Induction Of Experimental Autoimmune Encephalomyelitis Research Articles

Adipose-derived mesenchymal stem cells ameliorates experimental autoimmune encephalomyelitis via modulation of Th1/Th17 and expansion of Th2/Treg responses.

The most common central nervous system (CNS) inflammatory disease is multiple sclerosis (MS), modeled using experimental autoimmune encephalomyelitis (EAE). Mesenchymal stem cells (MSCs) exhibit potent immunomodulatory capabilities, including the suppression of immune cell functions and anti-inflammatory cytokine production. Female C57BL/6 mice (8-10 weeks old) were divided into three groups: 1. Control, 2. Allogeneic MSCs (ALO) treatment, and 3. Syngeneic MSCs (SYN) treatment. To induce EAE, myelin oligodendrocyte glycoprotein was injected subcutaneously with complete Freund's adjuvant, followed by intraperitoneal pertussis toxin. On Days 6 and 12 postimmunization, the treatment groups received intraperitoneal injections of 2 × 106 MSCs. Daily clinical and weight assessments were performed, and on Day 25, the mice were euthanized. At the end of the period, brain histological analysis was conducted to quantify lymphocyte infiltration. T-cell characteristics were determined usingenzyme-linked immunosorbent assay and Real-time polymerase chain reaction (RT-PCR). The assessment of transcription factor expression levels in the CNS was also performed using RT-PCR. Compared to the control group, both the allogeneic (ALO) and syngeneic (SYN) groups demonstrated significantly reduced disease progression. The maximum clinical scores for the control, ALO, and SYN groups were 4.4 ± 0.1, 2.4 ± 0.2, and 2.1 ± 0.2, respectively (ALO and SYN vs. Control: p < .001). In comparison to the control group, histological studies demonstrated that the allogeneic and syngeneic groups had less lymphocytic infiltration (ALO: 1.4 ± 0.1, SYN: 1.2 ± 0.2, and control: 2.8 ± 0.15; p < .001) and demyelination (ALO: 1.2 ± 0.15, SYN: 1.1 ± 0.1 and control: 2.9 ± 0.1, p < .001). ALO and SYN groups had lower expression of Th1 and Th17 cytokines and transcription factors (IFN-γ: 0.067, 0.051; STAT4: 0.189, 0.162; T-bet: 0.175, 0.163; IL-17: 0.074, 0.061; STAT3: 0.271, 0.253; ROR-γt: 0.163, 0.149, respectively) compared to the control group on Day 25 following EAE induction. Additionally, ALO and SYN groups compared to the control group, expressed more Th2 and Treg cytokines and transcription factors (IL-4: 4.25, 4.63; STAT6: 2.78, 2.96; GATA3: 2.91, 3.08; IL-27: 2.32, 2.46, IL-33: 2.71, 2.85; TGF-β: 4.8, 5.05; IL-10: 4.71, 4.93; CTLA-4: 7.72, 7.95; PD1: 4.12,4.35; Foxp3: 3.82,4.08, respectively). This research demonstrated that MSCs possess the potential to be a therapeutic option for MS and related CNS inflammatory disorders. Their immunomodulatory properties, coupled with the observed reductions in disease severity, lymphocytic infiltration, and demyelination, indicate that MSCs could play a crucial role in altering the course of MS by mitigating inflammatory immune responses and promoting regulatory immune processes. These findings open up new possibilities for the development of MSC-based therapies for MS, and further investigation and clinical trials may be warranted to explore their efficacy and safety in human patients.

MicroRNAs targeting TGF-β signaling exacerbate central nervous system autoimmunity by disrupting regulatory T cell development and function.

Transforming growth factor beta (TGF-β) signaling is essential for a balanced immune response by mediating the development and function of regulatory T cells (Tregs) and suppressing autoreactive T cells. Disruption of this balance can result in autoimmune diseases, including multiple sclerosis (MS). MicroRNAs (miRNAs) targeting TGF-β signaling have been shown to be upregulated in naïve CD4 T cells in MS patients, resulting in a limited in vitro generation of human Tregs. Utilizing the murine model experimental autoimmune encephalomyelitis, we show that perinatal administration of miRNAs, which target the TGF-β signaling pathway, enhanced susceptibility to central nervous system (CNS) autoimmunity. Neonatal mice administered with these miRNAs further exhibited reduced Treg frequencies with a loss in T cell receptor repertoire diversity following the induction of experimental autoimmune encephalomyelitis in adulthood. Exacerbated CNS autoimmunity as a result of miRNA overexpression in CD4 T cells was accompanied by enhanced Th1 and Th17 cell frequencies. These findings demonstrate that increased levels of TGF-β-associated miRNAs impede the development of a diverse Treg population, leading to enhanced effector cell activity, and contributing to an increased susceptibility to CNS autoimmunity. Thus, TGF-β-targeting miRNAs could be a risk factor for MS, and recovering optimal TGF-β signaling may restore immune homeostasis in MS patients.

Intrinsic ecto-5'-Nucleotidase/A1R Coupling may Confer Neuroprotection to the Cerebellum in Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is widely used animal model of multiple sclerosis (MS). The disease is characterized by demyelination and neurodegeneration triggered by infiltrated autoimmune cells and their interaction with astrocytes and microglia. While neuroinflammation is most common in the spinal cord and brainstem, it is less prevalent in the cerebellum, where it predisposes to rapid disease progression. Because the induction and progression of EAE are tightly regulated by adenosinergic signaling, in the present study we compared the adenosine-producing and -degrading enzymes, ecto-5'-nucleotidase (eN/CD73) and adenosine deaminase (ADA), as well as the expression levels of adenosine receptors A1R and A2AR subtypes in nearby areas around the fourth cerebral ventricle-the pontine tegmentum, the choroid plexus (CP), and the cerebellum. Significant differences in histopathological findings were observed between pontine tegmentum and cerebellum on the same horizontal section level. Reactive astrogliosis and massive infiltration of CD4 + cells and macrophages in CP and pontine tegmentum resulted in local demyelination. In cerebellum, there was no evidence of infiltrates, microgliosis and neuroinflammation at the same sectional level. In addition, Bergman glia showed no signs of reactive gliosis. As for adenosinergic signaling, significant upregulation of eN/CD73 was observed in all areas studied, but in association with different adenosine receptor subtypes. In CP and pons, overexpression of eN/CD73 was coupled with induction of A2AR, whereas in cerebellum, a modest increase in eN/CD73 in resident Bergman glia was accompanied by a strong induction of A1R in the same type of astrocytes. Thus, the presence of specialized astroglia and intrinsic differences in adenosinergic signaling may play a critical role in the differential regional susceptibility to EAE inflammation.

Short report of potential Myelinogenesis effects of taper up-off of opium tincture in rodent model of multiple sclerosis

Multiple Sclerosis (MS) is one of the most common demyelinating autoimmune diseases that affects the central nervous system and is characterized by major immune-mediated myelin and axonal damage or axonal loss explicable to the absence of myelin sheaths. Here we present the early findings of the gene expression study of myelinogenesis-related genes of MS rat models which were treated with a novel protocol of taper up-off of opium tincture. The study included normal Lewis rats, MS rat models by induction of experimental autoimmune encephalomyelitis (EAE) without treatment, and MS rat models with a novel protocol of taper up-off treatment of opium tincture called Dezhakam-step-time (DST) in different dosages. RNA was extracted and cDNA was synthesized from the spinal cord tissue. Gene expression analysis was conducted for eight genes as markers of myelinogenesis (OLIG1, OLIG2, MBP, MYRF, PLP1, PMP22, EGF, and UGT8) using the Real-time PCR. All eight genes were down-regulated in EAE models vs. healthy controls and all eight genes were up-regulated after the taper up-off treatment of opium tincture. The most over-expression of myelinogenesis-related genes was revealed at higher dosages of opium tincture. These are the early results of a gene expression study in a multiple sclerosis model treated with opium tincture. It seems that the opium tincture method may induce the activation of myelinogenesis in EAE models which could lead to a potential treatment for improvement of neural dysfunctions in MS patients.

Intranasal Resveratrol Nanoparticles Enhance Neuroprotection in a Model of Multiple Sclerosis.

Resveratrol is a natural polyphenol which has a very low bioavailability but whose antioxidant, anti-inflammatory and anti-apoptotic properties may have therapeutic potential for the treatment of neurodegenerative diseases such as multiple sclerosis (MS). Previously, we reported the oral administration of resveratrol nanoparticles (RNs) elicited a neuroprotective effect in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, at significantly lower doses than unconjugated resveratrol (RSV) due to enhanced bioavailability. Furthermore, we demonstrated that the intranasal administration of a cell-derived secretome-based therapy at low concentrations leads to the selective neuroprotection of the optic nerve in EAE mice. The current study sought to assess the potential selective efficacy of lower concentrations of intranasal RNs for attenuating optic nerve damage in EAE mice. EAE mice received either a daily intranasal vehicle, RNs or unconjugated resveratrol (RSV) for a period of thirty days beginning on the day of EAE induction. Mice were assessed daily for limb paralysis and weekly for visual function using the optokinetic response (OKR) by observers masked to treatment regimes. After sacrifice at day 30, spinal cords and optic nerves were stained to assess inflammation and demyelination, and retinas were immunostained to quantify retinal ganglion cell (RGC) survival. Intranasal RNs significantly increased RGC survival at half the dose previously shown to be required when given orally, reducing the risk of systemic side effects associated with prolonged use. Both intranasal RSV and RN therapies enhanced RGC survival trends, however, only the effects of intranasal RNs were significant. RGC loss was prevented even in the presence of inflammatory and demyelinating changes induced by EAE in optic nerves. The intranasal administration of RNs is able to reduce RGC loss independent of the inflammatory and demyelinating effects on the optic nerve and the spinal cord. The concentration of RNs needed to achieve neuroprotection is lower than previously demonstrated with oral administration, suggesting intranasal drug delivery combined with nanoparticle conjugation warrants further exploration as a potential neuroprotective strategy for the treatment of optic neuritis, alone as well as in combination with glucocorticoids.

Exploring the immune-modulating properties of boswellic acid in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a condition where the central nervous system loses its myelin coating due to autoimmune inflammation. The experimental autoimmune encephalomyelitis (EAE) simulates some aspects of human MS. Boswellic acids are natural compounds derived from frankincense extract, known for their anti-inflammatory properties. The purpose of this research was to investigate therapeutic potential of boswellic acids. Mice were divided into three groups: low-dose (LD), high-dose (HD), and control groups (CTRL). Following EAE induction, the mice received daily doses of boswellic acid for 25 days. Brain tissue damage, clinical symptoms, and levels of TGF-β, IFN-γ, and IL-17 cytokines in cell cultured supernatant of lymphocytes were assessed. Gene expression of transcription factors in brain was measured using real-time PCR. The levels of brain demyelination were significantly lower in the treatment groups compared to the CTRL group. Boswellic acid reduced the severity and duration of EAE symptoms. Furthermore, boswellic acid decreased the amounts of IFN-γ and IL-17, also the expression of T-bet and ROR-γt in brain. On the contrary, it increased the levels of TGF-β and the expression FoxP3 and GATA3. Our findings suggest that boswellic acids possess therapeutic potential for EAE by modulating the immune response and reducing inflammation.

Endoplasmic reticulum stress response in immune cells contributes to experimental autoimmune encephalomyelitis pathogenesis in rats

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3− (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1β, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

Ozanimod-mediated remission in experimental autoimmune encephalomyelitis is associated with enhanced activity of CNS CD27low/- NK cell subset.

Ozanimod (RPC1063) is an immunomodulator that has been recently approved by the FDA (2020) for the treatment of relapsing-remitting multiple sclerosis (RRMS). It is a selective agonist of the sphingosine-1-phophate receptors 1 and 5, expressed on naïve and central memory T and B cells, as well as natural killer (NK) cells, and is involved in lymphocyte trafficking. Oral administration of ozanimod was reported to result in rapid and reversible reduction in circulating lymphocytes in multiple sclerosis (MS) patients, however, only minimal effect on NK cells was observed. In this study, we sought to investigate the effect of ozanimod on NK cells and assess whether they play any role in ozanimod-induced remission in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Active EAE induction was done in C57BL/6 female mice, followed by daily oral treatment with ozanimod (0.6mg/kg) starting at disease onset (score 1). Flow cytometry of blood and CNS was performed 24 hours after the last oral dose of ozanimod treatment in diseased mice. Histological analysis of lumbar spinal cord was performed for evaluating the level of inflammation and demyelination. Depletion of peripheral NK cells was done using anti-NK1.1 mouse antibody (mAb) at day 5 post-EAE induction. Ozanimod was effective in reducing the clinical severity of EAE and reducing the percentage of autoreactive CD4+ and CD8+ T cells along with significant inhibition of lymphocyte infiltration into the spinal cord, accompanied by reversed demyelination. Furthermore, ozanimod treatment resulted in a significant increase in the frequency of total NK cells in the blood and CNS along with upregulation of the activating receptor NKG2D on CD27low/- NK cell subset in the CNS. The effectiveness of ozanimod treatment in inhibiting the progression of the disease was reduced when NK cells were depleted using anti-NK1.1 mAb. The current study demonstrated that ozanimod treatment significantly improved clinical symptoms in EAE mice. Ozanimod and anti-NK1.1 mAb appear to function in opposition to one another. Collectively, our data suggest that ozanimod-mediated remission is associated with an increased percentage of total NK cells and CD27low/- NK cells expressing the activating receptor, NKG2D in the CNS.

Complete Freund's adjuvant as a confounding factor in multiple sclerosis research.

Complete Freund's adjuvant (CFA) is used as a standard adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model in multiple sclerosis studies. Still, CFA induces glial activation and neuroinflammation on its own and provokes pain. In addition, as CFA contains Mycobacteria, an immune response against bacterial antigens is induced in parallel to the response against central nervous system antigens. Thus, CFA can be considered as a confounding factor in multiple sclerosis-related studies performed on EAE. Here, we discuss the effects of CFA in EAE in detail and present EAE variants induced in experimental animals without the use of CFA. We put forward CFA-free EAE variants as valuable tools for studying multiple sclerosis pathogenesis and therapeutic approaches.

A16 ALTERATIONS TO BOTH INTESTINAL BARRIER INTEGRITY AND THE MICROBIOTA CONSPIRE DURING AGING TO PROMOTE DISEASE PROGRESSION IN A MOUSE MODEL OF MULTIPLE SCLEROSIS

Abstract Background Multiple sclerosis (MS) is a chronic, inflammatory condition impacted by both genetic and environmental factors. Aged individuals have an increased risk of progressive multiple sclerosis (pMS), as well as altered microbiota community composition and elevated levels of circulating gut microbiota-derived metabolites due to compromised gut barrier function. Fecal microbiota transplantation (FMT) studies confirm that an MS-associated microbiome can potentiate disease onset and enhance neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Aims 1) Assess whether an aged microbiota alone is sufficient to promote age-associated progressive-like disease in our mouse model of EAE 2) Determine whether altered intestinal barrier integrity results in central nervous system-specific immune changes and/or disease course during EAE Methods To assess the impact of an aged microbiota on EAE outcomes we performed age-disparate human-derived FMT from young vs aged healthy co-habiting, first degree relatives into recipient mice. Then, to further interrogate the role of the gut-brain axis in the association of age and pMS we isolated the compromised intestinal barrier seen in aged individuals as a variable via two methods – Muc2-/- mice, which exhibit a defective mucus barrier, and DSS treated mice. Results Following adoptive transfer of young EAE-primed T cells, ‘young’ FMT recipients exhibited disease resolution while an ‘aged’ FMT lead to severe, non-remitting EAE relative to both controls and ‘young’ FMT recipients. This difference in outcomes was not seen in all donor pairs, suggesting that worsened EAE is not a universal feature of the aged microbiota. We collected cecal material, blood, and central nervous system (CNS) tissue from an age-differential FMT mouse pair and performed metabolomic analysis. We found robust differences in the metabolomic profile of young vs aged FMT recipients before and after EAE induction. Further, in both models of altered barrier integrity, we observed altered myeloid cell trafficking to and immune activation in the CNS, suggesting that the intestinal barrier itself plays a critical role in immune activation during EAE. Conclusions These findings suggest specific age-related microbiota-derived metabolites, which have increased access to the host system due to a leaky intestinal barrier, may be associated with increased risk of progressive disease and may represent promising therapeutic targets for MS. Funding Agencies CIHRMS Canada

Response Surface Methodology Approach to Optimize the Expression of Thioredoxin-MOG Fusion Protein

Background: The N-terminal domain of the myelin oligodendrocyte glycoprotein (MOG) has been shown to generate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. A considerable amount of MOG must be accessible for EAE induction. Here, for the first time, Response Surface Methodology-Box-Behnken (RSM-BBD) was employed to identify the ideal culture conditions for causing Escherichia coli (E. coli) BL21 to overproduce the Thioredoxin-MOG (Trx-MOG) fusion protein. The RSM method is a powerful, efficient, and reliable alternative to the One-Factor-At-A-Time (OFAT) method in optimizing process variables, allowing for a smaller number of experimental runs, investigating variable interaction, and being cheaper and less time-consuming. Methods: Here, using the 29 experimental assays, the direct and indirect effects of factors including post-induction time, IPTGinducer concentration, pre-induction optical density, and post-induction temperature on the protein expression level content were evaluated. Results: The proposed quadratic model demonstrated a significant effect of the two variables A (time) and C (temperature) on protein synthesis. An inducer concentration of 0.491 mM, the pre-induction optical density (OD600) of 0.8, and a temperature of 23 °C for 23.878 hours were found to be the best growth conditions for high yield Trx-MOG synthesis. The optimum protein concentration was attained (163.96 µg/mL) and was within the range of (200.04 µg/mL), which was the value predicted. Conclusion: The study concluded that RSM optimization effectively increased the production of Trx-MOG in E. coli, which could have the potential for large-scale fermentation.

Role of Signal-Transducing Adaptor Protein-1 for T Cell Activation and Pathogenesis of Autoimmune Demyelination and Airway Inflammation.

Signal-transducing adaptor protein (STAP)-1 is an adaptor protein that is widely expressed in T cells. In this article, we show that STAP-1 upregulates TCR-mediated T cell activation and T cell-mediated airway inflammation. Using STAP-1 knockout mice and STAP-1-overexpressing Jurkat cells, we found that STAP-1 enhanced TCR signaling, resulting in increased calcium mobilization, NFAT activity, and IL-2 production. Upon TCR engagement, STAP-1 binding to ITK promoted formation of ITK-LCK and ITK-phospholipase Cγ1 complexes to induce downstream signaling. Consistent with the results, STAP-1 deficiency reduced the severity of symptoms in experimental autoimmune encephalomyelitis. Single-cell RNA-sequencing analysis revealed that STAP-1 is essential for accumulation of T cells and Ifng and Il17 expression in spinal cords after experimental autoimmune encephalomyelitis induction. Th1 and Th17 development was also attenuated in STAP-1 knockout naive T cells. Taken together, STAP-1 enhances TCR signaling and plays a role in T cell-mediated immune disorders.

Thymoquinone improves experimental autoimmune encephalomyelitis by regulating both pro-inflammatory and anti-inflammatory cytokines.

Introduction Multiple sclerosis (MS) is an autoimmune condition marked by inflammation and the loss of myelin in the central nervous system (CNS). The aim of this research was to understand how Thymoquinone regulate the molecular and cellular processes involved in controlling experimental autoimmune encephalomyelitis (EAE), which is an animal model often used to study MS. MethodsFemale C57BL/6 mice were split into different groups receiving different doses (low, medium, and high) of Thymoquinone simultaneously with EAE induction. Clinical scores and other measurements were observed daily throughout the 25-day post immunization. We assessed lymphocyte infiltration and demyelination in the spinal cord through histological staining, analyzed T-cell profiles using ELISA, and quantified the expression levels of transcription factors in the CNS using Real-time PCR. Results Thymoquinone prevented the development of EAE. Histological experiments revealed only a small degree of leukocyte infiltration into the CNS. Thymoquinone resulted in a notable reduction in the generation of IFN-γ, IL-17, and IL-6, while simultaneously increasing the production of IL-4, IL-10, and TGF-β in Th2 and Treg cells. Results from Real-time PCR suggested Treatment with Thymoquinone decreased the expression of T-bet and ROR-γt while increasing the expression of Foxp3 and GATA3. Conclusion These findings showed that Thymoquinone could decrease both disease incidence and severity.

CRL4b Inhibition Ameliorates Experimental Autoimmune Encephalomyelitis Progression.

Multiple sclerosis, and its murine model experimental autoimmune encephalomyelitis (EAE), is a neurodegenerative autoimmune disease of the CNS characterized by T cell influx and demyelination. Similar to other autoimmune diseases, therapies can alleviate symptoms but often come with side effects, necessitating the exploration of new treatments. We recently demonstrated that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) aided in maintaining genome stability in proliferating T cells. In this study, we examined whether CRL4b was required for T cells to expand and drive EAE. Mice lacking Cul4b (Cullin 4b) in T cells had reduced EAE symptoms and decreased inflammation during the peak of the disease. Significantly fewer CD4+ and CD8+ T cells were found in the CNS, particularly among the CD4+ T cell population producing IL-17A, IFN-γ, GM-CSF, and TNF-α. Additionally, Cul4b-deficient CD4+ T cells cultured invitro with their wild-type counterparts were less likely to expand and differentiate into IL-17A- or IFN-γ-producing effector cells. When wild-type CD4+ T cells were activated invitro in the presence of the recently developed CRL4 inhibitor KH-4-43, they exhibited increased apoptosis and DNA damage. Treatment of mice with KH-4-43 following EAE induction resulted in stabilized clinical scores and significantly reduced numbers of T cells and innate immune cells in the CNS compared with control mice. Furthermore, KH-4-43 treatment resulted in elevated expression of p21 and cyclin E2 in T cells. These studies support that therapeutic inhibition of CRL4 and/or CRL4-related pathways could be used to treat autoimmune disease.

Tumor necrosis factor receptor 2 activation elicits sex-specific effects on cortical myelin proteins and functional recovery in a model of multiple sclerosis

Multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS), predominately affects females compared to males. Tumor necrosis factor (TNF), a pro-inflammatory cytokine, signaling through TNF receptor 1 contributes to inflammatory disease pathogenesis. In contrast, TNF receptor 2 signaling is neuroprotective. Current anti-TNF MS therapies are shown to be detrimental to patients due to pleiotropic effects on both pro- and anti-inflammatory functions. Using a non-pertussis toxin (nPTX) experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice, we systemically administered a TNFR2 agonist (p53-sc-mTNFR2) to investigate behavioral and pathophysiological changes in both female and male mice. Our data shows that TNFR2 activation alleviates motor and sensory symptoms in females. However, in males, the agonist only alleviates sensory symptoms and not motor. nPTX EAE induction in TNFR2 global knockout mice caused exacerbated motor symptoms in females along with an earlier day of onset, but not in males. Our data demonstrates that TNFR2 agonist efficacy is sex-specific for alleviation of motor symptoms, however, it effectively reduces mechanical hypersensitivity in both females and males. Altogether, these data support the therapeutic promise TNFR2 agonism holds as an MS therapeutic and, more broadly, to treat central neuropathic pain.

Upregulation of CD8+ regulatory T cells following liver-directed AAV gene therapy

Liver-directed AAV gene therapy represents a unique treatment modality for a host of diseases. This is due, in part, to the induction of tolerance to transgene products. Despite the plethora of recognized regulatory cells in the body, there is currently a lack of literature supporting the induction of non-CD4+ regulatory cells following hepatic AAV gene transfer. In this work, we show that CD8+ regulatory T cells are up-regulated in PBMCs of mice following capsid only and therapeutic transgene AAV administration. Further, we demonstrate that hepatic AAV gene transfer results in a significant increase in CD8+ regulatory T cells following experimental autoimmune encephalomyelitis induction. Notably, this response occurred only in therapeutic vector treated animals, not capsid only controls. Understanding the role these cells play in treatment efficacy will result in the development of improved AAV vectors that take advantage of the full gamut of regulatory cells within the body.

Effects of the mineralocorticoid receptor antagonist eplerenone in experimental autoimmune encephalomyelitis

There is growing evidence indicating that mineralocorticoid receptor (MR) expression influences a wide variety of functions in metabolic and immune response. The present study explored if antagonism of the MR reduces neuroinflammation in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE). Eplerenone (EPLE) (100 mg/kg dissolved in 30% 2-hydroxypropyl-β-cyclodextrin) was administered intraperitoneally (i.p.) daily from EAE induction (day 0) until sacrificed on day 17 post-induction. The MR blocker (a) significantly decreased the inflammatory parameters TLR4, MYD88, IL-1β, and iNOS mRNAs; (b) attenuated HMGB1, NLRP3, TGF-β mRNAs, microglia, and aquaporin4 immunoreaction without modifying GFAP. Serum IL-1β was also decreased in the EAE+EPLE group. Moreover, EPLE treatment prevented demyelination and improved clinical signs of EAE mice. Interestingly, MR was decreased and GR remained unchanged in EAE mice while EPLE treatment restored MR expression, suggesting that a dysbalanced MR/GR was associated with the development of neuroinflammation. Our results indicated that MR blockage with EPLE attenuated inflammation-related spinal cord pathology in the EAE mouse model of Multiple Sclerosis, supporting a novel therapeutic approach for immune-related diseases.

Midline-1 regulates effector T cell motility in experimental autoimmune encephalomyelitis via mTOR/microtubule pathway.

Background: Effector T cell activation, migration, and proinflammatory cytokine production are crucial steps in autoimmune disorders such as multiple sclerosis (MS). While several therapeutic approaches targeting T cell activation and proinflammatory cytokines have been developed for the treatment of autoimmune diseases, there are no therapeutic agents targeting the migration of effector T cells, largely due to our limited understanding of regulatory mechanisms of T cell migration in autoimmune disease. Here we reported that midline-1 (Mid1) is a key regulator of effector T cell migration in experimental autoimmune encephalomyelitis (EAE), a widely used animal model of MS. Methods: Mid1-/- mice were generated by Crispr-Cas9 technology. T cell-specific Mid1 knockout chimeric mice were generated by adoptive transfer of Mid1-/- T cells into lymphocyte deficient Rag2-/- mice. Mice were either immunized with MOG35-55 (active EAE) or received adoptive transfer of pathogenic T cells (passive EAE) to induce EAE. In vitro Transwell® assay or in vivo footpad injection were used to assess the migration of T cells. Results: Mid1 was significantly increased in the spinal cord of wild-type (Wt) EAE mice and disruption of Mid1 in T cells markedly suppressed the development of both active and passive EAE. Transcriptomic and flow cytometric analyses revealed a marked reduction in effector T cell number in the central nervous system of Mid1-/- mice after EAE induction. Conversely, an increase in the number of T cells was observed in the draining lymph nodes of Mid1-/- mice. Mice that were adoptively transferred with pathogenic Mid1-/- T cells also exhibited milder symptoms of EAE, along with a lower T cell count in the spinal cord. Additionally, disruption of Mid1 significantly inhibited T-cell migration both in vivo and in vitro. RNA sequencing suggests a suppression in multiple inflammatory pathways in Mid1-/- mice, including mTOR signaling that plays a critical role in cell migration. Subsequent experiments confirmed the interaction between Mid1 and mTOR. Suppression of mTOR with rapamycin or microtubule spindle formation with colcemid blunted the regulatory effect of Mid1 on T cell migration. In addition, mTOR agonists MHY1485 and 3BDO restored the migratory deficit caused by Mid1 depletion. Conclusion: Our data suggests that Mid1 regulates effector T cell migration to the central nervous system via mTOR/microtubule pathway in EAE, and thus may serve as a potential therapeutic target for the treatment of MS.

Unrestrained cleavage of Roquin-1 by MALT1 induces spontaneous T cell activation and the development of autoimmunity.

Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκBNS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.

Deletion of voltage-gated calcium channels in astrocytes decreases neuroinflammation and demyelination in a murine model of multiple sclerosis

The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination.