Oral administration of the nitroxide radical TEMPOL exhibits immunomodulatory and therapeutic properties in multiple sclerosis models.

Therapies with both immunomodulatory and neuroprotective properties are thought to have the greatest promise in reducing the severity and progression of multiple sclerosis (MS). Several reactive oxygen (ROS) and reactive nitrogen species (RNS) are implicated in inflammatory-mediated damage to the central nervous system (CNS) in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a stable nitroxide radical with potent antioxidant activity. The goal of our studies was to investigate the immunomodulatory effects and therapeutic potential of orally-delivered TEMPOL in the mouse EAE model. Mice receiving TEMPOL chow ad libitum for 2weeks prior to induction of active EAE showed delayed onset and reduced incidence of disease compared to control-fed animals. Reduced disease severity was associated with limited microglial activation and fewer inflammatory infiltrates. TEMPOL's effects were immunomodulatory, not immunosuppressive: T cells produced less interferon-γ and tumor necrosis factor-α, and TEMPOL-fed mice exhibited a shift towards TH2-type antibody responses. Both myeloid and myeloid-dendritic cells of TEMPOL-fed EAE animals had significantly lower levels of MHC class II expression than controls; CD40 was also significantly reduced. TEMPOL administration was associated with an enrichment of CD8+ T cell populations and CD4+FoxP3+ regulatory populations. TEMPOL reduced the severity of clinical disease when administered after the induction of disease, and also after the onset of clinical symptoms. To exclude effects on T cell priming in vivo, TEMPOL was tested with the passive transfer of encephalitogenic T cells and was found to reduce the incidence and peak severity of disease. Protection was associated with reduced infiltrates and a relative sparing of neurofilaments and axons. The ability of oral TEMPOL to reduce inflammation and axonal damage and loss demonstrate both anti-inflammatory and protective properties, with significant promise for the treatment of MS and related neurological disorders.

Myelin basic protein-specific TCR/HLA-DRB5*01:01 transgenic mice support the etiologic role of DRB5*01:01 in multiple sclerosis.

Genetic susceptibility to multiple sclerosis (MS) has been linked to the HLA-DR15 haplotype consisting of DRB1*15:01(DR2b) and DRB5*01:01(DR2a) alleles. Given almost complete linkage disequilibrium of the two alleles, recent studies suggested differential roles in susceptibility (DR2b) or protection from MS (DR2a). Our objective was to assess the potential contribution of DR2a to disease etiology in MS using a humanized model of autoimmunity. To assess the potential contribution of DR2a to disease etiology, we created DR2a humanized transgenic (Tg) mice and subsequently crossed them to Tg mice expressing TL3A6, an MS patient-derived myelin basic protein 83-99-specific TCR. In TL3A6/DR2a Tg mice, CD4 Tg T cells escape thymic and peripheral deletion and initiate spontaneous experimental autoimmune encephalomyelitis (EAE) at low rates, depending on the level of DR2a expression. The ability to induce active EAE was also increased in animals expressing higher levels of DR2a. Inflammatory infiltrates and neuronal damage were present throughout the spinal cord, consistent with a classical ascending EAE phenotype with minor involvement of the cerebellum, brainstem, and peripheral nerve roots in spontaneous, as well as actively induced, disease. These studies emphasize the pathologic contribution of the DR2a allele to the development of autoimmunity when expressed as the sole MHC class II molecule, as well as strongly argue for DR2a as a contributor to the CNS autoimmunity in MS.

Unique clinical and pathological features in HLA-DRB1*0401-restricted MBP 111-129-specific humanized TCR transgenic mice.

