Peak Of Experimental Autoimmune Encephalomyelitis Research Articles

Microglia and Dendritic Cells as a Source of IL-6 in a Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a complex autoimmune disease of central nervous system (CNS) characterized by the myelin sheath destruction and compromised nerve signal transmission. Understanding molecular mechanisms driving MS development is critical due to its early onset, chronic course, and therapeutic approaches based only on symptomatic treatment. Cytokines are known to play a pivotal role in the MS pathogenesis with interleukin-6 (IL-6) being one of the key mediators. This study investigates contribution of IL-6 produced by microglia and dendritic cells to the development of experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS. Mice with conditional inactivation of IL-6 in the CX3CR1+ cells, including microglia, or CD11c+ dendritic cells, displayed less severe symptoms as compared to their wild-type counterparts. Mice with microglial IL-6 deletion exhibited an elevated proportion of regulatory T cells and reduced percentage of pathogenic IFNγ-producing CD4+ T cells, accompanied by the decrease in pro-inflammatory monocytes in the CNS at the peak of EAE. At the same time, deletion of IL-6 from microglia resulted in the increase of CCR6+ T cells and GM-CSF-producing T cells. Conversely, mice with IL-6 deficiency in the dendritic cells showed not only the previously described increase in the proportion of regulatory T cells and decrease in the proportion of TH17 cells, but also reduction in the production of GM-CSF and IFNγ in the secondary lymphoid organs. In summary, IL-6 functions during EAE depend on both the source and localization of immune response: the microglial IL-6 exerts both pathogenic and protective functions specifically in the CNS, whereas the dendritic cell-derived IL-6, in addition to being critically involved in the balance of regulatory T cells and TH17 cells, may stimulate production of cytokines associated with pathogenic functions of T cells.

Altered T cell development in an animal model of multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system (CNS), leading to demyelination and axonal degeneration. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that has significantly improved our understanding of MS. Studies have observed early thymic involution in MS patients, suggesting the potential involvement of the thymus in CNS autoimmunity. However, our knowledge of the thymus's role in autoimmune disorders affecting the CNS remains limited. In this study, we examined the effects of EAE induction on thymopoiesis and observed alterations in T cell development. These changes were characterized by increased apoptosis and decreased proliferation of thymocytes at the EAE peak stage. We also identified a blockade in the transition from CD4−CD8− double-negative thymocytes to CD4+CD8+ double-positive cells, as evidenced by the accumulation of double-negative stage 1 thymocytes at both the EAE onset and peak stages. Furthermore, positive selection was disrupted in the thymus of EAE mice at both stages, leading to an elevated proportion and number of CD4+CD8− and CD4−CD8+ single-positive cells. Meanwhile, we observed an augmented production of regulatory T cells in the thymus of EAE mice. Moreover, peripheral blood analysis of EAE mice at the onset stage showed expanded T cell subsets but not at the peak stage. We also observed altered expression patterns in thymus-derived CD4+CD8− and CD4−CD8+ single-positive cells between MS patients and healthy controls. Our findings demonstrate a modified T cell development in EAE/MS, providing valuable insights into the potential of modulating thymic function as a targeted therapeutic approach to MS/EAE.

Farnesol brain transcriptomics in CNS inflammatory demyelination

BackgroundFarnesol (FOL) prevents the onset of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). ObjectiveWe examined the transcriptomic profile of the brains of EAE mice treated with daily oral FOL using next-generation sequencing (RNA-seq). MethodsTranscriptomics from whole brains of treated and untreated EAE mice at the peak of EAE was performed. ResultsEAE-induced mice, compared to naïve, healthy mice, overall showed increased expression in pathways for immune response, as well as an increased cytokine signaling pathway, with downregulation of cellular stress proteins. FOL downregulates pro-inflammatory pathways and attenuates the immune response in EAE. FOL downregulated the expression of genes involved in misfolded protein response, MAPK activation/signaling, and pro-inflammatory response. ConclusionThis study provides insight into the molecular impact of FOL in the brain and identifies potential therapeutic targets of the isoprenoid pathway in MS patients.

Interferon-gamma ameliorates experimental autoimmune encephalomyelitis by inducing homeostatic adaptation of microglia.

Compelling evidence has shown that interferon (IFN)-γ has dual effects in multiple sclerosis and in its animal model of experimental autoimmune encephalomyelitis (EAE), with results supporting both a pathogenic and beneficial function. However, the mechanisms whereby IFN-γ may promote neuroprotection in EAE and its effects on central nervous system (CNS)-resident cells have remained an enigma for more than 30 years. In this study, the impact of IFN-γ at the peak of EAE, its effects on CNS infiltrating myeloid cells (MC) and microglia (MG), and the underlying cellular and molecular mechanisms were investigated. IFN-γ administration resulted in disease amelioration and attenuation of neuroinflammation associated with significantly lower frequencies of CNS CD11b+ myeloid cells and less infiltration of inflammatory cells and demyelination. A significant reduction in activated MG and enhanced resting MG was determined by flow cytometry and immunohistrochemistry. Primary MC/MG cultures obtained from the spinal cord of IFN-γ-treated EAE mice that were ex vivo re-stimulated with a low dose (1 ng/ml) of IFN-γ and neuroantigen, promoted a significantly higher induction of CD4+ regulatory T (Treg) cells associated with increased transforming growth factor (TGF)-β secretion. Additionally, IFN-γ-treated primary MC/MG cultures produced significantly lower nitrite in response to LPS challenge than control MC/MG. IFN-γ-treated EAE mice had a significantly higher frequency of CX3CR1high MC/MG and expressed lower levels of program death ligand 1 (PD-L1) than PBS-treated mice. Most CX3CR1highPD-L1lowCD11b+Ly6G- cells expressed MG markers (Tmem119, Sall2, and P2ry12), indicating that they represented an enriched MG subset (CX3CR1highPD-L1low MG). Amelioration of clinical symptoms and induction of CX3CR1highPD-L1low MG by IFN-γ were dependent on STAT-1. RNA-seq analyses revealed that in vivo treatment with IFN-γ promoted the induction of homeostatic CX3CR1highPD-L1low MG, upregulating the expression of genes associated with tolerogenic and anti-inflammatory roles and down-regulating pro-inflammatory genes. These analyses highlight the master role that IFN-γ plays in regulating microglial activity and provide new insights into the cellular and molecular mechanisms involved in the therapeutic activity of IFN-γ in EAE.

Acute intermittent hypoxia alters disease course and promotes CNS repair including resolution of inflammation and remyelination in the experimental autoimmune encephalomyelitis model of MS.

Remyelination and neurodegeneration prevention mitigate disability in Multiple Sclerosis (MS). We have shown acute intermittent hypoxia (AIH) is a novel, non-invasive and effective therapy for peripheral nerve repair, including remyelination. Thus, we posited AIH would improve repair following CNS demyelination and address the paucity of MS repair treatments. AIH's capacity to enhance intrinsic repair, functional recovery and alter disease course in the experimental autoimmune encephalomyelitis (EAE) model of MS was assessed. EAE was induced by MOG35-55 immunization in C57BL/6 female mice. EAE mice received either AIH (10 cycles-5 min 11% oxygen alternating with 5 min 21% oxygen) or Normoxia (control; 21% oxygen for same duration) once daily for 7d beginning at near peak EAE disease score of 2.5. Mice were followed post-treatment for an additional 7d before assessing histopathology or 14d to examine maintenance of AIH effects. Alterations in histopathological correlates of multiple repair indices were analyzed quantitatively in focally demyelinated ventral lumbar spinal cord areas to assess AIH impacts. AIH begun at near peak disease significantly improved daily clinical scores/functional recovery and associated histopathology relative to Normoxia controls and the former were maintained for at least 14d post-treatment. AIH enhanced correlates of myelination, axon protection and oligodendrocyte precursor cell recruitment to demyelinated areas. AIH also effected a dramatic reduction in inflammation, while polarizing remaining macrophages/microglia toward a pro-repair state. Collectively, this supports a role for AIH as a novel non-invasive therapy to enhance CNS repair and alter disease course following demyelination and holds promise as a neuroregenerative MS strategy.

An IL-23/STAT4 axis regulates classical dendritic cell expansion and function in CNS autoimmunity

Abstract Signal transducer and activator of transcription 4 (STAT4) is a critical regulator of inflammation in autoimmunity by acting as a messenger for pro-inflammatory cytokines including IL-12 and IL-23. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, germ-line deletion of STAT4 in mice results in resistance to the development of paralysis. Here, we examined STAT4 cell-specific requirements in EAE. Using conditional Stat4mutant mice, we found that mice lacking Stat4in CD4 +T cells and CD11c +cells are resistant to EAE, while Lyz2 +cells are susceptible to EAE demonstrating cell type requirement for STAT4 in EAE. STAT4 is expressed and activated in CD11c +classical dendritic cells (cDCs) in the CNS at the peak of EAE. Stat4fl/flCD11cCre(Stat4ΔCD11c) mice exhibit a significant decrease of cDC expansion in the CNS, and this correlates with diminished numbers of T cells in the CNS and decreased inflammatory cytokine production. Adoptive transfer of wild-type DCs into Stat4ΔCD11crescues the development of EAE. In contrast, transferring Il23r−/−DCs to Stat4ΔCD11cdid not rescue EAE development. Transferred Il23r−/−DCs were retained in the lymph nodes suggesting that IL-23-STAT4 signaling in cDCs promotes their expansion in the CNS during neuroinflammation. CITE-seq analysis of CNS DCs from control and Stat4ΔCD11cmice identified STAT4-dependent genes in cDCs in EAE. Collectively, our results demonstrate that STAT4 in cDCs is required for their expansion in the CNS, the development of pro-inflammatory T cells, and the clinical symptoms of EAE. Thus, our study reveals previously unrecognized functions of STAT4 in cDCs that could identify novel therapeutic approaches for CNS autoimmunity.

Aldh1l1-cre/ERT2 promoter drives flox-mediated recombination in peripheral immune cells in addition to astrocytes (P2-3.007)

<h3>Objective:</h3> To characterize the cell specificity of <i>Aldh1l1-cre/ERT2-</i>driven recombination in the spleen and spinal cord of mice during peak experimental autoimmune encephalomyelitis (EAE). <h3>Background:</h3> Transgenic mouse models are deployed to study astrocyte-specific alterations in gene expression in <i>in vivo</i> models of neuroinflammation, stroke and neurodegenerative disease. The widely used <i>Aldh1L1-cre/ERT2</i> promoter demonstrates a high level of astrocyte specificity within the CNS though specificity outside the CNS has not yet been well characterized. <h3>Design/Methods:</h3> We compared the astrocyte specificity of two promoter lines, <i>Tg(Aldh1l1-cre/ERT2)1Khakh</i> and <i>Tg(Gfap-cre)73.12Mvs</i>, by crossing them to the <i>Gt(ROSA)26Sor</i> line, which contains a tdTomato sequence preceded by a <i>loxP</i>-flanked STOP cassette. Heterozygous mice (<i>Aldh1l1-cre/ERT2:ROSA</i> and <i>Gfap-cre</i>:<i>ROSA</i>) express promoter-driven tdTomato. Aldh1l1-cre/ERT2 was activated 2 weeks prior to EAE induction using 5 daily intraperitoneal (ip) injections of 100mg/kg tamoxifen in corn oil (20mg/ml). GFAP-cre is constitutive and does not require activation. EAE was induced at 8–10 postnatal weeks (<i>Aldh1l1-cre/ERT2:ROSA</i> and <i>Gfap-cre</i>:<i>ROSA</i>, n=3 each) with one dorsal lumbar subcutaneous injection of MOG_35–55 + Complete Freund’s Adjuvant (CFA) and two ip injections of 60ng pertussis (PTX) toxin at 2–5 and 20–25 hours. Spleen and spinal cords were processed for flow cytometry at 5 days from disease onset with spleens from healthy <i>Aldh1l1-cre/ERT2:ROSA</i> mice (n=3) as a control. Cells were stained for surface markers for neutrophils, macrophages, B cells and T cells and flow cytometry was measured on a Cytek Aurora Flow Cytometer and analyzed with FCS Express software (De Novo). <h3>Results:</h3> Multiple subpopulations of peripheral immune cells, including neutrophils, CD19⁺ B cells, CD4⁺ and CD8⁺ T cells, expressed <i>Aldh1l1-cre/ERT2</i> driven tdTomato. TdTomato positive immune cell populations were not present in <i>mGFAP-Cre tdTomato</i> mice. <h3>Conclusions:</h3> Aldh1l1-cre/ERT2 induces recombination in peripheral immune cells in addition to astrocytes. Gfap-cre does not and may therefore represent a more specific tool to target astrocytes in neuroinflammatory disease. <b>Disclosure:</b> Mario Amatruda has received personal compensation in the range of $50,000-$99,999 for serving as a Postdoctoral Fellow with The Moun Sinai. Mr. Villavicencio has nothing to disclose. The institution of Dr. Britton has received research support from Crohn’s and Colitis Foundation of America. Dr. Britton has received intellectual property interests from a discovery or technology relating to health care. The institution of Dr. Horng has received research support from National Institutes of Health . The institution of Dr. Horng has received research support from Jayne and Harvey Beker Foundation .

Rat Ovarian Function Is Impaired during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease affecting the CNS and occurring far more prevalently in women than in men. In both MS and its animal models, sex hormones play important immunomodulatory roles. We have previously shown that experimental autoimmune encephalomyelitis (EAE) affects the hypothalamic-pituitary-gonadal axis in rats of both sexes and induces an arrest in the estrous cycle in females. To investigate the gonadal status in female rats with EAE, we explored ovarian morphometric parameters, circulating and intraovarian sex steroid levels, and the expression of steroidogenic machinery components in the ovarian tissue. A prolonged state of diestrus was recorded during the peak of EAE, with maintenance of the corpora lutea, elevated intraovarian progesterone levels, and increased gene and protein expression of StAR, similar to the state of pseudopregnancy. The decrease in CYP17A1 protein expression was followed by a decrease in ovarian testosterone and estradiol levels. On the contrary, serum testosterone levels were slightly increased. With unchanged serum estradiol levels, these results point at extra-gonadal sites of sex steroid biosynthesis and catabolism as important regulators of their circulating levels. Our study suggests alterations in the function of the female reproductive system during central autoimmunity and highlights the bidirectional relationships between hormonal status and EAE.

Cannabidiol Attenuates In Vivo Leukocyte Recruitment to the Spinal Cord Microvasculature at Peak Disease of Experimental Autoimmune Encephalomyelitis.

Introduction: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by neuroinflammation leading to demyelination. The associated symptoms lead to a devastating decrease in quality of life. The cannabinoids and their derivatives have emerged as an encouraging alternative due to their management of symptom in MS. Objective: The aim of the study was to investigate the mechanism of action of cannabidiol (CBD), a nonpsychoactive cannabinoid, on molecular and cellular events associated with leukocyte recruitment induced by experimental autoimmune encephalomyelitis (EAE). Materials and Methods: C57BL/6 female mice were randomly assigned to the four experimental groups: C (control group), CBD (cannabidiol-treated group, 5 mg/kg i.p.; 14 days), EAE (experimental autoimmune encephalomyelitis-induced group), and EAE+CBD (experimental autoimmune encephalomyelitis-induced plus cannabidiol-treated group). Results: The results indicated that 5 mg/kg of CBD injected intraperitoneally between the 1st and 14th days of EAE could reduce the leukocyte rolling and adhesion into the spinal cord microvasculature as well cellular tissue infiltration. These results were supported by a decreased mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the spinal cord. Conclusion: Purified CBD reduces in vivo VCAM and ICAM-mediated leukocyte recruitment to the spinal cord microvasculature at EAE peak disease.

Brain inflammation induces alterations in glycosaminoglycan metabolism and subsequent changes in CS-4S and hyaluronic acid

It remains uncertain how brain glycosaminoglycans (GAGs) contribute to the progression of inflammatory disorders like multiple sclerosis (MS). We investigated here neuroinflammation-mediated changes in GAG composition and metabolism using the mouse model of experimental autoimmune encephalomyelitis (EAE) and sham-immunized mice as controls. Cerebellum, mid- and forebrain at different EAE phases were investigated using gene expression analysis (microarray and RT-qPCR) as well as HPLC quantification of CS and hyaluronic acid (HA). The cerebellum was the most affected brain region showing a downregulation of Bcan, Cspg5, and an upregulation of Dse, Gusb, Hexb, Dcn and Has2 at peak EAE. Upregulation of genes involved in GAG degradation as well as synthesis of HA and decorin persisted from onset to peak, and diminished at remission, suggesting a severity-related decrease in CS and increments in HA. Relative disaccharide quantification confirmed a 3.6 % reduction of CS-4S at peak and a normalization during remission, while HA increased in both phases by 26.1 % and 17.6 %, respectively. Early inflammatory processes led to altered GAG metabolism in early EAE stages and subsequent partially reversible changes in CS-4S and in HA. Targeting early modifications in CS could potentially mitigate progression of EAE/MS.

High K+ intake alleviates experimental autoimmune encephalomyelitis (EAE) and increases T regulatory cells

Multiple sclerosis is believed to be triggered by the interplay between the environmental and genetic factors. In contrast to the Paleolithic diet, the modern Western diet is high in Na+ and low in K+. The present study was undertaken to determine whether high K+ intake alleviated experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Treatment of C57BL/6 or SJL mice for 7 days with a 5 % K+ diet prior to induction of EAE and maintaining mice on the diet until the end of experiments delayed the onset, reduced the peak, and accelerated the recovery of EAE in both strains compared with mice on a control diet (0.7 % K+), whereas feeding C57BL/6 mice with a 0.1 % K+ diet did the opposite. High K+ intake increased the splenic Treg cell frequency in the pretreatment and peak EAE. Thus, high K+ intake attenuates EAE, possibly by increasing the Treg cells.

Ferroptosis as a mechanism of oligodendrocyte loss and demyelination in experimental autoimmune encephalomyelitis

Ferroptosis, distinct from necrosis, autophagy and apoptosis, is a unique form of regulated cell death，and is a potential pathogenic mechanism of neuronal loss and defunction in many neurodegenerative disorders. Recent studies have shown a presence of iron deposition in the central nervous system (CNS) of patients with multiple sclerosis (MS). However, whether ferroptosis is involved in the pathogenesis of MS remains unclear. In the present study, we tested certain classical biomarkers of ferroptosis in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, to substantiate the relationship between ferroptosis and oligodendrocyte (OL) loss and demyelination. Our results revealed decreased levels of key molecules in glutathione antioxidant mechanisms, including system xC (xCT) and glutathione peroxidase 4 (GPX4) in spinal cord of EAE mice, with evident lipid peroxidation in OLs. Moreover, transferrin receptor and ferritinophagy further catalyzed the generation of lipid reactive oxygen species through the fenton reaction, which induced OL death and demyelination at disease peak of EAE. This phenomenon was largely reversed by administering Fer-1, an inhibitor of ferritin phagocytosis, further validating the key role of ferritin phagocytosis in EAE. Taken together, these findings demonstrate that OL loss and demyelination may be induced in EAE through, at least in part, a mechanism of ferroptosis.

Anlotinib attenuates experimental autoimmune encephalomyelitis mice model of multiple sclerosis via modulating the differentiation of Th17 and Treg cells

Background In multiple sclerosis (MS), the imbalance between T helper (Th)-17 cells and regulatory T (Treg) cells are critical in autoimmune central nervous system (CNS) inflammation and demyelination. Experimental autoimmune encephalomyelitis (EAE) is an established mouse MS model and simulates MS at diverse levels. Objectives This study aims at investigating the impact of anlotinib on the clinical severity of EAE and CD4+ T cell differentiation. Materials and methods EAE-induced mice were treated with water (control) or 6 mg/kg anlotinib by gavage daily. At the peak of EAE, histopathological examination and flow cytometry analysis of CNS-infiltrating CD4+ T cells were performed. In vitro differentiation of CD4+ T cells under different conditions was detected by flow cytometry and quantitative real-time PCR. Finally, the impacts of anlotinib on the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the transcription levels of key genes involved in Th17 and Treg differentiation were tested. Results Anlotinib attenuated the clinical severity of EAE and changed the frequencies of CNS-infiltrating CD4+ T cell subsets. Anlotinib inhibited the differentiation of Th17 cells in vitro, decreased the phosphorylation of STAT3, and reduced the expression of Rorc. Anlotinib promoted the differentiation of Treg cells and upregulated the expression levels of CD39 and CD73. Discussion and conclusions Anlotinib alleviated the symptoms of EAE via inhibiting the Th17 cell differentiation and promoting Treg cell differentiation. Our study provides new opportunities for the exploitation of anlotinib as a therapeutic agent for the treatment of MS.

Versican promotes T helper 17 cytotoxic inflammation and impedes oligodendrocyte precursor cell remyelination

Remyelination failure in multiple sclerosis (MS) contributes to progression of disability. The deficient repair results from neuroinflammation and deposition of inhibitors including chondroitin sulfate proteoglycans (CSPGs). Which CSPG member is repair-inhibitory or alters local inflammation to exacerbate injury is unknown. Here, we correlate high versican-V1 expression in MS lesions with deficient premyelinating oligodendrocytes, and highlight its selective upregulation amongst CSPG members in experimental autoimmune encephalomyelitis (EAE) lesions modeling MS. In culture, purified versican-V1 inhibits oligodendrocyte precursor cells (OPCs) and promotes T helper 17 (Th17) polarization. Versican-V1-exposed Th17 cells are particularly toxic to OPCs. In NG2CreER:MAPTmGFP mice illuminating newly formed GFP+ oligodendrocytes/myelin, difluorosamine (peracetylated,4,4-difluoro-N-acetylglucosamine) treatment from peak EAE reduces lesional versican-V1 and Th17 frequency, while enhancing GFP+ profiles. We suggest that lesion-elevated versican-V1 directly impedes OPCs while it indirectly inhibits remyelination through elevating local Th17 cytotoxic neuroinflammation. We propose CSPG-lowering drugs as potential dual pronged repair and immunomodulatory therapeutics for MS.

CD4+c-Met+Itg\u03b14+ T cell subset promotes murine neuroinflammation

Objectivec-Met, a tyrosine kinase receptor, is the unique receptor for hepatocyte growth factor (HGF). The HGF/c-Met axis is reported to modulate cell migration, maturation, cytokine production, and antigen presentation. Here, we report that CD4+c-Met+ T cells are detected at increased levels in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS).Methodsc-Met expression by CD4+ T cells was analyzed mostly by flow cytometry and by immunohistochemistry from mice and human PBMCs. The in vivo role of CD4+c-Met+ T cells was assessed in EAE.ResultsCD4+c-Met+ T cells found in the CNS during EAE peak disease are characterized by a pro-inflammatory phenotype skewed towards a Th1 and Th17 polarization, with enhanced adhesion and transmigration capacities correlating with increased expression of integrin α4 (Itgα4). The adoptive transfer of Itgα4-expressing CD4+Vα3.2+c-Met+ T cells induces increased disease severity compared to CD4+Vα3.2+c-Met− T cells. Finally, CD4+c-Met+ T cells are detected in the brain of MS patients, as well as in the blood with a higher level of Itgα4. These results highlight c-Met as an immune marker of highly pathogenic pro-inflammatory and pro-migratory CD4+ T lymphocytes associated with neuroinflammation.

Different Impact of Gadopentetate and Gadobutrol on Inflammation-Promoted Retention and Toxicity of Gadolinium Within the Mouse Brain.

ObjectivesUsing a murine model of multiple sclerosis, we previously showed that repeated administration of gadopentetate dimeglumine led to retention of gadolinium (Gd) within cerebellar structures and that this process was enhanced with inflammation. This study aimed to compare the kinetics and retention profiles of Gd in inflamed and healthy brains after application of the macrocyclic Gd-based contrast agent (GBCA) gadobutrol or the linear GBCA gadopentetate. Moreover, potential Gd-induced neurotoxicity was investigated in living hippocampal slices ex vivo.Materials and MethodsMice at peak of experimental autoimmune encephalomyelitis (EAE; n = 29) and healthy control mice (HC; n = 24) were exposed to a cumulative dose of 20 mmol/kg bodyweight of either gadopentetate dimeglumine or gadobutrol (8 injections of 2.5 mmol/kg over 10 days). Magnetic resonance imaging (7 T) was performed at baseline as well as at day 1, 10, and 40 post final injection (pfi) of GBCAs. Mice were sacrificed after magnetic resonance imaging and brain and blood Gd content was assessed by laser ablation-inductively coupled plasma (ICP)-mass spectrometry (MS) and ICP-MS, respectively. In addition, using chronic organotypic hippocampal slice cultures, Gd-induced neurotoxicity was addressed in living brain tissue ex vivo, both under control or inflammatory (tumor necrosis factor α [TNF-α] at 50 ng/μL) conditions.ResultsNeuroinflammation promoted a significant decrease in T1 relaxation times after multiple injections of both GBCAs as shown by quantitative T1 mapping of EAE brains compared with HC. This corresponded to higher Gd retention within the EAE brains at 1, 10, and 40 days pfi as determined by laser ablation-ICP-MS. In inflamed cerebellum, in particular in the deep cerebellar nuclei (CN), elevated Gd retention was observed until day 40 after last gadopentetate application (CN: EAE vs HC, 55.06 ± 0.16 μM vs 30.44 ± 4.43 μM). In contrast, gadobutrol application led to a rather diffuse Gd content in the inflamed brains, which strongly diminished until day 40 (CN: EAE vs HC, 0.38 ± 0.08 μM vs 0.17 ± 0.03 μM). The analysis of cytotoxic effects of both GBCAs using living brain tissue revealed an elevated cell death rate after incubation with gadopentetate but not gadobutrol at 50 mM. The cytotoxic effect due to gadopentetate increased in the presence of the inflammatory mediator TNF-α (with vs without TNF-α, 3.15% ± 1.18% vs 2.17% ± 1.14%; P = 0.0345).ConclusionsIn the EAE model, neuroinflammation promoted increased Gd retention in the brain for both GBCAs. Whereas in the inflamed brains, efficient clearance of macrocyclic gadobutrol during the investigated time period was observed, the Gd retention after application of linear gadopentetate persisted over the entire observational period. Gadopentetate but not gadubutrol appeared to be neurotoxic in an ex vivo paradigm of neuronal inflammation.

NFAT5 contributes to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and decrease of T regulatory cells in female mice

Multiple sclerosis disproportionally affects women. The present study was undertaken to determine whether NFAT5 contributed to the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and if it did, whether the impact was sex associated. NFAT5 haplodeficiency reduced the disease severity only in female mice. This effect was associated with significant increases in frequency of T regulatory (Treg) cells in the CNS (from 1.45 ± 0.39% to 3.73 ± 0.94%) and spleen from (0.31 ± 0.06% to 0.94 ± 0.29%) without significantly affecting the CNS CD4+ subsets frequency. NFAT5 haploinsufficiency also significantly reduced the frequency of CD11c+CD8α+ dendritic cells in the female CNS. However, increase of their frequency in the CNS via intraperitoneal Flt3L injection at peak EAE had no significant effect on the disease courses. We conclude that NFAT5 contributes to pathogenesis of EAE in female mice, possibly through decreasing tissue specific frequency of Treg cells.

Sexual Dimorphism in Extracellular Matrix Composition and Viscoelasticity of the Healthy and Inflamed Mouse Brain.

Simple SummaryIn multiple sclerosis (MS), an autoimmune disease of the central nervous system that primarily affects women, gender differences in disease course and in brain softening have been reported. It has been shown that the molecular network found between the cells of the tissue, the extracellular matrix (ECM), influences tissue stiffness. However, it is still unclear if sex influences ECM composition. Therefore, here we investigated how brain ECM and stiffness differ between sexes in the healthy mouse, and in an MS mouse model. We applied multifrequency magnetic resonance elastography and gene expression analysis for associating in vivo brain stiffness with ECM protein content in the brain, such as collagen and laminin. We found that the cortex was softer in males than in females in both healthy and sick mice. Softening was associated with sex differences in expression levels of collagen and laminin. Our findings underscore the importance of considering sex when studying the constitution of brain tissue in health and disease, particularly when investigating the processes underlying gender differences in MS. Magnetic resonance elastography (MRE) has revealed sexual dimorphism in brain stiffness in healthy individuals and multiple sclerosis (MS) patients. In an animal model of MS, named experimental autoimmune encephalomyelitis (EAE), we have previously shown that inflammation-induced brain softening was associated with alterations of the extracellular matrix (ECM). However, it remained unclear whether the brain ECM presents sex-specific properties that can be visualized by MRE. Therefore, here we aimed at quantifying sexual dimorphism in brain viscoelasticity in association with ECM changes in healthy and inflamed brains. Multifrequency MRE was applied to the midbrain of healthy and EAE mice of both sexes to quantitatively map regional stiffness. To define differences in brain ECM composition, the gene expression of the key basement membrane components laminin (Lama4, Lama5), collagen (Col4a1, Col1a1), and fibronectin (Fn1) were investigated by RT-qPCR. We showed that the healthy male cortex expressed less Lama4, Lama5, and Col4a1, but more Fn1 (all p < 0.05) than the healthy female cortex, which was associated with 9% softer properties (p = 0.044) in that region. At peak EAE cortical softening was similar in both sexes compared to healthy tissue, with an 8% difference remaining between males and females (p = 0.006). Cortical Lama4, Lama5 and Col4a1 expression increased 2 to 3-fold in EAE in both sexes while Fn1 decreased only in males (all p < 0.05). No significant sex differences in stiffness were detected in other brain regions. In conclusion, sexual dimorphism in the ECM composition of cortical tissue in the mouse brain is reflected by in vivo stiffness measured with MRE and should be considered in future studies by sex-specific reference values.

NFIL3 deficiency alleviates EAE through regulating different immune cell subsets

IntroductionThe transcription factor NFIL3 exerts comprehensive effects on the immune system. Previous studies revealed that NFIL3 is related to the function and development of different immune cell subsets. Experimental autoimmune encephalomyelitis (EAE) is mediated by immune cells which results in inflammatory demyelination in the central nervous system (CNS). However, how NFIL3 affects EAE has not been thoroughly studied. ObjectivesThe current study aimed to investigate how NFIL3 affects EAE, especially the changes of T cells and dendritic cells as well as the crosstalk between them. MethodsWe used NFIL3-/- mice and C57BL/6J mice (wildtype) to establish MOG35-55-induced EAE. The clinical scores were recorded daily. The immune cells within and outside the CNS of EAE mice were analyzed by flow cytometry. Histology was used to evaluated the neuroinflammation and demyelination in the CNS. Besides, CD11c+ dendritic cells (DCs) were cocultured with T cells and the interplay was measured. ResultsAt the peak of EAE, Th17 cells decreased within the CNS accompanying with lower clinical scores and milder neuroinflammation and demyelination in NFIL3 knockout EAE mice. Outside the CNS, PD-1 and ICOS on CD4+T cells increased, whereas Th2, Th9, CD8+CD103+T cells and GM-CSF+CD4+T cells decreased. Besides, the pro-inflammatory capacity of NFIL3-/- CD11c+ dendritic cells was impaired while the anti-inflammatory capacity was promoted. ConclusionsThis study suggests that NFIL3 deficiency could alleviate MOG35-55-induced EAE through regulating different immune cell subsets, which is not only related with adaptive immunity and innate immunity, but also related with the cross-talk between them, especially CD4+ T cells and CD11c+ dendritic cells.

Expression of IL-20 Receptor Subunit β Is Linked to EAE Neuropathology and CNS Neuroinflammation.

Considerable clinical evidence supports that increased blood–brain barrier (BBB) permeability is linked to immune extravasation of CNS parenchyma during neuroinflammation. Although BBB permeability and immune extravasation are known to be provoked by vascular endothelial growth factor-A (i.e., VEGF-A) and C-X-C motif chemokine ligand 12 (CXCL12), respectively, the mechanisms that link both processes are still elusive. The interleukin-20 (i.e., IL-20) cytokine signaling pathway was previously implicated in VEGF-mediated angiogenesis and is known to induce cellular response by way of signaling through IL-20 receptor subunit β (i.e., IL-20RB). Dysregulated IL-20 signaling is implicated in many inflammatory pathologies, but it’s contribution to neuroinflammation has yet to be reported. We hypothesize that the IL-20 cytokine, and the IL cytokine subfamily more broadly, play a key role in CNS neuroinflammation by signaling through IL-20RB, induce VEGF activity, and enhance both BBB-permeability and CXCL12-mediated immune extravasation. To address this hypothesis, we actively immunized IL-20RB–/– mice and wild-type mice to induce experimental autoimmune encephalomyelitis (EAE) and found that IL-20RB–/– mice showed amelioration of disease progression compared to wild-type mice. Similarly, we passively immunized IL-20RB–/– mice and wild-type mice with myelin-reactive Th1 cells from either IL-20RB–/– and wild-type genotype. Host IL-20RB–/– mice showed lesser disease progression than wild-type mice, regardless of the myelin-reactive Th1 cells genotype. Using multianalyte bead-based immunoassay and ELISA, we found distinctive changes in levels of pro-inflammatory cytokines between IL-20RB–/– mice and wild-type mice at peak of EAE. We also found detectable levels of all cytokines of the IL-20 subfamily within CNS tissues and specific alteration to IL-20 subfamily cytokines IL-19, IL-20, and IL-24, expression levels. Immunolabeling of CNS region-specific microvessels confirmed IL-20RB protein at the spinal cord microvasculature and upregulation during EAE. Microvessels isolated from macaques CNS tissues also expressed IL-20RB. Moreover, we identified the expression of all IL-20 receptor subunits: IL-22 receptor subunit α-1 (IL-22RA1), IL-20RB, and IL-20 receptor subunit α (IL-20RA) in human CNS microvessels. Notably, human cerebral microvasculature endothelial cells (HCMEC/D3) treated with IL-1β showed augmented expression of the IL-20 receptor. Lastly, IL-20-treated HCMEC/D3 showed alterations on CXCL12 apicobasal polarity consistent with a neuroinflammatory status. This evidence suggests that IL-20 subfamily cytokines may signal at the BBB via IL-20RB, triggering neuroinflammation.