Mdivi-1, a mitochondrial fission inhibitor, modulates T helper cells and suppresses the development of experimental autoimmune encephalomyelitis.

BACKGROUND: Unrestrained activation of Th1 and Th17 cells is associated with the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). While inactivation of dynamin-related protein 1 (Drp1), a GTPase that regulates mitochondrial fission, can reduce EAE severity by protecting myelin from demyelination, its effect on immune responses in EAE has not yet been studied. METHODS: We investigated the effect of Mdivi-1, a small molecule inhibitor of Drp1, on EAE. Clinical scores, inflammation, demyelination and Drp1 activation in the central nervous system (CNS), and T cell responses in both CNS and periphery were determined. RESULTS: Mdivi-1 effectively suppressed EAE severity by reducing demyelination and cellular infiltration in the CNS. Mdivi-1 treatment decreased the phosphorylation of Drp1 (ser616) on CD4+ T cells, reduced the numbers of Th1 and Th17 cells, and increased Foxp3+ regulatory T cells in the CNS. Moreover, Mdivi-1 treatment effectively inhibited IFN-γ+, IL-17+, and GM-CSF+ CD4+ T cells, while it induced CD4+ Foxp3+ regulatory T cells in splenocytes by flow cytometry. CONCLUSIONS: Together, our results demonstrate that Mdivi-1 has therapeutic potential in EAE by modulating the balance between Th1/Th17 and regulatory T cells.

IL-12R beta 2 promotes the development of CD4+CD25+ regulatory T cells.

We have previously shown that mice lacking the IL-12-specific receptor subunit beta2 (IL-12Rbeta2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rbeta2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rbeta2(-/-) mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rbeta2-deficient mice to autoimmune diseases. T cells from IL-12Rbeta2(-/-) mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25(+)CD4(+) regulatory T cells (Tregs) in the thymus and spleen of IL-12Rbeta2(-/-) mice were comparable to those of WT mice. However, IL-12Rbeta2(-/-) mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-beta, as shown by significantly lower numbers of CD25(+)CD4(+) T cells that expressed Foxp3. Functionally, CD25(+)CD4(+) Tregs derived from IL-12Rbeta2(-/-) mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rbeta2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rbeta2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway.

Role of the innate immune system in autoimmune inflammatory demyelination.

Considerable research has been devoted to the role of the adaptive immune system in the pathogenesis of autoimmune inflammatory demyelination (AID). AID is thought to occur spontaneously in patients with multiple sclerosis (MS), a common cause of neurological disability. AID is also observed in the best characterized animal model of MS, experimental autoimmune encephalomyelitis (EAE). The adaptive immune system recognizes and responds to antigens via highly specific T-cell receptors. Myelin-reactive T-cells may initiate pathological immune responses that lead to central nervous system damage in MS and EAE. By contrast, the innate immune system recognizes evolutionarily conserved structures that are common to invading pathogens with high efficiency for rapid recognition and elimination of viruses, bacteria, and fungi. This recognition is mediated by pattern-recognition receptors such as Toll-like receptors (TLRs) expressed on cells of the innate immune system (dendritic cells and CNS-resident cells, such as microglia) that have the potential to activate autoimmune responses by inducing the production of inflammatory cytokines and chemokines. Conversely, the innate immune system can also regulate autoimmune inflammation by inducing the production of immunoregulatory molecules such as type I interferons, which are currently used in the treatment of MS. We review the evidence that TLRs can exacerbate or regulate AID and discuss the therapeutic potential of targeting either process.

Upregulation of chemokine receptor expression by IL-10/IL-4 in adult neural stem cells.

It has been shown that adoptively transferred adult neural stem cells (NSCs) ameliorate experimental autoimmune encephalomyelitis (EAE) by differentiating into myelin-forming cells. However, NSC migration into the lesion foci is inefficient and relatively slow, resulting in only modest therapeutic effect. A possible reason for the inefficient migration of NSCs could be the production of anti-inflammatory cytokines by these cells, which might in turn suppress their migration. To address this question we established subventricular zone-derived NSC neurospheres and determined the influence of IL-10 and IL-4 on chemokine receptor expression by these cells, and on their migration in response to chemokines relevant to EAE pathogenesis. We found that treatment with IL-4 and IL-10 upregulates surface adhesion molecule LFA-1 and chemokine receptors CXCR4 and CCR5 on NSCs. IL-10-treated NSCs exhibited significantly higher chemotaxis to the ligands of the above chemokine receptors than untreated cells. Treatment of NSC with IL-4 also resulted in a higher chemotaxis of these cells to RANTES. Thus, in vitro pretreatment with cytokines may be a useful approach to facilitate migration of NSCs into CNS inflammatory foci, producing stronger therapeutic effects. This approach has the potential to improve clinical outcomes of NSC-based therapies for neurological disorders such as multiple sclerosis.

Retinal ganglion cell damage induced by spontaneous autoimmune optic neuritis in MOG-specific TCR transgenic mice.

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are marked by inflammatory demyelinating lesions throughout the central nervous system, including optic nerve. Neuronal loss also occurs in EAE, including retinal ganglion cell (RGC) loss in eyes with optic neuritis, but the finding of RGC loss in relation to optic nerve inflammation differs in different EAE settings. Recently, Myelin oligodendrocyte glycoprotein (MOG)-specific TCR transgenic mice were found to develop spontaneous isolated optic neuritis in the absence of EAE. In the current study, the relationship of inflammation to retinal ganglion cell (RGC) loss during isolated optic neuritis is examined. RGCs of MOG-specific TCR transgenic mice were labeled with Flourogold and then treated with pertussis toxin (PT) or observed untreated. At various time points, RGCs were counted, retinas were TUNEL labeled, and optic nerves were examined for inflammatory cell infiltrates. 29% of untreated MOG-specific TCR transgenic mice developed periocular inflammation by 4 months of age, and 32% of optic nerves of TCR transgenic mice had histological lesions in the optic nerve. Incidence of histological optic neuritis was 20% at day 8 following injection of PT and increased to 48% by day 12, and 68% by day 16. In contrast, no RGC loss or TUNEL staining was detected in eyes with optic neuritis until day 12 in the mice injected with PT. A 28% reduction in RGC numbers at day 12 increased to 39% by day 16, and RGC loss of eyes with severe or massive inflammation was significantly higher than that of eyes with mild or moderate inflammation. No RGC loss occurred in TCR transgenic mouse eyes without optic neuritis. The fact that inflammation precedes RGC loss suggests that neuronal loss during optic neuritis occurs secondary to the inflammatory process in isolated optic neuritis.

Pathophysiology of interleukin-23 in experimental autoimmune encephalomyelitis.

Interleukin-23 (IL-23) is a heterodimeric cytokine that is composed of a p40 subunit, shared with the closely related cytokine IL-12, and a smaller IL-23p19 subunit. It belongs to a family of heterodimeric cytokines that also includes IL-12 and IL-27. Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease that serves as a model for multiple sclerosis, an inflammatory demyelinating disease of the central nervous system that is a frequent cause of disability in young adults. EAE is thought to be initiated by CD4+ T cells. The production of interferon-gamma and tumor necrosis factor-alpha (T helper 1 [Th1] phenotype) was considered a marker for the ability of such cells to induce disease. Consistent with this view, IL-12, a cytokine that induces the differentiation of Th1 cells, was considered essential for EAE susceptibility. However, it is now clear that IL-23 rather than IL-12 is required for EAE susceptibility. IL-23 induces a population of IL-17-producing cells that is more critically involved in EAE pathogenesis than Th1 cells. Here, we review the role of the IL-23 system in the pathophysiology of EAE.

Pathogenesis of neuroimmunologic diseases. Experimental models.

Animal models of autoimmune diseases have greatly improved our current understanding of the pathogenesis of human autoimmunity and have provided the potential for therapies based on manipulation of the immune system. In our laboratory, we have investigated the immunopathogenesis of autoimmune diseases of the nervous system and muscle. We have developed immune-based approaches for the suppression of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), and experimental autoimmune neuritis (EAN), a model for the Guillain-Barré syndrome (GBS). These approaches included induction of peripheral tolerance, immunotoxin targeting of activated T cells, and cytokine manipulations. In addition, we identified the antigen and characterized immunopathologically an autoimmune inflammatory disease of skeletal muscle, experimental autoimmune myositis (EAM), a model for the human inflammatory muscle disease polymyositis.

Magnetic resonance imaging of the cauda equina in Guillain-Barré syndrome.

