Myelin Oligodendrocyte Glycoprotein Peptide Research Articles

Therapeutic role of interferon-γ in experimental autoimmune encephalomyelitis is mediated through a tolerogenic subset of splenic CD11b+ myeloid cells

Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency of regulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased with no effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effects of IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either interleukin (IL)-10, transforming growth factor (TGF)-β or program death (PD)-1, revealed that the protective effects of IFN-γ in EAE were also dependent on TGF-β and PD-1, but not on IL-10, suggesting that IFN-γ might have an indirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency of a subset of splenic CD11b+ myeloid cells expressing TGF-β-Latency Associated Peptide (LAP) and program death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore, splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein (MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells mediated by secretion of TGF-β. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltration of mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic and therapeutic activities.

Lactate Dehydrogenase-Elevating Virus Infection Inhibits MOG Peptide Presentation by CD11b+CD11c+ Dendritic Cells in a Mouse Model of Multiple Sclerosis.

Infections may affect the course of autoimmune inflammatory diseases of the central nervous system (CNS), such as multiple sclerosis (MS). Infections with lactate dehydrogenase-elevating virus (LDV) protected mice from developing experimental autoimmune encephalomyelitis (EAE), a mouse counterpart of MS. Uninfected C57BL/6 mice immunized with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) experienced paralysis and lost weight at a greater rate than mice who had previously been infected with LDV. LDV infection decreased the presentation of the MOG peptide by CD11b+CD11c+ dendritic cells (DC) to pathogenic T lymphocytes. When comparing non-infected mice to infected mice, the histopathological examination of the CNS showed more areas of demyelination and CD45+ and CD3+, but not Iba1+ cell infiltration. These results suggest that the protective effect of LDV infection against EAE development is mediated by a suppression of myelin antigen presentation by a specific DC subset to autoreactive T lymphocytes. Such a mechanism might contribute to the general suppressive effect of infections on autoimmune diseases known as the hygiene hypothesis.

Role of autoreactive Tc17 cells in the pathogenesis of experimental autoimmune encephalomyelitis

AbstractBackgroundThe pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE—an animal model of MS) is primarily mediated by T cells. However, recent studies have only focused on interleukin (IL)‐17‐secreting CD4+ T‐helper cells, also known as Th17 cells. This study aimed to compare Th17 cells and IL‐17‐secreting CD8+ T‐cytotoxic cells (Tc17) in the context of MS/EAE.MethodsFemale C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptides 35–55 (MOG35–55), pertussis toxin, and complete Freund's adjuvant to establish the EAE animal model. T cells were isolated from the spleen (12–14 days postimmunization). CD4+ and CD8+ T cells were purified using isolation kit and then differentiated into Th17 and Tc17, respectively, using MOG35–55 and IL‐23. The secretion levels of interferon‐γ (IFN‐γ) and IL‐17 were measured via enzyme‐linked immunosorbent assay using cultured CD4+ and CD8+ T cell supernatants. The pathogenicity of Tc17 and Th17 cells was assessed through adoptive transfer (tEAE), with the clinical course assessed using an EAE score (0–5). Hematoxylin and eosin as well as Luxol fast blue staining were used to examine the spinal cord. Purified CD8+ CD3+ and CD4+ CD3+ cells differentiated into Tc17 and Th17 cells, respectively, were stimulated with MOG35–55 peptide for proliferation assays.ResultsThe results showed that Tc17 cells (15,951 ± 1985 vs. 55,709 ± 4196 cpm; p < 0.050) exhibited a weaker response to highest dose (20 μg/mL) MOG35–55 than Th17 cells. However, this response was not dependent on Th17 cells. After the 48 h stimulation, at the highest dose (20 μg/mL) of MOG35–55. Tc17 cells secreted lower levels of IFN‐γ (280.00 ± 15.00 vs. 556.67 ± 15.28 pg/mL, p < 0.050) and IL‐17 (102.67 ± 5.86 pg/mL vs. 288.33 ± 12.58 pg/mL; p < 0.050) than Th17 cells. Similar patterns were observed for IFN‐γ secretion at 96 and 144 h. Furthermore, Tc17 cell‐induced tEAE mice exhibited similar EAE scores to Th17 cell‐induced tEAE mice and also showed similar inflammation and demyelination.ConclusionThe degree of pathogenicity of Tc17 cells in EAE is lower than that of Th17 cells. Future investigation on different immune cells and EAE models is warranted to determine the mechanisms underlying MS.

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

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.

Pharmacological targeting of coagulation factor XI attenuates experimental autoimmune encephalomyelitis in mice.

Multiple sclerosis (MS) is the most common causes of non-traumatic disability in young adults worldwide. MS pathophysiologies include the formation of inflammatory lesions, axonal damage and demyelination, and blood brain barrier (BBB) disruption. Coagulation proteins, including factor (F)XII, can serve as important mediators of the adaptive immune response during neuroinflammation. Indeed, plasma FXII levels are increased during relapse in relapsing-remitting MS patients, and previous studies showed that reducing FXII levels was protective in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). Our objective was to determine if pharmacological targeting of FXI, a major substrate of activated FXII (FXIIa), improves neurological function and attenuates CNS damage in the setting of EAE. EAE was induced in male mice using murine myelin oligodendrocyte glycoprotein peptides combined with heat-inactivated Mycobacterium tuberculosis and pertussis toxin. Upon onset of symptoms, mice were treated every other day intravenously with anti-FXI antibody, 14E11, or saline. Disease scores were recorded daily until euthanasia for ex vivo analyses of inflammation. Compared to the vehicle control, 14E11 treatment reduced the clinical severity of EAE and total mononuclear cells, including CD11b+CD45high macrophage/microglia and CD4+ T cell numbers in brain. Following pharmacological targeting of FXI, BBB disruption was reduced, as measured by decreased axonal damage and fibrin(ogen) accumulation in the spinal cord. These data demonstrate that pharmacological inhibition of FXI reduces disease severity, immune cell migration, axonal damage, and BBB disruption in mice with EAE. Thus, therapeutic agents targeting FXI and FXII may provide a useful approach for treating autoimmune and neurologic disorders.

Tolerogenic Nanovaccine for Prevention and Treatment of Autoimmune Encephalomyelitis.

Herein, a tolerogenic nanovaccine is developed and tested on an animal model of multiple sclerosis. The nanovaccine is constructed to deliver the self-antigen, myelin oligodendrocyte glycoprotein (MOG) peptide, and dexamethasone on an abatacept-modified polydopamine core nanoparticle (AbaLDPN-MOG). AbaLDPN-MOG can target dendritic cells and undergo endocytosis followed by trafficking to lysosomes. AbaLDPN-MOG blocks the interaction between CD80/CD86 and CD28 in antigen-presenting cells and T cells, leading to decreased interferon gamma secretion. The subcutaneous administration of AbaLDPN-MOG to mice yields significant biodistribution to lymph nodes and, in experimental-autoimmune encephalomyelitis (EAE) model mice, increases the integrity of the myelin basic sheath and minimizes the infiltration of immune cells. EAE mice are treated with AbaLDPN-MOG before or after injection of the autoantigen, MOG. Preimmunization of AbaLDPN-MOG before the injection of MOG completely blocks the development of clinical symptoms. Early treatment with AbaLDPN-MOG at three days after injection of MOG also completely blocks the development of symptoms. Notably, treatment of EAE symptom-developed mice with AbaLDPN-MOG significantly alleviates the symptoms, indicating that the nanovaccine has therapeutic effects. Although AbaLDPN is used for MOG peptide delivery in the EAE model, the concept of AbaLDPN can be widely applied for the prevention and alleviation of other autoimmune diseases.

Chronic experimental autoimmune encephalomyelitis is an excellent model to study neuroaxonal degeneration in multiple sclerosis.

Animal models of multiple sclerosis (MS), specifically experimental autoimmune encephalomyelitis (EAE), have been used extensively to develop anti-inflammatory treatments. However, the similarity between MS and one particular EAE model does not end at inflammation. MS and chronic EAE induced in C57BL/6 mice using myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 share many neuropathologies. Beyond both having white matter lesions in spinal cord, both also have widespread neuropathology in the cerebral cortex, hippocampus, thalamus, striatum, cerebellum, and retina/optic nerve. In this review, we compare neuropathologies in each of these structures in MS with chronic EAE in C57BL/6 mice, and find evidence that this EAE model is well suited to study neuroaxonal degeneration in MS.

