Shuo Yu

Suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated T cells.

Excessive inflammation occurs during infection and autoimmunity in mice lacking the α-subunit of the interleukin 27 (IL-27) receptor. The molecular mechanisms underlying this increased inflammation are incompletely understood. Here we report that IL-27 upregulated IL-10 in effector T cells that produced interferon-γ and expressed the transcription factor T-bet but did not express the transcription factor Foxp3. These IFN-γ+T-bet+Foxp3− cells resembled effector T cells that have been identified as the main source of host-protective IL-10 during inflammation. IL-27-induced production of IL-10 was associated with less secretion of IL-17, and exogenous IL-27 reduced the severity of adoptively transferred experimental autoimmune encephalomyelitis by a mechanism dependent on IL-10. Our data show that IL-27-induced production of IL-10 by effector T cells contributes to the immunomodulatory function of IL-27.

IL-12p35-deficient mice are susceptible to experimental autoimmune encephalomyelitis : evidence for redundancy in the IL-12 system in the induction of central nervous system autoimmune demyelination

Experimental autoimmune encephalomyelitis (EAE) serves as a model for multiple sclerosis and is considered a CD4+, Th1 cell-mediated autoimmune disease. IL-12 is a heterodimeric cytokine, composed of a p40 and a p35 subunit, which is thought to play an important role in the development of Th1 cells and can exacerbate EAE. We induced EAE with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 (MOG35–55) in C57BL/6 mice and found that while IL-12p40-deficient (−/−) mice are resistant to EAE, IL-12p35−/− mice are susceptible. Typical spinal cord mononuclear cell infiltration and demyelination were observed in wild-type and IL-12p35−/− mice, whereas IL-12p40−/− mice had normal spinal cords. A Th1-type response to MOG35–55 was observed in the draining lymph node and the spleen of wild-type mice. A weaker MOG35–55-specific Th1 response was observed in IL-12p35−/− mice, with lower production of IFN-γ. By contrast, a Th2-type response to MOG35–55 correlated with disease resistance in IL-12p40−/− mice. Production of TNF-α by microglia, CNS-infiltrating macrophages, and CD4+ T cells was detected in wild-type and IL-12p35−/−, but not in IL-12p40−/−, mice. In addition, NO production was higher in IL-12p35−/− and wild-type mice than in IL-12p40−/− mice. These data demonstrate a redundancy of the IL-12 system in the induction of EAE and suggest that p40-related heterodimers, such as the recently cloned IL-23 (p40p19), may play an important role in disease pathogenesis.

Induction of Experimental Autoimmune Encephalomyelitis in IL-12 Receptor-β2-Deficient Mice: IL-12 Responsiveness Is Not Required in the Pathogenesis of Inflammatory Demyelination in the Central Nervous System

IL-12 is thought to be involved in the susceptibility to experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. IL-12 signals through a heterodimeric receptor (IL-12Rβ1/IL-12Rβ2), whose β2-chain is up-regulated on activated, autoreactive Th1 cells. Contrary to the expectation that the absence of IL-12Rβ2 would protect from EAE, we found that IL-12Rβ2-deficient mice developed earlier and more severe disease, with extensive demyelination and CNS inflammation. The inflammatory cells were mainly comprised of CD4+ T cells, monocyte/macrophages, and dendritic cells. Compared to wild-type mice, IL-12Rβ2-deficient mice exhibited significantly increased autoantigen-induced proliferative response and increased production of TNF-α, GM-CSF, IL-17, IL-18/IL-18Rα, and NO. In addition, we found significantly increased levels of IL-23p19 mRNA expression in spleen cells from immunized IL-12Rβ2−/− mice compared with wild-type mice. These findings indicate that IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS, and that, in the absence of IL-12Rβ2, increased IL-23 and other inflammatory molecules may be responsible for increased severity of EAE.

IDO Upregulates Regulatory T Cells via Tryptophan Catabolite and Suppresses Encephalitogenic T Cell Responses in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA–induced Tregs contribute to inhibition of Th17 cells. By using a DC–T cell coculture, we found that 3-HAA–treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28–stimulated naive CD4+ T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.

Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis

Adult neural stem cells (aNSCs) derived from the subventricular zone of the brain show therapeutic effects in EAE, an animal model of the chronic inflammatory neurodegenerative disease MS; however, the beneficial effects are modest. One critical weakness of aNSC therapy may be an insufficient antiinflammatory effect. Here, we demonstrate that i.v. or i.c.v. injection of aNSCs engineered to secrete IL-10 (IL-10-aNSCs), a potent immunoregulatory cytokine, induced more profound functional and pathological recovery from ongoing EAE than that with control aNSCs. IL-10-aNSCs exhibited enhanced antiinflammatory effects in the periphery and inflammatory foci in the CNS compared with control aNSCs, more effectively reducing myelin damage, a hallmark of MS. When compared with mice treated with control aNSCs, those treated with IL-10-aNSCs demonstrated differentiation of transplanted cells into greater numbers of oligodendrocytes and neurons but fewer astrocytes, thus enhancing exogenous remyelination and neuron/axonal growth. Finally, IL-10-aNSCs converted a hostile environment to one supportive of neurons/oligodendrocytes, thereby promoting endogenous remyelination. Thus, aNSCs engineered to express IL-10 show enhanced ability to induce immune suppression, remyelination, and neuronal repair and may represent a novel approach that can substantially improve the efficacy of neural stem cell-based therapy in EAE/MS.

Differential effect of IL-27 on developing versus committed Th17 cells.

IL-27 counters the effect of TGF-β+IL-6 on naive CD4+ T cells, resulting in near complete inhibition of de novo Th17 development. In contrast, little is known about the effect of IL-27 on already differentiated Th17 cells. A better understanding of how IL-27 regulates these cells is needed to evaluate the therapeutic potential of IL-27 in Th17 cells-associated diseases. In this study, we show that IL-27 had surprisingly little effect on committed Th17 cells, despite its expression of a functional IL-27R. Contrary to de novo differentiation of Th17 cells, IL-27 did not suppress expression of retinoid-related orphan receptor (ROR)γt or RORα in committed Th17 cells. Consistent with this finding, the frequency of committed Th17 cells and their cytokine secretion remained unaffected by IL-27. Both memory Th17 cells (CD4+CD25−CD62Llow) that developed in vivo and encephalitogenic Th17 cells infiltrating the CNS of mice developing experimental autoimmune encephalomyelitis produced similar amounts of IL-17A when reactivated with IL-23 in the absence and presence of exogenous IL-27. Finally, IL-27 failed to suppress encephalitogenicity of Th17 cells in an adoptive transfer of experimental autoimmune encephalomyelitis. Analysis ex vivo of transferred Th17 cells in the spleen and CNS of recipient mice showed that cells retained similar phenotype irrespective of whether cells were treated or not with IL-27. Our data demonstrate that in contrast to inhibition of de novo differentiation of Th17 cells, IL-27 has little or no effect on committed Th17 cells. These findings indicate that therapeutic applications of IL-27 might have a limited efficacy in inflammatory conditions where aggressive Th17 responses have already developed.

Role of IL-12 Receptor β1 in Regulation of T Cell Response by APC in Experimental Autoimmune Encephalomyelitis

IL-12 was thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. However, we have recently found that IL-12 responsiveness, via IL-12Rβ2, is not required in the induction of EAE. To determine the role of IL-12Rβ1, a key subunit for the responsiveness to both IL-12 and IL-23, in the development of autoimmune diseases, we studied EAE in mice deficient in this subunit of IL-12R. IL-12Rβ1−/− mice are completely resistant to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with an autoantigen-specific Th2 response. To study the mechanism underlying this Th2 bias, we cocultured purified CD4+ T cells and APCs of MOG-immunized mice. We demonstrate that IL-12Rβ1−/− APCs drive CD4+ T cells of both wild-type and IL-12Rβ1−/− mice to an Ag-induced Th2 phenotype, whereas wild-type APCs drive these CD4+ T cells toward a Th1 type. IL-12Rβ1−/− CD4+ T cells, in turn, appear to exert an immunoregulatory effect on the capacity of wild-type APCs to produce IFN-γ and TNF-α. Furthermore, decreased levels of IL-12p40, p35, and IL-23p19 mRNA expression were found in IL-12Rβ1−/− APCs, indicating an autocrine pathway of IL-12/IL-23 via IL-12Rβ1. IL-18 production and IL-18Rα expression are also significantly decreased in IL-12Rβ1−/− mice immunized with MOG. We conclude that in the absence of IL-12Rβ1, APCs play a prominent regulatory role in the induction of autoantigen-specific Th2 cells.

Evaluation of Bone Marrow- and Brain-Derived Neural Stem Cells in Therapy of Central Nervous System Autoimmunity

Adult subventricular zone (SVZ)-derived neural stem cells (NSCs) have therapeutic effects in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, SVZ precursor cells as a source of NSCs are not readily accessible for clinical application. In the present study, we demonstrate that NSCs derived from bone marrow (BM) cells exhibit comparable morphological properties as those derived from SVZ cells and possess a similar ability to differentiate into neurons, astrocytes, and oligodendrocytes both in vitro and in vivo. Importantly, both types of NSCs suppressed chronic experimental autoimmune encephalomyelitis to a comparable extent on transplantation. Mechanisms underlying the therapeutic effects of NSCs include immunomodulation in the periphery and the central nervous system (CNS), neuron/oligodendrocyte repopulation by transplanted cells, and enhanced endogenous remyelination and axonal recovery. Furthermore, we provide evidence for the trans-differentiation of transplanted BM-NSCs into neural cells in the CNS, while no fusion of these cells with host neural cells was detected. This is the first study that directly compares SVZ- versus BM-NSCs with regard to in vivo neural differentiation and anti-inflammatory and therapeutic effects on CNS inflammatory demyelination. Their virtually identical therapeutic potential, greater accessibility, and autologous properties make BM-NSCs a novel and highly applicable substitute for SVZ-NSCs in cell-based multiple sclerosis therapies.

Glucosamine Abrogates the Acute Phase of Experimental Autoimmune Encephalomyelitis by Induction of Th2 Response

Glucosamine, a natural glucose derivative and an essential component of glycoproteins and proteoglycans, has been safely used to relieve osteoarthritis in humans. Recent studies have shown that glucosamine also possesses immunosuppressive properties and is effective in prolonging graft survival in mice. Whether this reagent is effective in human multiple sclerosis (MS), an inflammatory demyelination in the CNS, is not known. We thus investigated the therapeutic effect of glucosamine on experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We demonstrated that oral, i.p., or i.v. administration of glucosamine significantly suppressed acute EAE, with reduced CNS inflammation and demyelination. A significant, albeit not strong, blockade of Th1 response and an up-regulation of Th2 cytokines (IL-5 and IL-10) are observed in the splenocytes of glucosamine-treated mice. Glucosamine also regulates IL-5 and IL-10 in vitro. As glucosamine is able to effectively suppress acute EAE, has low or absent toxicity, and has been safely used in humans orally, our study suggests a potential use for this drug alone or in combination with other disease-modifying immunotherapies to enhance their efficacy and reduce their doses in MS and possibly other autoimmune disorders. Furthermore, because glucosamine functions not simply as an immunosuppressant, but as a mild immunomodulator, administration of glucosamine provides a novel immunoregulatory approach for autoimmune disorders.

IL-12Rβ2 Promotes the Development of CD4+CD25+ Regulatory T Cells

We have previously shown that mice lacking the IL-12-specific receptor subunit β2 (IL-12Rβ2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rβ2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rβ2−/− mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rβ2-deficient mice to autoimmune diseases. T cells from IL-12Rβ2−/− mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25+CD4+ regulatory T cells (Tregs) in the thymus and spleen of IL-12Rβ2−/− mice were comparable to those of WT mice. However, IL-12Rβ2−/− mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-β, as shown by significantly lower numbers of CD25+CD4+ T cells that expressed Foxp3. Functionally, CD25+CD4+ Tregs derived from IL-12Rβ2−/− mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rβ2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rβ2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway.