Acute EAE Research Articles

Iguratimod Ameliorates the Severity of Secondary Progressive Multiple Sclerosis in Model Mice by Directly Inhibiting IL-6 Production and Th17 Cell Migration via Mitigation of Glial Inflammation.

We previously reported a novel secondary progressive multiple sclerosis (SPMS) model, progressive experimental autoimmune encephalomyelitis (pEAE), in oligodendroglia-specific Cx47-inducible conditional knockout (Cx47 icKO) mice. Based on our prior study showing the efficacy of iguratimod (IGU), an antirheumatic drug, for acute EAE treatment, we aimed to elucidate the effect of IGU on the SPMS animal model. We induced pEAE by immunizing Cx47 icKO mice with myelin oligodendrocyte glycoprotein peptide 35-55. IGU was orally administered from 17 to 50 days post-immunization. We also prepared a primary mixed glial cell culture and measured cytokine levels in the culture supernatant after stimulation with designated cytokines (IL-1α, C1q, TNF-α) and lipopolysaccharide. A migration assay was performed to evaluate the effect of IGU on the migration ability of T cells toward mixed glial cell cultures. IGU treatment ameliorated the clinical signs of pEAE, decreased the demyelinated area, and attenuated glial inflammation on immunohistochemical analysis. Additionally, IGU decreased the intrathecal IL-6 level and infiltrating Th17 cells. The migration assay revealed reduced Th17 cell migration and IL-6 levels in the culture supernatant after IGU treatment. Collectively, IGU successfully mitigated the clinical signs of pEAE by suppressing Th17 migration through inhibition of IL-6 production by proinflammatory-activated glial cells.

Biological roles of myeloid cell subsets during CNS inflammation (P2-3.005)

<h3>Objective:</h3> We designed an experimental platform, utilizing single-cell transcriptomic and epigenomic profiling. We employed this system to interrogate single-cell data for subset-specific signatures. <h3>Background:</h3> Myeloid cells (MC) of the central nervous system (CNS) constitute a heterogeneous population that includes A) yolk sac-derived CNS microglia, B) bone marrow-derived resident MC and C) infiltrating bone marrow-derived MC. While bone marrow and yolk sac progenies differ in origin, their protein expression exhibit significant overlap. Current canonical myeloid markers, lack the specificity required for use as disease biomarkers or therapeutic targets. <h3>Design/Methods:</h3> We created chimeras in congenic animals by transplantation of bone marrow from CD45.1⁺ donors into sub-lethally irradiated CD45.2⁺ recipients. Following active EAE, CD11b⁺ MC were collected from CNS at the peak of the disease. Fluorescent-activated cell sorting (FACS) separated CD45.1⁺ MC from CD45.2⁺ MC. Each sample was analyzed simultaneously for single-cell gene expression (scRNA-seq) and epigenetic signatures (scATAC-seq) via the 10X Genomics Multiomic assay. We employed bioinformatics software (Seurat v4.0) for analysis and visualization of data profiling over 20,000 cells. <h3>Results:</h3> Bone marrow derived (CD45.1+) or microglial and CNS-resident (CD45.2+) MC were both present in the CNS tissue during acute EAE. Analyses of single-cell transcriptomic and ATAC-seq data, independently distinguished common clusters of MC derived entirely from the CD45.2+ cells. Unsupervised annotation, recognized these cells as microglia. CD45.1+ MC and microglia exhibited extensive gene expression overlap; however, differential gene expression analysis and logistic regression modeling yielded a signature set of genes within the microglia accurately distinguished microglia from bone-marrow-derived MC in independent datasets. <h3>Conclusions:</h3> Single-cell data recapitulates the subtle differences in the otherwise comparable gene expression patterns among microglia and bone marrow-derived MC. These patterns shed light on the subset-specific biological roles and may present as superior substitutes for the suboptimal canonical myeloid markers. <b>Disclosure:</b> Navid Manouchehri has nothing to disclose. The institution of an immediate family member of Dr. Salinas has received research support from Veterans Health Administration. Rehana Hussain has nothing to disclose. Dr. Stuve has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for EMD Serono. Dr. Stuve has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for Novartis. Dr. Stuve has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for Roche Genentech. Dr. Stuve has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for TG Therapeutics. Dr. Stuve has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for TG Therapeutics. Dr. Stuve has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for EMD Serono. Dr. Stuve has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for VYNE. Dr. Stuve has received personal compensation in the range of $5,000-$9,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Therapeutic Advances in Neurological Diseases. Dr. Stuve has received research support from US Department of Veterans Affairs. Dr. Stuve has received research support from National Multiple Sclerosis Society (US). Dr. Stuve has received research support from Exalys. Dr. Stuve has received research support from Merck KGaA.

