Immunodominance in the TCR repertoire of a [corrected] TCR peptide-specific CD4+ Treg population that controls experimental autoimmune encephalomyelitis

Immunodominance in self-Ag-reactive pathogenic CD4(+) T cells has been well established in several experimental models. Although it is clear that regulatory lymphocytes (Treg) play a crucial role in the control of autoreactive cells, it is still not clear whether immunodominant CD4(+) Treg clones are also involved in control of autoreactivity. We have shown that TCR-peptide-reactive CD4(+) and CD8(+) Treg play an important role in the spontaneous recovery and resistance from reinduction of experimental autoimmune encephalomyelitis in B10.PL mice. We report, by sequencing of the TCR alpha- and beta-chain associated with CD4(+) Treg, that the TCR repertoire is limited and the majority of CD4(+) Treg use the TCR Vbeta14 and Valpha4 gene segments. Interestingly, sequencing and spectratyping data of cloned and polyclonal Treg populations revealed that a dominant public CD4(+) Treg clonotype expressing Vbeta14-Jbeta1.2 with a CDR3 length of 7 aa exists in the naive peripheral repertoire and is expanded during the course of recovery from experimental autoimmune encephalomyelitis. Furthermore, a higher frequency of CD4(+) Treg clones in the naive repertoire correlates with less severity and more rapid spontaneous recovery from disease in parental B10.PL or PL/J and (B10.PL x PL/J)F(1) mice. These findings suggest that unlike the Ag-nonspecific, diverse TCR repertoire among the CD25(+)CD4(+) Treg population, TCR-peptide-reactive CD4(+) Treg involved in negative feedback regulation of autoimmunity use a highly limited TCR V-gene repertoire. Thus, a selective set of immunodominant Treg as well as pathogenic T cell clones can be targeted for potential intervention in autoimmune disease conditions.     

Synaptic plasticity in multiple sclerosis and in experimental autoimmune encephalomyelitis

Approximately half of all patients with multiple sclerosis (MS) experience cognitive dysfunction, including learning and memory impairment. Recent studies suggest that hippocampal pathology is involved, although the mechanisms underlying these deficits remain poorly understood. Evidence obtained from a mouse model of MS, the experimental autoimmune encephalomyelitis (EAE), suggests that in the hippocampus of EAE mice long-term potentiation (LTP) is favoured over long-term depression in response to repetitive synaptic activation, through a mechanism dependent on enhanced IL-1β released from infiltrating lymphocytes or activated microglia. Facilitated LTP during an immune-mediated attack might underlie functional recovery, but also cognitive deficits and excitotoxic neurodegeneration. Having identified that pro-inflammatory cytokines such as IL-1β can influence synaptic function and integrity in early MS, it is hoped that new treatments targeted towards preventing synaptic pathology can be developed.     

Cross-reactivity of myelin basic protein-specific T cells with multiple microbial peptides: experimental autoimmune encephalomyelitis induction in TCR transgenic mice.

Activation of autoreactive T cells is a crucial event in the pathogenesis of autoimmune diseases. Cross-reactivity between microbial and self Ags (molecular mimicry) is one hypothesis that could explain the activation of autoreactive T cells. We have systematically examined this hypothesis in experimental autoimmune encephalomyelitis using mice bearing exclusively myelin basic protein (MBP)-specific T cells (designated T+ alpha-). A peptide substitution analysis was performed in which each residue of the MBPAc1-11 peptide was exchanged by all 20 naturally occurring amino acids. This allowed the definition of the motif (supertope) that is recognized by the MBPAc1-11-specific T cells. The supertope was used to screen protein databases (SwissProt and TREMBL). By the search, 832 peptides of microbial origin were identified and synthesized. Of these, 61 peptides induced proliferation of the MBPAc1-11-specific transgenic T cells in vitro. Thus, the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only two native MBP-residues were sufficient to activate MBPAc1-11-specific T cells in vitro, and experimental autoimmune encephalomyelitis could be induced by immunizing mice with a mimic peptide with only four native MBP residues.     

Fcgamma receptor-ligating complexes improve the course of experimental autoimmune encephalomyelitis by enhancing basal Th2 responses

IL-12p40 and macrophages are essential for the induction of disease in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. In this paper, we show that treatment of mice with opsonized erythrocytes, which have been shown to ligate Fcgamma receptors on macrophages and alter their cytokine profile, significantly delayed the onset of experimental autoimmune encephalomyelitis. This protection correlated to the induction of Th2 responses by autoreactive T cells, enhanced basal systemic responses and a significant downregulation of IL-12p40 and nitric oxide synthase-2, but not IFN-gamma expression. IL-4 was essential for the protection by opsonized erythrocytes as the effects of treatment were eliminated in IL-4-deficient mice. Together these studies suggest that the ligation of Fcgamma receptors can modify the development of autoimmune disease by altering macrophage activation and enhancing Th2 responses.     

Impaired cell volume regulation in taurine deficient cultured astrocytes

Taurine concentration was reduced by 40 and 65%, respectively in rat cerebellar astrocytes grown in a chemically defined medium or in culture medium containing a blocker of taurine transport (GES). Cell volume in these taurine deficient cells was 10%–16% higher than in controls. When challenged by hyposmotic conditions, astrocytes release taurine and this efflux contributes to the volume regulatory decrease observed in these cells. Taurine deficient astrocytes showed a less efficient volume recovery as compared to controls with normal taurine levels. Exposed to 50% hyposmotic medium, astrocytes with normal taurine concentration recovered 60% of their original volume whereas taurine deficient cells recovered only 30–35%. Similarly, in 30% hyposmotic medium, taurine deficient astrocytes recovered only 40% as compared to 75% in controls. No compensatory increases in the efflux of other osmolytes (free amino acids or potassium) were observed during regulatory volume decrease in taurine deficient astrocytes.     

The inflamed central nervous system drives the activation and rapid proliferation of Foxp3+ regulatory T cells

Resolution of experimental autoimmune encephalomyelitis requires a large cohort of Foxp3(+) regulatory T cells (Tregs) within the CNS. In this study, we have used the passive transfer of murine experimental autoimmune encephalomyelitis using myelin-reactive T cells to study the development of this Treg response. Rapid proliferation of Tregs within the CNS (which is not seen in lymphoid organs) drives a switch in the balance of CNS proliferation from T effectors to Tregs, correlating with recovery. This proliferative burst drives a local over-representation of Vbeta8(+) Tregs in the CNS, indicative of an oligoclonal expansion. There is also evidence for a small, but detectable, myelin oligodendrocyte glycoprotein-reactive Treg component expanded without prior immunization. Furthermore, CNS-derived Tregs, taken during recovery, suppressed the proliferation of CNS-derived effectors in response to myelin oligodendrocyte glycoprotein. Under these conditions, Tregs could also limit the level of IFN-gamma production, but not IL-17 production, by CNS-derived effectors. These data establish the CNS as an environment that permits extensive Treg proliferation and are the first to demonstrate Treg expansion specifically within the tissues during the natural resolution of autoimmune inflammation.     

Effects of complement C5 on apoptosis in experimental autoimmune encephalomyelitis

Complement activation is involved in the initiation of Ab-mediated inflammatory demyelination in experimental autoimmune encephalomyelitis (EAE). At a sublytic dose, the C5b-9 membrane attack complex protects oligodendrocytes (OLG) from apoptosis. Using C5-deficient (C5-d) mice, we previously showed a dual role for C5: enhancement of inflammatory demyelination in acute EAE, and promotion of remyelination during recovery. In this study, we investigated the role of C5 in apoptosis in myelin-induced EAE. In acute EAE, C5-d and C5-sufficient (C5-s) mice had similar numbers of total apoptotic cells, whereas C5-s had significantly fewer than C5-d during recovery. In addition, although both groups of mice displayed TUNEL(+) OLG, there were significantly fewer in C5-s than in C5-d during both acute EAE and recovery. Gene array and immunostaining of apoptosis-related genes showed that Fas ligand expression was higher in C5-s. In C5-s mice, Fas(+) cells were also higher than in C5-d mice in acute EAE; however, these cells were significantly reduced during recovery. Together, these findings are consistent with the role of C5, possibly by forming the membrane attack complex, in limiting OLG apoptosis in EAE, thus promoting remyelination during recovery.     

Plasma levels and renal handling of endogenous amino acids in snakes: a comparative study

Plasma levels of 22 endogenous amino acids were measured by ion-exchange chromatography in four species of snakes: Thamnophis sirtalis, T. radix, Aipysurus laevis, and Python molurus. Despite considerable interspecific variation in the amino acid composition, all species showed relatively high plasma concentrations of histidine, a feature apparently unique to reptiles. The renal handling of these amino acids was studied by renal clearance methods. As in other vertebrates, net tubular absorption of filtered amino acids predominated. However, net tubular secretion of taurine, cysteic acid and/or phosphoserine and beta-alanine was observed, with taurine being the predominant amino acid secreted. The percentage reabsorption of the total amino acids filtered by the snake kidneys ranged from 79 to 95%. Evidence for the postrenal absorption of amino acids in these reptiles is presented. In species that normally undergo hibernation (Thamnophis spp.), the ability of the kidney to reabsorb amino acids was depressed by cold acclimation. Cold acclimation significantly decreased plasma levels of all amino acids except taurine, whose concentration increased. The increase in plasma taurine level may have resulted from cellular osmoregulation. Under these conditions, renal excretion of taurine increased concomitantly with the increase in plasma taurine concentration.     

Direct and indirect effects of microbiota-derived metabolites on neuroinflammation in multiple sclerosis

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are highly influenced by changes in the microbiota and of microbiota-derived metabolites, including short chain fatty acids, bile acids, and tryptophan derivatives. This review will discuss the effects of microbiota-derived metabolites on neuroinflammation driven by central nervous system-resident cells and peripheral immune cells, and their influence on outcomes of EAE and MS.     

Deficiency of fatty acid-binding proteins in mice confers protection from development of experimental autoimmune encephalomyelitis

Fatty acid-binding proteins (FABPs) act as intracellular receptors for a variety of hydrophobic compounds, enabling their diffusion within the cytoplasmic compartment. Recent studies have demonstrated the ability of FABPs to simultaneously regulate metabolic and inflammatory pathways. We investigated the role of adipocyte FABP and epithelial FABP in the development of experimental autoimmune encephalomyelitis to test the hypothesis that these FABPs impact adaptive immune responses and contribute to the pathogenesis of autoimmune disease. FABP-deficient mice exhibited a lower incidence of disease, reduced clinical symptoms of experimental autoimmune encephalomyelitis and dramatically lower levels of proinflammatory cytokine mRNA expression in CNS tissue as compared with wild-type mice. In vitro Ag recall responses of myelin oligodendrocyte glycoprotein 35-55-immunized FABP(-/-) mice showed reduced proliferation and impaired IFN-gamma production. Dendritic cells deficient for FABPs were found to be poor producers of proinflammatory cytokines and Ag presentation by FABP(-/-) dendritic cells did not promote proinflammatory T cell responses. This study reveals that metabolic-inflammatory pathway cross-regulation by FABPs contributes to adaptive immune responses and subsequent autoimmune inflammation.     

Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system

Immune regulation of autoimmune disease can function at two sites: at the secondary lymphoid organs or in the target organ itself. In this study, we investigated the natural resolution of autoimmune pathology within the CNS using murine experimental autoimmune encephalomyelitis (EAE). Recovery correlates with the accumulation of IL-10-producing CD4+CD25+ T cells within the CNS. These CD4+CD25+ cells represent as many as one in three of CD4+ cells in the CNS during recovery, they are FoxP3+ and express other markers associated with regulatory cells (CTLA-4, GITR, and alpha(E)beta7), and they have regulatory function ex vivo. Depletion of CD25+ cells inhibits the natural recovery from EAE. Also, depletion of CD25+ cells after recovery removes the resistance to reinduction of EAE observed in this model. Furthermore, passive transfer of CNS-derived CD4+CD25+ cells in low numbers provides protection from EAE in recipient mice. These are the first data demonstrating the direct involvement of CD4+CD25+ regulatory T cells in the natural resolution of autoimmune disease within the target organ.     

Cutting edge: central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis

Plasmacytoid dendritic cells (pDCs) have both stimulatory and regulatory effects on T cells. pDCs are a major CNS-infiltrating dendritic cell population during experimental autoimmune encephalomyelitis but, unlike myeloid dendritic cells, have a minor role in T cell activation and epitope spreading. We show that depletion of pDCs during either the acute or relapse phases of experimental autoimmune encephalomyelitis resulted in exacerbation of disease severity. pDC depletion significantly enhanced CNS but not peripheral CD4(+) T cell activation, as well as IL-17 and IFN-gamma production. Moreover, CNS pDCs suppressed CNS myeloid dendritic cell-driven production of IL-17, IFN-gamma, and IL-10 in an IDO-independent manner. The data demonstrate that pDCs play a critical regulatory role in negatively regulating pathogenic CNS CD4(+) T cell responses, highlighting a new role for pDCs in inflammatory autoimmune disease.     

The role of macrophage subpopulations in autoimmune disease of the central nervous system

Summary In this review the role of various subpopulations of macrophages in the pathogenesis of experimental autoimmune encephalomyetitis is discussed. Immunohistochemistry with macrophage markers shows that in this disease different populations of macrophages (i.e. perivascular cells, microglia and infiltrating blood-borne macrophages) are present in the central nervous system. These subpopulations partially overlap in some functional activity while other activities seem to be restricted to a distinct subpopulation, indicating that these subpopulations have different roles in the pathogenesis of encephalomyelitis. The studies discussed in this review reveal that immunocytochemical and morphological studies, combined with new techniques such asin situ nick translation and experimental approaches like the use of bone marrow chimeras and macrophage depletion techniques, give valuable information about the types and functions of cells involved in central nervous system inflammation. The review is divided in three parts. In the first part the experimental autoimmune encephalomyelitis model is introduced. The second part gives an overview of the origin, morphology and functions of the various subpopulations. In the third part the role of these subpopulations is discussed in relation to the various stages (i.e. preclinical, clinical and recovery) of the experimental disease.     

GluR2-free alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors intensify demyelination in experimental autoimmune encephalomyelitis

We adopted a genetic approach to test the importance of edited GluR2-free, Ca(2+)-permeable, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the pathophysiology of experimental autoimmune encephalomyelitis, an inflammatory demyelinative disorder resembling multiple sclerosis. Initial studies showed that oligodendroglial lineage cells from mice lacking functional copies of the gene encoding the GluR3 AMPA receptor subunit (Gria3) had a diminished capacity to assemble edited GluR2-free AMPA receptors, and were resistant to excitotoxicity in vitro. Neurological deficits and spinal cord demyelination elicited by immunization with myelin oligodendrocyte glycoprotein peptide were substantially milder in these Gria3 mutant mice than in their wild-type littermates. These results support the hypothesis that oligodendroglial excitotoxicity mediated by AMPA receptors that do not contain edited GluR2 subunits contributes to demyelination in experimental autoimmune encephalomyelitis, and suggest that inhibiting these Ca(2+)-permeable AMPA receptors would be therapeutic in multiple sclerosis.     

Changes in plasma and urinary taurine and amino acids in runners immediately and 24 h after a marathon

Summary. Changes in urinary and plasma taurine and amino acids have been evaluated in trained runners competing in the Rotterdam Marathon, 1998, both immediately after completing the event and 24 h after recovery. There were significant changes in the urinary amino acids excretion, the majority showing a significant decrease both immediately at the completion of the Marathon and after 24 h recovery. In contrast urinary taurine excretion increased immediately post Marathon, although not significantly as the range of results was wide. Such changes in urinary taurine correlated with percentage changes in plasma creatine kinase both immediately post race, (r = 0.972, P < 0.001), and 24 h later (r = 0.872, P < 0.001), possibly indicating that the source of the taurine was muscle. Significant correlations between the individual values for urinary and plasma amino acids in all of the athletes were calculated for taurine (r = 0.528), glycine (r = 0.853), threonine (r = 0.749), alanine (r = 0.747), serine (r = 0.620), glutamine (0.614), arginine (r = 0.507), histidine (r = 0.470) and valine (r = 0.486). Changes in the mean plasma concentrations of amino acids were comparable to our previously published data (Ward et al., 1999) the majority showing significant decreases immediately and 24 h post Marathon, such an adaptation being due primarily to their utilisation for gluconeogenesis. However, in contrast, the mean taurine concentrations were significantly elevated both post race, P < 0.01 and after 24 h, P < 0.05. The physiological response by the muscle to exhaustive exercise, particularly with regard to changes in plasma and urinary taurine concentrations remain to be elucidated, but is probably related to muscle function impairment. The increase in taurine urinary excretion could be used as an indicator of muscle damage occurring during exhaustive exercise. Whether taurine supplementation would minimise such changes is an interesting scientific question and merits investigation. Received January 6, 2000 / Accepted February 1, 2000     

Comparison of biliary excretion and metabolism of lithocholic acid and its sulfate and glucuronide conjugates in rats

Biliary excretion and biotransformation of tracer doses of [14C]lithocholic acid and its sulfate and glucuronide intravenously injected into bile-drainaged rats were compared. Biliary excretion efficiency was in the order of unconjugate sulfate glucuronide and all conjugates were completely excreted into bile within 60 min after injection. Only tracer doses of radioactivity were found in the liver and urine. About 90% of radiolabeled bile acids in bile were conjugated with taurine immediately after injection of lithocholic acid, whereas lithocholic acid-glucuronide was only partly conjugated with taurine all the time (less than 6%) and excreted into bile mainly as native compound. In the first 10 min, 66% of lithocholic acid-sulfate was conjugated with taurine and it gradually proceeded up to 87%. Hydroxylation at C-6 and C-7 positions of lithocholic acid proceeded time-dependently up to 45%. No hydroxylation was observed with lithocholic acid-sulfate or glucuronide. Differences of biliary excretion rate of these conjugates may be one of the reasons for the delayed decrease of sulfated and glucuronidated bile acids in serum after bile drainage to patients with obstructive jaundice of during the recovery of acute hepatitis than non-esterified bile acids.     

Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production

Males are less susceptible than females to experimental autoimmune encephalomyelitis and many other autoimmune diseases. Gender differences in cytokine production have been observed in splenocytes of experimental autoimmune encephalomyelitis mice stimulated with myelin proteins and may underlie gender differences in susceptibility. As these differences should not be limited to responses specific for myelin proteins, gender differences in cytokine production upon stimulation with Ab to CD3 were examined, and the mechanisms were delineated. Splenocytes from male mice stimulated with Ab to CD3 produced more IL-10 and IL-4 and less IL-12 than those from female mice. Furthermore, splenocytes from dihydrotestosterone (DHT)-treated female mice produced more IL-10 and less IL-12 than those from placebo-treated female mice, whereas there was no difference in IL-4. IL-12 knockout mice were then used to determine whether changes in IL-10 production were mediated directly by testosterone vs indirectly by changes in IL-12. The results of these experiments favored the first hypothesis, because DHT treatment of female IL-12 knockout mice increased IL-10 production. To begin to delineate the mechanism by which DHT may be acting, the cellular source of IL-10 was determined. At both the RNA and protein levels, IL-10 was produced primarily by CD4+ T lymphocytes. CD4+ T lymphocytes were then shown to express the androgen receptor, raising the possibility that testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production. In vitro experiments demonstrated increased IL-10 production following treatment of CD4+ T lymphocytes with DHT. Thus, testosterone can act directly via androgen receptors on CD4+ T lymphocytes to increase IL-10 gene expression.     

Roles of TNF-related apoptosis-inducing ligand in experimental autoimmune encephalomyelitis

TRAIL, the TNF-related apoptosis-inducing ligand, induces apoptosis of tumor cells, but not normal cells; the roles of TRAIL in nontransformed tissues are unknown. Using a soluble TRAIL receptor, we examined the consequences of TRAIL blockade in an animal model of multiple sclerosis. We found that chronic TRAIL blockade in mice exacerbated experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. The exacerbation was evidenced primarily by increases in disease score and degree of inflammation in the CNS. Interestingly, the degree of apoptosis of inflammatory cells in the CNS was not affected by TRAIL blockade, suggesting that TRAIL may not regulate apoptosis of inflammatory cells in experimental autoimmune encephalomyelitis. By contrast, myelin oligodendrocyte glycoprotein-specific Th1 and Th2 cell responses were significantly enhanced in animals treated with the soluble TRAIL receptor. Based on these observations, we conclude that unlike TNF, which promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis and prevents activation of autoreactive T cells.     

Cutting edge: Attenuated experimental autoimmune encephalomyelitis in eta-1/osteopontin-deficient mice

Recent studies indicate that early T lymphocyte activation 1 (Eta-1), also known as osteopontin, is a cytokine contributing to the development of Th1 immunity. In the present report, the role of Eta-1 in experimental autoimmune encephalomyelitis (EAE), a disease associated with Th1 immunity, was examined by analysis of disease progression in Eta-1-deficient (Eta-1-/-) mice. Although incidence and onset of peptide-induced EAE were found to be similar in Eta-1-/- and Eta-1+/+ mice, Eta-1-/- mice displayed significantly lower mean maximal clinical score and faster recovery without spontaneous relapses. Accordingly, decreased inflammatory infiltration and demyelination were observed in the spinal cords of Eta-1-/- mice. Furthermore, in comparison to Eta-1+/+, Eta-1-/- CD4+ T cells had reduced expression of IFN-gamma and TNF-alpha upon ex vivo restimulation. Taken together, these results suggest that Eta-1 may sustain autoimmune responses by assisting in maintenance of Th1 immunity during EAE.