Experimental Autoimmune Encephalopathy Research Articles

In Utero Injections of Nanoparticle Encapsulated Myelin Oligodendrocyte Glycoprotein Peptides Prevent Experimental Autoimmune Encephalopathy Via Central Tolerance

IntroductionIn utero hematopoietic cell transplantation (IUHCT) of allogenic bone marrow mononuclear cells (MNC) results in successful engraftment across immune barriers. This is secondary to immune tolerance that is acquired due to the presence of donor cells in the thymus prior to E17. We have demonstrated that immune tolerance to specific proteins is possible by injecting nanoparticle encapsulated peptides which are taken up by dendritic cells and presented in the thymus.The aim of this study is to show that in utero injections of nanoparticle encapsulated antigen can produce tolerance to that antigen. We demonstrate this by preventing the autoimmune disease Experimental Autoimmune Encephalopathy (EAE, the mouse equivalent of multiple sclerosis) by injecting NPs loaded with the peptide Myelin Oligodendrocyte Glycoprotein (35-55) (MOG35-55.)MethodsIn utero injections of nanoparticles were performed in C57BL/6 mice at E14 intravenously through the vitelline vein. Post-natal boosters were injected subcutaneously at 2, 4, 6, and 8 weeks of age. Disease inducing injections of MOG with Complete Freud's Adjuvant (CFA) was given at 8 1/7 weeks. Intraperitoneal pertussis toxin (PT) was given on the same day as the MOG/CFA and again 48 hours later. Positive controls got MOG/CFA and PT injections. Negative controls got CFA without MOG as well as the PT injections.Mice were monitored for paralysis using the 5-point EAE scoring system for 4 weeks after the MOG/CFA injection. Spines and brains were harvested 2 weeks after the disease inducing MOG/CFA injection. Spines and brains were filtered and then MNCs were isolated using a Percoll gradient. MNCs were stained for CD45, CD19, CD11b, CD3, CD4, CD8, and I-A(b) MOG35-55 tetramer. Cells were then analyzed on the FACSAria™.Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children's Hospital of Philadelphia.ResultsMice that received MOG nanoparticles prenatally along with boosters (Group A) developed disease with less frequency than mice that received MOG nanoparticles postnatally along with boosters (Group B) or positive controls (Group D) (52.0% vs 87.5% vs 90.0% p<0.005) supporting the theory that antigen needs to be expressed prior to E17 in the thymus to develop tolerance. Mice that were injected prenatally with MOG nanoparticles and did not receive booster injections (Group C) also did as poorly as positive controls (85.72% vs 90.0%) suggesting that continued antigen expression is necessary to maintain tolerance. Group A also had significantly lower EAE scores on days 15 to 28 compared to Group B, Group C, and the positive control group (p<0.005).Spinal cord and brain analysis of the Group A mice showed significantly fewer CD45 cells compared to the positive control but not the negative control (133.2±52.0 vs 6062.5±1166.9 vs 79.8±15.0 p<0.0001.) The same is seen with CD3 cells (45.6±17.0 vs 4727.3±1283.0 vs 21.3±2.8 p<0.0001) and CD4 cells (12.6±5.7 vs 3339.6912.1 vs 11.8±0.8 p<0.0001.) Finally, the same is seen with CD4+ Tetramer+ cells (0.7±0.3 vs 379.1±156.6 vs 2.9±1.0 p<0.0001) showing that there is central tolerance to the MOG peptide.ConclusionBy decreasing the frequency and average severity of EAE and demonstrating the lack of CD4+ Tetramer+ cells in the spine, we have shown that autoimmune disease can be prevented by central tolerance to the target antigen. We have demonstrated that this central tolerance is possible with prenatal injections of nanoparticle encapsulated peptide. We have also shown that continued antigen presentation is necessary to maintain central tolerance. We hope that nanoparticle encapsulated peptides could provide a possible pharmacological benefit for numerous autoimmune diseases in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Suppression of Experimental Autoimmune Encephalomyelitis by ILT3.Fc.

Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS that is characterized by demyelination, axonal loss, gliosis, and inflammation. The murine model of MS is the experimental autoimmune encephalopathy (EAE) induced by immunization of mice with myelin oligodendrocyte glycoprotein (MOG)35-55 Ig-like transcript 3 (ILT3) is an inhibitory cell surface receptor expressed by tolerogenic human dendritic cells. In this study, we show that the recombinant human ILT3.Fc protein binds to murine immune cells and inhibits the release of proinflammatory cytokines that cause the neuroinflammatory process that result in paralysis. Administration of ILT3.Fc prevents the rapid evolution of the disease in C57BL/6 mice and is associated with a profound reduction of proliferation of MOG35-55-specific Th1 and Th17 cells. Inhibition of IFN-γ and IL-17A in mice treated with ILT3.Fc is associated with delayed time of onset of the disease and its evolution to a peak clinical score. Neuropathological analysis shows a reduction in inflammatory infiltrates and demyelinated areas in the brains and spinal cords of treated mice. These results indicate that inhibition of Th1 and Th17 development provides effective suppression of EAE and suggests the feasibility of a clinical approach based on the use of ILT3.Fc for treatment of MS. Furthermore, our results open the way to further studies on the effect of the human ILT3.Fc protein in murine experimental models of autoimmunity and cancer.

Experimental autoimmune encephalopathy (EAE)-induced hippocampal neuroinflammation and memory deficits are prevented with the non-opioid TLR2/TLR4 antagonist (+)-naltrexone

Multiple sclerosis (MS) is associated with burdensome memory impairments and preclinical literature suggests that these impairments are linked to neuroinflammation. Previously, we have shown that toll-like receptor 4 (TLR4) antagonists, such as (+)-naltrexone [(+)-NTX], block neuropathic pain and associated spinal inflammation in rats. Here we extend these findings to first demonstrate that (+)-NTX blocks TLR2 in addition to TLR4. Additionally, we examined in two rat strains whether (+)-NTX could attenuate learning and memory disturbances and associated neuroinflammation using a low-dose experimental autoimmune encephalomyelitis (EAE) model of MS. EAE is the most commonly used experimental model for the human inflammatory demyelinating disease, MS. This low-dose model avoided motor impairments that would confound learning and memory measurements. Fourteen days later, daily subcutaneous (+)-NTX or saline injections began and continued throughout the study. Contextual and auditory-fear conditioning were conducted at day 21 to assess hippocampal and amygdalar function. With this low-dose model, EAE impaired long-term, but not short-term, contextual fear memory; both long-term and short-term auditory-cued fear memory were spared. This was associated with increased mRNA for hippocampal interleukin-1β (IL-1β), TLR2, TLR4, NLRP3, and IL-17 and elevated expression of the microglial marker Iba1 in CA1 and DG regions of the hippocampus, confirming the neuroinflammation observed in higher-dose EAE models. Importantly, (+)-NTX completely prevented the EAE-induced memory impairments and robustly attenuated the associated proinflammatory effects. These findings suggest that (+)-NTX may exert therapeutic effects on memory function by dampening the neuroinflammatory response in the hippocampus through blockade of TLR2/TLR4. This study suggests that TLR2 and TLR4 antagonists may be effective at treating MS-related memory deficits.

Antibody mediated targeting of autoimmune cells in multiple sclerosis

Multiple Sclerosis (MS) is a debilitating autoimmune disorder of the central nervous system (CNS) affecting over 77,000 Canadians, with a prevalence rate of almost one out of 300 Canadians, with the highest rates in Saskatchewan. Myelination of neuronal axons is essential for the normal and rapid electrical conduction along an axon. Currently there are few feasible options to treat MS. Here, we propose a novel therapy that specifically targets aberrant immune cells.We created a trimolecular peptide (TPC) that combines and antigen sequence (myelin oligodendrocyte glycoprotein; MOG 35‐55), a modified type IV secretatory system (TIVSS) peptide, and an apoptotic protein. As the TPC displays MOG, the TPC is hypothesized to only bind to immune cells that are actively seeking MOG (e.g. in MS). Since the TPC contains TIVSS, the cell that bound the antigen (e.g. MOG 35‐55) is forced to take up the TPC. Lastly, the apoptotic protein, which is now inside the cell, causes the cell to undergo apoptosis. Thus eliminating the aberrant cell.In order to test this TPC compound, we induced experimental autoimmune encephalopathy (EAE) using MOG 35‐55. Test compound was administered 15 days post EAE induction. Administration of the TPC resulted in the suppression of autoimmune cells, as determined via flocytometry. We also found significant improvements in motor function as well as a reduction in MS plaque formation in the cerebellum. By targeting the pathogenic immune response whilst leaving the patient’s normal immune response intact, we believe this strategy will lead to improved clinical outcomes without the side effects seen in current MS therapies.Support or Funding InformationSaskatchewan Health Research Foundation

Identification of 14-dehydroergosterol as a novel anti-inflammatory compound inducing tolerogenic dendritic cells

Tolerogenic dendritic cells (DCs) have the ability to induce regulatory T cells and play an important role in preventing chronic inflammatory and autoimmune diseases. We have identified a novel compound, 14-dehydroergosterol, from Koji, a Japanese traditional food material fermented with fungi. 14-dehydroergosterol is an ergosterol analogue with a conjugated double bond, but the activity of 14-dehydroergosterol is much higher than that of ergosterol. 14-dehydroergosterol induces the conversion of murine bone marrow (BM)-derived DCs and differentiated DCs into tolerogenic DCs, in which the production of IL-12 is suppressed and that of IL-10 is increased. In a co-culture experiment, DCs treated with 14-dehydroergosterol induced the conversion of naïve CD4-positive T cells into regulatory T cells. In a murine model of multiple sclerosis, experimental autoimmune encephalopathy, 14-dehydroergosterol suppressed the clinical score and inflammatory responses of myeloid DCs and T cells to myelin oligodendrocyte glycoprotein. 14-dehydroergosterol-treated human DCs induced from PBMCs also showed a tolerogenic phenotype. This is the first report to identify a novel compound, 14-dehydroergosterol, that induces DCs to convert to a tolerogenic type. 14-dehydroergosterol is contained in various fermented foods based on Koji, so 14-dehydroergosterol might be a helpful aid to prevent chronic inflammatory and autoimmune diseases.

WWL70 attenuates PGE2 production derived from 2-arachidonoylglycerol in microglia by ABHD6-independent mechanism

Backgroundα/β-Hydrolase domain 6 (ABHD6) is one of the major enzymes for endocannabinoid 2-arachidonoylglycerol (2-AG) hydrolysis in microglia cells. Our recent studies have shown that a selective ABHD6 inhibitor WWL70 has anti-inflammatory and neuroprotective effects in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 in the neuroinflammatory response and the mechanisms by which WWL70 suppresses inflammation has not yet been elucidated in reactive microglia.MethodsThe hydrolytic activity and the levels of 2-AG in BV2 cells were measured by radioactivity assay and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthases in microglia treated with lipopolysaccharide (LPS) with/without WWL70 was determined by western blot and quantitative RT-PCR. The conversion of 2-AG to PGE2 or PGE2-glyceryl ester (PGE2-G) was assessed by enzyme-linked immunoassay (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE2 production was assessed using pharmacological inhibitors and small interfering RNA (siRNA). The effect of WWL70 on PGE2 biosynthesis activity in the microsome fraction from BV2 cells and experimental autoimmune encephalopathy (EAE) mouse brain was also examined.ResultsWe found that WWL70 suppressed PGE2 production in LPS-activated microglia via cannabinoid receptor-independent mechanisms, although intracellular levels of 2-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT182, an alternative ABHD6 inhibitor failed to suppress PGE2 production. WWL70 attenuated the expression of COX-2 and PGES-1/2 leading to the downregulation of the biosynthetic pathways of PGE2 and PGE2-G. Moreover, PGE2 production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 also targets PGE2 biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model.ConclusionsWWL70 is an anti-inflammatory therapeutic agent capable of inhibiting PGE2 and PGE2-G production, primarily due to its reduction of COX-2 and microsomal PGES-1/2 expression and their PGE2 biosynthesis activity in microglia cells, as well as in the EAE mouse brain.

Spinster 2, a sphingosine-1-phosphate transporter, plays a critical role in inflammatory and autoimmune diseases.

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite that regulates numerous processes important for immune responses. S1P is made within cells and must be transported out of cells to exert its effects through activation of 5 specific cell surface GPCRs in an autocrine or paracrine fashion. Spinster 2 (Spns2) transports S1P out of cells, and its deletion in mice reduces circulating levels of S1P, alters immune cell trafficking, and induces lymphopenia. Here we examined the effects of Spns2 deletion on adaptive immune responses and in autoimmune disease models. Airway inflammation and hypersensitivity as well as delayed-type contact hypersensitivity were attenuated in Spns2(-/-) mice. Similarly, Spns2 deletion reduced dextran sodium sulfate- and oxazolone-induced colitis. Intriguingly, Spns2(-/-) mice were protected from the development of experimental autoimmune encephalopathy, a model of the autoimmune disease multiple sclerosis. Deletion of Spns2 also strongly alleviated disease development in collagen-induced arthritis. These results point to a broad role for Spns2-mediated S1P transport in the initiation and development of adaptive immune related disorders.

Adenosine 2A receptor agonism: A single intrathecal administration attenuates motor paralysis in experimental autoimmune encephalopathy in rats.

A single intrathecal dose of adenosine 2A receptor (A2AR) agonist was previously reported to produce a multi-week reversal of allodynia in two different models of neuropathic pain in addition to downregulating glial activation markers in the spinal cord. We aimed to determine whether a single intrathecal administration of an A2AR agonist was able to attenuate motor symptoms induced by experimental autoimmune encephalopathy. Two A2AR agonists (CGS21680 and ATL313) significantly attenuated progression of motor symptoms following a single intrathecal administration at the onset of motor symptoms. OX-42, a marker of microglial activation, was significantly attenuated in the lumbar spinal cord following A2AR administration compared to vehicle. Therefore, A2AR agonists attenuate motor symptoms of EAE by acting on A2AR in the spinal cord.

T Cell Apoptosis and Induction of Foxp3+ Regulatory T Cells Underlie the Therapeutic Efficacy of CD4 Blockade in Experimental Autoimmune Encephalomyelitis

The pathogenesis of multiple sclerosis requires the participation of effector neuroantigen-specific T cells. Thus, T cell targeting has been proposed as a promising therapeutic strategy. However, the mechanism underlying effective disease prevention following T cell targeting remains incompletely known. We found, using several TCR-transgenic strains, that CD4 blockade is effective in preventing experimental autoimmune encephalopathy and in treating mice after the disease onset. The mechanism does not rely on direct T cell depletion, but the anti-CD4 mAb prevents the proliferation of naive neuroantigen-specific T cells, as well as acquisition of effector Th1 and Th17 phenotypes. Simultaneously, the mAb favors peripheral conversion of Foxp3(+) regulatory T cells. Pre-existing effector cells, or neuroantigen-specific cells that undergo cell division despite the presence of anti-CD4, are committed to apoptosis. Therefore, protection from experimental autoimmune encephalopathy relies on a combination of dominant mechanisms grounded on regulatory T cell induction and recessive mechanisms based on apoptosis of neuropathogenic cells. We anticipate that the same mechanisms may be implicated in other T cell-mediated autoimmune diseases that can be treated or prevented with Abs targeting T cell molecules, such as CD4 or CD3.

The role of herpesvirus entry mediator as a negative regulator of T cell–mediated responses

Herpesvirus entry mediator (HVEM), a TNF receptor superfamily member, has been previously described as a T cell costimulatory receptor. Surprisingly, HVEM-/- T cells showed enhanced responses to in vitro concanavalin A (ConA) stimulation when compared with WT T cells. Consistent with these findings, HVEM-/- mice exhibited increased morbidity and mortality as compared with WT mice in a model of ConA-mediated T cell-dependent autoimmune hepatitis. HVEM-/- mice produced higher levels of multiple cytokines, which were dependent on the presence of CD4+ T cells. Furthermore, HVEM-/- mice were more susceptible to MOG peptide-induced experimental autoimmune encephalopathy, and they showed increased T cell proliferation and cytokine production in response to antigen-specific challenge. Taken together, our data revealed an unexpected regulatory role of HVEM in T cell-mediated immune responses and autoimmune diseases.

The role of herpesvirus entry mediator as a negative regulator of T cell–mediated responses

Herpesvirus entry mediator (HVEM), a TNF receptor superfamily member, has been previously described as a T cell costimulatory receptor. Surprisingly, HVEM-/- T cells showed enhanced responses to in vitro concanavalin A (ConA) stimulation when compared with WT T cells. Consistent with these findings, HVEM-/- mice exhibited increased morbidity and mortality as compared with WT mice in a model of ConA-mediated T cell-dependent autoimmune hepatitis. HVEM-/- mice produced higher levels of multiple cytokines, which were dependent on the presence of CD4+ T cells. Furthermore, HVEM-/- mice were more susceptible to MOG peptide-induced experimental autoimmune encephalopathy, and they showed increased T cell proliferation and cytokine production in response to antigen-specific challenge. Taken together, our data revealed an unexpected regulatory role of HVEM in T cell-mediated immune responses and autoimmune diseases.