Discovery of selective urokinase plasminogen activator (uPA) inhibitors as a potential treatment for multiple sclerosis

We report here the design and synthesis of a novel series of benzylamines that are potent and selective inhibitors of uPA with promising oral availability in rat. Further evaluation of one representative (ZK824859) of the new structural class showed that this compound lowered clinical scores when dosed in either acute or chronic mouse EAE models, suggesting that uPA inhibitors of this type could be useful for the treatment of multiple sclerosis.

TRH Administered by Mouth Inhibits Acute EAE (P05.168)

TRH Administered by Mouth Inhibits Acute EAE (P05.168)

Ingested (oral) thyrotropin releasing factor (TRH) inhibits EAE

BackgroundIngested immunoactive proteins type I IFN, SIRS peptide 1–21, α-MSH, ACTH, SST inhibit clinical attacks and inflammation in acute EAE by decreasing Th1-like cytokines, increasing Th2-like cytokines or increasing Treg cell frequencies. ObjectiveWe examined whether another protein, thyrotropin releasing factor (TRH), would have similar anti-inflammatory effects in EAE after oral administration. Design/methodsB6 mice were immunized with MOG peptide 35–55 and gavaged with control saline or TRH during ongoing disease. Splenocytes from mock fed or TRH fed mice were adoptively transferred into active MOG peptide 35–55 immunized recipient mice during ongoing disease. ResultsIngested (oral) TRH inhibited ongoing disease and decreased inflammation. Adoptively transferred cells from TRH fed donors protected against actively induced disease and decreased inflammation. In actively fed mice, oral TRH decreased IL-17 and TNF-α cytokines in both the spleen and the CNS. In recipients of donor cells from TRH fed mice there was a reduction of Th1 and Th17 and induction of Th2-like IL-13 cytokines in both the spleen and CNS. Oral TRH decreased clinical score and decreased inflammatory foci in both actively fed and recipients of actively fed mice. There was no significant increase in Treg cell frequencies in actively fed or recipients of TRH fed donor cells. ConclusionsIngested (orally administered) TRH can inhibit clinical disease, inhibit CNS inflammation by decreasing Th1-like, Th17 and TNF-α cytokines and increasing Th2-like cytokines (IL-13) in the CNS.

Ingested (oral) neuropeptide Y inhibits EAE

BackgroundIngested immunoactive proteins, type I IFN, SIRS peptide 1–21, α-MSH, ACTH, and SST inhibit clinical attacks and inflammation in acute EAE by decreasing Th1-like cytokines, increasing Th2-like cytokines or increasing Treg cell frequencies. ObjectiveWe examined whether another protein, neuropeptide Y, would have similar anti-inflammatory effects in EAE after oral administration. Design/methodsB6 mice were immunized with MOG peptide 35–55 and gavaged with control saline or NPY during ongoing disease. Splenocytes from mock fed or NPY fed mice were adoptively transferred into active MOG peptide 35–55 immunized recipient mice during ongoing disease. ResultsIngested (oral) NPY inhibited ongoing disease, and decreased inflammation. Adoptively transferred cells from NPY fed donors protected against actively induced disease and decreased inflammation. In actively fed mice, oral NPY decreased Th1-like cytokines and increased Th2-like IL-13 cytokines in both the spleen and the CNS. In recipients of donor cells from NPY fed mice there was a reduction of Th1 and Th17 and induction of Th2-like IL-13 cytokines in both the spleen and CNS. Oral NPY decreased clinical score and decreased inflammatory foci in both actively fed and recipients of actively fed mice. There was no significant increase in Treg cell frequencies in actively fed or recipients of NPY fed donor cells. ConclusionsIngested (orally administered) NPY can inhibit clinical disease, inhibit CNS inflammation by decreasing Th17 and Th1-like cytokines and increasing Th2-like cytokines in the CNS.

Ingested (Oral) TRH and Neuropeptide Y Inhibit Acute EAE (P05.115)

Ingested (Oral) TRH and Neuropeptide Y Inhibit Acute EAE (P05.115)

PS1-30 Ingested (oral) SST inhibits acute and passive EAE

PS1-30 Ingested (oral) SST inhibits acute and passive EAE

VEGF and angiogenesis in acute and chronic MOG (35–55) peptide induced EAE

An increased expression of vascular endothelial growth factor (VEGF) is associated with demyelinated lesions in both multiple sclerosis (MS) and its model (EAE), implicating changes in vasculature as a potential component of CNS plaque formation. The purpose of this study was to investigate the vascular changes in acute and chronic EAE in C57BL/6 mice induced with myelin oligodendrocyte glycoprotein (MOG 35–55) peptide. We investigated the functional contribution of VEGF to acute and chronic EAE by treating immunized mice with SU5416 (Semaxinib), a potent and selective inhibitor of VEGF receptor 2 (VEGFR2). Animals received seven daily injections of SU5416 (50 mg/kg) or vehicle beginning on the day after disease onset (acute study) or on day 45 post-immunization (chronic study). Spinal cord sections were collected on the day of sacrifice. Modulation of angiogenic gene expression was determined using RNA isolated from 4 acute and 4 non-immunized controls. MOG peptide induction produced extensive demyelination, immune cell infiltration, tissue laminin deposits, and axonal loss in lesions. VEGF expression was extensively increased in the acute mice, which correlated positively with clinical score. In the acute study, SU5416 treatment produced a significant clinical improvement versus vehicle controls ( p < 0.001), with less demyelination (− 37%) and cellular infiltration (− 23%) in the spinal cord ( p < 0.05). Treated animals also had significantly fewer blood vessels per section than controls (56.1 ± 6.1 v. 81.6 ± 11.5, p < 0.05), and significantly reduced laminin abnormalities (28.9% of lesion area v. 46.8%, p < 0.05). There was no improvement in clinical score or tissue pathology, and no difference in vessel number or lesion laminin expression, when SU5416 was administered during the chronic disease (all p > 0.05). In the acute study only, VEGF staining correlated with demyelination and the extent of cellular infiltration in both control ( r = 0.723, r = 0.665) and treated ( r = 0.681, r = 0.487) animals (all p < 0.05). Laminin staining in lesion areas was strongly correlated with tissue pathology for all animals in both the acute and chronic study (all p < 0.001). Vascular alterations in MOG peptide-induced EAE in the mouse are accompanied by increased lesion-specific levels of VEGF, extensive laminin deposits in the tissue and altered transcription of numerous angiogenic factors. In the microarray studies, acute mice showed a significant increase in several angiogenic RNA transcripts, six of which were verified by RT-PCR, alanyl aminopeptidase, caspase 8, Hif1a, MMP-19, plasminogen activator inhibitor, and thrombospondin1.

Differential effects of decoy chemokine (7ND) gene therapy on acute, biphasic and chronic autoimmune encephalomyelitis: Implication for pathomechanisms of lesion formation

Multiple sclerosis (MS) exhibits several clinical subtypes such as the relapsing–remitting (RR) and secondary progressive (SP) forms. In accordance with this, formation of demyelinating plaques in the central nervous system (CNS) occurs by different mechanisms. In the present study, we induced acute, biphasic and chronic (RR or SP) EAE in rats and examined the effects of decoy chemokine (7ND) gene therapy, which inhibits the migration of macrophages, to address the above issue. Interestingly, it was demonstrated that the clinical signs of acute EAE and the first attack of biphasic EAE were minimally affected, whereas chronic EAE and the relapse of biphasic EAE were completely suppressed with 7ND treatment. In the CNS, the number of infiltrating macrophages was reduced in all the stages of the three types of EAE. These findings suggest that in acute EAE and in the first attack of biphasic EAE, where anti-macrophage migration therapy was almost ineffective, pathogenic T cells are mainly involved in lesion formation. In contrast, the relapse of biphasic EAE and chronic EAE macrophages play a major role in the disease process. Thus, the mechanisms of lesion formation are not uniform and immunotherapy should be performed on the basis of information about the pathomechanisms of autoimmune diseases.

Immunomodulation by neural stem cells

Neural (stem) cell transplantation has been proposed as a means of cell replacement therapy. Multipotential neural precursor cells (NPCs) that expand in floating spheres, and are (partially) committed to a glial fate, showed excellent remyelinating properties in a focal, chemically induced demyelinated lesion in the rat spinal cord. When transplanted into the CNS of rodents with acute and chronic EAE the NPCs were attracted by the inflammatory process to migrate exclusively into inflamed white matter but not into adjacent gray matter. Following magnetic labeling, mouse NPCs and human ESC-derived neural precursors' migration was detected by high-resolution magnetic resonance images. Intraventricular transplantation of neural spheres attenuated brain inflammation in acute and chronic EAE, reduced the clinical severity of disease, and reduced demyelination and axonal pathology. Intravenous (IV) NPC injection also inhibited EAE and reduced CNS inflammation and tissue injury. However, NPCs did not enter the CNS but were transiently found in lymph nodes and spleen, where they inhibited the activation and proliferation of T cells and markedly reduced their encephalitogenicity. Thus, IV administration of neural precursors inhibits EAE by a peripheral immunosuppression, involving a profound bystander inhibitory effect of NPCs on T cell activation and proliferation in lymph nodes. In conclusion, neural precursor cells exert an immunomodulatory effect that inhibits CNS inflammation. Cell therapy in MS should be optimized to utilize both regenerative and immunologic properties of the cells.

