Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

The V-region disease hypothesis: evidence from autoimmune encephalomyelitis.

Experimental allergic encephalomyelitis has been shown to have an immunological basis. In fact, the disease can be induced by T cells specific for myelin basic protein, a molecule found in abundance in the central nervous system. In this article, Ellen Heber-Katz and Hans Acha-Orbea discuss the T-cell receptor (TCR) repertoire of the encephalitogenic T-cell response, and show that a limited V gene pool, in fact a single V beta and two V alpha families, are being used by the PL/J and B10.PL mice and by every rat strain examined, even though the antigenic determinants and the major histocompatibility complex (MHC) molecules are different in all cases. This extraordinary finding suggests that the TCR is involved in encephalitogenicity in a way that not only involves the recognition of antigen in association with MHC, but also as an effector molecule that results in encephalitis. If this is true, it implies that TCRs, in general, play more than one role in mammalian physiology.

TL1A-DR3 interaction regulates Th17 cell function and Th17-mediated autoimmune disease.

T helper type 17 (Th17) cells play an important pathogenic function in autoimmune diseases; their regulation, however, is not well understood. We show that the expression of a tumor necrosis factor receptor family member, death receptor 3 (DR3; also known as TNFRSF25), is selectively elevated in Th17 cells, and that TL1A, its cognate ligand, can promote the proliferation of effector Th17 cells. To further investigate the role of the TL1A-DR3 pathway in Th17 regulation, we generated a TL1A-deficient mouse and found that TL1A(-/-) dendritic cells exhibited a reduced capacity in supporting Th17 differentiation and proliferation. Consistent with these data, TL1A(-/-) animals displayed decreased clinical severity in experimental autoimmune encephalomyelitis (EAE). Finally, we demonstrated that during EAE disease progression, TL1A was required for the optimal differentiation as well as effector function of Th17 cells. These observations thus establish an important role of the TL1A-DR3 pathway in promoting Th17 cell function and Th17-mediated autoimmune disease.

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression.

Via a transcription factor, Foxp3, immunoregulatory CD4(+)CD25(+) T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2-IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1-2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2-IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex-incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides

OBJECTIVE: A distinct subset of proinflammatory CD4+ T cells that produce interleukin-17 was recently identified. These cells are implicated in different autoimmune disease models, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, but their involvement in human autoimmune disease has not yet been clearly established. The purpose of this study was to assess the frequency and functional properties of Th17 cells in healthy donors and in patients with different autoimmune diseases. METHODS: Peripheral blood was obtained from 10 psoriatic arthritis (PsA), 10 ankylosing spondylitis (AS), 10 rheumatoid arthritis (RA), and 5 vitiligo patients, as well as from 25 healthy donors. Synovial tissue samples from a separate group of patients were also evaluated (obtained as paraffin-embedded sections). Peripheral blood cells were analyzed by multiparameter flow cytometry and immunohistochemistry. Cytokine production was examined by enzyme-linked immunosorbent assay and intracellular cytokine staining using specific monoclonal antibodies. Synovial tissue was examined for infiltrating T cells by immunohistochemical analysis. RESULTS: We found increased numbers of circulating Th17 cells in the peripheral blood of patients with seronegative spondylarthritides (PsA and AS), but not in patients with RA or vitiligo. In addition, Th17 cells from the spondylarthritis patients showed advanced differentiation and were polyfunctional in terms of T cell receptor-driven cytokine production. CONCLUSION: These observations suggest a role of Th17 cells in the pathogenesis of certain human autoimmune disorders, in particular the seronegative spondylarthritides.

Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies.

CD62L(high) Treg cells with superior immunosuppressive properties accumulate within the CNS during remissions of EAE.

The role of regulatory T cell populations within the CNS in the regulation of CNS-autoimmunity is controversial. We show that during recovery from relapsing remitting experimental autoimmune encephalomyelitis, regulatory T cells accumulate within the CNS that express high levels of CD62L. These CD62L(high) Treg cells express increased amounts of CTLA-4, ICOS and TGF-β and are more potent than CD62L(low) Treg cells in suppressing proliferation and inducing apoptosis in effector T cells. CD62L(high) Treg cells thus represent a population of Treg cells that display superior immunosuppressive properties and accumulate in the CNS during recovery from CNS-autoimmunity.

Effects of the PPAR-beta agonist GW501516 in an in vitro model of brain inflammation and antibody-induced demyelination.

BACKGROUND: Brain inflammation plays a central role in numerous brain pathologies, including multiple sclerosis (MS). Microglial cells and astrocytes are the effector cells of neuroinflammation. They can be activated also by agents such as interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Peroxisome proliferator-associated receptor (PPAR) pathways are involved in the control of the inflammatory processes, and PPAR-beta seems to play an important role in the regulation of central inflammation. In addition, PPAR-beta agonists were shown to have trophic effects on oligodendrocytes in vitro, and to confer partial protection in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In the present work, a three-dimensional brain cell culture system was used as in vitro model to study antibody-induced demyelination and inflammatory responses. GW 501516, a specific PPAR-beta agonist, was examined for its capacity to protect from antibody-mediated demyelination and to prevent inflammatory responses induced by IFN-gamma and LPS. METHODS: Aggregating brain cells cultures were prepared from embryonal rat brain, and used to study the inflammatory responses triggered by IFN-gamma and LPS and by antibody-mediated demyelination induced by antibodies directed against myelin-oligodendrocyte glycoprotein (MOG). The effects of GW 501516 on cellular responses were characterized by the quantification of the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), inducible NO synthase (i-NOS), PPAR-beta, PPAR-gamma, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and high molecular weight neurofilament protein (NF-H). GFAP expression was also examined by immunocytochemistry, and microglial cells were visualized by isolectin B4 (IB4) and ED1 labeling. RESULTS: GW 501516 decreased the IFN-gamma-induced up-regulation of TNF-alpha and iNOS in accord with the proposed anti-inflammatory effects of this PPAR-beta agonist. However, it increased IL-6 m-RNA expression. In demyelinating cultures, reactivity of both microglial cells and astrocytes was observed, while the expression of the inflammatory cytokines and iNOS remained unaffected. Furthermore, GW 501516 did not protect against the demyelination-induced changes in gene expression. CONCLUSION: Although GW 501516 showed anti-inflammatory activity, it did not protect against antibody-mediated demyelination. This suggests that the protective effects of PPAR-beta agonists observed in vivo can be attributed to their anti-inflammatory properties rather than to a direct protective or trophic effect on oligodendrocytes.

Minocycline promotes remyelination in aggregating rat brain cell cultures after interferon-γ plus lipopolysaccharide-induced demyelination.

Minocycline has been shown to inhibit microglia reactivity, and to decrease the severity and progression of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. It remained to be examined whether minocycline was also able to promote remyelination. In the present study, myelinating aggregating brain cell cultures were used as a model to study the effects of minocycline on microglial reactivity, demyelination, and remyelination. Cultures were treated simultaneously with two inflammatory agents, interferon-γ (IFN-γ) and lipopolysaccharide (LPS), which caused an inflammatory response accompanied by demyelination. The inflammatory response was characterized by microglial reactivity, upregulation of inflammatory cytokines and iNOS, and increased phophorylation of P38 and P44/42 mitogen activated protein (MAP) kinases. Minocycline inhibited microglial reactivity, and attenuated the increased phophorylation of P38 and P44/42 MAP kinases. Demyelination, determined by a decrease in myelin basic protein (MBP) content and immunoreactivity 48 h after the treatment with the inflammatory agents, was not prevented by minocycline. However, 1 week after demyelination was assessed, the MBP content was restored in presence of minocycline, indicating that remyelination was promoted. Concomitantly, in cultures treated with minocycline, the markers of oligodendrocyte precursors cells (OPCs) and immature oligodendrocytes NG2 and O4, respectively, were decreased compared to cultures treated with the inflammatory agents only. These results suggest that minocycline attenuates microglial reactivity and favors remyelination by enhancing the differentiation of OPCs and immature oligodendrocytes.

IL-27 Induces Th17 Differentiation in the Absence of STAT1 Signaling.

It is known that differentiation of Th17 cells is promoted by activation of STAT3 and inhibited by activation of STAT1. Although both transcription factors are activated by several cytokines, including IL-6, IL-21, and IL-27, each of these cytokines has a very different effect on Th17 differentiation, ranging from strong induction (IL-6) to strong inhibition (IL-27). To determine the molecular basis for these differences, we measured STAT3 and STAT1 activation profiles for IL-6, IL-21, and IL-27, as well as for cytokine pairs over time. We found that the ratio of activated STAT3/activated STAT1 is crucial in determining whether cytokines promote or inhibit Th17 differentiation. IL-6 and IL-21 induced p-STAT3/p-STAT1 ratios > 1, leading to the promotion of Th17 differentiation, whereas IL-27 or IL-6+IL-27 induced p-STAT3/p-STAT1 ratios < 1, resulting in inhibition of Th17 differentiation. Consistent with these findings, we show that IL-27 induces sufficient p-STAT3 to promote Th17 differentiation in the absence of STAT1. Furthermore, IL-27-induced STAT1-deficient T cells were indistinguishable from bona fide highly proinflammatory Th17 cells because they induced severe experimental autoimmune encephalomyelitis upon adoptive transfer. Our results suggest that the ratio of p-STAT3/p-STAT1 induced by a cytokine or cytokine pairs can be used to predict whether they induce a competent Th17-differentiation program.