We report three patients (two children and one adult) with Guillain-Barré syndrome and magnetic resonance imaging evidence of gadolinium enhancement of the cauda equina and lumbar nerve roots. All three patients exhibited symmetric ascending paralysis and areflexia, and two (one child, one adult) suffered urinary incontinence and retention. Similar enhancement has been observed in patients with chronic inflammatory demyelinating polyneuropathy and suggests proximal nerve inflammation. Magnetic resonance imaging in Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy may have diagnostic utility.

Anti-interleukin-12 antibody: potential role in preventing relapses of multiple sclerosis.

Multiple sclerosis is an inflammatory demyelinating disorder of the central nervous system. Immunological evidence from patients with multiple sclerosis and experimental models suggests that the cytokine interleukin-12 (IL-12) plays an important role in the pathogenesis of inflammatory demyelination. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, antibodies that block IL-12 can prevent relapses of the disease. Anti-IL-12 antibodies present a novel approach for preventing relapses in multiple sclerosis.

Serial evoked potential studies and MRI imaging in chronic progressive multiple sclerosis.

Measurements of serial evoked potential latencies and plaque burden on MRI scans are often obtained during clinical studies of multiple sclerosis patients to provide additional information to the disability-based primary endpoints. The ideal laboratory-based marker of progression would be expected to significantly change over the time period of study. Serial visual (VEP) and brainstem auditory evoked potentials (BAEP) and MRI scans of 11 chronic progressive MS patients were obtained over a 1.5 year period in a clinical study. Over this period, there was no significant change in disability as measured by the Kurtzke EDSS, Ambulation Index or Neurological Rating Score. The VEP P100 significantly progressed over the period of study. However, the MRI T(2) plaque burden and BAEP I-V intrapeak latency did not significantly progress over the 1.5 years. We conclude that, in chronic progressive MS, serial visual evoked potential tests may complement standard disability-based endpoints to assess disease progression.

IL-12 reverses the suppressive effect of the CD40 ligand blockade on experimental autoimmune encephalomyelitis (EAE).

Blockade of the CD40 ligand (CD40L)-CD40 interaction suppresses experimental autoimmune encephalomyelitis (EAE). Since this interaction induces IL-12, an essential cytokine for EAE induction, we hypothesized that CD40L blockade may suppress EAE through IL-12 inhibition. Here we show that exogenous IL-12 abolishes the ability of anti-CD40L monoclonal antibodies to prevent EAE. Anti-IL-12 antibodies prevent this reversal and protect from EAE. These results show that IL-12 is sufficient to overcome CD40L blockade and suggest that, of the multiple consequences of the CD40L-CD40 interaction, IL-12 induction is an essential one for induction of EAE.

Different mechanisms of inflammation induced in virus and autoimmune-mediated models of multiple sclerosis in C57BL6 mice.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the human central nervous system (CNS). Neurotropic demyelinating strain of MHV (MHV-A59 or its isogenic recombinant strain RSA59) induces MS-like disease in mice mediated by microglia, along with a small population of T cells. The mechanism of demyelination is at least in part due to microglia-mediated myelin stripping, with some direct axonal injury. Immunization with myelin oligodendrocyte glycoprotein (MOG) induces experimental autoimmune encephalomyelitis (EAE), a mainly CD4(+) T-cell-mediated disease, although CD8(+) T cells may play a significant role in demyelination. It is possible that both autoimmune and nonimmune mechanisms such as direct viral toxicity may induce MS. Our study directly compares CNS pathology in autoimmune and viral-induced MS models. Mice with viral-induced and EAE demyelinating diseases demonstrated similar patterns and distributions of demyelination that accumulated over the course of the disease. However, significant differences in acute inflammation were noted. Inflammation was restricted mainly to white matter at all times in EAE, whereas inflammation initially largely involved gray matter in acute MHV-induced disease and then is subsequently localized only in white matter in the chronic disease phase. The presence of dual mechanisms of demyelination may be responsible for the failure of immunosuppression to promote long-term remission in many MS patients.

The protease inhibitor, Bowman-Birk Inhibitor, suppresses experimental autoimmune encephalomyelitis: a potential oral therapy for multiple sclerosis.

Available treatments for multiple sclerosis (MS) require frequent injections and have significant side effects. Proteases generated during inflammation are involved in the induction of tissue damage during inflammatory demyelination in the central nervous system (CNS). The Bowman-Birk Inhibitor (BBI), a soy-derived protease inhibitor with anti-carcinogenic and anti-inflammatory properties, has been shown to be well tolerated in clinical trials for pre-cancerous conditions, such as oral leukoplakia and the inflammatory disease, ulcerative colitis. We hypothesized that BBI may modulate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The BBI concentrate (BBIC), a soybean extract enriched in BBI, was administered to myelin basic protein (MBP)-immunized Lewis rats by gastric gavage in different treatment regimens, during the induction or the effector phase of disease. BBIC significantly delayed disease onset and suppressed disease severity, clinically and pathologically, in all treatment protocols. Both in vitro and ex vivo, BBIC inhibited MBP-specific proliferation of lymph node cells. BBIC reduced the activity of matrix metalloproteinase (MMP)-2 and -9 in spleen cell supernatants and was detected in the CNS of treated rats. BBIC suppresses EAE, it can be administered orally, and it is safe and relatively inexpensive. It may have a therapeutic role in patients with MS.

Pathogenesis of immune-mediated neuropathies.

A variety of peripheral neuropathies are believed to be immune-mediated. Acute inflammatory demyelinating polyneuropathy or Guillain-Barré syndrome (GBS) is the prototype of these neuropathies. GBS is characterized by acute progressive motor weakness of the extremities and of bulbar and facial musculature. Deep tendon reflexes are reduced or absent, and sensory symptoms are mild. Respiratory failure and autonomic dysfunction may be seen. The cerebrospinal fluid shows increased protein and no or very few cells. The nerve conduction velocity is slowed, and the pathology shows segmental demyelination with mononuclear cell infiltration. Studies from man and experimental animals suggest an immunologic basis for demyelination of the peripheral nerves in GBS, but the mechanism is not well understood. Experimental allergic neuritis, an animal model of GBS, is induced in laboratory animals by immunization with myelin P2 protein, some peptides of P2 protein, and galactocerebroside. The animals develop weakness and show electrophysiologic and pathologic features similar to GBS. P2-reactive T cells and antigalactocerebroside antisera can adoptively transfer experimental allergic neuritis. Various antibodies to peripheral nerve myelin and circulating immune complexes have been found in patients with GBS. The target antigen(s) for these antibodies are not well understood, but neutral glycolipids cross-reactive with Forssman antigen and gangliosides are possible candidates. The mainstay of therapy is the management of the paralyzed patient. Steroids are ineffective. Plasmapheresis, especially early in the course of the disease, can shorten the duration of paralysis and intubation. Results from a multicenter study in the Netherlands demonstrate the efficacy of high-dose immune globulin therapy in GBS.(ABSTRACT TRUNCATED AT 250 WORDS)

P2-reactive T cells in inflammatory demyelination of the peripheral nerve.

Lewis rats immunized with P2 protein, a 14.5-kDa protein of the peripheral nerve myelin, develop experimental allergic neuritis, a paralytic disorder with clinical, histologic, and electrophysiologic features similar to those of human Guillain-Barré syndrome (GBS). T cells reactive to P2 protein or a peptide corresponding to 53-78 residues of the protein can transfer the disease to naive animals. The mechanisms by which these T cells induce demyelination are not well understood; however, they may induce inflammation and demyelination in the nerves by production of Th1 cytokines. Th2 cytokines may lead to suppression of the inflammation and eventual recovery. There is no conclusive evidence that P2 protein plays a role in the pathogenesis of GBS, with or without association with Campylobacter jejuni; however, studies of the immunopathogenesis of P2 protein-induced experimental allergic neuritis are important for understanding the pathogenesis of inflammatory demyelination in the peripheral nerves, the hallmark of GBS.

Guillain-Barré syndrome: clinical and immunological aspects.

Immune-mediated PNS disorders comprise a significant segment of diseases of the nervous system. Studies on GBS as a prototype of these disorders and its experimental model EAN have helped to elucidate some of the mechanisms responsible for myelin injury in the PNS. These mechanisms, although partially understood have been useful in implementing therapies such as plasmapheresis and IVIG and various other immunomodulators. The question of whether an infectious agent such as a virus can directly damage the myelin sheath and/or Schwann cells or whether the agent triggers an immune response against self through antigenic mimicry remains unanswered. The association between C. jejuni infection and GBS has opened new areas of investigation in understanding the immunopathogenesis of the disease. Similar observations with other environmental factors may be made in the future, pointing to the possibility that GBS may not be caused by a single agent but could be the result of an immunological attack on the PNS myelin assembly by a variety of agents or factors. Regardless of the etiology, if the myelin injury is aggravated by product of the immune cells, such as various cytokines, neutralization of these factors could help lessen the burden of injury to the nerves. Future research in autoimmune disorders of the PNS needs to focus on identifying environmental factors that directly, or indirectly through antigen mimicry, damage the PNS myelin. In parallel, further understanding of the immunopathogenesis by dissecting the immunological phenomenon at the systemic and local levels, especially the role of cytokines, growth factors, and adhesion molecules will pave the way for more rational therapies, even if the causative factors are not known. Studies in laboratory animals have demonstrated the efficacy of selective immunotherapy through modulation of the trimolecular complex, i.e., T cell receptor, MHC/molecule, and antigen. Immunological tolerance, presumably through deletion of autoreactive clones, clonal anergy, or active suppression, has proven effective in animals. Other modes of immunotherapy such as nonspecific depletion of T or B cells or down-regulation of activated cells have also been shown to abolish or decrease the severity of experimental autoimmune neurological disorders, including EAN. These immunotherapeutic modalities may become applicable to human autoimmune neuropathies.

Urinary free kappa light chain levels in chronic progressive multiple sclerosis.

Previous studies have shown elevated levels of free kappa light chains (FKLC) in the urine of patients with relapsing-remitting multiple sclerosis (MS). The levels correlated well with clinical relapses, indicating that they could serve as useful markers of disease activity. In this study, we performed monthly measurements of urinary FKLC in patients with the chronic progressive form of MS, and correlated them with clinical indicators of disease activity, as expressed by Kurtzke's expanded disability status scale (EDSS) and brain magnetic resonance imaging (MRI). We noted wide fluctuations in the FKLC levels, which correlated poorly with EDSS scores or MRI changes. Longer follow-up periods may be needed before definite conclusions can be drawn.

Chemokines and peripheral nerve demyelination.

It has been speculated that beta-chemokines play a pivotal role in the development of peripheral nervous system (PNS) disorders characterized by mononuclear cell infiltration. In experimental allergic neuritis (EAN), an animal model for human Guillain-Barré syndrome (GBS) with mononuclear cell infiltration, we found by quantitative PCR that beta-chemokine messages were upregulated during the active stage. Moreover, an increase in the monocyte chemoattractant protein-1 (MCP-1) message was found in the preclinical stage of EAN, suggesting the critical role of MCP-1 for inducing mononuclear cell infiltrations in this model. Since many cell lineages other than immune cells can produce chemokines, this early upregulation of MCP-1 may be mediated by non-immune cells, probably endothelia or Schwann cells. To date, apart from MCP-1, only RANTES (Regulated on activation, normal T cell expressed and secreted) and macrophage inflammatory protein (MIP)-1alpha have been examined in EAN and found to have similar kinetics of induction. Therefore, understanding the regulation of production of these chemokines as well as mechanisms of inhibiting chemokine/receptor interactions in the PNS may ultimately lead to disease-specific therapy for GBS and related demyelinating disorders.

Chemokines and chemokine receptors in the pathogenesis of multiple sclerosis.

In recent years we have seen growing evidence for the role of chemokines in the pathogenesis of several infectious and non-infectious inflammatory CNS disease states, including Multiple Sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE). An increase in proinflammatory chemokines has been associated with demyelinating lesions and clinical neurological dysfunction in patients with MS; these chemokines could be potential targets for MS therapy. Besides a clearly defined role in mediating leukocyte migration, these and other chemokines may act as immunoregulatory molecules in the driving to Th1/Th2 responses, switch of cytokine profiles, and the induction of tolerance. Since chemokine receptors have now been identified on macrophages, microglia, astrocytes, and endothelial cells as well as neurons in the CNS, chemokine/receptor interactions may mediate functional responses in a variety of CNS cell types during the course of inflammatory disease states. Therefore, clarification of the roles of chemokines and their receptors in the pathogenesis of EAE and MS will be useful in establishing immunotherapeutic strategies for these neurological autoimmune disorders.