Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Myeloid caspase-8 restricts RIPK3-dependent proinflammatory IL-1ß production and CD4 T cell activation in autoimmune demyelination.

Caspase-8 functions at the crossroad of programmed cell death and inflammation. Here, using genetic approaches and the experimental autoimmune encephalomyelitis model of inflammatory demyelination, we identified a negative regulatory pathway for caspase-8 in infiltrated macrophages whereby it functions to restrain interleukin (IL)-1ß-driven autoimmune inflammation. Caspase-8 is partially activated in macrophages/microglia in active lesions of multiple sclerosis. Selective ablation of Casp8 in myeloid cells, but not microglia, exacerbated autoimmune demyelination. Heightened IL-1ß production by caspase-8-deficient macrophages underlies exacerbated activation of encephalitogenic T cells and production of GM-CSF and interferon-γ. Mechanistically, IL-1ß overproduction by primed caspase-8-deficient macrophages was mediated by RIPK1/RIPK3 through the engagement of NLRP3 inflammasome and was independent of cell death. When instructed by autoreactive CD4 T cells in the presence of antigen, caspase-8-deficient macrophages, but not their wild-type counterparts, released significant amount of IL-1ß that in turn acted through IL-1R to amplify T cell activation. Moreover, the worsened experimental autoimmune encephalomyelitis progression in myeloid Casp8 mutant mice was completely reversed when Ripk3 was simultaneously deleted. Together, these data reveal a functional link between T cell-driven autoimmunity and inflammatory IL-1ß that is negatively regulated by caspase-8, and suggest that dysregulation of the pathway may contribute to inflammatory autoimmune diseases, such as multiple sclerosis.

N-Acylethanolamine-Hydrolyzing Acid Amidase Inhibition, but Not Fatty Acid Amide Hydrolase Inhibition, Prevents the Development of Experimental Autoimmune Encephalomyelitis in Mice.

N-acylethanolamines (NAEs) are endogenous bioactive lipids reported to exert anti-inflammatory and neuroprotective effects mediated by cannabinoid receptors and peroxisome proliferator-activated receptors (PPARs), among others. Therefore, interfering with NAE signaling could be a promising strategy to decrease inflammation in neurological disorders such as multiple sclerosis (MS). Fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA) are key modulators of NAE levels. This study aims to investigate and compare the effect of NAAA inhibition, FAAH inhibition, and dual inhibition of both enzymes in a mouse model of MS, namely the experimental autoimmune encephalomyelitis (EAE). Our data show that NAAA inhibition strongly decreased the hallmarks of the pathology. Interestingly, FAAH inhibition was less efficient in decreasing inflammatory hallmarks despite the increased NAE levels. Moreover, the inhibition of both NAAA and FAAH, using a dual-inhibitor or the co-administration of NAAA and FAAH inhibitors, did not show an added value compared to NAAA inhibition. Furthermore, our data suggest an important role of decreased activation of astrocytes and microglia in the effects of NAAA inhibition on EAE, while NAAA inhibition did not affect T cell recall. This work highlights the beneficial effects of NAAA inhibition in the context of central nervous system inflammation and suggests that the simultaneous inhibition of NAAA and FAAH has no additional beneficial effect in EAE.

Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4.

Perturbed expression of microRNAs (miRs) has been reported in different diseases including autoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR-25-3p and its targets in the central nervous system (CNS) tissue from mice with experimental autoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets in activated macrophages and splenocytes. EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyte glycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR-25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We next established primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR-25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and anti-CD3/anti-CD28 antibodies, respectively. MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression of phosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak of the disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR-25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significant upregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR-25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show any alterations in miR-25-3p expression compared with vehicle-treated cells. Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators during autoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.

PRMT5 Promotes Cyclin E1 and Cell Cycle Progression in CD4 Th1 Cells and Correlates With EAE Severity.

Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5's link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4+ T cells. The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS.

Murine endometrial-derived mesenchymal stem cells suppress experimental autoimmune encephalomyelitis depending on indoleamine-2,3-dioxygenase expression.