Abstract 5 Human Umbilical Cord Blood-Derived Cell Therapy Product, DUOC-01, Promotes Remyelination by Driving the Differentiation of Oligodendrocyte Progenitor Cells

IntroductionDUOC-01 is a macrophage-like cell therapy product manufactured by culturing banked human umbilical cord blood cells under GMP conditions. Currently, the safety of DUOC-01 is being tested as a bridging therapy in children with demyelinating leukodystrophies undergoing unrelated donor umbilical cord blood transplantation after myeloablative conditioning. DUOC-01 protects against loss of function in several preclinical models with demyelinating conditions of the central nervous system, making it an attractive therapy for patients with multiple sclerosis (MS) who experience destruction of myelin sheaths as pathology of their disease. The mechanism by which DUOC-01 promotes remyelination and if it directly influences oligodendrocyte lineage cells is untested.ObjectiveUsing multiple systems (primary oligodendrocyte precursor cell [OPC] cultures, in vitro cerebellar slice cultures, and experimental autoimmune encephalomyelitis [EAE], a mouse model of MS), we examined how DUOC-01 influences numerous steps of pathology and recovery.MethodsUsing a brain slice culture, we added DUOC-01 to the lysophosphatidylcholine (LPC)-treated slices. We quantified myelinated axons by assessing percent co-localization of myelin basic protein and neurofilament in the control, LPC, and LPC+DUOC-01 groups. To test the DUOC-01 effect in the EAE model, we immunized C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in complete Freund’s adjuvant. To match clinical protocols, we incubated DUOC-01 in Ringer’s lactate with hydrocortisone (HC) for 2 hours at room temperature. At the onset of EAE disease symptoms, we injected DUOC-01 into the cerebrospinal fluid by a single intra-cisterna magna injection, then recorded clinical scores daily for 2 weeks. To test if DUOC-01 could directly affect OPCs, we set up a primary OPC culture isolated from neonatal mice and added DUOC-01 treatment to the culture.ResultsIn the cerebellar slice model, we demonstrated a higher number of myelinated neuron fibers in the DUOC-treated group compared with the LPC-treated group. In the EAE model, compared with mice injected with Ringer’s or HC+Ringer’s, mice injected with DUOC-01 derived clinical benefit with lower clinical scores. In the primary OPC culture, the DUOC-01 treatment drove the maturation of OPC to become myelin producing oligodendrocytes.DiscussionOur data suggest that DUOC-01 could be beneficial in treating MS and other diverse neurological demyelinating conditions.

Antioxidant effect of grape seed extract corrects experimental autoimmune encephalomyelitis behavioral dysfunctions, demyelination, and glial activation.

Background and purposeMultiple sclerosis (MS), a multifactorial autoimmune disease of the central nervous system (CNS), is characterized by demyelination and chronic inflammation, as well as axonal and neuronal loss. There is no cure for MS, and despite a significant improvement in the therapeutic management of patients during the last 20 years, some symptoms are still resistant to treatment, and the evolution of the disease to progressive form seems still ineluctable. The etiology of MS is complex and still not fully understood. However, inflammation is a major driver of physiopathology and oxidative stress contributes to CNS lesions and promotes existing inflammatory response. Plant polyphenols are endowed with many therapeutic benefits through alleviating oxidative stress and inflammation, thus providing neuroprotection in MS. We presently evaluated the curative effect of grape seed extract (GSE) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS.Experimental approachSix-week-old C57Bl/6J females were subjected to the EAE paradigm (using myelin oligodendrocyte glycoprotein peptide fragment (35-55), complete Freund’s adjuvant, and pertussis toxin) and then chronically treated with GSE from day 10 to day 30 post-induction. Clinical score and body weight were monitored daily, while evaluation of sensitive, motor, cognitive, and anxiety-related behaviors was performed weekly. Then, the GSE effect was evaluated on whole brain and spinal cord samples through the evaluation of oxidative stress damage, antioxidant capacities, myelin alteration, astroglial and microglial proliferation, and sirtuin expression.Key resultsGrape seed extract curative chronic treatment corrected the clinical course of EAE, as well as the mechanical hypersensitivity, and avoided the development of EAE mouse thermal cold allodynia. The neuropathological evaluation showed that GSE reduced oxidative stress in the brain and spinal cord by decreasing the lipid and protein oxidation through correction of the three main antioxidant enzyme activities, namely, superoxide dismutase, catalase, and glutathione peroxidase, as well as restoring normal myelin protein expression and correcting microglial and astroglial protein overexpression and sirtuin downregulation.Conclusion and implicationsThese data strongly support GSE as an effective therapeutic approach in MS treatment.

