Immunodominant T Cell Determinants of Aquaporin-4, the Autoantigen Associated with Neuromyelitis Optica

Autoantibodies that target the water channel aquaporin-4 (AQP4) in neuromyelitis optica (NMO) are IgG1, a T cell-dependent Ig subclass. However, a role for AQP4-specific T cells in this CNS inflammatory disease is not known. To evaluate their potential role in CNS autoimmunity, we have identified and characterized T cells that respond to AQP4 in C57BL/6 and SJL/J mice, two strains that are commonly studied in models of CNS inflammatory diseases. Mice were immunized with either overlapping peptides or intact hAQP4 protein encompassing the entire 323 amino acid sequence. T cell determinants identified from examination of the AQP4 peptide (p) library were located within AQP4 p21-40, p91-110, p101-120, p166-180, p231-250 and p261-280 in C57BL/6 mice, and within p11-30, p21-40, p101-120, p126-140 and p261-280 in SJL/J mice. AQP4-specific T cells were CD4+ and MHC II-restricted. In recall responses to immunization with intact AQP4, T cells responded primarily to p21-40, indicating this region contains the immunodominant T cell epitope(s) for both strains. AQP4 p21-40-primed T cells secreted both IFN-γ and IL-17. The core immunodominant AQP4 21-40 T cell determinant was mapped to residues 24-35 in C57BL/6 mice and 23-35 in SJL/J mice. Our identification of the AQP4 T cell determinants and characterization of its immunodominant determinant should permit investigators to evaluate the role of AQP4-specific T cells in vivo and to develop AQP4-targeted murine NMO models

Passive experimental autoimmune encephalomyelitis in C57BL/6 with MOG: evidence of involvement of B cells

Experimental autoimmune encephalomyelitis (EAE) is the most relevant animal model to study demyelinating diseases such as multiple sclerosis. EAE can be induced by active (active EAE) or passive (at-EAE) transfer of activated T cells in several species and strains of rodents. However, histological features of at-EAE model in C57BL/6 are poorly described. The aim of this study was to characterize the neuroinflammatory and neurodegenerative responses of at-EAE in C57BL/6 mice by histological techniques and compare them with that observed in the active EAE model. To develop the at-EAE, splenocytes from active EAE female mice were harvested and cultured in presence of MOG 35-55 and IL-12, and then injected intraperitoneally in recipient female C57BL6/J mice. In both models, the development of EAE was similar except for starting before the onset of symptoms and presenting a higher EAE cumulative score in the at-EAE model. Spinal cord histological examination revealed an increased glial activation as well as more extensive demyelinating areas in the at-EAE than in the active EAE model. Although inflammatory infiltrates composed by macrophages and T lymphocytes were found in the spinal cord and brain of both models, B lymphocytes were significantly increased in the at-EAE model. The co-localization of these B cells with IgG and their predominant distribution in areas of demyelination would suggest that IgG-secreting B cells are involved in the neurodegenerative processes associated with at-EAE

A Comparative Approach Linking Molecular Dynamics of Altered Peptide Ligands and MHC with In Vivo Immune Responses

The recognition of peptide in the context of MHC by T lymphocytes is a critical step in the initiation of an adaptive immune response. However, the molecular nature of the interaction between peptide and MHC and how it influences T cell responsiveness is not fully understood.We analyzed the immunological consequences of the interaction of MHC class II (I-Au) restricted 11-mer peptides of myelin basic protein with amino acid substitutions at position 4. These mutant peptides differ in MHC binding affinity, CD4+ T cell priming, and alter the severity of peptide-induced experimental allergic encephalomyelitis. Using molecular dynamics, a computational method of quantifying intrinsic movements of proteins at high resolution, we investigated conformational changes in MHC upon peptide binding. We found that irrespective of peptide binding affinity, MHC deformation appears to influence costimulation, which then leads to effective T cell priming and disease induction. Although this study compares in vivo and molecular dynamics results for three altered peptide ligands, further investigation with similar complexes is essential to determine whether spatial rearrangement of peptide-MHC and costimulatory complexes is an additional level of T cell regulation

PEG Minocycline-Liposomes Ameliorate CNS Autoimmune Disease

Minocycline is an oral tetracycline derivative with good bioavailability in the central nervous system (CNS). Minocycline, a potent inhibitor of matrix metalloproteinase (MMP)-9, attenuates disease activity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Potential adverse effects associated with long-term daily minocycline therapy in human patients are concerning. Here, we investigated whether less frequent treatment with long-circulating polyethylene glycol (PEG) minocycline liposomes are effective in treating EAE.Performing in vitro time kinetic studies of PEG minocycline-liposomes in human peripheral blood mononuclear cells (PBMCs), we determined that PEG minocycline-liposome preparations stabilized with CaCl(2) are effective in diminishing MMP-9 activity. Intravenous injections of PEG minocycline-liposomes every five days were as effective in ameliorating clinical EAE as daily intraperitoneal injections of minocycline. Treatment of animals with PEG minocycline-liposomes significantly reduced the number of CNS-infiltrating leukocytes, and the overall expression of MMP-9 in the CNS. There was also a significant suppression of MMP-9 expression and proteolytic activity in splenocytes of treated animals, but not in CNS-infiltrating leukocytes. Thus, leukocytes gaining access to the brain and spinal cord require the same absolute amount of MMP-9 in all treatment groups, but minocycline decreases the absolute cell number.Our data indicate that less frequent injections of PEG minocycline-liposomes are an effective alternative pharmacotherapy to daily minocycline injections for the treatment of CNS autoimmune diseases. Also, inhibition of MMP-9 remains a promising treatment target in EAE and patients with MS

Amniotic Epithelial Cells from the Human Placenta Potently Suppress a Mouse Model of Multiple Sclerosis

Human amniotic epithelial cells (hAEC) have stem cell-like features and immunomodulatory properties. Here we show that hAEC significantly suppressed splenocyte proliferation in vitro and potently attenuated a mouse model of multiple sclerosis (MS). Central nervous system (CNS) CD3+ T cell and F4/80+ monocyte/macrophage infiltration and demyelination were significantly reduced with hAEC treatment. Besides the known secretion of prostaglandin E2 (PGE2), we report the novel finding that hAEC utilize transforming growth factor-β (TGF-β) for immunosuppression. Neutralization of TGF-β or PGE2 in splenocyte proliferation assays significantly reduced hAEC-induced suppression. Splenocytes from hAEC-treated mice showed a Th2 cytokine shift with significantly elevated IL-5 production. While transferred CFSE-labeled hAEC could be detected in the lung, none were identified in the CNS or in lymphoid organs. This is the first report documenting the therapeutic effect of hAEC in a MS-like model and suggest that hAEC may have potential for use as therapy for MS

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

‘Multi-Epitope-Targeted’ Immune-Specific Therapy for a Multiple Sclerosis-Like Disease via Engineered Multi-Epitope Protein Is Superior to Peptides

Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS) yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and “epitope spread”, have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such “multi-epitope-targeting” approach in murine experimental autoimmune encephalomyelitis (EAE) associated with a single (“classical”) or multiple (“complex”) anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc) encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as “multi-epitope-targeting” agents. Y-MSPc was superior to peptide(s) in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells). Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of “classical” or “complex EAE” or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a “multi-epitope-targeting” strategy is required for effective immune-specific therapy of organ-specific autoimmune diseases associated with complex and dynamic pathogenic autoimmunity, such as MS; our data further demonstrate that the “multi-epitope-targeting” approach to therapy is optimized through specifically designed multi-epitope-proteins, rather than myelin peptide cocktails, as “multi-epitope-targeting” agents. Such artificial multi-epitope proteins can be tailored to other organ-specific autoimmune diseases

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference