Oligodendrocyte-Specific Deletion of FGFR1 Reduces Cerebellar Inflammation and Neurodegeneration in MOG35-55-Induced EAE.

Multiple sclerosis (MS) is a chronic inflammatory and degenerative disease of the central nervous system (CNS). MS commonly affects the cerebellum causing acute and chronic symptoms. Cerebellar signs significantly contribute to clinical disability, and symptoms such as tremor, ataxia, and dysarthria are difficult to treat. Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in demyelinating pathologies such as MS. In autopsy tissue from patients with MS, increased expression of FGF1, FGF2, FGF9, and FGFR1 was found in lesion areas. Recent research using mouse models has focused on regions such as the spinal cord, and data on the expression of FGF/FGFR in the cerebellum are not available. In recent EAE studies, we detected that oligodendrocyte-specific deletion of FGFRs results in a milder disease course, less cellular infiltrates, and reduced neurodegeneration in the spinal cord. The objective of this study was to characterize the role of FGFR1 in oligodendrocytes in the cerebellum. Conditional deletion of FGFR1 in oligodendrocytes (Fgfr1ind-/-) was achieved by tamoxifen application, EAE was induced using the MOG35-55 peptide. The cerebellum was analyzed by histology, immunohistochemistry, and western blot. At day 62 p.i., Fgfr1ind-/- mice showed less myelin and axonal degeneration compared to FGFR1-competent mice. Infiltration of CD3(+) T cells, Mac3(+) cells, B220(+) B cells and IgG(+) plasma cells in cerebellar white matter lesions (WML) was less in Fgfr1ind-/-mice. There were no effects on the number of OPC or mature oligodendrocytes in white matter lesion (WML). Expression of FGF2 and FGF9 associated with less myelin and axonal degeneration, and of the pro-inflammatory cytokines IL-1ß, IL-6, and CD200 was downregulated in Fgfr1ind-/- mice. The FGF/FGFR signaling protein pAkt, BDNF, and TrkB were increased in Fgfr1ind-/- mice. These data suggest that cell-specific deletion of FGFR1 in oligodendrocytes has anti-inflammatory and neuroprotective effects in the cerebellum in the EAE disease model of MS.

Pro-inflammatory T helper 17 directly harms oligodendrocytes in neuroinflammation.

T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes, and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate, which induced cell stress and changes in biosynthesis of cholesterol and lipids, as revealed by single-cell RNA-sequencing analysis. Finally, exposure to glutamate decreased myelination, whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities in MS.

CNS Autoimmune Responses in BCMA-Deficient Mice Provide Insight for the Failure of Atacicept in MS.

OBJECTIVE: B cells have emerged as a therapeutic target for MS. Anti-CD20 antibodies, which deplete B cells, are effective therapies for MS. However, atacicept (TACI-Fc), which blocks BAFF and APRIL and reduces B cells, unexpectedly exacerbates MS. We tested the hypothesis that B cell maturation antigen (BCMA), a receptor for BAFF and APRIL, plays a role in the paradoxical effects of anti-CD20 antibody and TACI-Fc using experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced in wild-type (BCMA+/+) and BCMA-deficient (BCMA-/-) mice with an immunization of rodent myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Treatment with anti-CD20 antibody, TACI-Fc, and isotype controls was administered by intraperitoneal injections. CNS infiltration was evaluated by histology; immune cell phenotypes were evaluated by flow cytometry; MOG-specific antibodies were determined by ELISA. Mixed bone marrow chimeras and cell culture assays were used to identify the specific subsets of immune cells affected by BCMA deficiency. RESULTS: First, we found that BCMA-/- mice had more severe EAE compared with BCMA+/+ mice and the increased disease was associated with elevated anti-MOG B-cell responses. Second, we found that anti-CD20 therapy attenuated EAE in BCMA-/- mice but not in BCMA+/+ mice. Third, TACI-Fc attenuated EAE in BCMA+/+ mice but not in BCMA-/- mice. Mixed bone marrow chimeric and cell culture experiments demonstrated that BCMA deficiency elevates inflammatory B-cell responses but inhibits inflammatory responses in macrophages. CONCLUSIONS: BCMA has multifaceted roles during inflammation that affects therapeutic efficacies of anti-CD20 and TACI-Fc in EAE. Our results from BCMA-deficient mice provide insights into the failure of atacicept in MS.

Anti-inflammatory Activity of Caspian Cobra (Naja naja oxiana) Snake Venom on the Serum Level of Interleukin-27 and Histopathological Changes in Myelin Oligodendrocyte Glycoprotein-experimental Autoimmune Encephalomyelitisinduced Mice.

Multiple sclerosis (MS) is considered a chronic disease of the central nervous system, with a strong neurodegenerative component. The exact mechanism of MS is not clear. However, the therapeutic strategies for controlling MS are based on immune modulation and inflammation control. Regarding this, the present study was conducted to investigate the influence of snake venom on the suppression of the immune system after the induction of experimental autoimmune encephalomyelitis (EAE) in mice. For this purpose, C57BL/6 female mice, divided into three groups, were selected to be induced by EAE. Groups 2 and 3 received flank injection with the emulsion of myelin oligodendrocyte glycoprotein (MOG 35-55), as well as complete Freund adjuvant, followed by the administration of pertussis toxin. Furthermore, the treatment group, as an immune-modulator, received cobra venom (CV) after EAE induction. The mice were then evaluated daily based on clinical symptoms, weight changes (within 26 days), histopathological analysis, and serum levels of interleukin 27 (IL-27) for neurological motor deficits. The clinical signs of MOG-EAE in C57BL/6 mice began 9-14 days post-immunization. Histopathological results also revealed that CV-treated EAE mice, compared to the untreated EAE group, witnessed a significant reduction in the intensity of inflammatory cells in parenchymal sections. Furthermore, the increase of IL-27 levels was significant in the CV-treated group (P=0.001), compared with those in the EAE and control groups. Based on results obtained in the present study, it may be concluded that Naja naja oxiana snake venom is a potential immunomodulatory agent that can be effective in the treatment of MS.

Paricalcitol improves experimental autoimmune encephalomyelitis (EAE) by suppressing inflammation via NF-κB signaling.

Multiple sclerosis (MS) is known as an autoimmune disease in the central nervous system (CNS) characterized by motor deficits, pain, fatigue, cognitive impairment, and sensory and visual dysfunction. MS is considered to be resulted from significant inflammatory response. Paricalcitol (Pari) is a vitamin D2 analogue, which has been indicated to show anti-inflammatory activities in kidney and heart diseases. In the present study, if Pari could ameliorate the experimental autoimmune encephalomyelitis (EAE) was investigated. Here, the C57BL/6 mice were immunized using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). Subsequently, Pari was intraperitoneally injected into the mice. As for in vitro analysis, RAW264.7 and Jurkat cells were incubated with Pari together with corresponding stimulus. The results indicated that Pari administration reduced the paralytic severity, neuropathology and apoptosis in MOG-treated mice compared to the MOG single group. Pari also exhibited a significantly inhibitory effect on immune cell infiltration, glial cell activation, expression of pro-inflammatory factors and the activation of nuclear factor κB (NF-κB). The expression of pro-inflammatory regulators and the translocation of NF-κB from cytoplasm into nuclear in RAW264.7 and Jurkat cells under specific stimulation was clearly down-regulated by Pari incubation. Furthermore, we found that suppressing NF-κB with its inhibitor combined with Pari could further reduce the expression of pro-inflammatory factors and associated proteins. These data illustrated that Pari could diminish MOG-triggered EAE, as well as macrophages and T cells activation through blocking NF-κB activation. Collectively, Pari might have therapeutic effects in mouse models with MS.

Ac-SDKP ameliorates the progression of experimental autoimmune encephalomyelitis via inhibition of ER stress and oxidative stress in the hippocampus of C57BL/6 mice.

Despite the attention given to the treatment of multiple sclerosis (MS), still no certain cure is available. The main purpose of MS drugs is acting against neuroinflammation which underlies the pathology of MS. Neuroinflammation is associated with endoplasmic reticulum (ER) stress that mediates neural apoptosis. In the present study, we hypothesized that the tetrapeptide N-acetyl-ser-asp-lys-pro (Ac-SDKP) with the previously described anti-fibrotic effects might have anti-inflammatory, anti-oxidative and anti-ER stress roles in the hippocampus. We used myelin oligodendrocyte glycoprotein (MOG) to induce experimental autoimmune encephalomyelitis (EAE), a widely-accepted animal model of MS, in C57BL/6 mice. The protein levels of ER stress-related molecules including caspase-12, C/EBP homologous protein (CHOP), and protein disulfide isomerase (PDI) in the hippocampus were examined by immunoblotting. Hence, reactive oxygen species (ROS) production, lipid peroxidation and antioxidant capacity of the hippocampus were studied. Moreover, hippocampal morphology changes, leukocytes infiltration, and the levels of IL-6 and IL-1ß pro-inflammatory cytokines were evaluated. Our results displayed that Ac-SDKP down regulates caspase-12 and CHOP expression in the hippocampus-resident oligodendrocytes of EAE mice. Further, treatment with Ac-SDKP decreased oxidative stress markers and caspase-3 activation in the hippocampus of EAE mice. According to our findings, Ac-SDKP showed beneficial effects against ER stress and oxidative stress in addition to inflammation in the hippocampus of EAE mice. The present study provides the basis for further research on the therapeutic applications of Ac-SDKP to reduce ER stress and oxidative stress-induced apoptosis in neurodegenerative disorders.

γδ T Cell-Secreted XCL1 Mediates Anti-CD3-Induced Oral Tolerance.

Oral tolerance is defined as the specific suppression of cellular and/or humoral immune responses to an Ag by prior administration of the Ag through the oral route. Although the investigation of oral tolerance has classically involved Ag feeding, we have found that oral administration of anti-CD3 mAb induced tolerance through regulatory T (Treg) cell generation. However, the mechanisms underlying this effect remain unknown. In this study, we show that conventional but not plasmacytoid dendritic cells (DCs) are required for anti-CD3-induced oral tolerance. Moreover, oral anti-CD3 promotes XCL1 secretion by small intestine lamina propria γδ T cells that, in turn, induces tolerogenic XCR1+ DC migration to the mesenteric lymph node, where Treg cells are induced and oral tolerance is established. Consistent with this, TCRδ-/- mice did not develop oral tolerance upon oral administration of anti-CD3. However, XCL1 was not required for oral tolerance induced by fed Ags, indicating that a different mechanism underlies this effect. Accordingly, oral administration of anti-CD3 enhanced oral tolerance induced by fed MOG35-55 peptide, resulting in less severe experimental autoimmune encephalomyelitis, which was associated with decreased inflammatory immune cell infiltration in the CNS and increased Treg cells in the spleen. Thus, Treg cell induction by oral anti-CD3 is a consequence of the cross-talk between γδ T cells and tolerogenic DCs in the gut. Furthermore, anti-CD3 may serve as an adjuvant to enhance oral tolerance to fed Ags.

A TLR-CXCL1 pathway in DRG neurons induces neutrophil accumulation in the DRG and mechanical allodynia in EAE mice.

Multiple sclerosis (MS) is a potentially disabling disease of the central nervous system. Approximately half of the patients with MS experience severe pain; however, currently available therapeutics provide only insufficient relief. The mechanisms underlying the generation of neuropathic pain in patients with MS are not fully understood. Recently, we found that neutrophil elastase from accumulated neutrophils in the dorsal root ganglion (DRG) sensitizes DRG neurons and induces mechanical allodynia in a mouse model of experimental autoimmune encephalomyelitis (EAE). However, the mechanism underlying neutrophil accumulation in the DRG after myelin oligodendrocyte glycoprotein (MOG35-55, immunogenic peptide) immunization remains unclear. Here, we found that C-X-C motif ligand 1 (CXCL1) was upregulated in DRG neurons after MOG35-55 immunization. Increased expression of CXCL1 protein was also observed in primary cultured DRG neurons treated with MOG35-55, which was mediated through toll-like receptor 4 (TLR4). Gene silencing of TLR4 or CXCL1 in DRG neurons significantly attenuated neutrophil accumulation in the DRG and mechanical allodynia during the preclinical phase of EAE (around day 5 after immunization). Our results thus suggest that a TLR4-CXCL1 pathway in DRG neurons triggers neutrophil recruitment in the DRG and subsequent mechanical allodynia in response to MOG35-55.

Autoimmune encephalomyelitis in NOD mice is not initially a progressive multiple sclerosis model.

OBJECTIVE: Despite progress in treating relapsing multiple sclerosis (MS), effective inhibition of nonrelapsing progressive MS is an urgent, unmet, clinical need. Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE), provide valuable tools to examine the mechanisms contributing to disease and may be important for developing rational therapeutic approaches for treatment of progressive MS. It has been suggested that myelin oligodendrocyte glycoprotein (MOG) peptide residues 35-55 (MOG35-55 )-induced EAE in nonobese diabetic (NOD) mice resembles secondary progressive MS. The objective was to determine whether the published data merits such claims. METHODS: Induction and monitoring of EAE in NOD mice and literature review. RESULTS: It is evident that the NOD mouse model lacks validity as a progressive MS model as the individual course seems to be an asynchronous, relapsing-remitting neurodegenerative disease, characterized by increasingly poor recovery from relapse. The seemingly progressive course seen in group means of clinical score is an artifact of data handling and interpretation. INTERPRETATION: Although MOG35-55 -induced EAE in NOD mice may provide some clues about approaches to block neurodegeneration associated with the inflammatory penumbra as lesions form, it should not be used to justify trials in people with nonactive, progressive MS. This adds further support to the view that drug studies in animals should universally adopt transparent raw data deposition as part of the publication process, such that claims can adequately be interrogated. This transparency is important if animal-based science is to remain a credible part of translational research in MS.

Neuroinflammation and B-Cell Phenotypes in Cervical and Lumbosacral Regions of the Spinal Cord in Experimental Autoimmune Encephalomyelitis in the Absence of Pertussis Toxin.

OBJECTIVES: The active experimental autoimmune encephalomyelitis (EAE) model is often initiated using myelin oligodendrocyte glycoprotein (MOG) immunization followed by pertussis toxin (PTX) to study multiple sclerosis. However, PTX inactivates G protein-coupled receptors, and with increasing knowledge of the role that various G protein-coupled receptors play in immune homeostasis, it is valuable to establish neuroimmune endpoints for active EAE without PTX. METHODS: Female C57BL/6 mice were immunized with MOG35-55 peptide in Complete Freund's Adjuvant and neuroinflammation, including central nervous system B-cell infiltration, was compared to saline-injected mice. Since it was anticipated that disease onset would be slower and less robust than EAE in the presence of PTX, both cervical and lumbosacral sections of the spinal cord were evaluated. RESULTS: Immunohistochemical analysis showed that EAE without PTX induced immune infiltration, CCL2 and VCAM-1 upregulation. Demyelination in the cervical region correlated with the infiltration of CD19+ B cells in the cervical region. There was upregulation of IgG, CD38, and PDL1 on B cells in cervical and lumbosacral regions of the spinal cord in EAE without PTX. Interestingly, IgG was expressed predominantly by CD19- cells. CONCLUSIONS: These data demonstrate that many neuroimmune endpoints are induced in EAE without PTX and although clinical disease is mild, this can be used as an autoimmune model when PTX inactivation of G protein-coupled receptors is not desired.

RRAS2 shapes the TCR repertoire by setting the threshold for negative selection.

Signal strength controls the outcome of αß T cell selection in the thymus, resulting in death if the affinity of the rearranged TCR is below the threshold for positive selection, or if the affinity of the TCR is above the threshold for negative selection. Here we show that deletion of the GTPase RRAS2 results in exacerbated negative selection and above-normal expression of positive selection markers. Furthermore, Rras2-/- mice are resistant to autoimmunity both in a model of inflammatory bowel disease (IBD) and in a model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). We show that MOG-specific T cells in Rras2-/- mice have reduced affinity for MOG/I-Ab tetramers, suggesting that enhanced negative selection leads to selection of TCRs with lower affinity for the self-MOG peptide. An analysis of the TCR repertoire shows alterations that mostly affect the TCRα variable (TRAV) locus with specific VJ combinations and CDR3α sequences that are absent in Rras2-/- mice, suggesting their involvement in autoimmunity.

Acylated and deacylated quillaja saponin-21 adjuvants have opposite roles when utilized for immunization of C57BL/6 mice model with MOG35-55 peptide.

BACKGROUND: The majority of patients with multiple sclerosis (MS) suffer from central neuropathic pain (CNP). Using experimental autoimmune encephalomyelitis (EAE) model, only a few experiments were performed to assess pain behaviors in MS. To address this issue, complete Freund's adjuvant (CFA) was replaced with an acylated triterpene glycoside saponin adjuvant named quillaja saponin-21 (QS-21) to develop CNP in the EAE mouse model. The deacylated form of QS-21, named QT-0101, has been suggested to have an immunomodulatory effect. Thus, QT-0101 was used as a vaccine adjuvant to modulate the immune system against myelin oligodendrocyte glycoprotein (MOG35-55) antigen. METHODS: In this study, C57BL/6 mice, except for mice in the negative control (PBS) and MOG groups, were divided into three groups and immunized by MOG35-55 emulsified with CFA, QS-21, or QT-0101 adjuvants, respectively. Thermal hyperalgesia, as a CNP clinical manifestation, through the Hot Plate test and the clinical signs, was assessed for 60 days after immunization. On days 21 and 60, mice were sacrificed and the frequency of TCD4+, TCD8+, IL-17+, IL-4+, and CD25+/FoxP3+ cells population in the total splenocytes population was assessed by flow cytometry. Infiltration of Leukocytes into the brain and demyelination of white matter were also evaluated by histopathologic studies. RESULTS: Our results revealed that unlike the MOG+QT-0101 group, the MOG+QS-21 and MOG+CFA groups represented clinical symptoms that mimic the mild relapsing-remitting and monophasic models, respectively. Thermal hyperalgesia, as a CNP clinical manifestation, developed in the bilateral hind paws in the MOG+CFA and MOG+QS-21 mice groups during the onset of neurologic deficits, but it is maintained until completion of the study only in MOG+QS-21 mice group. The frequency of TCD4+, TCD8+ and IL-17+ cells population in the MOG+QS-21 and MOG+CFA mice groups, as well as IL-4+ and CD25+/Foxp3+ cells population in the MOG+QT-0101 mice group, significantly increased in comparison with the PBS mice group. Infiltration of inflammatory cells increased significantly in the MOG+QS-21 and MOG+CFA mice groups compared with the PBS mice group. Demyelination of white matter was identified significantly only in the MOG+CFA mice group compared with the PBS mice group. CONCLUSION: These results showed that QS-21 is a suitable adjuvant for the establishment of a mild relapsing-remitting EAE model for CNP development and open a new avenue to future pre-clinical and clinical research studies related to CNP treatment. Nevertheless, QT-0101 seems to have the potential to act as a vaccine adjuvant with immunomodulatory property against auto-antigens.

VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1ß in macrophages.

Hyperactivation of the NLRP3 inflammasome contributes to the pathogenesis of multiple diseases, but the mechanisms underlying transcriptional regulation of Nlrp3 remain elusive. We demonstrate here that macrophages lacking V-set and immunoglobulin domain-containing 4 (Vsig4) exhibit significant increases in Nlrp3 and Il-1ß transcription, caspase-1 activation, pyroptosis, and interleukin-1ß (IL-1ß) secretion in response to NLRP3 inflammasome stimuli. VSIG4 interacts with MS4A6D in the formation of a surface signaling complex. VSIG4 occupancy triggers Ser232 and Ser235 phosphorylation in MS4A6D, leading to activation of JAK2-STAT3-A20 cascades that further results in nuclear factor κB suppression and Nlrp3 and Il-1ß repression. Exaggerated NLRP3 and IL-1ß expression in Vsig4-/- mice is accountable for deleterious disease severity in experimental autoimmune encephalomyelitis (EAE) and resistance to dextran sulfate sodium (DSS)-induced colitis. The agonistic VSIG4 antibodies (VG11), acting through NLRP3 and IL-1ß suppression, show significant therapeutic efficacy in mouse EAE. These findings highlight VSIG4 as a prospective target for treating NLRP3-associated inflammatory disorders.

[Mechanism of immune tolerance induced by soluble myelin oligodendrocyte glycoprotein in mice with experimental autoimmune encephalomyelitis].

Objective To explore the mechanism of immune tolerance induced by soluble MOG35-55 (MOG) peptide in mice with experimental autoimmune encephalomyelitis (EAE). Methods EAE mice were randomly divided into three groups, MOG, OVA and control groups, which were injected intraperitoneally with MOG, OVA peptide and PBS, respectively, from day 6 to day 16 after EAE induction. Lymphocytes in the spleen and CNS were enumerated and their phenotypes and function were analyzed by flow cytometry to explore the role of T cell migration in MOG-induced EAE tolerance. Next, CD11b+ antigen presenting cells (APCs) in the spleen and CNS infiltration were analyzed by flow cytometry to evaluate their maturation, and the role of mature APCs in blocking MOG-CD4+ T cells trafficking to CNS was determined by immunofluorescence technique. Results MOG trapped effector T cells in the spleen and protected mice from EAE. MOG triggered the maturation of splenic APCs, while prevented the maturation of CNS-infiltrating APCs. MOG-loaded APCs could interact with MOG reactive CD4+ T cells and limit their migration. Conclusion MOG can protect mice from EAE by inducing the maturation of splenic APCs that interact with MOG-T cells and trap them in the periphery.

Lack of junctional adhesion molecule (JAM)-B ameliorates experimental autoimmune encephalomyelitis.

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) autoaggressive CD4+ T cells cross the blood-brain barrier (BBB) and cause neuroinflammation. Therapeutic targeting of CD4+ T-cell trafficking into the CNS by blocking α4-integrins has proven beneficial for the treatment of MS but comes with associated risks, probably due to blocking CD8+ T cell mediated CNS immune surveillance. Our recent observations show that CD8+ T cells also rely on α4ß1-integrins to cross the BBB. Besides vascular cell adhesion molecule-1 (VCAM-1), we identified junctional adhesion molecule-B (JAM-B) as a novel vascular α4ß1-integrin ligand involved in CD8+ T-cell migration across the BBB. This prompted us to investigate, if JAM-B also mediates CD4+ T-cell migration across the BBB. We first ensured that encephalitogenic T cells can bind to JAM-B in vitro and next compared EAE pathogenesis in JAM-B-/- C57BL/6J mice and their wild-type littermates. Following immunization with MOGaa35-55 peptide, JAM-B-/- mice developed ameliorated EAE compared to their wild-type littermates. At the same time, we isolated higher numbers of CD45+ infiltrating immune cells from the CNS of JAM-B-/- C57BL/6J mice suffering from EAE. Immunofluorescence staining revealed that the majority of CD45+ inflammatory cells accumulated in the leptomeningeal and perivascular spaces of the CNS behind the BBB but do not gain access to the CNS parenchyma. Trapping of CNS inflammatory cells was not due to increased inflammatory cell proliferation. Neither a loss of BBB integrity or BBB polarity potentially affecting local chemokine gradients nor a lack of focal gelatinase activation required for CNS parenchymal immune cell entry across the glia limitans could be detected in JAM-B-/- mice. Lack of a role for JAM-B in the effector phase of EAE was supported by the observation that we did not detect any role for JAM-B in EAE pathogenesis, when EAE was elicited by in vitro activated MOG aa35-55-specific CD4+ effector T cells. On the other hand, we also failed to demonstrate any role of JAM-B in in vivo priming, proliferation or polarization of MOGaa35-55-specific CD4+ T cells in peripheral immune organs. Finally, our study excludes expression of and thus a role for JAM-B on peripheral and CNS infiltrating myeloid cells. Taken together, although endothelial JAM-B is not required for immune cell trafficking across the BBB in EAE, in its absence accumulation of inflammatory cells mainly in CNS leptomeningeal spaces leads to amelioration of EAE.

Functional network analysis reveals biological roles of lncRNAs and mRNAs in MOG35-55 specific CD4+T helper cells.

Long non-coding RNAs have the potential to regulate immune responses. Their impact on multiple sclerosis has remained elusive. For illustrating their roles in experimental autoimmune encephalomyelitis (EAE) pathogenesis, we investigated the differential expression of lncRNAs and mRNAs in CD4+Th cells obtained from myelin oligodendrocytic glycoprotein35-55(MOG35-55)-induced EAE and complete Freund's adjuvant (CFA) controls. We observed differential expression of 1112 lncRNAs and 519 mRNAs in CD4+Th cells. The functional network showed lncRNAs had the capacity to modulate EAE pathogenesis via regulating many known EAE regulators such as Ptpn6. Predicting the function of lncRNAs demonstrated that dysregulated lncRNAs were closely associated with the development of EAE. These dysregulated lncRNAs may have function in EAE and they could be novel biomarkers and therapeutic targets of EAE. However, the precise mechanisms and biological functions of these specific lncRNAs in EAE pathogenesis require further study.

DRα1-MOG-35-55 Reduces Permanent Ischemic Brain Injury.

Stroke induces a catastrophic immune response that involves the global activation of peripheral leukocytes, especially T cells. The human leukocyte antigen-DRα1 domain linked to MOG-35-55 peptide (DRα1-MOG-35-55) is a partial major histocompatibility complex (MHC) class II construct which can inhibit neuroantigen-specific T cells and block binding of the cytokine/chemokine macrophage migration inhibitory factor (MIF) to its CD74 receptor on monocytes and macrophages. Here, we evaluated the therapeutic effect of DRα1-MOG-35-55 in a mouse model of permanent distal middle cerebral artery occlusion (dMCAO). DRα1-MOG-35-55 was administered to WT C57BL/6 mice by subcutaneous injection starting 4 h after the onset of ischemia followed by three daily injections. We demonstrated that DRα1-MOG-35-55 post treatment significantly reduced brain infarct volume, improved functional outcomes, and inhibited the accumulation of CD4+ and CD8+ T cells and expression of pro-inflammatory cytokines in the ischemic brain 96 h after dMCAO. In addition, DRα1-MOG-35-55 treatment shifted microglia/macrophages in the ischemic brain to a beneficial M2 phenotype without changing their total numbers in the brain or blood. This study demonstrates for the first time the therapeutic efficacy of the DRα1-MOG-35-55 construct in dMCAO across MHC class II barriers in C57BL/6 mice. This MHC-independent effect obviates the need for tissue typing and will thus greatly expedite treatment with DRα1-MOG-35-55 in human stroke subjects. Taken together, our findings suggest that DRα1-MOG-35-55 treatment may reduce ischemic brain injury by regulating post-stroke immune responses in the brain and the periphery.

Down-regulation of neuronal L1 cell adhesion molecule expression alleviates inflammatory neuronal injury.

In multiple sclerosis (MS), the immune cell attack leads to axonal injury as a major cause for neurological disability. Here, we report a novel role of the cell adhesion molecule L1 in the crosstalk between the immune and nervous systems. L1 was found to be expressed by CNS axons of MS patients and human T cells. In MOG35-55-induced murine experimental neuroinflammation, CD4+ T cells were associated with degenerating axons in the spinal cord, both expressing L1. However, neuronal L1 expression in the spinal cord was reduced, while levels of the transcriptional repressor REST (RE1-Silencing Transcription Factor) were up-regulated. In PLP139-151-induced relapsing-remitting neuroinflammation, L1 expression was low at the peak stage of disease, reached almost normal levels in the remission stage, but decreased again during disease relapse indicating adaptive expression regulation of L1. In vitro, activated CD4+ T cells caused contact-dependent down-regulation of L1, up-regulation of its repressor REST and axonal injury in co-cultured neurons. T cell adhesion to neurons and axonal injury were prevented by an antibody blocking L1 suggesting that down-regulation of L1 ameliorates neuroinflammation. In support of this hypothesis, antibody-mediated blocking of L1 in C57BL/6 mice as well as neuron-specific depletion of L1 in synapsinCre × L1fl/fl mice reduces disease severity and axonal pathology despite unchanged immune cell infiltration of the CNS. Our data suggest that down-regulation of neuronal L1 expression is an adaptive process of neuronal self-defense in response to pro-inflammatory T cells, thereby alleviating immune-mediated axonal injury.

Zinc transporter 3 (ZnT3) gene deletion reduces spinal cord white matter damage and motor deficits in a murine MOG-induced multiple sclerosis model.

The present study aimed to evaluate the role of zinc transporter 3 (ZnT3) on multiple sclerosis (MS) pathogenesis. Experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis, was induced by immunization with myelin oligodendrocyte glycoprotein (MOG35-55) in female mice. Three weeks after the initial immunization, demyelination, immune cell infiltration and blood brain barrier (BBB) disruption in the spinal cord were analyzed. Clinical signs of EAE first appeared on day 11 and reached a peak level on day 19 after the initial immunization. ZnT3 gene deletion profoundly reduced the daily clinical score of EAE. The ZnT3 gene deletion-mediated inhibition of the clinical course of EAE was accompanied by suppression of inflammation and demyelination in the spinal cord. The motor deficit accompanying neuropathological changes associated with EAE were mild in ZnT3 gene deletion mice. This reduction in motor deficit was accompanied by coincident reductions in demyelination and infiltration of encephalitogenic immune cells including CD4+ T cells, CD8+ T cells, CD20+ B cells and F4/80+ microglia in the spinal cord. These results demonstrate that ZnT3 gene deletion inhibits the clinical features and neuropathological changes associated with EAE. ZnT3 gene deletion also remarkably inhibited formation of EAE-associated aberrant synaptic zinc patches, matrix metalloproteinases-9 (MMP-9) activation and BBB disruption. Therefore, amelioration of EAE-induced clinical and neuropathological changes by ZnT3 gene deletion suggests that vesicular zinc may be involved in several steps of MS pathogenesis.

Pathways and gene networks mediating the regulatory effects of cannabidiol, a nonpsychoactive cannabinoid, in autoimmune T cells.

BACKGROUND: Our previous studies showed that the non-psychoactive cannabinoid, cannabidiol (CBD), ameliorates the clinical symptoms in mouse myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis model of multiple sclerosis (MS) as well as decreases the memory MOG35-55-specific T cell (TMOG) proliferation and cytokine secretion including IL-17, a key autoimmune factor. The mechanisms of these activities are currently poorly understood. METHODS: Herein, using microarray-based gene expression profiling, we describe gene networks and intracellular pathways involved in CBD-induced suppression of these activated memory TMOG cells. Encephalitogenic TMOG cells were stimulated with MOG35-55 in the presence of spleen-derived antigen presenting cells (APC) with or without CBD. mRNA of purified TMOG was then subjected to Illumina microarray analysis followed by ingenuity pathway analysis (IPA), weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) elucidation of gene interactions. Results were validated using qPCR and ELISA assays. RESULTS: Gene profiling showed that the CBD treatment suppresses the transcription of a large number of proinflammatory genes in activated TMOG. These include cytokines (Xcl1, Il3, Il12a, Il1b), cytokine receptors (Cxcr1, Ifngr1), transcription factors (Ier3, Atf3, Nr4a3, Crem), and TNF superfamily signaling molecules (Tnfsf11, Tnfsf14, Tnfrsf9, Tnfrsf18). "IL-17 differentiation" and "IL-6 and IL-10-signaling" were identified among the top processes affected by CBD. CBD increases a number of IFN-dependent transcripts (Rgs16, Mx2, Rsad2, Irf4, Ifit2, Ephx1, Ets2) known to execute anti-proliferative activities in T cells. Interestingly, certain MOG35-55 up-regulated transcripts were maintained at high levels in the presence of CBD, including transcription factors (Egr2, Egr1, Tbx21), cytokines (Csf2, Tnf, Ifng), and chemokines (Ccl3, Ccl4, Cxcl10) suggesting that CBD may promote exhaustion of memory TMOG cells. In addition, CBD enhanced the transcription of T cell co-inhibitory molecules (Btla, Lag3, Trat1, and CD69) known to interfere with T/APC interactions. Furthermore, CBD enhanced the transcription of oxidative stress modulators with potent anti-inflammatory activity that are controlled by Nfe2l2/Nrf2 (Mt1, Mt2a, Slc30a1, Hmox1). CONCLUSIONS: Microarray-based gene expression profiling demonstrated that CBD exerts its immunoregulatory effects in activated memory TMOG cells via (a) suppressing proinflammatory Th17-related transcription, (b) by promoting T cell exhaustion/tolerance, (c) enhancing IFN-dependent anti-proliferative program, (d) hampering antigen presentation, and (d) inducing antioxidant milieu resolving inflammation. These findings put forward mechanism by which CBD exerts its anti-inflammatory effects as well as explain the beneficial role of CBD in pathological memory T cells and in autoimmune diseases.