Paralysis of CD4 +CD25 + regulatory T cell response in chronic autoimmune encephalomyelitis

Increasing evidence strongly suggest that CD4 +CD25 + regulatory T (Treg) cells play a pivotal role in suppressing the development of autoimmune diseases. However, it remains poorly understood how these cells are involved in the persistence of, or recovery from, the diseases. In the present study, we examined the role of CD4 +CD25 + Treg cells in chronic EAE and compared the results with those obtained in acute EAE. In EAE lesions, CD25 + cells decreased rapidly at the beginning of chronic EAE, whereas these cells were maintained at high levels during the recovery from acute EAE. The number of Foxp3 +CD4 +CD25 + Treg and levels of Foxp3 mRNA in the lymphoid organ were significantly lower in chronic EAE. Importantly, the regulatory function of individual CD4 +CD25 + Treg cells was maintained in animals with chronic EAE. Furthermore, adoptive transfer of activated CD4 +CD25 + Treg cells suppressed the development of chronic EAE. These findings suggest that impairment of the CD4 +CD25 + Treg response is critical for development of chronic autoimmune diseases, and can be adjustable by autologous Treg transplantation.

Ingested (oral) SIRS peptide 1–21 inhibits acute EAE by inducing Th 2-like cytokines

Objective Ingested type I IFN inhibits clinical attacks, relapses and inflammation in murine chronic relapsing EAE by inhibiting Th 1-like cytokines. Type I IFN activates human suppressor T cells that produce SIRS. Methods We examined whether oral (ingested) SIRS peptide inhibits EAE by decreasing Th 1-like cytokines. Results Parenteral SIRS peptide 1–21 showed a significant inhibition of disease severity in murine EAE. Ingested SIRS peptide at 10 and 100 μg SIRS peptide showed a significant inhibition of disease severity but also a prolonged delay in the onset of disease compared to placebo. There were significantly less inflammatory foci in the SIRS peptide fed group compared to the control mock fed group. Splenocytes from SIRS peptide 1–21 fed mice showed increased production of Th 2-like CD30L, IL-13, TCA-3 cytokines/chemokines and decreased production of Th 1-like cytokine lymphotactin. Interpretation Ingested (oral) SIRS peptide significantly inhibits both clinical EAE and inflammation predominately via counter-regulatory type 2-like cytokines/chemokines IL-13, CD30L and TCA-3.

Induction and blockage of oligodendrogenesis by differently activated microglia in an animal model of multiple sclerosis

The role of activated microglia (MG) in demyelinating neurodegenerative diseases such as multiple sclerosis is controversial. Here we show that high, but not low, levels of IFN-gamma (a cytokine associated with inflammatory autoimmune diseases) conferred on rodent MG a phenotype that impeded oligodendrogenesis from adult neural stem/progenitor cells. IL-4 reversed the impediment, attenuated TNF-alpha production, and overcame blockage of IGF-I production caused by IFN-gamma. In rodents with acute or chronic EAE, injection of IL-4-activated MG into the cerebrospinal fluid resulted in increased oligodendrogenesis in the spinal cord and improved clinical symptoms. The newly formed oligodendrocytes were spatially associated with MG expressing MHC class II proteins and IGF-I. These results point to what we believe to be a novel role for MG in oligodendrogenesis from the endogenous stem cell pool.

Motor evoked potentials in a mouse model of chronic multiple sclerosis

We tested cortical motor evoked potentials (cMEPs) as a quantitative marker for in vivo monitoring of corticospinal tract damage in a murine multiple sclerosis model (experimental autoimmune encephalomyelitis, EAE). The cMEPs, previously standardized in naive C57BL/6 developing and adult mice, were studied longitudinally in adult EAE mice. Central conduction times (CCTs) increased significantly shortly before the earliest clinical signs developed (10 days postimmunization, dpi), with peak delay in acute EAE (20-40 dpi). In clinically stable disease (80 dpi), CCTs did not increase further, but cMEP amplitude declined progressively, with complete loss in >80% of mice at 120 dpi. Increase in CCT correlated with presence of inflammatory infiltrates and demyelination in acute EAE, whereas small or absent cMEPs were associated with continuing axonal damage in clinically-stabilized disease and beyond (>80 dpi). These results demonstrate that cMEPs are a useful method for monitoring corticospinal tract function in chronic-progressive EAE, and provide insight into the pathological substrate of the condition.

Effects of pixantrone on immune-cell function in the course of acute rat experimental allergic encephalomyelitis

Pixantrone is an immunesuppressor similar to mitoxantrone but with lower cardiotoxicity. We evaluated the effect of pixantrone on B cells and lymphomononuclear cells in the course of acute EAE. Pixantrone reduced the number of B cells and suppressed myelin basic protein (MBP) specific IgG production. In vitro, pixantrone induced apoptosis of rat B lymphocytes in a way similar to mitoxantrone. In addition, pixantrone inhibited antigen specific and mitogen induced lymphomononuclear cell proliferation, as well as IFN-γ production, during EAE. These findings suggest a similar mechanism of action for pixantrone and mitoxantrone on the effector function of lymphomonocyte B and T cells.

In vitro metabolism and in vivo pharmacokinetics of quinoline 3-carboxamide derivatives in various species

The metabolism of a group of quinoline 3-carboxamide derivatives was evaluated in liver microsomes from various species. In addition, metabolism data were compared with in vivo pharmacokinetics in the mouse. The studied compounds were metabolized by cytochrome P450 enzymes. Microsomal clearance was low and seemed independent of a substituent in the quinoline moiety, whereas clearance was enhanced when an ethyl group replaced the methyl group at the carboxamide position. A similar metabolism with hydroxylated and dealkylated metabolites was found in the various species, with quantitative differences due to substituent. As predicted from the in vitro studies, in vivo pharmacokinetics showed low clearance and thus high exposure of the parent compounds in the mouse. The therapeutic effect seen in the acute EAE mouse model for these related compounds seems dependent on the high exposure of parent compound rather than formation of any potentially active metabolites.

OPL124 MRI and histological analysis of iron labeled macrophages during alpha 4 blockade in acute EAE

OPL124 MRI and histological analysis of iron labeled macrophages during alpha 4 blockade in acute EAE

Amelioration of CR-EAE with lisofylline: effects on mRNA levels of IL-12 and IFN-γ in the CNS

CR-EAE is a Th1-mediated inflammatory autoimmune demyelinating disease of the CNS and serves as a model of human multiple sclerosis. Our previous studies have shown the protective effect of orally administered lisofylline in the prevention of active and passively induced acute EAE. In our present studies we have examined the efficacy and mechanism of action of lisofylline on CR-EAE. Lisofylline decreased the number and severity of paralytic attacks in mice with relapsing EAE. The reduction of clinical disease correlated with decreased levels of mRNA levels of IFN-γ but not of mRNA levels of IL-12. These studies suggest that lisofylline may be an effective therapeutic for established Th1 mediated autoimmune disease and that it acts by blocking IL-12R signaling and not IL-12 production in vivo.

Immunity to heat shock proteins and neurological disorders of women.

Stress or heat shock proteins are constitutively expressed in normal CNS tissues in a variety of cell types (oligodendrocytes, astrocytes, and neurons). Their presence may protect cells from various stresses, such as hypoxia, anoxia, and excessive excitatory stimulation. Increased amounts of hsp are expressed in various cells of the CNS during acute toxic-metabolic states and in chronic degenerative and inflammatory diseases. Increased expression of hsp may lead to immune responses to these proteins. Antibodies to mycobacterial hsp bind to normal human myelin and to oligodendrocytes in regions of MS demyelination. Cellular immune responses to hsp occur with increased frequency and magnitude in persons with MS, especially those with recent onset of disease. In addition, there are populations of T cells expressing gamma/delta T cells in the brains and spinal fluids of persons with MS, suggesting an in situ immune response to hsps. Humoral immune responses to hsp are found in CSF, but no disease specificity has been documented. Some myelin proteins have sequence homology with particular hsps. One instance is the homology between a peptide of mycobacterial Hsp65 and the myelin protein CNP. Our data on EAE suggest that immune responses to either cross-reactive hsp epitopes or whole hsp can modify the course of both acute and chronic relapsing EAE. In addition, the severity and frequency of environmental exposure to infectious agents can modify the course of EAE, possibly by altering the patterns of immune response to hsp. Finally, tolerance to the small hsp, alpha B-crystallin, a putative autoantigen in persons with MS, alters the course of relapsing EAE, supporting its role in chronic, autoimmune CNS disease. Modifying immune responses to hsp may be a potential new treatment option for persons with MS.