Gamma delta T cells regulate the extent and duration of inflammation in the central nervous system by a Fas ligand-dependent mechanism

Gamma delta T cells have been shown to regulate immune responses associated with inflammation, but the mechanism of this regulation is largely unknown. Using the experimental autoimmune encephalomyelitis (EAE) model of the human CNS autoimmune disease multiple sclerosis, we demonstrate that gamma delta T cells are important regulators of CNS inflammation. This was shown using gamma delta T cell-deficient mice that were unable to recover from EAE. The chronic disease was accompanied by a prolonged presence of both macrophages and lymphocytes in the CNS. This extended inflammatory response was due to alterations in both cell proliferation and death. In mice lacking gamma delta T cells, proliferation of encephalitogenic T cells was 3-fold higher, and caspase activity, indicating apoptosis, was 2-fold lower compared with those in control mice recovering from EAE. gamma delta T cell-deficient mice reconstituted with wild-type gamma delta T cells recovered from EAE and resolved inflammation in the CNS, whereas mice reconstituted with Fas ligand-dysfunctional gamma delta T cells did not. Thus, gamma delta T cells regulate both inflammation in the CNS and disease recovery via Fas/Fas ligand-induced apoptosis of encephalitogenic T cells, and a quick resolution of inflammation in the CNS is essential to prevent permanent damage to the CNS resulting in chronic disease.     

Gamma delta T cell regulation of IFN-gamma production by central nervous system-infiltrating encephalitogenic T cells: correlation with recovery from experimental autoimmune encephalomyelitis

Interferon-gamma has been shown to be important for the resolution of inflammation associated with CNS autoimmunity. Because one of the roles of gamma delta T cells is the regulation of inflammation, we asked whether gamma delta T cells were able to regulate CNS inflammation using the autoimmune disease mouse model experimental autoimmune encephalomyelitis (EAE). We show that the presence of gamma delta T cells was needed to promote the production of IFN-gamma by both CD4 and CD8 T cells in the CNS before the onset of EAE. This regulation was shown to be independent of the ability of gamma delta T cells to produce IFN-gamma, and was specific to T cells in the CNS, as no alterations in IFN-gamma production were detectable in gamma delta T cell-deficient mice in the spleen and lymph nodes of mice with EAE or following immunization. Analysis of TCR gamma delta gene usage in the CNS showed that the only TCR delta V gene families present in the CNS before EAE onset are from the DV7s6 and DV105s1 gene families. We also show that the primary IFN-gamma-producing cells in the CNS are the encephalitogenic T cells, and that gamma delta T cell-deficient mice are unable to resolve EAE disease symptoms like control mice, thus exhibiting a long-term chronic disease course similar to that observed in IFN-gamma-deficient mice. These data suggest that CNS resident gamma delta T cells promote the production of IFN-gamma by encephalitogenic T cells in the CNS, which is ultimately required for the recovery from EAE.     

Chloroquine Treatment Enhances Regulatory T Cells and Reduces the Severity of Experimental Autoimmune Encephalomyelitis

Background

The modulation of inflammatory processes is a necessary step, mostly orchestrated by regulatory T (Treg) cells and suppressive Dendritic Cells (DCs), to prevent the development of deleterious responses and autoimmune diseases. Therapies that focused on adoptive transfer of Treg cells or their expansion in vivo achieved great success in controlling inflammation in several experimental models. Chloroquine (CQ), an anti-malarial drug, was shown to reduce inflammation, although the mechanisms are still obscure. In this context, we aimed to access whether chloroquine treatment alters the frequency of Treg cells and DCs in normal mice. In addition, the effects of the prophylactic and therapeutic treatment with CQ on Experimental Autoimmune Encephalomyelitis (EAE), an experimental model for human Multiple Sclerosis, was investigated as well.

Methodology/Principal Findings

EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG_35–55) peptide. C57BL/6 mice were intraperitoneally treated with chloroquine. Results show that the CQ treatment provoked an increase in Treg cells frequency as well as a decrease in DCs. We next evaluated whether prophylactic CQ administration is capable of reducing the clinical and histopathological signs of EAE. Our results demonstrated that CQ-treated mice developed mild EAE compared to controls that was associated with lower infiltration of inflammatory cells in the central nervous system CNS) and increased frequency of Treg cells. Also, proliferation of MOG_35–55-reactive T cells was significantly inhibited by chloroquine treatment. Similar results were observed when chloroquine was administrated after disease onset.

Conclusion

We show for the first time that CQ treatment promotes the expansion of Treg cells, corroborating previous reports indicating that chloroquine has immunomodulatory properties. Our results also show that CQ treatment suppress the inflammation in the CNS of EAE-inflicted mice, both in prophylactic and therapeutic approaches. We hypothesized that the increased number of regulatory T cells induced by the CQ treatment is involved in the reduction of the clinical signs of EAE.     

Ingested (oral) rituximab inhibits EAE

BackgroundBlocking CD20 can inhibit autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA).ObjectiveWe examined whether an antibody against CD20, rituximab (RTX) (Rituxan®), used clinically in oncology, MS and RA would have similar anti-inflammatory effects in EAE after oral administration.Design/methodsB6 mice were immunized with MOG peptide 35–55 and gavaged with control saline or RTX during ongoing disease. Splenocytes or CD4⁺ T cells from control fed or RTX fed mice were adoptively transferred into active MOG peptide 35–55 immunized recipient mice during ongoing disease. Actively fed and recipient mice were examined for disease inhibition, inflammation, and cytokine responses.ResultsIngested (oral) RTX inhibited ongoing disease and decreased inflammation. Adoptively transferred cells from RTX fed donors protected against actively induced disease and decreased inflammation. There was a decrease in Th1-like cytokines IFN-γ and IL-12, IL-17 and TNF-α in active fed and adoptively treated recipients without upregulation of counter-regulatory cytokines.ConclusionsIngested (orally administered) RTX can inhibit disease, CNS inflammation, decrease pro-inflammatory IL-17 and Th1-like cytokines without increases in Th2-like anti-inflammatory cytokines.     

IFN-gamma shapes immune invasion of the central nervous system via regulation of chemokines

Dynamic interplay between cytokines and chemokines directs trafficking of leukocyte subpopulations to tissues in autoimmune inflammation. We have examined the role of IFN-gamma in directing chemokine production and leukocyte infiltration to the CNS in experimental autoimmune encephalomyelitis (EAE). BALB/c and C57BL/6 mice are resistant to induction of EAE by immunization with myelin basic protein. However, IFN-gamma-deficient (BALB/c) and IFN-gammaR-deficient (C57BL/6) mice developed rapidly progressing lethal disease. Widespread demyelination and disseminated leukocytic infiltration of spinal cord were seen, unlike the focal perivascular infiltrates in SJL/J mice. Gr-1+ neutrophils predominated in CNS, and CD4+ T cells with an activated (CD69+, CD25+) phenotype and eosinophils were also present. RANTES and macrophage chemoattractant protein-1, normally up-regulated in EAE, were undetectable in IFN-gamma- and IFN-gammaR-deficient mice. Macrophage inflammatory protein-2 and T cell activation gene-3, both neutrophil-attracting chemokines, were strongly up-regulated. There was no induction of the Th2 cytokines, IL-4, IL-10, or IL-13. RNase protection assays and RT-PCR showed the prevalence of IL-2, IL-3, and IL-15, but no increase in IL-12p40 mRNA levels in IFN-gamma- or IFN-gammaR-deficient mice with EAE. Lymph node cells from IFN-gamma-deficient mice proliferated in response to myelin basic protein, whereas BALB/c lymph node cells did not. These findings show a regulatory role for IFN-gamma in EAE, acting on T cell proliferation and directing chemokine production, with profound implications for the onset and progression of disease.     

Essential role of CD8+CD122+ regulatory T cells in the recovery from experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is one of the best-documented animal models of autoimmune disease. We examined the role of CD8+CD122+ regulatory T cells, which we previously identified as naturally occurring regulatory T cells that effectively regulate CD8+ T cells, in EAE. Depletion of CD8+CD122+ regulatory T cells by in vivo administration of anti-CD122 mAb resulted in persistent EAE symptoms. Transfer of CD8+CD122+ regulatory T cells into EAE mice at the peak EAE score clearly improved symptoms, indicating an important role of CD8+CD122+ regulatory T cells in the recovery phase of EAE. This was further confirmed by an increase and a decrease in the number of infiltrating T cells in the CNS and T cell cytokine production in mice that were depleted of or complemented with CD8+CD122+ cells. Furthermore, transfer of preactivated CD8+CD122+ regulatory T cells resulted in diminished EAE symptoms, especially in the recovery phase of EAE. These results elucidate the essential role of CD8+CD122+ regulatory T cells in the recovery phase of EAE and suggest the preventive effect of preactivated CD8+CD122+ regulatory T cells for EAE.     

Estriol generates tolerogenic dendritic cells in vivo that protect against autoimmunity

Chronic inflammation contributes to numerous diseases, and regulation of inflammation is crucial for disease control and resolution. Sex hormones have potent immunoregulatory abilities. Specifically, estrogen influences immune cells and inflammation, which contributes to the sexual dimorphism of autoimmunity and protection against disease seen during pregnancy in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although long thought to act primarily on T cells, recent evidence demonstrated that myeloid cells, such as dendritic cells (DCs), are essential in mediating estrogen's protective effects. Estriol (E3), a pregnancy-specific estrogen, has therapeutic efficacy in MS and EAE, and we evaluated whether E3 could act exclusively through DCs to protect against the inflammatory autoimmune disease EAE. Levels of activation markers (CD80 and CD86) and inhibitory costimulatory markers (PD-L1, PD-L2, B7-H3, and B7-H4) were increased in E3 DCs. E3 DCs had decreased proinflammatory IL-12, IL-23, and IL-6 mRNA expression, increased immunoregulatory IL-10 and TGF-β mRNA expression, and a decreased ratio of IL-12/IL-10 protein production. Importantly, transfer of E3 DCs to mice prior to active induction of EAE protected them from developing EAE through immune deviation to a Th2 response. This protection was apparent, even in the face of in vitro and in vivo inflammatory challenge. In summary, our results showed that E3 generates tolerogenic DCs, which protect against the inflammatory autoimmune disease EAE. Targeted generation of tolerogenic DCs with immunomodulatory therapeutics, such as E3, has potential applications in the treatment of numerous autoimmune and chronic inflammatory diseases.     

Ingested (oral) tocilizumab inhibits EAE

BackgroundBlocking the activity of IL-6 can inhibit autoimmune diseases such as rheumatoid arthritis and Crohn’s disease.ObjectiveWe examined whether an antibody against IL-6, tocilizumab (TCZ) (Actemra®), used clinically in rheumatoid arthritis (RA) would have similar anti-inflammatory effects in EAE after oral administration.Design/methodB6 mice were immunized with MOG peptide 35–55 and gavaged with control saline or TCZ during ongoing disease. Splenocytes, CD4⁺ T cells or macrophages/monocyte lineage cells (CD11b⁺) from control fed or TCZ fed mice were adoptively transferred into active MOG peptide 35–55 immunized recipient mice during ongoing disease. Actively fed and recipient mice were examined for disease inhibition, inflammation, and cytokine responses.ResultsIngested (oral) TCZ inhibited ongoing disease and decreased inflammation. Adoptively transferred cells from TCZ fed donors protected against actively induced disease and decreased inflammation. There was a decrease in IL-6 in actively treated spleen, decrease in TNF-α, Th1-like cytokine IL-12 and increase in Th2-like cytokine IL-10 in active fed and adoptively treated recipients.ConclusionsIngested (orally administered) TCZ can inhibit disease, CNS inflammation, decrease pro-inflammatory Th1-like cytokines and increase Th2-like anti-inflammatory cytokines.     

Transplantation of Autologous Adipose Stem Cells Lacks Therapeutic Efficacy in the Experimental Autoimmune Encephalomyelitis Model

Multiple sclerosis (MS), characterized by chronic inflammation, demyelination, and axonal damage, is a complicated neurological disease of the human central nervous system. Recent interest in adipose stromal/stem cell (ASCs) for the treatment of CNS diseases has promoted further investigation in order to identify the most suitable ASCs. To investigate whether MS affects the biologic properties of ASCs and whether autologous ASCs from MS-affected sources could serve as an effective source for stem cell therapy, cells were isolated from subcutaneous inguinal fat pads of mice with established experimental autoimmune encephalomyelitis (EAE), a murine model of MS. ASCs from EAE mice and their syngeneic wild-type mice were cultured, expanded, and characterized for their cell morphology, surface antigen expression, osteogenic and adipogenic differentiation, colony forming units, and inflammatory cytokine and chemokine levels in vitro. Furthermore, the therapeutic efficacy of the cells was assessed in vivo by transplantation into EAE mice. The results indicated that the ASCs from EAE mice displayed a normal phenotype, typical MSC surface antigen expression, and in vitro osteogenic and adipogenic differentiation capacity, while their osteogenic differentiation capacity was reduced in comparison with their unafflicted control mice. The ASCs from EAE mice also demonstrated increased expression of pro-inflammatory cytokines and chemokines, specifically an elevation in the expression of monocyte chemoattractant protein-1 and keratin chemoattractant. In vivo, infusion of wild type ASCs significantly ameliorate the disease course, autoimmune mediated demyelination and cell infiltration through the regulation of the inflammatory responses, however, mice treated with autologous ASCs showed no therapeutic improvement on the disease progression.     

Repetitive pertussis toxin promotes development of regulatory T cells and prevents central nervous system autoimmune disease

Bacterial and viral infections have long been implicated in pathogenesis and progression of multiple sclerosis (MS). Incidence and severity of its animal model experimental autoimmune encephalomyelitis (EAE) can be enhanced by concomitant administration of pertussis toxin (PTx), the major virulence factor of Bordetella pertussis. Its adjuvant effect at the time of immunization with myelin antigen is attributed to an unspecific activation and facilitated migration of immune cells across the blood brain barrier into the central nervous system (CNS). In order to evaluate whether recurring exposure to bacterial antigen may have a differential effect on development of CNS autoimmunity, we repetitively administered PTx prior to immunization. Mice weekly injected with PTx were largely protected from subsequent EAE induction which was reflected by a decreased proliferation and pro-inflammatory differentiation of myelin-reactive T cells. Splenocytes isolated from EAE-resistant mice predominantly produced IL-10 upon re-stimulation with PTx, while non-specific immune responses were unchanged. Longitudinal analyses revealed that repetitive exposure of mice to PTx gradually elevated serum levels for TGF-β and IL-10 which was associated with an expansion of peripheral CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg). Increased frequency of Treg persisted upon immunization and thereafter. Collectively, these data suggest a scenario in which repetitive PTx treatment protects mice from development of CNS autoimmune disease through upregulation of regulatory cytokines and expansion of CD4(+)CD25(+)FoxP3(+) Treg. Besides its therapeutic implication, this finding suggests that encounter of the immune system with microbial products may not only be part of CNS autoimmune disease pathogenesis but also of its regulation.     

The ICOS molecule plays a crucial role in the development of mucosal tolerance

The ICOS molecule stimulates production of the immunoregulatory cytokine IL-10, suggesting an important role for ICOS in controlling IL-10-producing regulatory T cells and peripheral T cell tolerance. In this study we investigate whether ICOS is required for development of oral, nasal, and high dose i.v. tolerance. Oral administration of encephalitogenic myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide to ICOS-deficient (ICOS-/-) mice did not inhibit experimental autoimmune encephalomyelitis (EAE), T cell proliferation, or IFN-gamma production, in striking contrast to wild-type mice. Similarly, intranasal administration of MOG(35-55) before EAE induction suppressed EAE and T cell responses in wild-type, but not in ICOS-/-, mice. In contrast, ICOS-/- mice were as susceptible as wild-type mice to high dose tolerance. These results indicate that ICOS plays an essential and specific role in mucosal tolerance and that distinct costimulatory pathways differentially regulate different forms of peripheral tolerance. Surprisingly, CD4+ cells from MOG-fed wild-type and ICOS-/- mice could transfer suppression to wild-type recipients, indicating that functional regulatory CD4+ cells can develop in the absence of ICOS. However, CD4+ T cells from MOG-fed wild-type mice could not transfer suppression to ICOS-/- recipients, suggesting that ICOS may have a key role in controlling the effector functions of regulatory T cells. These results suggest that stimulating ICOS may provide an effective therapeutic approach for promoting mucosal tolerance.     

SAP suppresses the development of experimental autoimmune encephalomyelitis in C57BL/6 mice

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated disease of the central nervous system. Serum amyloid P component (SAP) is a highly conserved plasma protein named for its universal presence in amyloid deposits. Here we report that SAP-transgenic mice had unexpectedly attenuated EAE due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35-55 in complete Freund's adjuvant, SAP-transgenic mice showed reduced spinal cord inflammation with lower severity of EAE attacks as compared with control C57BL/6 mice. However, in SAP-Knockout mice, the severity of EAE is enhanced. Adoptive transfer of Ag-restimulated T cells from wild type to SAP-transgenic mice, or transfer of SAP-transgenic Ag-restimulated T cells to control mice, induced milder EAE. T cells from MOG-primed SAP-transgenic mice showed weak proliferative responses. Furthermore, in SAP-transgenic mice, there is little infiltration of CD45-positive cells in the spinal cord. In vitro, SAP suppressed the secretion of interleukin-2 stimulated by P-selectin and blocked P-selectin binding to T cells. Moreover, SAP could change the affinity between α4-integrin and T cells. These data suggested that SAP could antagonize the development of the acute phase of inflammation accompanying EAE by modulating the function of P-selectin.     

A novel IL-10-independent regulatory role for B cells in suppressing autoimmunity by maintenance of regulatory T cells via GITR ligand

B cells are important for the regulation of autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), B cells are required for spontaneous recovery in acute models. Production of IL-10 by regulatory B cells has been shown to modulate the severity EAE and other autoimmune diseases. Previously, we suggested that B cells regulated the number of CD4(+)Foxp3(+) T regulatory cells (Treg) in the CNS during EAE. Because Treg suppress autoimmune responses, we asked whether B cells control autoimmunity by maintenance of Treg numbers. B cell deficiency achieved either genetically (μMT) or by depletion with anti-CD20 resulted in a significant reduction in the number of peripheral but not thymic Treg. Adoptive transfer of WT B cells into μMT mice restored both Treg numbers and recovery from EAE. When we investigated the mechanism whereby B cells induce the proliferation of Treg and EAE recovery, we found that glucocorticoid-induced TNF ligand, but not IL-10, expression by B cells was required. Of clinical significance is the finding that anti-CD20 depletion of B cells accelerated spontaneous EAE and colitis. Our results demonstrate that B cells play a major role in immune tolerance required for the prevention of autoimmunity by maintenance of Treg via their expression of glucocorticoid-induced TNFR ligand.     

Synthetic peptides representing sequence 39 to 59 of rat V beta 8 TCR fail to elicit regulatory T cells reactive with V beta 8 TCR on rat encephalitogenic T cells.

Subpathogenic doses of syngeneic autoreactive T cells protect experimental animals against associated autoimmune disease. Preferential use of the TCR of encephalitogenic T cells suggests that this molecule serves as the target for immunoregulation in experimental autoimmune encephalomyelitis (EAE). Whether peptides derived from the V beta 8 of the rat TCR elicit regulatory T cells and produce the same vaccinating effect against EAE as do whole T cells remains unknown. Here we show that immunization of Lewis rats with V beta 8(39-59), a peptide representing residues 39 to 59 of the rat V beta 8 TCR, does not induce the production of regulatory T cells reactive to the intact TCR V beta 8 containing this sequence. Moreover, animals that had recovered from both actively induced EAE and transferred EAE did not generate regulatory T cells that recognized the V beta 8(39-59) peptide. Further, transfusion of large doses of peptide-specific T cells did not protect the animals from EAE. Our results suggest that the V beta 8(39-59) peptide may comprise so-called cryptic epitopes, which function as immunogens only when dissociated from large protein complexes.     

Cinnamon Ameliorates Experimental Allergic Encephalomyelitis in Mice via Regulatory T Cells: Implications for Multiple Sclerosis Therapy

Upregulation and/or maintenance of regulatory T cells (Tregs) during an autoimmune insult may have therapeutic efficacy in autoimmune diseases. Although several immunomodulatory drugs and molecules are available, most present significant side effects over long-term use. Cinnamon is a commonly used natural spice and flavoring material used for centuries throughout the world. Here, we have explored a novel use of cinnamon powder in protecting Tregs and treating the disease process of experimental allergic encephalomyelitis (EAE), an animal model of MS. Oral feeding of cinnamon (Cinnamonum verum) powder suppresses clinical symptoms of relapsing-remitting EAE in female PLP-TCR transgenic mice and adoptive transfer mouse model. Cinnamon also inhibited clinical symptoms of chronic EAE in male C57/BL6 mice. Dose-dependent study shows that cinnamon powder at a dose of 50 mg/kg body wt/d or higher significantly suppresses clinical symptoms of EAE in mice. Accordingly, oral administration of cinnamon also inhibited perivascular cuffing, maintained the integrity of blood-brain barrier and blood-spinal cord barrier, suppressed inflammation, normalized the expression of myelin genes, and blocked demyelination in the central nervous system of EAE mice. Interestingly, cinnamon treatment upregulated Tregs via reduction of nitric oxide production. Furthermore, we demonstrate that blocking of Tregs by neutralizing antibodies against CD25 abrogates cinnamon-mediated protection of EAE. Taken together, our results suggest that oral administration of cinnamon powder may be beneficial in MS patients and that no other existing anti-MS therapies could be so economical and trouble-free as this approach.     

Kit (W-sh) mice develop earlier and more severe experimental autoimmune encephalomyelitis due to absence of immune suppression

Mast cells (MCs) have been thought to play a pathogenic role in the development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, an immunoregulatory function of these cells has recently been suggested. We investigated the role of MCs in EAE using the W(-sh) mouse strain, which is MC deficient. W(-sh) mice developed earlier and more severe clinical and pathological disease with extensive demyelination and inflammation in the CNS. The inflammatory cells were mainly composed of CD4(+) T cells, monocyte/macrophages, neutrophils, and dendritic cells. Compared with wild-type mice, MC-deficient mice exhibited an increased level of MCP-1/CCR2 and CD44 expression on CD4(+) T cells in addition to decreased production of regulatory T cells, IL-4, IL-5, IL-27, and IL-10. We also found that levels of IL-17, IFN-γ, and GM-CSF were significantly increased in peripheral lymphocytes from immunized W(-sh) mice compared with those in peripheral lymphocytes from wild-type mice. Reconstitution of W(-sh) mice downregulated susceptibility to EAE, which correlated with MC recruitment and regulatory T cell activation in the CNS. These findings indicate that responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS and that, in the absence of MCs, increased MCP-1, CCR2, IL-17, IFN-γ, CD44, and other inflammatory molecules may be responsible for increased severity of EAE.