
,
Yin
Y.
.
2016
.
mTORC1 signaling and IL-17 expression: defining pathways and possible therapeutic targets.
Eur. J. Immunol.
46
:
291
299
.
54
Yu
C. R.
,
Mahdi
R. M.
,
Ebong
S.
,
Vistica
B. P.
,
Gery
I.
,
Egwuagu
C. E.
.
2003
.
Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells.
J. Biol. Chem.
278
:
29752
29759
.
55
Delgoffe
G. M.
,
Pollizzi
K. N.
,
Waickman
A. T.
,
Heikamp
E.
,
Meyers
D. J.
,
Horton
M. R.
,
Xiao
B.
,
Worley
P. F.
,
Powell
J. D.
.
2011
.
The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2.
Nat. Immunol.
12
:
295
303
.
56
Heikamp
E. B.
,
Patel
C. H.
,
Collins
S.
,
Waickman
A.
,
Oh
M. H.
,
Sun
I. H.
,
Illei
P.
,
Sharma
A.
,
Naray-Fejes-Toth
A.
,
Fejes-Toth
G.
, et al
.
2014
.
The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex.
Nat. Immunol.
15
:
457
464
.
57
Huang
K.
,
Fingar
D. C.
.
2014
.
Growing knowledge of the mTOR signaling network.
Semin. Cell Dev. Biol.
36
:
79
90
.
58
Carbone
F.
,
De Rosa
V.
,
Carrieri
P. B.
,
Montella
S.
,
Bruzzese
D.
,
Porcellini
A.
,
Procaccini
C.
,
La Cava
A.
,
Matarese
G.
.
2014
.
Regulatory T cell proliferative potential is impaired in human autoimmune disease. [Published erratum appears in 2014 Nat. Med. 20: 220.]
Nat. Med.
20
:
69
74
.
59
Zeng
H.
,
Yang
K.
,
Cloer
C.
,
Neale
G.
,
Vogel
P.
,
Chi
H.
.
2013
.
mTORC1 couples immune signals and metabolic programming to establish T(reg)-cell function.
Nature
499
:
485
490
.
60
Mercalli
A.
,
Calavita
I.
,
Dugnani
E.
,
Citro
A.
,
Cantarelli
E.
,
Nano
R.
,
Melzi
R.
,
Maffi
P.
,
Secchi
A.
,
Sordi
V.
,
Piemonti
L.
.
2013
.
Rapamycin unbalances the polarization of human macrophages to M1.
Immunology
140
:
179
190
.
61
Proud
C. G.
2002
.
Regulation of mammalian translation factors by nutrients.
Eur. J. Biochem.
269
:
5338
5349
.
62
Durán
R. V.
,
Oppliger
W.
,
Robitaille
A. M.
,
Heiserich
L.
,
Skendaj
R.
,
Gottlieb
E.
,
Hall
M. N.
.
2012
.
Glutaminolysis activates Rag-mTORC1 signaling.
Mol. Cell
47
:
349
358
.
63
Zheng
Y.
,
Delgoffe
G. M.
,
Meyer
C. F.
,
Chan
W.
,
Powell
J. D.
.
2009
.
Anergic T cells are metabolically anergic.
J. Immunol.
183
:
6095
6101
.
64
Moon
Y. W.
,
Hajjar
J.
,
Hwu
P.
,
Naing
A.
.
2015
.
Targeting the indoleamine 2,3-dioxygenase pathway in cancer.
J. Immunother. Cancer
3
:
51
.
65
Mbongue
J. C.
,
Nicholas
D. A.
,
Zhang
K.
,
Kim
N. S.
,
Hamilton
B. N.
,
Larios
M.
,
Zhang
G.
,
Umezawa
K.
,
Firek
A. F.
,
Langridge
W. H.
.
2015
.
Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.
PLoS One
10
:
e0118562
.

L.N. has no conflicts of interest to disclose. J.C. is a board member of Merck-Serono Argentina, Novartis Argentina, Genzyme Latin America, Genzyme Global, Biogen-Idec Latin America, and Merck-Serono Latin America. J.C. has received reimbursement for developing educational presentations for Merck-Serono Argentina, Merck-Serono Latin America, Biogen-Idec Argentina, Genzyme Argentina, Novartis Argentina, Novartis Latin America, Novartis Global, and TEVA Argentina, as well as professional travel/accommodations stipends.

Supplementary data