Less mortality but more relapses in experimental allergic encephalomyelitis in CD8-/- mice.

Mice lacking in CD8 were generated from homologous recombination in embryonal stem cells at the CD8 locus and bred with the experimental allergic encephalomyelitis (EAE)-susceptible PL/JH-2u through four backcross generations to investigate the role of CD8+ T cells in this model of multiple sclerosis. The disease onset and susceptibility were similar to those of wild-type mice. However, the mutant mice had a milder acute EAE, reflected by fewer deaths, but more chronic EAE, reflected by a higher frequency of relapse. This suggests that CD8+ T lymphocytes may participate as both effectors and regulators in this animal model.

Limited heterogeneity of autoantigens and T cells in autoimmune diseases?

For many induced and spontaneous autoimmune diseases, a predominant role for T cells in the organ-specific destruction process has been shown. In one of the induced models of autoimmunity, experimental allergic encephalomyelitis (EAE), a very small heterogeneity of T-cell receptor (TcR) molecules is expressed by the pathogenic T cells in both rats and mice. Contrary to induced autoimmune diseases, little is known about the autoantigens recognized by these autoimmune T cells and the heterogeneity of their TcR in spontaneous autoimmune diseases. The aim of this work was to establish a system which allows characterization of relevant autoantigens in spontaneous insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice. A completely different approach was taken to characterize the gene products of the minor lymphocyte stimulatory (Mls) loci. These gene products are responsible for the clonal elimination or the clonal stimulation of T cells expressing particular TcR V beta genes and therefore could be implicated in induction of autoimmune diseases by oligoclonal T-cell populations. The finding that Mls antigens are encoded by retroviral sequences leads to the hypothesis that viruses could be the inducing agents of autoimmune diseases.

Nitric oxide synthase 2 is required for conversion of pro-fibrogenic inflammatory CD133+ progenitors into F4/80+ macrophages in experimental autoimmune myocarditis.

AIMS: Experimental autoimmune myocarditis (EAM) model mirrors important mechanisms of inflammatory dilated cardiomyopathy (iDCM). In EAM, inflammatory CD133(+) progenitors are a major cellular source of cardiac myofibroblasts in the post-inflammatory myocardium. We hypothesized that exogenous delivery of macrophage-colony-stimulating factor (M-CSF) can stimulate macrophage lineage differentiation of inflammatory progenitors and, therefore, prevent their naturally occurring myofibroblast fate in EAM. METHODS AND RESULTS: EAM was induced in wild-type (BALB/c) and nitric oxide synthase 2-deficient (Nos2(-/-)) mice and CD133(+) progenitors were isolated from inflamed hearts. In vitro, M-CSF converted inflammatory CD133(+) progenitors into nitric oxide-producing F4/80(+) macrophages and prevented transforming growth factor-β-mediated myofibroblast differentiation. Importantly, only a subset of heart-infiltrating CD133(+) progenitors expresses macrophage-specific antigen F4/80 in EAM. These CD133(+)/F4/80(hi) cells show impaired myofibrogenic potential compared with CD133(+)/F4/80(-) cells. M-CSF treatment of wild-type mice with EAM at the peak of disease markedly increased CD133(+)/F4/80(hi) cells in the myocardium, and CD133(+) progenitors isolated from M-CSF-treated mice failed to differentiate into myofibroblasts. In contrast, M-CSF was not effective in converting CD133(+) progenitors from inflamed hearts of Nos2(-/-) mice into macrophages, and M-CSF treatment did not result in increased CD133(+)/F4/80(hi) cell population in hearts of Nos2(-/-) mice. Accordingly, M-CSF prevented post-inflammatory fibrosis and left ventricular dysfunction in wild-type but not in Nos2(-/-) mice. CONCLUSION: Active and NOS2-dependent induction of macrophage lineage differentiation abrogates the myofibrogenic potential of heart-infiltrating CD133(+) progenitors. Modulating the in vivo differentiation fate of specific progenitors might become a novel approach for the treatment of inflammatory heart diseases.

Protective effect of intravitreal administration of tresperimus, an immunosuppressive drug, on experimental autoimmune uveoretinitis.

PURPOSE: To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS: In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS: Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.