Experimental Autoimmune Encephalomyelitis Mouse Model Research Articles

Enhanced Therapeutic Effects of Human Mesenchymal stem Cells Transduced with Secreted Klotho in a Murine Experimental Autoimmune Encephalomyelitis Model.

Treatment of multiple sclerosis (MS) remains a major challenge. The aim of this study was to evaluate the therapeutic potential of mesenchymal stem cells (MSCs) engineered with secreted Klotho (SKL) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. EAE was induced in mice. MSCs or MSCs engineered with SKL (SKL-MSCs) were administered to EAE mice at the onset of disease. Hematoxylin-eosin and luxol fast blue staining were performed to evaluate histopathological changes. Expression of pro-inflammatory (TNF-α, IFN-γ, and IL-17) and anti-inflammatory (IL-10) cytokines was determined in the spinal cord using real-time PCR. Spinal cords were then processed for immunohistochemistry of the aforementioned cytokines. The frequencies of Th1, Th17, and regulatory T (Treg) cells were evaluated by flow cytometry of the spleen. The results showed that SKL-MSCs decreased clinical scores and reduced demyelination and inflammatory infiltration in the spinal cord more significantly than MSCs. Compared to MSCs, SKL-MSCs also exhibited a more profound capability of decreasing expression of TNF-α, IFN-γ, and IL-17 and increasing expression of IL-10 in the spinal cord with an enhanced homing to the inflamed tissue. Moreover, SKL-MSCs decreased the frequencies of Th1 and Th17 cells and increased the frequency of Treg cells in the spleen more potently than MSCs. Taken together, these findings demonstrate that SKL overexpression enhances the therapeutic potential of MSCs, as evidenced by significantly improved disease severity, decreased inflammation and tissue damage in the spinal cord, and a promoted shift in the Th17/Treg balance towards the anti-inflammatory Treg side in the EAE mice.

Screening, Deconvolution and Parallel Synthesis of Trisubstituted Piperazine and Trisubstituted 2,3-diketopierazine Libraries for the Rapid Identification of Antagonists of the Nuclear Retinoic Acid Receptor-related Orphan Receptor Gamma (RORγ)

Background: Genetic studies support a key role for RORγ and RORα in the differentiation of proinflammatory Th17 cells, and a growing body of evidence suggests a pathogenic role for Th17 in several autoimmune diseases, including MS, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, and psoriasis. RORγ antagonists have been shown to suppress Th17 differentiation and delay the onset of disease in an experimental autoimmune encephalomyelitis mouse model of MS. Objective: Given the high therapeutic interest of RORγ antagonists and the promising activity of currently known ligands, small molecules with higher potency and receptor selectivity (in particular within the ROR family) are highly desirable. We used our small molecule compound library to discover, characterize, and optimize novel RORγ antagonists for the treatment of autoimmune diseases from Mixture-based Combinatorial Chemical Libraries. Methods: We screened the FIU collection of small molecule libraries (>30 million compounds) composed of 75 molecular scaffolds systematically arranged in positional scanning and scaffold ranking formats. We identified scaffolds that selectively inhibit the binding of RORγ, RORγ, and RORβ but not RORα, and others that function as antagonists of all three receptors. Results: The deconvolution of selected PS-SCL mixtures led to the identification of novel chemical entities, trisubstituted piperazine and diketopiperazine that function as RORγ antagonists. Conclusion: The screening of a large complex library led to the rapid identification of novel trisubstituted piperazine and diketopiperazine antagonists of the nuclear retinoic acid receptor-related orphan receptor gamma (RORγ).

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.

Targeted-Neuroinflammation Mitigation Using Inflammasome-Inhibiting Nanoligomers is Therapeutic in an Experimental Autoimmune Encephalomyelitis Mouse Model.

Multiple sclerosis (MS) is a debilitating autoimmune disease that impacts millions of patients worldwide, disproportionately impacting women (4:1), and often presenting at highly productive stages of life. This disease affects the spinal cord and brain and is characterized by severe neuroinflammation, demyelination, and subsequent neuronal damage, resulting in symptoms like loss of mobility. While untargeted and pan-immunosuppressive therapies have proven to be disease-modifying and manage (or prolong the time between) symptoms in many patients, a significant fraction are unable to achieve remission. Recent work has suggested that targeted neuroinflammation mitigation through selective inflammasome inhibition can offer relief to patients while preserving key components of immune function. Here, we show a screening of potential therapeutic targets using inflammasome-inhibiting Nanoligomers (NF-κB1, TNFR1, TNF-α, IL-6) that meet or far-exceed commercially available small-molecule counterparts like ruxolitinib, MCC950, and deucravacitinib. Using the human brain organoid model, top Nanoligomer combinations (NF-κB1 + TNFR1: NI111, and NF-κB1 + NLRP3: NI112) were shown to significantly reduce neuroinflammation without any observable negative impact on organoid function. Further testing of these top Nanoligomer combinations in an aggressive experimental autoimmune encephalomyelitis (EAE) mouse model for MS using intraperitoneal (IP) injections showed that NF-κB1 and NLRP3 targeting Nanoligomer combination NI112 rescues mice without observable loss of mobility or disability, minimal inflammation in brain and spinal cord histology, and minimal to no immune cell infiltration of the spinal cord and no demyelination, similar to or at par with mice that received no EAE injections (negative control). Mice receiving NI111 (NF-κB1 + TNFR1) also showed reduced neuroinflammation compared to saline (sham)-treated EAE mice and at par/similar to other inflammasome-inhibiting small molecule treatments, although it was significantly higher than NI112 leading to subsequent worsening clinical outcomes. Furthermore, treatment with an oral formulation of NI112 at lower doses showed a significant reduction in EAE severity, albeit with higher variance owing to administration and formulation/fill-and-finish variability. Overall, these results point to the potential of further development and testing of these inflammasome-targeting Nanoliogmers as an effective neuroinflammation treatment for multiple neurodegenerative diseases and potentially benefit several patients suffering from such debilitating autoimmune diseases like MS.

Neuroprotection by acrolein sequestration through exogenously applied scavengers and endogenous enzymatic enabling strategies in mouse EAE model

We have previously shown that the pro-oxidative aldehyde acrolein is a critical factor in MS pathology. In this study, we found that the acrolein scavenger hydralazine (HZ), when applied from the day of induction, can suppress acrolein and alleviate motor and sensory deficits in a mouse experimental autoimmune encephalomyelitis (EAE) model. Furthermore, we also demonstrated that HZ can alleviate motor deficits when applied after the emergence of MS symptoms, making potential anti-acrolein treatment a more clinically relevant strategy. In addition, HZ can reduce both acrolein and MPO, suggesting a connection between acrolein and inflammation. We also found that in addition to HZ, phenelzine (PZ), a structurally distinct acrolein scavenger, can mitigate motor deficits in EAE when applied from the day of induction. This suggests that the likely chief factor of neuroprotection offered by these two structurally distinct acrolein scavengers in EAE is their common feature of acrolein neutralization. Finally, up-and-down regulation of the function of aldehyde dehydrogenase 2 (ALDH2) in EAE mice using either a pharmacological or genetic strategy led to correspondent motor and sensory changes. This data indicates a potential key role of ALDH2 in influencing acrolein levels, oxidative stress, inflammation, and behavior in EAE. These findings further consolidate the critical role of aldehydes in the pathology of EAE and its mechanisms of regulation. This is expected to reinforce and expand the possible therapeutic targets of anti-aldehyde treatment to achieve neuroprotection through both endogenous and exogenous manners.

Regulatory T cells use heparanase to access IL-2 bound to extracellular matrix in inflamed tissue

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

The mechanism by which cannabidiol (CBD) suppresses TNF-α secretion involves inappropriate localization of TNF-α converting enzyme (TACE)

Cannabidiol (CBD) is a phytocannabinoid derived from Cannabis sativa that exerts anti-inflammatory mechanisms. CBD is being examined for its putative effects on the neuroinflammatory disease, multiple sclerosis (MS). One of the major immune mediators that propagates MS and its mouse model experimental autoimmune encephalomyelitis (EAE) are macrophages. Macrophages can polarize into an inflammatory phenotype (M1) or an anti-inflammatory phenotype (M2a). Therefore, elucidating the impact on macrophage polarization with CBD pre-treatment is necessary to understand its anti-inflammatory mechanisms. To study this effect, murine macrophages (RAW 264.7) were pre-treated with CBD (10 µM) or vehicle (ethanol 0.1 %) and were either left untreated (naive; cell media only), or stimulated under M1 (IFN-γ + lipopolysaccharide, LPS) or M2a (IL-4) conditions for 24 hr. Cells were analyzed for macrophage polarization markers, and supernatants were analyzed for cytokines and chemokines. Immunofluorescence staining was performed on M1-polarized cells for the metalloprotease, tumor necrosis factor-α-converting enzyme (TACE), as this enzyme is responsible for the secretion of TNF-α. Overall results showed that CBD decreased several markers associated with the M1 phenotype while exhibiting less effects on the M2a phenotype. Significantly, under M1 conditions, CBD increased the percentage of intracellular and surface TNF-α but decreased secreted TNF-α. This phenomenon might be mediated by TACE as staining showed that CBD sequestered TACE intracellularly. CBD also prevented RelA nuclear translocation. These results suggest that CBD may exert its anti-inflammatory effects by reducing M1 polarization and decreasing TNF-α secretion via inappropriate localization of TACE and RelA.

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.

Effects of oral and intranasal resveratrol nanoparticle administration on neurodegeneration and neuronal dysfunction in a model of multiple sclerosis

Aims/Purpose: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system. Immunomodulatory treatments seek to reduce inflammation in MS but have limited effect on neurodegeneration. Resveratrol is a natural polyphenol with antioxidant and anti‐inflammatory properties which could be neuroprotective in MS. The purpose of this study is to compare the oral and intranasal administration of resveratrol nanoparticles (RN) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS with ocular and neurological endpoints.Methods: RN were formulated using a thin rehydration technique. In the 1st experiment, 16.9 mg/kg RN (n = 6) or equivalent vehicle (n = 5) were given orally to EAE mice daily for 30 days. In the 2nd experiment, 8.44 mg/kg RN (n = 6) or vehicle (n = 6) were given intranasally to EAE mice daily for 30 days. EAE mice were assessed daily for clinical signs of ascending paralysis and weekly for visual function. After sacrifice at day 30, retinas were immunostained for brn3a to count retinal ganglion cells (RGC), and spinal cords and optic nerves were stained by H&E and luxol fast blue to assess inflammation and demyelination.Results: 16.9 mg/kg oral RN treatment reduced RGC loss compared to vehicle (4164 ± 406 vs 2422 ± 401 RGC/1.56 mm2, p < 0.05). Similarly, 8.44 mg/kg RN given intranasally reduced RGC loss compared to vehicle (3569 ± 241 vs 2280 ± 329 RGC/1.56 mm2, p < 0.05). Oral and intranasal RN showed similar neuroprotective effects (4164 ± 406 vs 3569 ± 241 RGC/1.56 mm2, p > 0.05). Although neither route of administration showed a reduction of ascending paralysis, 16.9 mg/kg oral RN reduced the severity of paralysis compared to vehicle and 8.44 mg/kg intranasal RN (survival analysis with median survival 29.5 vs 19.0 and 19.0 days, p < 0.05). Likewise, the severity of visual function loss was reduced by the administration of 16.9 mg/kg oral RN compared to vehicle and 8.44 mg/kg intranasal RN (median survival > 28.0 vs 28.0 and 21.0 days, p < 0.05). Neither oral nor intranasal administration showed a significant reduction of spinal cord or optic nerve inflammation or demyelination.Conclusions: RN were able to reduce RGC loss independent of their administration route, but oral administration demonstrated additional neuroprotective effects by reducing paralysis severity.

MiR-142-3p is a Critical Modulator of TNF-mediated Neuronal Toxicity in Multiple Sclerosis.

TNF-dependent synaptotoxicity contributes to the neuronal damage occurring in patients with Multiple Sclerosis (pwMS) and its mouse model Experimental Autoimmune Encephalomyelitis (EAE). Here, we investigated miR-142-3p, a synaptotoxic microRNA induced by inflammation in EAE and MS, as a potential downstream effector of TNF signalling. Electrophysiological recordings, supported by molecular, biochemical and histochemical analyses, were performed to explore TNF-synaptotoxicity in the striatum of EAE and healthy mice. MiR-142 heterozygous (miR-142 HE) mice and/or LNA-anti miR-142-3p strategy were used to verify the TNF-miR-142-3p axis hypothesis. The cerebrospinal fluid (CSF) of 151 pwMS was analysed to evaluate possible correlation between TNF and miR-142-3p levels and their impact on clinical parameters (e.g. progression index (PI), age-related clinical severity (gARMSS)) and MRI measurements at diagnosis (T0). High levels of TNF and miR-142-3p were detected in both EAE striatum and MS-CSF. The TNF-dependent glutamatergic alterations were prevented in the inflamed striatum of EAE miR-142 HE mice. Accordingly, TNF was ineffective in healthy striatal slices incubated with LNA-anti miR- 142-3p. However, both preclinical and clinical data did not validate the TNF-miR-142-3p axis hypothesis, suggesting a permissive neuronal role of miR-142-3p on TNF-signalling. Clinical data showed a negative impact of each molecule on disease course and/or brain lesions and unveiled that their high levels exert a detrimental synergistic effect on disease activity, PI and white matter lesion volume. We propose miR-142-3p as a critical modulator of TNF-mediated neuronal toxicity and suggest a detrimental synergistic action of these molecules on MS pathology.

A B cell-driven EAE mouse model reveals the impact of B cell-derived cytokines on CNS autoimmunity.

In multiple sclerosis (MS), pathogenic T cell responses are known to be important drivers of autoimmune inflammation. However, increasing evidence suggests an additional role for B cells, which may contribute to pathogenesis via antigen presentation and production of proinflammatory cytokines. However, these B cell effector functions are not featured well in classical experimental autoimmune encephalomyelitis (EAE) mouse models. Here, we compared properties of myelin oligodendrocyte glycoprotein (MOG)-specific and polyclonal B cells and developed an adjuvant-free cotransfer EAE mouse model, where highly activated, MOG-specific induced germinal center B cells provide the critical stimulus for disease development. We could show that high levels of MOG-specific immunoglobulin G (IgGs) are not required for EAE development, suggesting that antigen presentation and activation of cognate T cells by B cells may be important for pathogenesis. As our model allows for B cell manipulation prior to transfer, we found that overexpression of the proinflammatory cytokine interleukin (IL)-6 by MOG-specific B cells leads to an accelerated EAE onset accompanied by activation/expansion of the myeloid compartment rather than a changed T cell response. Accordingly, knocking out IL-6 or tumor necrosis factor α in MOG-specific B cells via CRISPR-Cas9 did not affect activation of pathogenic T cells. In summary, we generated a tool to dissect pathogenic B cell effector function in EAE development, which should improve our understanding of pathogenic processes in MS.

Bone marrow mesenchymal stem cells to ameliorate experimental autoimmune encephalomyelitis via modifying expression patterns of miRNAs.

Clinical and experimental studies highlighted the significant therapeutic role of Mesenchymal stem cells (MSCs) in neurodegenerative diseases. MSCs possess potent immunomodulatory properties by releasing exosomes, which generate a suitable microenvironment. microRNAs (miRNAs), as one of several effective bioactive molecules of exosomes, influence cellular communication and activities in recipient cells. Recent studies revealed that miRNAs could control the progression of multiple sclerosis (MS) via differentiation and function of T helper cells (Th). Here, we investigated the therapeutic effects of syngeneic-derived BM-MSC in experimental autoimmune encephalomyelitis (EAE) mouse model of MS by evaluating expression profile of miRNAs, pro- and anti-inflammatory in serum and brain tissues. Three-time scheme groups (6th day, 6th & 12th days, and 12th day, of post-EAE induction) were applied to determine the therapeutic effects of intraperitoneally received 1*106 of BM-MSCs. The expression levels of mature isoforms of miR-193, miR-146a, miR-155, miR-21, and miR-326 showed that BM-MSCs treatment attenuated the EAE clinical score and reduced clinical inflammation as well as demyelination. The improved neurological functional outcome associated with enhanced expression of miR-193 and miR-146a, but decreased expression levels of miR-155, miR-21, and miR-326 were followed by suppressing effects on Th1/Th17 immune responses (reduced levels of IFN-γand IL-17 cytokine expression) and induction of Treg cells, immunoregulatory responses (increase of IL-10, TGF-β, and IL-4) in treatment groups. Our findings suggest that BM-MSCs administration might change expression patterns of miRNAs and downstream interactions followed by immune system modulation. However, there is a need to carry out future human clinical trials and complementary experiments.

Comparison Study of the Influence of Caffeic Acid Phenethyl Ester and Mitoxantrone in Experimental Autoimmune Encephalomyelitis Balb/C Mice Model

A common neurodegenerative condition that still presents clinical challenges is Multiple Sclerosis (MS). Effective multiple sclerosis treatments are sorely needed in clinical settings. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of multiple sclerosis, a T-cell-mediated disease. Active T-cells differentiate into the Th9 and Th17 subsets, which are controlled by NF-kB and produce the proinflammatory cytokines IL9 and IL17. Because these cytokines are crucial to the pathophysiology of EAE, they have been used as targets for MS therapy. Caffeic acid phenethyl ester (CAPE) is an active ingredient of propolis that has been shown to have immunomodulatory and anti-inflammatory activities. Mitoxantrone is a synthetic antineoplastic agent and cytotoxic immunosuppressive effect used to treat MS. The study aimed to determine whether the two medications have superior efficacy and effect in the treatment of EAE mouse model MS compared to the other. After inducing EAE in mice, CAPE and mitoxantrone were administered to evaluate this therapeutic effectiveness. ELISA was used to measure IL9, IL17 levels and the activity of NF-kBp56. H&E was used to evaluate cell infiltration T lymphocytes for histopathology of spinal cord tissue. Molecular docking was performed to predict the interaction between CAPE and a cytokine. We found that CAPE has a sufficient effect of reducing the level of IL9, IL17, active NF-kBp56, and inflammatory cell infiltration T-lymphocytes in all groups of mice EAE treated with CAPE. In contrast, mitoxantrone reduced cytokines and cell infiltration, so EAE mice treated with both compounds were observed more improvement than other groups. Based on our findings, two medications demonstrated the same efficacy and effect in EAE mice model MS., whereas CAPE did not statistically reach a significant value. While the combination of two medications has the optimal effect.

MAP kinase inhibitor PD98059 regulates Th1, Th9, Th17, and natural T regulatory cells in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis

Experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS), provides significant insights into the mechanisms that initiate and drive autoimmunity. MS is a chronic autoimmune disease of the central nervous system, characterized by inflammatory infiltration associated with demyelination. T lymphocyte cells play a crucial role in MS, whereas natural T regulatory (nTreg) cells prevent autoimmune inflammation by suppressing lymphocyte activity. This study sought to investigate the role of PD98059, a selective MAP kinase inhibitor, in Th1, Th9, Th17, and nTreg cells using the SJL/J mouse model of EAE. Following EAE development, the mice were intraperitoneally administered PD98059 (5 mg/kg for two weeks) daily. We evaluated the effects of PD98059 on Th1 (IFN-γ and T-bet), Th9 (IL-9 and IRF4), Th17 (IL-17A and RORγT), and nTreg (FoxP3 and Helios) cells in the spleen using flow cytometry. Moreover, we explored the effects of PD98059 on the IFN-γ, T-bet, IL-9, IRF4, IL-17A, RORγT, FoxP3, and Helios mRNA and protein levels in brain tissues using qRT-PCR and Western blot analyses. PD98059 treatment significantly decreased the proportion of CD4+IFN-γ+, CD4+T-bet+, CD4+IL-9+, CD4+IRF4+, CD4+IL-17A+, CD4+RORγT+, CD4+IL-17A+, and CD4+RORγT+ cells while increasing that of CD4+FoxP3+ and CD4+Helios+ cells. In addition, PD98059 administration decreased the mRNA and protein levels of IFN-γ, T-bet, IL-9, IRF4, IL-17A, and RORγT but increased those of FoxP3 and Helios in the brain tissue of EAE mice. Our findings suggest that PD98059 corrects immune dysfunction in EAE mice, which is concurrent with the modulation of multiple signaling pathways.

Oral vancomycin treatment suppresses gut trypsin activity and preserves intestinal barrier function during EAE

Studies have reported increased intestinal permeability in multiple sclerosis (MS) patients and its mouse model experimental autoimmune encephalomyelitis (EAE). However, the mechanisms driving increased intestinal permeability that in turn exacerbate neuroinflammation during EAE remain unclear. Here we showed that vancomycin preserved the integrity of the intestinal barrier, while also suppressing gut trypsin activity, enhancing the relative abundance of specific Lactobacilli and ameliorating disease during EAE. Furthermore, Lactobacilli enriched in the gut of vancomycin-treated EAE mice at day 3 post immunization negatively correlated with gut trypsin activity and EAE severity. In untreated EAE mice, we observed increased intestinal permeability and increased intestinal protease activated receptor 2 (PAR2) expression at day 3 post immunization. Prior studies have shown that trypsin increases intestinal permeability by activating PAR2. Our results suggest that the interaction between intestinal PAR2 and trypsin may be a key modulator of intestinal permeability and disease severity during EAE.

Novel therapeutic for multiple sclerosis protects white matter function in EAE mouse model.

Multiple sclerosis (MS) is a chronic demyelinating disease with prominent axon dysfunction. Our previous studies in an MS mouse model, experimental autoimmune encephalomyelitis (EAE), demonstrated that major histocompatibility complex Class II constructs can reverse clinical signs of EAE. These constructs block binding and downstream signaling of macrophage migration inhibitory factors (MIF-1/2) through CD74, thereby inhibiting phosphorylation of extracellular signal-regulated kinase (ERK) activation and tissue inflammation and promoting remyelination. To directly assess the effects of a novel third generation construct, DRhQ, on axon integrity in EAE, we compared axon conduction properties using electrophysiology on corpus callosum slices and optic nerves. By using two distinct white matter (WM) tracts, we aimed to assess the impact of the EAE and the benefit of DRhQ on myelinated and unmyelinated axons as well as to test the clinical value of DRhQ on demyelinating lesions in CC and optic myelitis. Our study found that EAE altered axon excitability, delayed axon conduction and slowed spatiotemporal summation correlated with diffuse astrocyte and microglia activation. Because MS predisposes patients to stroke, we also investigated and showed that vulnerability to WM ischemia is increased in the EAE MS mouse model. Treatment with DRhQ after the onset of EAE drastically inhibited microglial and astrocyte activation, improved functional integrity of the myelinated axons and enhanced recovery after ischemia. These results demonstrate that DRhQ administered after the onset of EAE promotes WM integrity and function, and reduces subsequent vulnerability to ischemic injury, suggesting important therapeutic potential for treatment of progressive MS.