Amino acid residues 111-129 represent an immunodominant epitope of myelin basic protein (MBP) in humans with human leukocyte antigen (HLA)-DRB1*0401 allele(s). The MBP 111-129-specific T cell clone MS2-3C8 was repeatedly isolated from a patient with multiple sclerosis (MS), suggesting an involvement of MS2-3C8 T cells in the pathogenesis. To address the pathogenic potential of the MS2-3C8 T cell clone, we generated transgenic (Tg) mice expressing its T cell receptor and restriction element, HLA-DRB1*0401, to examine the pathogenic characteristics of MS2-3C8 Tg T cells by adoptive transfer into HLA-DRB1*0401 Tg mice. In addition to the ascending paralysis typical of experimental autoimmune encephalomyelitis, mice displayed dysphagia due to restriction in jaw and tongue movements and abnormal gait. In accordance with the clinical phenotype, infiltrates of MS2-3C8 Tg T cells and inflammatory lesions were predominantly located in the brainstem and the cranial nerve roots in addition to the spinal cord and spinal nerve roots. Together, these data suggest a pathogenic role of MBP-specific T cells in inflammatory demyelination within the brainstem and cranial nerve roots during the progression of MS. This notion may help to explain the clinical and pathological heterogeneity of MS.

Different development of myelin basic protein agonist- and antagonist-specific human TCR transgenic T cells in the thymus and periphery.

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111-129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111-129 of MBP (MBP(111-129)) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that approximately 50% of human MBP(111-129) (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP(111-129) (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP(111-129) (MBP122:Lys). We found that MBP(111-129) (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP(111-129) TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8(+) T cells develop along with MS2-3C8 TCR Tg CD4(+) T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4(+) and CD8(+) T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.

Disease progression after bone marrow transplantation in a model of multiple sclerosis is associated with chronic microglial and glial progenitor response.

Multiple sclerosis (MS), the most common nontraumatic cause of neurologic disability in young adults in economically developed countries, is characterized by inflammation, gliosis, demyelination, and neuronal degeneration in the CNS. Bone marrow transplantation (BMT) can suppress inflammatory disease in a majority of patients with MS but retards clinical progression only in patients treated in the early stages of the disease. Here, we applied BMT in a mouse model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE), and investigated the kinetics of reconstitution of the immune system in the periphery and in the CNS using bone marrow cells isolated from syngeneic donors constitutively expressing green fluorescent protein. This approach allowed us to dissect the contribution of donor cells to the turnover of resident microglia and to the pathogenesis of observed disease relapses after BMT. BMT effectively blocked or delayed EAE development when mice were treated early in the course of the disease but was without effect in mice with chronic disease. We found that there is minimal overall replacement of host microglia with donor cells in the CNS and that newly transplanted cells do not appear to contribute to disease progression. In contrast, EAE relapses are accompanied by the robust activation of endogenous microglial and macroglial cells, which further involves the maturation of endogenous Olig2 glial progenitor cells into reactive astrocytes through the cytoplasmic translocation of Olig2 and the expression of CD44 on the cellular membrane. The observed maturation of large numbers of reactive astrocytes from glial progenitors and the chronic activation of host microglial cells have relevance for our understanding of the resident glial response to inflammatory injury in the CNS. Our data indicate that reactivation of a local inflammatory process after BMT is sustained predominantly by endogenous microglia/macrophages.

Limited repertoire of HLA-DRB1*0401-restricted MBP111-129-specific T cells in HLA-DRB1*0401 Tg mice and their pathogenic potential.

Since myelin basic protein (MBP)111-129 is an immunodominant epitope in humans carrying HLA-DRB1*0401, we investigated the encephalitogenic potential of HLA-DRB1*0401-restricted MBP111-129-specific T cells using HLA-DRB1*0401/DRA*0101 transgenic (Tg) mice. Although we could not detect the primary recall response to MBP111-129 peptide after immunization of HLA-DRB1*0401/DRA*0101 Tg mice with human MBP, V beta 10(+) and V beta 2(+) HLA-DRB1*0401-restricted MBP111-129-specific T cells proliferated after restimulation of the lymph node cells with human MBP111-129 in vitro. The V beta 2(+) T cell line recognized only human MBP111-129 in the context of HLA-DRB1*0401, while the V beta 10(+) T cell line recognized both the human and murine MBP111-129 epitopes. Therefore, we examined the encephalitogenic potential of the V beta 10(+) T cell line in HLA-DRB1*0401/DRA*0101 Tg mice by adoptive transfer experiments. The V beta 10(+) T cell line induced mild EAE and inflammatory lesions were observed in the spinal cord and the brainstem. In the spinal cord, the inflammation was observed in the peripheral nerve roots as well as in the CNS. These data suggest the pathogenic potential of HLA-DRB1*0401-restricted MBP111-129-specific T cells in humans.