Cellular therapy with mesenchymal stem cells (MSCs) is a huge challenge for scientists, as little translational relevance has been achieved. However, many studies using MSCs have proved their suppressive and regenerative capacity. Thus, there is still a need for a better understanding of MSCs biology and the establishment of newer protocols, or to test unexplored tissue sources. Here, we demonstrate that murine endometrial-derived MSCs (meMSCs) suppress Experimental Autoimmune Encephalomyelitis (EAE). MSC-treated animals had milder disease, with a significant reduction in Th1 and Th17 lymphocytes in the lymph nodes and in the central nervous system (CNS). This was associated with increased Il27 and Cyp1a1 expression, and presence of IL-10-secreting T CD4+ cells. At EAE peak, animals had reduced CNS infiltrating cells, histopathology and demyelination. qPCR analysis evidenced the down-regulation of several pro-inflammatory genes and up-regulation of indoleamine-2,3-dioxygenase (IDO). Consistently, co-culturing of WT and IDO-/- meMSCs with T CD4+ cells evidenced the necessity of IDO on the suppression of encephalitogenic lymphocytes, and IDO-/- meMSCs were not able to suppress EAE. In addition, WT meMSCs stimulated with IL-17A and IFN-γ increased IDO expression and secretion of kynurenines in vitro, indicating a negative feedback loop. Pathogenic cytokines were increased when CD4+ T cells from AhR-/- mice were co-cultured with WT meMSC. In summary, our research evidences the suppressive activity of the unexplored meMSCs population, and shows the mechanism depends on IDO-kynurenines-Aryl hydrocarbon receptor (AhR) axis. To our knowledge this is the first report evidencing that the therapeutic potential of meMSCs relying on IDO expression.

Myeloid ATP Citrate Lyase Regulates Macrophage Inflammatory Responses In Vitro Without Altering Inflammatory Disease Outcomes.

Macrophages are highly plastic, key regulators of inflammation. Deregulation of macrophage activation can lead to excessive inflammation as seen in inflammatory disorders like atherosclerosis, obesity, multiple sclerosis and sepsis. Targeting intracellular metabolism is considered as an approach to reshape deranged macrophage activation and to dampen the progression of inflammatory disorders. ATP citrate lyase (Acly) is a key metabolic enzyme and an important regulator of macrophage activation. Using a macrophage-specific Acly-deficient mouse model, we investigated the role of Acly in macrophages during acute and chronic inflammatory disorders. First, we performed RNA sequencing to demonstrate that Acly-deficient macrophages showed hyperinflammatory gene signatures in response to acute LPS stimulation in vitro. Next, we assessed endotoxin-induced peritonitis in myeloid-specific Acly-deficient mice and show that, apart from increased splenic Il6 expression, systemic and local inflammation were not affected by Acly deficiency. Also during obesity, both chronic low-grade inflammation and whole-body metabolic homeostasis remained largely unaltered in mice with Acly-deficient myeloid cells. Lastly, we show that macrophage-specific Acly deletion did not affect the severity of experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis. These results indicate that, despite increasing inflammatory responses in vitro, macrophage Acly deficiency does not worsen acute and chronic inflammatory responses in vivo. Collectively, our results indicate that caution is warranted in prospective long-term treatments of inflammatory disorders with macrophage-specific Acly inhibitors. Together with our earlier observation that myeloid Acly deletion stabilizes atherosclerotic lesions, our findings highlight that therapeutic targeting of macrophage Acly can be beneficial in some, but not all, inflammatory disorders.

Increase in Autoantibodies-Abzymes with Peroxidase and Oxidoreductase Activities in Experimental Autoimmune Encephalomyelitis Mice during the Development of EAE Pathology.

The exact mechanisms of multiple sclerosis (MS) development are still unknown, but the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice is associated with the violation of bone marrow hematopoietic stem cells (HSCs) differentiation profiles associated with the production of harmful for human's autoantibodies hydrolyzing myelin basic protein, myelin oligodendrocyte glycoprotein (MOG35-55), and DNA. It was shown that IgGs from the sera of healthy humans and autoimmune patients oxidize many different compounds due to their H2O2-dependent peroxidase and oxidoreductase activity in the absence of H2O2. Here we first analyzed the change in the relative redox activities of IgGs antibodies from the blood of C57BL/6 mice over time at different stages of the EAE development. It was shown that the peroxidase activity of mice IgGs in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is on average 6.9-fold higher than the oxidoreductase activity. The peroxidase activity of IgGs increased during the spontaneous development of EAE during 40 days, 1.4-fold. After EAE development acceleration due to mice immunization with MOG35-55 (5.3-fold), complexes of bovine DNA with methylated bovine serum albumin (DNA-metBSA; 3.5-fold), or with histones (2.6-fold), the activity was increased much faster. The increase in peroxidase activity after mice immunization with MOG35-55 and DNA-metBSA up to 40 days of experiments was relatively gradual, while for DNA-histones complex was observed its sharp increase at the acute phase of EAE (14-20 days). All data show that IgGs' redox activities can play an important role in the protection of mice from toxic compounds and oxidative stress.

Chondroitin sulfate N-acetylgalactosyltransferase-1 knockout shows milder phenotype in experimental autoimmune encephalomyelitis than in wild type.

Proteoglycans (PGs) are one of the main components in the extracellular matrix of the central nervous system. Chondroitin sulfate (CS) is a glycosaminoglycan (GAG), which is composed of major PGs. Similar to keratin sulfate (KS), another GAG, CS inhibits axon regeneration. However, the influence of these GAGs on the pathogenicity of neuroimmunological diseases is unclear. Here, we induced experimental autoimmune encephalomyelitis (EAE) in mice lacking CS N-acetylgalactosaminyltransferase-1 (CSGalNAcT1-KO), an important enzyme for CS synthesis. In our study, CSGalNAcT1-KO mice showed milder EAE symptoms than those in wild-type (WT) mice. The recall response of antigen-specific lymphocytes showed that CSGalNAcT1-KO-derived lymphocytes had a milder cell proliferation response than that in WT-derived lymphocytes. These results suggest that CS contributes toward the induction phase of EAE. We previously performed EAE experiments in GlcNAc-6-O-sulfotransferase KO (GlcNAc6ST-KO) and C6ST1-KO mice, which had reduced KS and reduced CS-C, respectively. EAE in CSGalNAcT1-KO mice was more similar to that in GlcNAc6ST-KO mice than in C6ST1-KO mice. In conclusion, the distinct GAG sugar chains are associated with severe or mild phenotypes of EAE and are therefore potential new therapeutic targets for neuroimmunological diseases, including multiple sclerosis.

Involvement of Indoleamine-2,3-Dioxygenase and Kynurenine Pathway in Experimental Autoimmune Encephalomyelitis in Mice.

The experimental autoimmune encephalomyelitis (EAE) is a model that mimics multiple sclerosis in rodents. Evidence has suggested that the activation of indoleamine-2,3-dioxygenase (IDO), the rate-limiting enzyme in the kynurenine pathway (KP), plays a crucial role in inflammation-related diseases. The present study aimed to investigate the involvement of the inflammatory process and KP components in a model of EAE in mice. To identify the role of KP in EAE pathogenesis, mice received IDO inhibitor (INCB024360) at a dose of 200 mg/kg (per oral) for 25 days. We demonstrated that IDO inhibitor mitigated the clinical signs of EAE, in parallel with the reduction of cytokine levels (brain, spinal cord, spleen and lymph node) and ionized calcium-binding adaptor protein-1 (Iba-1) gene expression in the central nervous system of EAE mice. Besides, IDO inhibitor causes a significant decrease in the levels of tryptophan, kynurenine and neurotoxic metabolites of KP, such as 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) in the prefrontal cortex, hippocampus, spinal cord, spleen and lymph node of EAE mice. The mRNA expression and enzyme activity of IDO and kynurenine 3-monooxygenase (KMO) were also reduced by IDO inhibitor. These findings indicate that the inflammatory process concomitant with the activation of IDO/KP is involved in the pathogenic mechanisms of EAE. The modulation of KP is a promising target for novel pharmacological treatment of MS.

CD226 Attenuates Treg Proliferation via Akt and Erk Signaling in an EAE Model.

Cluster of differentiation 226 (CD226) molecules play a crucial role in the activation of effector CD4+ T cells during the immune response process, but a cell-intrinsic function of CD226 in CD4+ T subsets is not clear. In this study, we showed that Cd226-/- mice were resistant to myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) with highly expressed IL-10+CD4+ T cells and downregulated IL-17A+CD4+ T cells when compared with wild-type (WT) mice. Th17 cell infiltration into the central nervous system (CNS) was largely decreased in the absence of CD226 during EAE. CD226 deficiency facilitated the proliferation of regulatory T cells (Tregs), with increased numbers of Tregs observed in EAE mice, and supported the elevated induced regulatory T cell (iTregs) proliferation in vitro. The Akt and Erk signaling pathways were shown to be involved in Cd226-/- Treg proliferation and function in vivo and in vitro. These findings collectively indicate that CD226 is a key molecule regulating the Treg-mediated suppression of autoimmune responses by inhibiting Treg proliferation. Thus, the results of this study identify additional mechanisms by which CD226 regulates Treg functions in EAE and supports the potential therapeutic effects of anti-CD226 molecules on autoimmune diseases.

Thimet Oligopeptidase Biochemical and Biological Significances: Past, Present, and Future Directions.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15, THOP1) is a metallopeptidase ubiquitously distributed in mammalian tissues. Beyond its previously well characterized role in major histocompatibility class I (MHC-I) antigen presentation, the recent characterization of the THOP1 C57BL6/N null mice (THOP1-/-) phenotype suggests new key functions for THOP1 in hyperlipidic diet-induced obesity, insulin resistance and non-alcoholic liver steatosis. Distinctive levels of specific intracellular peptides (InPeps), genes and microRNAs were observed when comparing wild type C57BL6/N to THOP1-/- fed either standard or hyperlipidic diets. A possible novel mechanism of action was suggested for InPeps processed by THOP1, which could be modulating protein-protein interactions and microRNA processing, thus affecting the phenotype. Together, research into the biochemical and biomedical significance of THOP1 suggests that degradation by the proteasome is a step in the processing of various proteins, not merely for ending their existence. This allows many functional peptides to be generated by proteasomal degradation in order to, for example, control mRNA translation and the formation of protein complexes.

Inhibition of Bruton's tyrosine kinase interferes with pathogenic B-cell development in inflammatory CNS demyelinating disease.

Anti-CD20-mediated B-cell depletion effectively reduces acute multiple sclerosis (MS) flares. Recent data shows that antibody-mediated extinction of B cells as a lasting immune suppression, harbors the risk of developing humoral deficiencies over time. Accordingly, more selective, durable and reversible B-cell-directed MS therapies are needed. We here tested inhibition of Bruton's tyrosine kinase (BTK), an enzyme centrally involved in B-cell receptor signaling, as the most promising approach in this direction. Using mouse models of MS, we determined that evobrutinib, the first BTK inhibiting molecule being developed, dose-dependently inhibited antigen-triggered activation and maturation of B cells as well as their release of pro-inflammatory cytokines. Most importantly, evobrutinib treatment functionally impaired the capacity of B cells to act as antigen-presenting cells for the development of encephalitogenic T cells, resulting in a significantly reduced disease severity in mice. In contrast to anti-CD20, BTK inhibition silenced this key property of B cells in MS without impairing their frequency or functional integrity. In conjunction with a recent phase II trial reporting that evobrutinib is safe and effective in MS, our mechanistic data highlight therapeutic BTK inhibition as a landmark towards selectively interfering with MS-driving B-cell properties.

N-Acylethanolamine Acid Amidase contributes to disease progression in a mouse model of multiple sclerosis.

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these NaaaCD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in NaaaCD11b+ than in control mice. NaaaCD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS.

Interleukin-1 promotes autoimmune neuroinflammation by suppressing endothelial heme oxygenase-1 at the blood-brain barrier.

The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we studied the role of IL-1 signaling in blood-brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes Vcam1, Icam1 and Ackr1 (Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation.

Deubiquitinating enzymes (DUBs): DoUBle-edged swords in CNS autoimmunity.

Multiple sclerosis (MS) is the most common autoimmune disease of the CNS. The etiology of MS is still unclear but it is widely recognized that both genetic and environmental factors contribute to its pathogenesis. Immune signaling and responses are critically regulated by ubiquitination, a posttranslational modification that is promoted by ubiquitinating enzymes and inhibited by deubiquitinating enzymes (DUBs). Genome-wide association studies (GWASs) identified that polymorphisms in or in the vicinity of two human DUB genes TNFAIP3 and USP18 were associated with MS susceptibility. Studies with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, have provided biological rationale for the correlation between these DUBs and MS. Additional studies have shown that other DUBs are also involved in EAE by controlling distinct cell populations. Therefore, DUBs are emerging as crucial regulators of MS/EAE and might become potential therapeutic targets for the clinical treatment of MS.

Positive allosteric modulation of indoleamine 2,3-dioxygenase 1 restrains neuroinflammation.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.

Oxidative Stress and Lymphocyte Alterations in Chronic Relapsing Experimental Allergic Encephalomyelitis in the Rat Hippocampus and Protective Effects of an Ethanolamine Phosphate Salt.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) exhibits neuropathological and immunological dysfunctions similar to those found in multiple sclerosis (MS) and has been used as an animal model of MS. Inflammatory infiltrates and oxidative stress have been linked to the development of both diseases. Ethanolamine plasmalogen derivates have been shown to be powerful antioxidants and immunomodulators. Therefore, the objective of this study was to analyse inflammatory infiltrates, the state of the oxidative defences and the possible protective effects of calcium, magnesium and phosphate ethanolamine (EAP) in the CR-EAE rat hippocampus. To this aim, we evaluated, by immunohistochemistry, T cell infiltrates, Iba-1+ (a marker of activated microglia) immunoreactivity and TUNEL (+) cells. We also measured the protein levels and activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione reductase (GR). In addition, reduced (GSH) and oxidized (GSSG) glutathione levels, lipid peroxidation and cholesterol as well as desmosterol content were determined. We found an increase in T cell infiltrates and Iba1+ immunoreactivity, lipid peroxidation, SOD, GP and GR activities as well as enhanced cholesterol levels and a decrease in CAT activity, GSH and desmosterol levels in the first and second attack in the CR-EAE rat hippocampus. Pretreatment of CR-EAE rats with EAP led to a delay in the onset of the clinical signs of the disease as well as a decrease in inflammatory infiltrates and alterations of the antioxidant defences in the hippocampus. Altogether, the present results suggest a protective role of EAP in the CR-EAE rat hippocampus.

Pharmacological Inhibition of Acid Sphingomyelinase Ameliorates Experimental Autoimmune Encephalomyelitis.

BACKGROUND/AIMS: Multiple sclerosis (MS) is one of the most common autoimmune disorders of the central nervous system (CNS) and the leading cause of neurological disability among young adults in the Western world. We have previously shown that the acid sphingomyelinase plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: We induced adoptively transferred EAE in wildtype and acid sphingomyelinase-deficient mice. In addition, we immunized mice with MOGaa35-55 to induce active EAE and treated the mice with amitriptyline, a functional inhibitor of the acid sphingomyelinase. We investigated symptoms of EAE, blood-brain barrier integrity and neuroinflammation. RESULTS: In the model of adoptively transferred EAE we demonstrate that expression of acid sphingomyelinase in the recipients rather than on transferred encephalitogenic T cells contributes to the clinical development of EAE symptoms. To test if pharmacological targeting of acid sphingomyelinase can be explored for the development of novel therapies for MS, we inhibited acid sphingomyelinase with amitriptyline in mice in which EAE was induced by active immunization. We demonstrate that pharmacological inhibition of acid sphingomyelinase using amitriptyline protects against the development of EAE and markedly attenuates the characteristic detrimental neuroinflammatory response. CONCLUSION: The studies identify the acid sphingomyelinase as a novel therapeutic target for treating MS patients.

Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation.

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.