Remibrutinib (LOU064) inhibits neuroinflammation driven by B cells and myeloid cells in preclinical models of multiple sclerosis

BackgroundBruton’s tyrosine kinase (BTK) is a key signaling node in B cell receptor (BCR) and Fc receptor (FcR) signaling. BTK inhibitors (BTKi) are an emerging oral treatment option for patients suffering from multiple sclerosis (MS). Remibrutinib (LOU064) is a potent, highly selective covalent BTKi with a promising preclinical and clinical profile for MS and other autoimmune or autoallergic indications.MethodsThe efficacy and mechanism of action of remibrutinib was assessed in two different experimental autoimmune encephalomyelitis (EAE) mouse models for MS. The impact of remibrutinib on B cell-driven EAE pathology was determined after immunization with human myelin oligodendrocyte glycoprotein (HuMOG). The efficacy on myeloid cell and microglia driven neuroinflammation was determined in the RatMOG EAE. In addition, we assessed the relationship of efficacy to BTK occupancy in tissue, ex vivo T cell response, as well as single cell RNA-sequencing (scRNA-seq) in brain and spinal cord tissue.ResultsRemibrutinib inhibited B cell-dependent HuMOG EAE in dose-dependent manner and strongly reduced neurological symptoms. At the efficacious oral dose of 30 mg/kg, remibrutinib showed strong BTK occupancy in the peripheral immune organs and in the brain of EAE mice. Ex vivo MOG-specific T cell recall response was reduced, but not polyclonal T cell response, indicating absence of non-specific T cell inhibition. Remibrutinib also inhibited RatMOG EAE, suggesting that myeloid cell and microglia inhibition contribute to its efficacy in EAE. Remibrutinib did not reduce B cells, total Ig levels nor MOG-specific antibody response. In brain and spinal cord tissue a clear anti-inflammatory effect in microglia was detected by scRNA-seq. Finally, remibrutinib showed potent inhibition of in vitro immune complex-driven inflammatory response in human microglia.ConclusionRemibrutinib inhibited EAE models by a two-pronged mechanism based on inhibition of pathogenic B cell autoreactivity, as well as direct anti-inflammatory effects in microglia. Remibrutinib showed efficacy in both models in absence of direct B cell depletion, broad T cell inhibition or reduction of total Ig levels. These findings support the view that remibrutinib may represent a novel treatment option for patients with MS.

Histamine H4 Receptor Agonist, 4-Methylhistamine, Aggravates Disease Progression and Promotes Pro-Inflammatory Signaling in B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model.

We sought to assess the impact of 4-Methylhistamine (4-MeH), a specific agonist targeting the Histamine H4 Receptor (H4R), on the progression of experimental autoimmune encephalomyelitis (EAE) and gain insight into the underlying mechanism. EAE is a chronic autoimmune, inflammatory, and neurodegenerative disease of the central nervous system (CNS) characterized by demyelination, axonal damage, and neurodegeneration. Over the past decade, pharmacological research into the H4R has gained significance in immune and inflammatory disorders. For this study, Swiss Jim Lambert EAE mice were treated with 4-MeH (30 mg/kg/day) via intraperitoneal administration from days 14 to 42, and the control group was treated with a vehicle. Subsequently, we evaluated the clinical scores. In addition, flow cytometry was employed to estimate the impact of 4-Methylhistamine (4-MeH) on NF-κB p65, GM-CSF, MCP-1, IL-6, and TNF-α within CD19+ and CXCR5+ spleen B cells. Additionally, we investigated the effect of 4-MeH on the mRNA expression levels of Nf-κB p65, Gmcsf, Mcp1, Il6, and Tnfα in the brain of mice using RT-PCR. Notably, the clinical scores of EAE mice treated with 4-MeH showed a significant increase compared with those treated with the vehicle. The percentage of cells expressing CD19+NF-κB p65+, CXCR5+NF-κB p65+, CD19+GM-CSF+, CXCR5+GM-CSF+, CD19+MCP-1+, CXCR5+MCP-1+, CD19+IL-6+, CXCR5+IL-6+, CD19+TNF-α+, and CXCR5+TNF-α+ exhibited was more pronounced in 4-MeH-treated EAE mice when compared to vehicle-treated EAE mice. Moreover, the administration of 4-MeH led to increased expression of NfκB p65, Gmcsf, Mcp1, Il6, and Tnfα mRNA in the brains of EAE mice. This means that the H4R agonist promotes pro-inflammatory mediators aggravating EAE symptoms. Our results indicate the harmful role of H4R agonists in the pathogenesis of MS in an EAE mouse model.

Proteolipid Protein-Induced Mouse Model of Multiple Sclerosis Requires B Cell-Mediated Antigen Presentation.

The pathogenic role B cells play in multiple sclerosis is underscored by the success of B cell depletion therapies. Yet, it remains unclear how B cells contribute to disease, although it is increasingly accepted that mechanisms beyond Ab production are involved. Better understanding of pathogenic interactions between B cells and autoreactive CD4 T cells will be critical for novel therapeutics. To focus the investigation on B cell:CD4 T cell interactions invivo and invitro, we previously developed a B cell-dependent, Ab-independent experimental autoimmune encephalomyelitis (EAE) mouse model driven by a peptide encompassing the extracellular domains of myelin proteolipid protein (PLPECD). In this study, we demonstrate that B cell depletion significantly inhibited PLPECD-induced EAE disease, blunted PLPECD-elicited delayed-type hypersensitivity reactions invivo, and reduced CD4 T cell activation, proliferation, and proinflammatory cytokine production. Further, PLPECD-reactive CD4 T cells sourced from B cell-depleted donor mice failed to transfer EAE to naive recipients. Importantly, we identified B cell-mediated Ag presentation as the critical mechanism explaining B cell dependence in PLPECD-induced EAE, where bone marrow chimeric mice harboring a B cell-restricted MHC class II deficiency failed to develop EAE. B cells were ultimately observed to restimulate significantly higher Ag-specific proliferation from PLP178-191-reactive CD4 T cells compared with dendritic cells when provided PLPECD peptide in head-to-head cultures. We therefore conclude that PLPECD-induced EAE features a required pathogenic B cell-mediated Ag presentation function, providing for investigable B cell:CD4 T cell interactions in the context of autoimmune demyelinating disease.

TREM-2 Drives Development of Multiple Sclerosis by Promoting Pathogenic Th17 Polarization.

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease, mediated by pathogenic T helper 17 (Th17) cells. However, the therapeutic effect is accompanied by the fluctuation of the proportion and function of Th17 cells, which prompted us to find the key regulator of Th17 differentiation in MS. Here, we demonstrated that the triggering receptor expressed on myeloid cells 2 (TREM-2), a modulator of pattern recognition receptors on innate immune cells, was highly expressed on pathogenic CD4-positive T lymphocyte (CD4+ T) cells in both patients with MS and experimental autoimmune encephalomyelitis (EAE) mouse models. Conditional knockout of Trem-2 in CD4+ T cells significantly alleviated the disease activity and reduced Th17 cell infiltration, activation, differentiation, and inflammatory cytokine production and secretion in EAE mice. Furthermore, with Trem-2 knockout in vivo experiments and in vitro inhibitor assays, the TREM-2/zeta-chain associated protein kinase 70 (ZAP70)/signal transducer and activator of transcription 3 (STAT3) signal axis was essential for Th17 activation and differentiation in EAE progression. In conclusion, TREM-2 is a key regulator of pathogenic Th17 in EAE mice, and this sheds new light on the potential of this therapeutic target for MS.