Novel small molecule IL-6 inhibitor suppresses autoreactive Th17 development and promotes Treg development.

Multiple sclerosis (MS) is the leading cause of non-traumatic neurological disability in the United States in young adults, but current treatments are only partially effective, making it necessary to develop new, innovative therapeutic strategies. Myelin-specific interleukin (IL)-17-producing T helper type 17 (Th17) cells are a major subset of CD4 T effector cells (Teff ) that play a critical role in mediating the development and progression of MS and its mouse model, experimental autoimmune encephalomyelitis (EAE), while regulatory T cells (Treg ) CD4 T cells are beneficial for suppressing disease. The IL-6/signal transducer and activator of transcription 3 (STAT-3) signaling pathway is a key regulator of Th17 and Treg cells by promoting Th17 development and suppressing Treg development. Here we show that three novel small molecule IL-6 inhibitors, madindoline-5 (MDL-5), MDL-16 and MDL-101, significantly suppress IL-17 production in myelin-specific CD4 T cells in a dose-dependent manner in vitro. MDL-101 showed superior potency in suppressing IL-17 production compared to MDL-5 and MDL-16. Treatment of myelin-specific CD4 T cells with MDL-101 in vitro reduced their encephalitogenic potential following their subsequent adoptive transfer. Furthermore, MDL-101 significantly suppressed proliferation and IL-17 production of anti-CD3-activated effector/memory CD45RO+ CD4+ human CD4 T cells and promoted human Treg development. Together, these data demonstrate that these novel small molecule IL-6 inhibitors have the potential to shift the Teff  : Treg balance, which may provide a novel therapeutic strategy for ameliorating disease progression in MS.

Impact of suppressing retinoic acid-related orphan receptor gamma t (ROR)γt in ameliorating central nervous system autoimmunity.

Multiple sclerosis (MS) is an immune-mediated chronic central nervous system (CNS) disease affecting more than 400 000 people in the United States. Myelin-reactive CD4 T cells play critical roles in the formation of acute inflammatory lesions and disease progression in MS and experimental autoimmune encephalomyelitis (EAE), a well-defined mouse model for MS. Current MS therapies are only partially effective, making it necessary to develop more effective therapies that specifically target pathogenic myelin-specific CD4 T cells for MS treatment. While suppressing T-bet, the key transcription factor in T helper type 1 (Th1) cells, has been demonstrated to be beneficial in prevention and treatment of EAE, the therapeutic potential of retinoic acid-related orphan receptor gamma t (ROR)γt, the key transcription factor for Th17 cells, has not been well-characterized. In this study, we characterized the correlation between RORγt expression and other factors affecting T cell encephalitogenicity and evaluated the therapeutic potential of targeting RORγt by siRNA inhibition of RORγt. Our data showed that RORγt expression correlates with interleukin (IL)-17 production, but not with the encephalitogenicity of myelin-specific CD4 T cells. IL-23, a cytokine that enhances encephalitogenicity, does not enhance RORγt expression significantly. Additionally, granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, which correlate with the encephalitogenicity of different myelin-specific CD4 T cell populations, do not correlate with RORγt. More importantly, inhibiting RORγt expression in myelin-specific CD4 T cells with an siRNA does not reduce disease severity significantly in adoptively transferred EAE. Thus, RORγt is unlikely to be a more effective therapeutic target for ameliorating pathogenicity of encephalitogenic CD4 T cells.

Cytokine-induced immune deviation as a therapy for inflammatory autoimmune disease.

The properties and outcome of an immune response are best predicted by the lymphokine phenotype of the responding T cells. Cytokines produced by CD4+ T helper type 1 (Th1) T cells mediate delayed type hypersensitivity (DTH) and inflammatory responses, whereas cytokines produced by Th2 T cells mediate helper T cell functions for antibody production. To determine whether induction of Th2-like cells would modulate an inflammatory response, interleukin 4 (IL-4) was administered to animals with experimental allergic encephalomyelitis (EAE), a prototypic autoimmune disease produced by Th1-like T cells specific for myelin basic protein (MBP). IL-4 treatment resulted in amelioration of clinical disease, the induction of MBP-specific Th2 cells, diminished demyelination, and inhibition of the synthesis of inflammatory cytokines in the central nervous system (CNS). Modulation of an immune response from one dominated by excessive activity of Th1-like T cells to one dominated by the protective cytokines produced by Th2-like T cells may have applicability to the therapy of certain human autoimmune diseases.

T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis.

Encounters with antigen can stimulate T cells to become activated and proliferate, become nonresponsive to antigen, or to die. T cell death was shown to be a physiological response to interleukin-2-stimulated cell cycling and T cell receptor reengagement at high antigen doses. This feedback regulatory mechanism attenuates the immune response by deleting a portion of newly dividing, antigen-reactive T cells. This mechanism deleted autoreactive T cells and abrogated the clinical and pathological signs of autoimmune encephalomyelitis in mice after repetitive administration of myelin basic protein.

Decreased dependence of myelin basic protein-reactive T cells on CD28-mediated costimulation in multiple sclerosis patients. A marker of activated/memory T cells.

Although multiple sclerosis (MS) patients and healthy individuals have similar frequencies of myelin basic protein (MBP)-specific T cells, the activation state of these cells has not been well characterized. Therefore, we investigated the dependence of MBP-reactive T cells on CD28-mediated costimulation in MS patients, healthy controls, and stroke patients. MBP-reactive T cells from healthy controls and stroke patients failed to proliferate efficiently when costimulation was blocked using anti-CD28, consistent with a naive T cell response. In contrast, MBP-specific T cell proliferation was not inhibited, or was only partially inhibited when CD28-mediated costimulation was blocked in MS patients. Blockade of CD28 failed to inhibit tetanus toxoid-specific T cell proliferation in both the controls and MS patients, demonstrating that memory cells are not dependent on CD28-mediated costimulation. Limiting dilution analysis indicated that the frequency of MBP-reactive T cells was significantly decreased in healthy controls compared with MS patients when CD28-mediated costimulation was blocked. These data suggest that MBP-reactive T cells are more likely to have been activated in vivo and/or differentiated into memory T cells in MS patients compared with controls, indicating that these cells may be participating in the pathogenesis of MS.

Regulation of experimental autoimmune encephalomyelitis with insulin-like growth factor (IGF-1) and IGF-1/IGF-binding protein-3 complex (IGF-1/IGFBP3).

Insulin-like growth factor (IGF)-1 is a cytokine that promotes oligodendrocyte development and myelin production. This study investigated whether treatment of chronic, relapsing murine experimental autoimmune encephalomyelitis (EAE) with IGF-1 or IGF-1 associated with its binding protein, IGFBP3, altered the course of disease. Administration of IGF-1/IGFBP3 (1-100 mg/kg per day) delayed the onset of disease in a dose-dependent manner and histologic examination showed a delay in inflammatory cells entering the central nervous system. However, once signs of EAE developed, disease was enhanced in the mice that had been given the highest dose of IGF-1/IGFBP3. Treatment with IGF-1/IGFBP3 after the onset of signs resulted in a severe relapse. Administration of free IGF-1 (10 mg/kg per day) provided mild protection when given before disease onset, but did not significantly alter the course of disease if given after disease onset. Possible mechanisms that could explain the altered disease in IGF-1/IGFBP3-treated mice included (a) IGF-1/IGFBP3 administration delayed the onset of EAE by downregulating ICAM-1 gene expression in the central nervous system, and (b) IGF-1/IGFBP3 treatment of EAE resulted in more severe disease due to enhanced expansion of encephalitogenic T cells. Although IGF-1 may enhance remyelination, these results indicate that administration of IGF-1 associated with IGFBP3 may also accentuate autoimmune demyelinating disease.

Distinct roles for B7-1 (CD-80) and B7-2 (CD-86) in the initiation of experimental allergic encephalomyelitis.

The activation and differentiation of T cells require both antigen/MHC recognition and costimulatory signals. The present studies examined the role of B7-1 (CD80) and B7-2 (CD86) costimulation in the prototypic autoimmune disorder, experimental allergic encephalomyelitis (EAE). In adoptively transferred EAE, in vitro activation of myelin basic protein (MBP)-specific lymph node cells was inhibited by the combination of anti-CD80 plus anti-CD86, but not individually. However, in actively induced disease, one injection of anti-CD80 significantly reduced disease, while anti-CD86 exacerbated disease. Interestingly, one injection of CTLA-4Ig suppressed disease, while multiple injections resulted in enhanced disease. Thus, the costimulation provided by B7-1 molecules appears to be important for the development of encephalitogenic T cells. The enhanced disease caused by multiple injections of CTLA-4Ig or a single injection of anti-CD86 suggests an inhibitory function for CD86 interaction with its counterreceptors CD28 and CTLA-4 in EAE. Alternatively, these results are consistent with an essential timing requirement for the coordinated interaction of B7 and CD28 family receptors, and that disruption of this critical timing can have opposing results on the outcome of an immune response.

Characterizing the mechanisms of progression in multiple sclerosis: evidence and new hypotheses for future directions.

Major advancements have been achieved in our ability to diagnose multiple sclerosis (MS) and to commence treatment intervention with agents that can favorably affect the disease course. Although MS exacerbations and the emergence of disability constitute the more conspicuous aspects of the disease process, evidence has confirmed that most of the disease occurs on a constitutive and occult basis. Disease-modifying therapies appear to be modest in the magnitude of their treatment effects, particularly in the progressive stage of the disease. Therapeutic strategies currently used for MS primarily target the inflammatory cascade. Several potential mechanisms appear to be involved in the progression of MS. Characterizing these mechanisms will result in a better understanding of the various forms of the disorder and how to effectively treat its clinical manifestations. It is our objective within this 2-part series on progression in MS to offer both evidence-based observations and hypothesis-driven expert perspectives on what constitutes the cause of progression in MS. We have chosen areas of inquiry that appear to have been most productive in helping us to better conceptualize the landscape of what MS looks like pathologically, immunologically, neuroscientifically, radiographically, and genetically. We have attempted to advance hypotheses focused on a deeper understanding of what contributes to the progression of this illness and to illustrate new technical capabilities that are catalyzing novel research initiatives targeted at achieving a more complete understanding of progression in MS.

B7-mediated costimulation can either provoke or prevent clinical manifestations of experimental allergic encephalomyelitis.

T-cell activation requires signalling provided by ligation of the T-cell receptor for antigen (TCR) and a second antigen (Ag) nonspecific signal, known as costimulation. The B7 receptors, CD80 (B7-1) and CD86 (B7-2), on the Ag-presenting cell (APC), interact with T-cell CD28 or CTLA-4 to deliver a costimulatory signal, which is particularly important for Th1 activation. Experimental allergic encephalomyelitis (EAE) is an autoimmune disorder, induced by Th1 cells directed against myelin antigens that provides an in vivo model for studying the role of B7-mediated costimulation in the induction of a pathological immune response. Using a soluble fusion protein ligand for the B7 receptors, as well as specific monoclonal antibodies specific for either CD80 or CD86, it has been demonstrated that B7 costimulation plays a prominent role in determining clinical disease outcome in EAE. Here we review recent data indicating that a paradoxical exacerbation of disease as well as the expected amelioration of disease can occur with costimulatory receptor blockade.

MRI characteristics of the MLF in MS patients with chronic internuclear ophthalmoparesis.

OBJECTIVE: The authors imaged the medial longitudinal fasciculus (MLF) in 58 patients with MS and chronic internuclear ophthalmoparesis (INO) to determine which MRI technique best shows the characteristic lesion associated with this ocular motor syndrome. METHODS: Using quantitative infrared oculography, the authors determined the ratios of abduction to adduction for velocity and acceleration, to confirm the presence of INO and to determine the severity of MLF dysfunction in 58 patients with MS and INO. Conventional MRI techniques, including proton density imaging (PDI), T2-weighted imaging, and fluid-attenuated inversion recovery (FLAIR) imaging, were used to ascertain which technique best shows MLF lesions within the brainstem tegmentum. T1-weighted imaging was performed to determine the frequency of brainstem tegmentum hypointensities. RESULTS: All patients studied had evidence of an MLF lesion hyperintensity on PDI, whereas T2-weighted imaging and FLAIR imaging showed these lesions in 88% and 48% of patients, respectively. With PDI, dorsomedial tegmentum lesions were seen in the pons in 93% of patients and in the midbrain of 66% of patients. Lesions were observed at both locations in 59% of patients. One patient had an MLF lesion with a corresponding T1 hypointensity. CONCLUSIONS: PDI best shows the MLF lesion in patients with MS and INO.

Vertigo in MS: utility of positional and particle repositioning maneuvers.

A 4-year experience with new-onset vertigo in a university-based MS population was retrospectively reviewed. Of 1,153 patients with MS, 25 could be clinically evaluated during the vertiginous episode. Of these, 13 (52%) were diagnosed with benign paroxysmal positioning vertigo and eight (32%) had acute MS exacerbations with corresponding lesions within the brainstem. All patients diagnosed with benign paroxysmal positioning vertigo were treated successfully with particle repositioning maneuvers.

Impaired renal function in progressive multiple sclerosis.

The authors analyzed renal function in 25 patients with progressive MS. The mean glomerular filtration rate (GFR) was 92 mL/min/1.73 m(2), compared to the predicted GFR of 110 (p < 0.001). Nine of the 25 (36%) patients had abnormally low GFR (<90). The mean serum creatinine for patients with MS was lower than predicted normal values and poorly estimated GFR using standard equations. These data document impaired renal function in patients with progressive MS and have implications for treatment of these patients with potentially nephrotoxic drugs.

Phase 1 trial of transforming growth factor beta 2 in chronic progressive MS.

Transforming growth factor (TGF)-beta2 is a pleiotropic cytokine associated with remissions in multiple sclerosis (MS) and amelioration of allergic encephalomyelitis. We assessed the safety of TGF-beta2 in an open-label trial of 11 patients with secondary progressive (SP) MS. Five patients had a reversible decline in the glomerular filtration rate. There was no change in expanded disability status scale or MRI lesions during treatment. Systemic TGF-beta2 may be associated with reversible nephrotoxicity, and further investigation of its therapeutic potential in MS should be performed with caution.

Copolymer-1-induced inhibition of antigen-specific T cell activation: interference with antigen presentation.

Copolymer-1 (Cop-1) has been shown to inhibit in vivo development of experimental allergic encephalomyelitis (EAE) in animals and has been reported to have some therapeutic benefit in relapsing/remitting multiple sclerosis (MS). The mechanism by which Cop-1 acts in vivo is not known. The present study demonstrates that Cop-1 inhibits the in vitro response of several antigen-specific murine T cell hybridomas restricted to I-A, and to a lesser extent, I-E. The ability of human myelin basic protein (MBP)-specific T cell lines (TCL) to lyse targets in the context of three HLA-DR types associated with MS was also impaired by Cop-1. The results suggest that the observed inhibition was due to competition between Cop-1 and nominal antigen for the class II major histocompatibility complex (MHC) peptide binding site.

Cytokine-regulated adhesion between encephalitogenic T lymphocytes and cerebrovascular endothelial cells.

Adhesive interactions between murine cerebrovascular endothelial cells (EC) which comprise the blood-brain barrier (BBB) and myelin basic protein (MBP)-specific encephalitogenic T lymphocytes were investigated. Adhesion was assessed by measuring the percent attachment of 51Cr-labeled T cells to EC monolayers. The basal level adhesion (20-35%) was significantly up-regulated by treating EC with recombinant murine gamma interferon (IFN-gamma), interleukin-1 alpha (IL-1 alpha) and/or tumor necrosis factor-alpha (TNF alpha). The ability of these cytokines to modulate adhesion was dose- and time-dependent and could be detected as early as 1 h after treatment. The expression of intercellular adhesion molecule-1 (ICAM-1) by EC was examined by immunofluorescence staining and ELISA. Although all unstimulated EC cultures expressed ICAM-1, treatment of EC with the above cytokines dramatically up-regulated the level of ICAM-1 expression in a dose- and time-dependent fashion similar to that observed in the adhesion assays. Treatment of EC with transforming growth factor-beta 1 (TGF beta) down-regulated the level of T cell adhesion on untreated EC in a dose-dependent manner. Pretreatment of EC with TGF beta also partially inhibited the up-regulation of adhesion induced by IFN-gamma, IL-1 alpha and/or TNF alpha. TGF beta had no effect on the up-regulation of ICAM-1 expression induced by IFN-gamma, IL-1 alpha and/or TNF alpha. These results indicate that in addition to ICAM-1, other molecules may be involved in adhesion of encephalitogenic T cells to the EC comprising the cerebral vasculature.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 2.

It had been demonstrated previously that the administration of transforming growth factor-beta 1 (TGF-beta 1) reduced the clinical severity of experimental allergic encephalomyelitis (EAE). Treatment with the related immunosuppressive molecule, TGF-beta 2, resulted in similar inhibition of T cell activation and proliferation in vitro. Long-term treatment was effective in reducing clinical severity of EAE and the number of relapses in mice receiving either myelin basic protein- or peptide-91-103-specific T cell lines. When examined histologically, mice that had received TGF-beta 2 demonstrated significantly less inflammation and demyelination in the central nervous system. Examination of other organs demonstrated no pathology or deleterious side effects from long-term TGF-beta 2 therapy. These findings have relevance for the use of TGF-beta 2 as a therapeutic agent for the human demyelinating disease, multiple sclerosis.

Longitudinal study of myelin basic protein-specific T-cell receptors during the course of multiple sclerosis.

This study analyzed the stability of the myelin basic protein (MBP)-specific T-cell receptor (TCR) repertoire during the course of multiple sclerosis (MS) in three patients who were monitored for three years by gadolinium-enhanced magnetic resonance imaging. Bulk-culture T-cell lines (TCLs) were generated from 3-4 time points for each patient, including times of active and quiescent disease. TCR analysis of these TCLs indicated that both the V alpha and V beta usage was similar over time for each patient. Sequencing of TCRs demonstrated conserved complementarity-determining region 3 (CDR3) sequences within TCLs that expressed the same V alpha segment over time, although the J alpha usage was different for each TCR. This indicates that the population of MBP-reactive T-cells is changing during the course of MS, but that host and/or environmental factors may be selecting T-cells with particular MHC/peptide binding domains.

The role of costimulation in autoimmune demyelination.

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated, autoimmune disorder characterized by central nervous system (CNS) inflammation and demyelination, features reminiscent of the human disease, multiple sclerosis (MS). In addition to the signal the encephalitogenic T cell receives through the T cell receptor (TCR), a second signal, termed costimulation, is required for complete T cell activation. The B7 family of cell surface molecules expressed on antigen presenting cells (APC) is capable of providing this second signal to T cells via two receptors, CD28 and CTLA-4. Our studies have shown that costimulation provided by B7 molecules to its ligand CD28 is important in the initiation of the autoimmune response in EAE. Further, it appears the costimulation provided by B7-1 is important in disease development, while B7-2 may play an important regulatory role. We and others later showed that B7/CTLA-4 interaction plays a critical role in down-regulating the immune response. Previous work has shown that activated T cells and T cells of a memory phenotype are less dependent on costimulation than naive T cells. T cells reactive with myelin components that are involved in the pathogenesis of EAE and possibly MS would be expected to have been activated as part of the disease process. Building upon our prior work in the EAE model, we have tested the hypothesis that myelin-reactive T cells, which are relevant to the pathogenesis of CNS inflammatory demyelination, can be distinguished from naive myelin-reactive T cells by a lack of dependence upon costimulation for activation and that the costimulatory requirements of these myelin-reactive T cells change during the course of disease. Our studies in the EAE model have also addressed the mechanisms of extrathymic (peripheral) T cell tolerance following intravenous (i.v. ) administration of high dose antigen. It is believed that TCR signaling in the absence of costimulation is a vital component of peripheral tolerance mechanisms. However, recent evidence suggests that peripheral tolerance of antigen-specific T cells induced in vivo may require CTLA-4 engagement of the tolerized T cells. We have begun to examine the molecular mechanisms of tolerance induction following intravenous and intraperitoneal administration of myelin antigens in the EAE model and test the hypothesis that tolerance induction is dependent on the B7:CD28/CTLA-4 pathway. The results from our studies will enhance our understanding of the role that myelin-reactive T cells may play in the pathogenesis of MS. We have determined that MBP-reactive T cells in MS patients are less dependent upon CD28 costimulation than in normal controls, suggesting that these T cells were previously primed in vivo. Characterization of these CD28-independent myelin-specific T cells will have broad implications for a variety of immunologically based therapies in diseases such as MS.

Retrovirus mediated gene transfer of the self antigen MBP into the bone marrow of mice alters resistance to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a prototypic model of organ specific autoimmunity. MHC class II restricted T-cells directed against myelin basic protein (MBP) have been shown to cause EAE in susceptible strains of mice. We have asked whether the introduction of a gene encoding an autoantigen (MBP) into the hematopoetic stem cells of mice would result in tolerance to that protein. We have introduced cDNA encoding the 21.5 kDa isoform of MBP into the hematopoetic stem cells of B10.PL (73NS), SJL, and B10 mice by retrovirus-mediated gene transfer. Our experiments show expression of proviral MBP in peripheral blood and thymus following transplantation of genetically modified stem cells. Such expression does not result in deletion of MBP-specific T cells or tolerance to MBP, nor does it alter susceptibility to MBP-induced EAE in susceptible strains B10.PL and SJL. However, retrovirus-mediated gene transfer resulted in resistant B10 mice developing mild EAE. This report demonstrates that autoreactive MBP-specific T cells can be selected in the presence of endogenous antigen or an MBP-encoding retrovirus.

Therapeutic potential of phosphodiesterase type 4 inhibition in chronic autoimmune demyelinating disease.

It was recently demonstrated that selective phosphodiesterase type 4 (PDE4) inhibition suppresses the clinical manifestations of acute experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), and inhibits the production of tumor necrosis factor-alpha (TNF-alpha), a pathogenetically central cytokine. Since the most common presentation of MS in humans is a relapsing-remitting course, we investigated the therapeutic potential of PDE4 inhibition in the relapsing-remitting EAE model of the SJL mouse. Administration of rolipram, the prototypic PDE4 inhibitor, reduced the clinical signs of EAE during both the initial episode of disease and subsequent relapses. In parallel, there was marked reduction of demyelination and also less inflammation throughout the central nervous system (CNS) of rolipram-treated animals. Gene expression of proinflammatory cytokines in the CNS was reduced in most of the rolipram-treated animals. Additional experiments demonstrated that PDE4 inhibition acted principally by inhibiting the secretion of Th1 cytokines, however, the encephalitogenic potential of myelin basic protein-specific T cells was not impaired. Our findings suggest that PDE4 inhibitors are a promising cytokine-directed therapy in chronic demyelinating disease.