Genetic analysis of CNS autoimmunity using the diversity of the Collaborative Cross reveals unique phenotypes and mechanisms

Abstract Multiple Sclerosis (MS) is a complex disease with remarkable heterogeneity in disease course and progression, the genetic basis of which remains obscure. Here we leveraged the Collaborative Cross (CC) - a highly genetically diverse mouse strain panel - and myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) induced experimental autoimmune encephalomyelitis (EAE), to model genetics of MS disease course. 33 CC strains were selected based on compatible MHC haplotypes (H2b and H2g7), which captured a wide spectrum of distinct EAE phenotypes, compared with typical chronic EAE in C57BL/6 mice. CC028 mice exhibited severe and rapidly progressing disease. In contrast, several strains, including CC011 and CC040, were highly resistant to EAE. Sex differences in EAE course were observed in 4 strains, including CC042. Remitting-relapsing EAE was observed in 4 strains, including CC002. In addition to classical EAE clinical signs (ascending paralysis), we identified two strains, CC004 and CC083 that exhibited high incidence of axial rotary (AR)-EAE, including profound ataxia, head tilt, and axial rotation. Preliminary quantitative trait locus (QTL) analysis revealed a distinct linkage pattern in each sex for classical EAE severity, with several emerging peaks on chromosome (Chr) 2, 8, 12, 18, and X in females, Chr10 in males, and Chr4 in both sexes. QTL analysis of AR-EAE severity revealed a narrow interval on Chr18 (39.2–41.0Mb) passing suggestive linkage significance at the 90% confidence threshold. Experiments are ongoing to determine the immunopathologic basis of distinct genetically controlled EAE phenotypes in CC strains of interest. Supported by grants from NIH (R21 AI145306-01) and the National MS Society (RG-1901-33309)

Effect of Rapamycin on MOG-Reactive Immune Cells and Lipopolysaccharide-Activated Microglia: An In Vitro Approach for Screening New Therapies for Multiple Sclerosis.

Rapamycin is an immunomodulatory drug that has been evaluated in preclinical and clinical trials as a disease-modifying therapy for multiple sclerosis (MS). In this study, we evaluated the in vitro effect of rapamycin on immune cells pivotally involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), which is an animal model to study MS. Splenocytes and central nervous system (CNS)-mononuclear cells obtained from EAE mice were stimulated with a myelin oligodendrocyte glycoprotein peptide, whereas the microglial BV-2 cell line was activated with LPS. The 3 immune cell types were simultaneously treated with rapamycin, incubated, and then used to analyze cytokines, transcription factors, and activation markers. Rapamycin reduced IL-17 production, TBX21, and RORc expression by splenic and CNS cell cultures. IFN-γ and TNF-α production were also decreased in CNS cultures. This treatment also decreased TNF-α, IL-6, MHC II, CD40, and CD86 expression by BV-2 cells. These results indicated that in vivo immunomodulatory activity of rapamycin in MS and EAE was, in many aspects, reproduced by in vitro assays done with cells derived from the spleen and the CNS of EAE mice. This procedure could constitute a screening strategy for choosing drugs with therapeutic potential for MS.

B cell-dependent EAE induces visual deficits in the mouse with similarities to human autoimmune demyelinating diseases

BackgroundIn the field of autoimmune demyelinating diseases, visual impairments have extensively been studied using the experimental autoimmune encephalomyelitis (EAE) mouse model, which is classically induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35–55). However, this model does not involve B cells like its human analogs. New antigens have thus been developed to induce a B cell-dependent form of EAE that better mimics human diseases.MethodsThe present study aimed to characterize the visual symptoms of EAE induced with such an antigen called bMOG. After the induction of EAE with bMOG in C57BL/6J mice, visual function changes were studied by electroretinography and optomotor acuity tests. Motor deficits were assessed in parallel with a standard clinical scoring method. Histological examinations and Western blot analyses allowed to follow retinal neuron survival, gliosis, microglia activation, opsin photopigment expression in photoreceptors and optic nerve demyelination. Disease effects on retinal gene expression were established by RNA sequencing.ResultsWe observed that bMOG EAE mice exhibited persistent loss of visual acuity, despite partial recovery of electroretinogram and motor functions. This loss was likely due to retinal inflammation, gliosis and synaptic impairments, as evidenced by histological and transcriptomic data. Further analysis suggests that the M-cone photoreceptor pathway was also affected.ConclusionTherefore, by documenting visual changes induced by bMOG and showing similarities to those seen in diseases such as multiple sclerosis and neuromyelitis optica, this study offers a new approach to test protective or restorative ophthalmic treatments.

Treatment of Experimental Autoimmune Encephalomyelitis with an Inhibitor of Phosphodiesterase-8 (PDE8).

After decades of development, inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice for the treatment of inflammatory disorders, with three PDE4 inhibitors being in clinical use as therapeutics for psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease and atopic dermatitis. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. We have previously demonstrated a role for the PDE8A-Raf-1 kinase complex in the regulation of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) activated CD4+ effector T cell adhesion and locomotion by a mechanism that differs from PDE4 activity. In this study, we explored the in vivo treatment of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) induced in mice immunized with MOG using the PDE8-selective inhibitor PF-04957325. For treatment in vivo, mice with EAE were either subcutaneously (s.c.) injected three times daily (10 mg/kg/dose), or were implanted subcutaneously with Alzet mini-osmotic pumps to deliver the PDE8 inhibitor (15.5 mg/kg/day). The mice were scored daily for clinical signs of paresis and paralysis which were characteristic of EAE. We observed the suppression of the clinical signs of EAE and a reduction of inflammatory lesion formation in the CNS by histopathological analysis through the determination of the numbers of mononuclear cells isolated from the spinal cord of mice with EAE. The PDE8 inhibitor treatment reduces the accumulation of both encephalitogenic Th1 and Th17 T cells in the CNS. Our study demonstrates the efficacy of targeting PDE8 as a treatment of autoimmune inflammation in vivo by reducing the inflammatory lesion load.

In-Depth Characterization of Somatic and Orofacial Sensitive Dysfunctions and Interfering-Symptoms in a Relapsing-Remitting Experimental Autoimmune Encephalomyelitis Mouse Model.

Among the many symptoms (motor, sensory, and cognitive) associated with multiple sclerosis (MS), chronic pain is a common disabling condition. In particular, neuropathic pain symptoms are very prevalent and debilitating, even in early stages of the disease. Unfortunately, chronic pain still lacks efficient therapeutic agents. Progress is needed (i) clinically by better characterizing pain symptoms in MS and understanding the underlying mechanisms, and (ii) preclinically by developing a more closely dedicated model to identify new therapeutic targets and evaluate new drugs. In this setting, new variants of experimental autoimmune encephalomyelitis (EAE) are currently developed in mice to exhibit less severe motor impairments, thereby avoiding confounding factors in assessing pain behaviors over the disease course. Among these, the optimized relapsing-remitting EAE (QuilA-EAE) mouse model, induced using myelin oligodendrocyte glycoprotein peptide fragment (35–55), pertussis toxin, and quillaja bark saponin, seems very promising. Our study sought (i) to better define sensitive dysfunctions and (ii) to extend behavioral characterization to interfering symptoms often associated with pain during MS, such as mood disturbances, fatigue, and cognitive impairment, in this optimized QuilA-EAE model. We made an in-depth characterization of this optimized QuilA-EAE model, describing for the first time somatic thermal hyperalgesia associated with mechanical and cold allodynia. Evaluation of orofacial pain sensitivity showed no mechanical or thermal allodynia. Detailed evaluation of motor behaviors highlighted slight defects in fine motor coordination in the QuilA-EAE mice but without impact on pain evaluation. Finally, no anxiety-related or cognitive impairment was observed during the peak of sensitive symptoms. Pharmacologically, as previously described, we found that pregabalin, a treatment commonly used in neuropathic pain patients, induced an analgesic effect on mechanical allodynia. In addition, we showed an anti-hyperalgesic thermal effect on this model. Our results demonstrate that this QuilA-EAE model is clearly of interest for studying pain symptom development and so could be used to identify and evaluate new therapeutic targets. The presence of interfering symptoms still needs to be further characterized.

The C-type lectin receptor Clec1A plays an important role in the development of experimental autoimmune encephalomyelitis by enhancing antigen presenting ability of dendritic cells and inducing inflammatory cytokine IL-17.

Clec1A, a member of C-type lectin receptor family, has a carbohydrate recognition domain in its extracellular region, but no known signaling motif in the cytoplasmic domain. Clec1a is highly expressed in endothelial cells and weakly in dendritic cells. Although this molecule was reported to play an important role in the host defense against Aspergillus fumigatus by recognizing 1,8-dihydroxynaphthalene-melanin on the fungal surface, the roles of this molecule in un-infected animals remain to be elucidated. In this study, we found that Clec1a−/− mice develop milder symptoms upon induction of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The maximum disease score was significantly lower, and demyelination and inflammation of the spinal cord were much milder in Clec1a−/− mice compared to wild-type mice. No abnormality was detected in the immune cell composition in the draining lymph nodes and spleen on day 10 and 16 after EAE induction. Recall memory T cell proliferation after restimulation with myelin oligodendrocyte glycoprotein peptide (MOG35–55) in vitro was decreased in Clec1a−/− mice, and antigen presenting ability of Clec1a−/− dendritic cells was impaired. Interestingly, RNA-Seq and RT-qPCR analyses clearly showed that the expression of inflammatory cytokines including Il17a, Il6 and Il1b was greatly decreased in Clec1a−/− mice after induction of EAE, suggesting that this reduced cytokine production is responsible for the amelioration of EAE in Clec1a−/− mice. These observations suggest a novel function of Clec1A in the immune system.

Evaluation of some blood parameters in the experimental autoimmune encephalomyelitis mouse model

Multiple sclerosis (MS) is a chronic neuroinflammatory demyelinating disorder of the central nervous system with unclear exact etiology. The experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice is the most common animal model for MS sharing many clinical and pathophysiological features to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. The current study was planned to evaluate the effect of EAE on hematologic and plasma total protein and albumin levels in C57BL/6 mice.
 EAE was induced with myelin oligodendrocyte glycoprotein (MOG35-55) peptide in the female C57BL/6 mice. The EAE clinically caused paralyzed tail, hind limb paresis, and uncoordinated movement in the mice. The EAE-induced mice hematologically had a mild increase in white blood cell count without altering neutrophil-lymphocyte ratio but no change in vital hematological parameters such as red blood cell count, packed cell volume, and hemoglobin level. Moreover, the EAE produced a rise in the plasma total protein level and an attenuation in plasma albumin level in the mice.
 In conclusion, our findings show that the EAE model in mice might not cause any significant change hematologically, except a slight increase in the white blood cell count, and might produce changes in the plasma protein level. The EAE-induced blood parameter effects, as the findings of the current study, could take consideration in terms of understanding the pathophysiology of the disease and developing a novel therapeutic approach for the disease.

Experimental Autoimmune Encephalomyelitis in the Mouse.

This article details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Additionally, active induction of EAE in the C57BL/6 strain using myelin oligodendrocyte glycoprotein (MOG) peptide is also discussed. Detailed materials and methods required for the purification of both PLP and MBP are described, and a protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains. © 2021 Wiley Periodicals LLC. Basic Protocol: Active induction of EAE with PLP, MBP, and MOG protein or peptide Alternate Protocol: Adoptive induction of EAE with PLP-, MBP-, or MOG-specific lymphocytes Support Protocol 1: Purification of proteolipid protein Support Protocol 2: Purification of myelin basic protein Support Protocol 3: Isolation of CNS-infiltrating lymphocytes.

SKI Expression Suppresses Pathogenic Th17 Cell Response and Mitigates Experimental Autoimmune Encephalomyelitis.

Pathogenic Th17 cells are critically involved in many autoimmune diseases, while non-pathogenic Th17 cells are more immune regulatory. Understanding the mechanisms of the induction and maintenance of pathogenic Th17 cells will benefit the development of therapeutic treatments of related diseases. We have shown that the transforming growth factor-β (TGFβ) induced SKI degradation and dissociation from Smad4 complex is a prerequisite for TGFβ-induced Th17 cell differentiation. However, it is unclear whether and how SKI regulates pathogenic Th17 differentiation, which does not require TGFβ cytokine. Here we showed that SKI expression was downregulated during pathogenic Th17 cell differentiation and the ectopic expression of SKI abrogated the differentiation of pathogenic Th17 cells. Functionally, using a knock-in mouse model, we found ectopic SKI expression specifically in T cells prevented myelin oligodendrocyte glycoprotein peptide (MOG33–55) induced experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. We further revealed that induced SKI expression in already differentiated pathogenic Th17 cells reduced the maintenance of Th17 program and ameliorated EAE in an adoptive T cell transfer model. Therefore, our study provides valuable insights of targeting SKI to modulate pathogenic Th17 cell function and treat Th17-related diseases.