Interleukin-9 protects from microglia- and TNF-mediated synaptotoxicity in experimental multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a progressive neurodegenerative disease of the central nervous system characterized by inflammation-driven synaptic abnormalities. Interleukin-9 (IL-9) is emerging as a pleiotropic cytokine involved in MS pathophysiology. METHODS: Through biochemical, immunohistochemical, and electrophysiological experiments, we investigated the effects of both peripheral and central administration of IL-9 on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. RESULTS: We demonstrated that both systemic and local administration of IL-9 significantly improved clinical disability, reduced neuroinflammation, and mitigated synaptic damage in EAE. The results unveil an unrecognized central effect of IL-9 against microglia- and TNF-mediated neuronal excitotoxicity. Two main mechanisms emerged: first, IL-9 modulated microglial inflammatory activity by enhancing the expression of the triggering receptor expressed on myeloid cells-2 (TREM2) and reducing TNF release. Second, IL-9 suppressed neuronal TNF signaling, thereby blocking its synaptotoxic effects. CONCLUSIONS: The data presented in this work highlight IL-9 as a critical neuroprotective molecule capable of interfering with inflammatory synaptopathy in EAE. These findings open new avenues for treatments targeting the neurodegenerative damage associated with MS, as well as other inflammatory and neurodegenerative disorders of the central nervous system.

CD22 blockade aggravates EAE and its role in microglia polarization.

AIMS: Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease. Microglia are reportedly involved in the pathogenesis of MS. However, the key molecules that control the inflammatory activity of microglia in MS have not been identified. METHODS: Experimental autoimmune encephalomyelitis (EAE) mice were randomized into CD22 blockade and control groups. The expression levels of microglial CD22 were measured by flow cytometry, qRT-PCR, and immunofluorescence. The effects of CD22 blockade were examined via in vitro and in vivo studies. RESULTS: We detected increased expression of microglial CD22 in EAE mice. In addition, an in vitro study revealed that lipopolysaccharide upregulated the expression of CD22 in microglia and that CD22 blockade modulated microglial polarization. Moreover, an in vivo study demonstrated that CD22 blockade aggravated EAE in mice and promoted microglial M1 polarization. CONCLUSION: Collectively, our study indicates that CD22 may be protective against EAE and may play a critical role in the maintenance of immune homeostasis in EAE mice.

CSF1R antagonism results in increased supraspinal infiltration in EAE.

BACKGROUND: Colony stimulating factor 1 receptor (CSF1R) signaling is crucial for the maintenance and function of various myeloid subsets. CSF1R antagonism was previously shown to mitigate clinical severity in experimental autoimmune encephalomyelitis (EAE). The associated mechanisms are still not well delineated. METHODS: To assess the effect of CSF1R signaling, we employed the CSF1R antagonist PLX5622 formulated in chow (PLX5622 diet, PD) and its control chow (control diet, CD). We examined the effect of PD in steady state and EAE by analyzing cells isolated from peripheral immune organs and from the CNS via flow cytometry. We determined CNS infiltration sites and assessed the extent of demyelination using immunohistochemistry of cerebella and spinal cords. Transcripts of genes associated with neuroinflammation were also analyzed in these tissues. RESULTS: In addition to microglial depletion, PD treatment reduced dendritic cells and macrophages in peripheral immune organs, both during steady state and during EAE. Furthermore, CSF1R antagonism modulated numbers and relative frequencies of T effector cells both in the periphery and in the CNS during the early stages of the disease. Classical neurological symptoms were milder in PD compared to CD mice. Interestingly, a subset of PD mice developed atypical EAE symptoms. Unlike previous studies, we observed that the CNS of PD mice was infiltrated by increased numbers of peripheral immune cells compared to that of CD mice. Immunohistochemical analysis showed that CNS infiltrates in PD mice were mainly localized in the cerebellum while in CD mice infiltrates were primarily localized in the spinal cords during the onset of neurological deficits. Accordingly, during the same timepoint, cerebella of PD but not of CD mice had extensive demyelinating lesions, while spinal cords of CD but not of PD mice were heavily demyelinated. CONCLUSIONS: Our findings suggest that CSF1R activity modulates the cellular composition of immune cells both in the periphery and within the CNS, and affects lesion localization during the early EAE stages.

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund's adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into naïve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS). Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides feedback on the extent of overall CNS injury in live mice.

Administration methods and dosage of poly(lactic acid)-glycol intervention to myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalitis mice.

BACKGROUND: Myelin oligodendrocyte glycoprotein-associated disorders (MOGAD) is an autoimmune central nervous system disease. Antigen-specific immune tolerance using nanoparticles such as Polylactic-co-glycolic acid (PLGA) have recently been used as a new therapeutic tolerization approach for CNS autoimmune diseases. We examined whether MOG1-125 conjugated with PLGA could induce MOG-specific immune tolerance in an experimental autoimmune encephalitis (EAE) mouse model. EAE was induced in sixty C57BL/6 J wild-type mice using MOG1-125 peptide with complete Freund's Adjuvant. The mice were divided into 12 groups (n = 5 each) to test the ability of MOG1-125 conjugated PLGA intervention to mitigate the severity or improve the outcomes from EAE with and without rapamycin compared to antigen alone or PLGA alone. EAE score and serum MOG-IgG titers were compared among the interventions.Kindly check and confirm the processed Affiliation “4” is appropriate.I confirmed the Aff 4.Affiliation: Corresponding author information have been changed to present affiliation. Kindly check and confirm.I checked and confirmed the Corresponding author's information. RESULTS: Mice with EAE that were injected intraperitoneally with MOG1-125 conjugated PLGA + rapamycin complex showed dose-dependent mitigation of EAE score. Intraperitoneal and intravenous administration resulted in similar clinical outcomes, whereas 80% of mice treated with subcutaneous injection had a recurrence of clinical score worsening after approximately 1 week. Although there was no significant difference in EAE scores between unconjugated-PLGA and MOG-conjugated PLGA, serum MOG-IgG tended to decrease in the MOG-conjugated PLGA group compared to controls. CONCLUSION: Intraperitoneal administration of PLGA resulted in dose-dependent and longer-lasting immune tolerance than subcutaneous administration. The induction of immune tolerance using PLGA may represent a future therapeutic option for patients with MOGAD.

Increased regulatory activity of intestinal innate lymphoid cells type 3 (ILC3) prevents experimental autoimmune encephalomyelitis severity.

Experimental autoimmune encephalomyelitis (EAE) induced in inbred rodents, i.e., genetically identical animals kept under identical environmental conditions, shows variable clinical outcomes. We investigated such variations of EAE in Dark Agouti rats immunized with spinal cord homogenate and identified four groups: lethal, severe, moderate, and mild, at day 28 post immunization. Higher numbers of CD4+ T cells, helper T cells type 1 (Th1) and 17 (Th17) in particular, were detected in the spinal cord of the severe group in comparison with the moderate group. In addition, increased proportion of Th1 and Th17 cells, and heightened levels of interferon (IFN)-γ and interleukin (IL)-6 were detected in the small intestine lamina propria of the severe group. A selective agonist of free fatty acid receptor type 2 (Ffar2) applied orally in the inductive phase of EAE shifted the distribution of the disease outcomes towards milder forms. This effect was paralleled with potentiation of intestinal innate lymphoid cells type 3 (ILC3) regulatory properties, and diminished Th1 and Th17 cell response in the lymph nodes draining the site of immunization. Our results suggest that different clinical outcomes in DA rats are under determinative influence of intestinal ILC3 activity during the inductive phase of 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.

A Novel Sensory Wave (P25) in Myelin Oligodendrocyte Glycoprotein-induced Experimental Autoimmune Encephalomyelitis Murine Model.

Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) is a murine model for multiple sclerosis. This model is characterized by chronic and progressive demyelination, leading to impairment of motor function and paralysis. While the outcomes of the disease, including impaired motor function and immunological changes, are well-characterized, little is known about the impact of EAE on the electrophysiology of the motor and sensory systems. In this study, we assessed evoked potentials as a quantitative marker for in vivo monitoring of nervous system damage. Motor-evoked potentials (MEPs) and sensory-evoked potentials (SEPs) were first standardized in naïve C57BL mice and studied thoroughly in EAE mice. The duration of MEPs and the number of connotative potentials increased significantly alongside an increase in temporal SEP amplitudes. Moreover, a new SEP wave was identified in naïve animals, which significantly increased in MOG-induced EAE animals with no or mild symptoms (clinical score 0-2, 0-5 scale). This wave occurred ∼25 milliseconds poststimulation, thus named p25. P25 was correlated with increased vocalization and was also reduced in amplitude following treatment with morphine. As the EAE score progressed (clinical score 3-4, 0-5 scale), the amplitude of MEPs and SEPs decreased drastically. Our results demonstrate that desynchronized neural motor activity, along with hypersensitivity in the early stages of EAE, leads to a complete loss of motor and sensory functions in the late stages of the disease. The findings also suggest an increase in p25 amplitude before motor deficits appear, indicating SEP as a predictive marker for disease progression. PERSPECTIVE: This article assesses p25, a new sensory electrophysiology wave that correlates with pain-related behavior in MOG-induced EAE mice and appears prior to the clinical symptoms. Motor electrophysiology correlates with traditional motor behavior scoring and histology.

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

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

Carnosine synthase deficiency aggravates neuroinflammation in multiple sclerosis.

Multiple sclerosis (MS) pathology features autoimmune-driven neuroinflammation, demyelination, and failed remyelination. Carnosine is a histidine-containing dipeptide (HCD) with pluripotent homeostatic properties that is able to improve outcomes in an animal MS model (EAE) when supplied exogenously. To uncover if endogenous carnosine is involved in, and protects against, MS-related neuroinflammation, demyelination or remyelination failure, we here studied the HCD-synthesizing enzyme carnosine synthase (CARNS1) in human MS lesions and two preclinical mouse MS models (EAE, cuprizone). We demonstrate that due to its presence in oligodendrocytes, CARNS1 expression is diminished in demyelinated MS lesions and mouse models mimicking demyelination/inflammation, but returns upon remyelination. Carns1-KO mice that are devoid of endogenous HCDs display exaggerated neuroinflammation and clinical symptoms during EAE, which could be partially rescued by exogenous carnosine treatment. Worsening of the disease appears to be driven by a central, not peripheral immune-modulatory, mechanism possibly linked to impaired clearance of the reactive carbonyl acrolein in Carns1-KO mice. In contrast, CARNS1 is not required for normal oligodendrocyte precursor cell differentiation and (re)myelin to occur, and neither endogenous nor exogenous HCDs protect against cuprizone-induced demyelination. In conclusion, the loss of CARNS1 from demyelinated MS lesions can aggravate disease progression through weakening the endogenous protection against neuroinflammation.

Cannabis and Cannabinoids in Multiple Sclerosis: From Experimental Models to Clinical Practice-A Review.

BACKGROUND: As far as 80% of people diagnosed with multiple sclerosis (MS) experience disabling symptoms in the course of the disease, such as spasticity and neuropathic pain. As first-line symptomatic therapy is associated with important adverse reactions, cannabinoids have become increasingly popular among patients with MS. This review intends to provide an overview of the evidence of the role of cannabinoids in treating symptoms related to MS and to encourage further research on this matter. AREAS OF UNCERTAINTY: To date, the evidence supporting the role of cannabis and its derivatives in alleviating the MS-related symptoms comes only from studies on experimental models of demyelination. To the best of our knowledge, relatively few clinical trials inquired about the therapeutic effects of cannabinoids on patients with MS, with variable results. DATA SOURCES: We conducted a literature search through PubMed and Google Scholar from the beginning until 2022. We included articles in English describing the latest findings regarding the endocannabinoid system, the pharmacology of cannabinoids, and their therapeutic purpose in MS. RESULTS: Evidence from preclinical studies showed that cannabinoids can limit the demyelination process, promote remyelination, and have anti-inflammatory properties by reducing immune cell infiltration of the central nervous system in mice with experimental autoimmune encephalomyelitis. Moreover, it has been established that experimental autoimmune encephalomyelitis mice treated with cannabinoids experienced a significant reduction of symptoms and slowing of the disease progression. Given the complexity of human immune and nervous systems, cannabinoids did not have the anticipated effects on human subjects. However, data obtained from clinical trials showed some beneficial results of cannabinoids as a single or as add-on therapy in reducing the spasticity and pain related to MS. CONCLUSION: Considering their various mechanisms of action and good tolerability, cannabinoids remain an interesting therapy for spasticity and chronic pain related to MS.

Low sulfated heparan sulfate mimetic differentially affects repair in immune-mediated and toxin-induced experimental models of demyelination.

There is an urgent need for therapies that target the multicellular pathology of central nervous system (CNS) disease. Modified, nonanticoagulant heparins mimic the heparan sulfate glycan family and are known regulators of multiple cellular processes. In vitro studies have demonstrated that low sulfated modified heparin mimetics (LS-mHeps) drive repair after CNS demyelination. Herein, we test LS-mHep7 (an in vitro lead compound) in experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. In EAE, LS-mHep7 treatment resulted in faster recovery and rapidly reduced inflammation which was accompanied by restoration of animal weight. LS-mHep7 treatment had no effect on remyelination or on OLIG2 positive oligodendrocyte numbers within the corpus callosum in the cuprizone model. Further in vitro investigation confirmed that LS-mHep7 likely mediates its pro-repair effect in the EAE model by sequestering inflammatory cytokines, such as CCL5 which are upregulated during immune-mediated inflammatory attacks. These data support the future clinical translation of this next generation modified heparin as a treatment for CNS diseases with active immune system involvement.

Effects of perinatal exposure to bisphenol A or S in EAE model of multiple sclerosis.

Epidemiological studies support the idea that multiple sclerosis (MS) is a multifactorial disease, overlapping genetic, epigenetic, and environmental factors. A better definition of environmental risks is critical to understand both etiology and the sex-related differences of MS. Exposure to endocrine-disrupting compounds (EDCs) fully represents one of these risks. EDCs are natural or synthetic exogenous substances (or mixtures) that alter the functions of the endocrine system. Among synthetic EDCs, exposure to bisphenol A (BPA) has been implicated in the etiology of MS, but to date, controversial data has emerged. Furthermore, nothing is known about bisphenol S (BPS), one of the most widely used substitutes for BPA. As exposure to bisphenols will not disappear soon, it is necessary to clarify their role also in this pathological condition defining their role in disease onset and course in both sexes. In this study, we examined, in both sexes, the effects of perinatal exposure to BPA and BPS in one of the most widely used mouse models of MS, experimental autoimmune encephalomyelitis (EAE). Exposure to bisphenols seemed to be particularly deleterious in males. In fact, both BPA- and BPS-treated males showed anticipation of the disease onset and an increased motoneuron loss in the spinal cord. Overall, BPA-treated males also displayed an exacerbation of EAE course and an increase in inflammation markers in the spinal cord. Analyzing the consequences of bisphenol exposure on EAE will help to better understand the role of both xenoestrogens and endogenous estrogens on the sexually dimorphic characteristics of MS.

Citrullinated human and murine MOG35-55 display distinct biophysical and biochemical behavior.

The peptide spanning residues 35 to 55 of the protein myelin oligodendrocyte glycoprotein (MOG) has been studied extensively in its role as a key autoantigen in the neuroinflammatory autoimmune disease multiple sclerosis. Rodents and nonhuman primate species immunized with this peptide develop a neuroinflammatory condition called experimental autoimmune encephalomyelitis, often used as a model for multiple sclerosis. Over the last decade, the role of citrullination of this antigen in the disease onset and progression has come under increased scrutiny. We recently reported on the ability of these citrullinated MOG35-55 peptides to aggregate in an amyloid-like fashion, suggesting a new potential pathogenic mechanism underlying this disease. The immunodominant region of MOG is highly conserved between species, with the only difference between the murine and human protein, a polymorphism on position 42, which is serine in mice and proline for humans. Here, we show that the biophysical and biochemical behavior we previously observed for citrullinated murine MOG35-55 is fundamentally different for human and mouse MOG35-55. The citrullinated human peptides do not show amyloid-like behavior under the conditions where the murine peptides do. Moreover, we tested the ability of these peptides to stimulate lymphocytes derived from MOG immunized marmoset monkeys. While the citrullinated murine peptides did not produce a proliferative response, one of the citrullinated human peptides did. We postulate that this unexpected difference is caused by disparate antigen processing. Taken together, our results suggest that further study on the role of citrullination in MOG-induced experimental autoimmune encephalomyelitis is necessary.

A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

Treatment with MDL 72527 Ameliorated Clinical Symptoms, Retinal Ganglion Cell Loss, Optic Nerve Inflammation, and Improved Visual Acuity in an Experimental Model of Multiple Sclerosis.

Multiple Sclerosis (MS) is a highly disabling neurological disease characterized by inflammation, neuronal damage, and demyelination. Vision impairment is one of the major clinical features of MS. Previous studies from our lab have shown that MDL 72527, a pharmacological inhibitor of spermine oxidase (SMOX), is protective against neurodegeneration and inflammation in the models of diabetic retinopathy and excitotoxicity. In the present study, utilizing the experimental autoimmune encephalomyelitis (EAE) model of MS, we determined the impact of SMOX blockade on retinal neurodegeneration and optic nerve inflammation. The increased expression of SMOX observed in EAE retinas was associated with a significant loss of retinal ganglion cells, degeneration of synaptic contacts, and reduced visual acuity. MDL 72527-treated mice exhibited markedly reduced motor deficits, improved neuronal survival, the preservation of synapses, and improved visual acuity compared to the vehicle-treated group. The EAE-induced increase in macrophage/microglia was markedly reduced by SMOX inhibition. Upregulated acrolein conjugates in the EAE retina were decreased through MDL 72527 treatment. Mechanistically, the EAE-induced ERK-STAT3 signaling was blunted by SMOX inhibition. In conclusion, our studies demonstrate the potential benefits of targeting SMOX to treat MS-mediated neuroinflammation and vision loss.

The innovative animal monitoring device for experimental autoimmune encephalomyelitis ("I AM D EAE"): A more detailed evaluation for improved results.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is the most widely used animal model for multiple sclerosis (MS). It is a rapid model, commonly induced in rodents. Even if EAE does not replicate all MS characteristics, it is appropriate to investigate the development of the disease, including the immune and neuroinflammatory aspects. Besides, EAE has also been shown to be a relevant model for pre-clinical studies, as several drugs effective in the model are beneficial for MS patients. However, despite its widespread use, there is no consensus on the clinical assessment of animals. Most researchers perform a daily evaluation and classify them on a 5-point scale, but many authors also use in-between scores or apply other systems. Besides, among the 5-point scale, different score definitions are used, and most of them do not recapitulate the signs or symptoms each animal can show. Thus, based on our experience with EAE, the aim of the present work was to develop a new scoring system. METHODS: We designed the "I AM D EAE" tool that independently evaluates 9 different items - an innovative and detailed scoring system, yet simple for non-experts to use. The new scale was tested in EAE-induced mice at three experiments, and different evaluators assessed the animals blindly. RESULTS: The "I AM D EAE" scoring system highly correlates to the commonly used 5-point scale and, importantly, it enables a more detailed evaluation. CONCLUSIONS: Considering its high reproducibility and inter-rater reliability, "I AM D EAE" is a useful tool for EAE monitoring.

Transmembrane protein 119 is neither a specific nor a reliable marker for microglia.

Microglia are the resident innate immune cells of the central nervous system (CNS) parenchyma. To determine the impact of microglia on disease development and progression in neurodegenerative and neuroinflammatory diseases, it is essential to distinguish microglia from peripheral macrophages/monocytes, which are eventually equally recruited. It has been suggested that transmembrane protein 119 (TMEM119) serves as a reliable microglia marker that discriminates resident microglia from blood-derived macrophages in the human and murine brain. Here, we investigated the validity of TMEM119 as a microglia marker in four in vivo models (cuprizone intoxication, experimental autoimmune encephalomyelitis (EAE), permanent filament middle cerebral artery occlusion (fMCAo), and intracerebral 6-hydroxydopamine (6-OHDA) injections) as well as post mortem multiple sclerosis (MS) brain tissues. In all applied animal models and post mortem MS tissues, we found increased densities of ionized calcium-binding adapter molecule 1+ (IBA1+ ) cells, paralleled by a significant decrease in TMEM119 expression. In addition, other cell types in peripheral tissues (i.e., follicular dendritic cells and brown adipose tissue) were also found to express TMEM119. In summary, this study demonstrates that TMEM119 is not exclusively expressed by microglia nor does it label all microglia, especially under cellular stress conditions. Since novel transgenic lines have been developed to label microglia using the TMEM119 promotor, downregulation of TMEM119 expression might interfere with the results and should, thus, be considered when working with these transgenic mouse models.

The innate immune receptor Nlrp12 suppresses autoimmunity to the retina.

BACKGROUND: Nod-like receptors (NLRs) are critical to innate immune activation and induction of adaptive T cell responses. Yet, their role in autoinflammatory diseases of the central nervous system (CNS) remains incompletely defined. The NLR, Nlrp12, has been reported to both inhibit and promote neuroinflammation in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), where its T cell-specific role has been investigated. Uveitis resulting from autoimmunity of the neuroretina, an extension of the CNS, involves a breach in immune privilege and entry of T cells into the eye. Here, we examined the contribution of Nlrp12 in a T cell-mediated model of uveitis, experimental autoimmune uveitis (EAU). METHODS: Mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20) emulsified in Complete Freund's adjuvant, CFA. Uveitis was evaluated by clinical and histopathological scoring, and comparisons were made in WT vs. Nlrp12-/- mice, lymphopenic Rag1-/- mice reconstituted with WT vs. Nlrp12-/- CD4+ T cells, or among bone marrow (BM) chimeric mice. Antigen-specific Th-effector responses were evaluated by ELISA and intracellular cytokine staining. Cellular composition of uveitic eyes from WT or Nlrp12-/- mice was compared using flow cytometry. Expression of Nlrp12 and of cytokines/chemokines within the neuroretina was evaluated by immunoblotting and quantitative PCR. RESULTS: Nlrp12-/- mice developed exacerbated uveitis characterized by extensive vasculitis, chorioretinal infiltrates and photoreceptor damage. Nlrp12 was dispensable for T cell priming and differentiation of peripheral Th1 or Th17 cells, and uveitis in immunodeficient mice reconstituted with either Nlrp12-/- or WT T cells was similar. Collectively, this ruled out T cells as the source of Nlrp12-mediated protection to EAU. Uveitic Nlrp12-/- eyes had more pronounced myeloid cell accumulation than uveitic WT eyes. Transplantation of Nlrp12-/- BM resulted in increased susceptibility to EAU regardless of host genotype, but interestingly, a non-hematopoietic origin for Nlrp12 function was also observed. Indeed, Nlrp12 was found to be constitutively expressed in the neuroretina, where it suppressed chemokine/cytokine induction. CONCLUSIONS: Our data identify a combinatorial role for Nlrp12 in dampening autoimmunity of the neuroretina. These findings could provide a pathway for development of therapies for uveitis and potentially other autoinflammatory/autoimmune diseases of the CNS.

Antibiotic treatment during pregnancy and lactation in dams exacerbates clinical symptoms and inflammatory responses in offspring with experimental autoimmune encephalomyelitis.

A growing body of evidence demonstrates that an imbalance in the intensive communication between gut microbiota and the host might be associated with immune-related disorders such as multiple sclerosis. This study set out to determine whether antibiotic treatment during pregnancy and lactation can affect the onset and severity of clinical symptoms and inflammatory responses in offspring with experimental autoimmune encephalomyelitis (EAE; a mouse model of multiples sclerosis). Female C57BL/6 mice received antibiotics or vehicles during pregnancy and lactation, then their offspring were induced with EAE in adulthood. We also measured interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon-gamma (IFN-γ), IL-17A, IL-10, and transforming growth factor (TGF)-ß in the serum of offspring. The findings indicate that antibiotic treatment in dams significantly exacerbated the severity of EAE clinical symptoms in both male and female offspring. However, antibiotic treatment only accelerated the onset of EAE disease in male but not female offspring. We did not find any significant changes in cytokines in non-EAE male and female offspring treated with antibiotics. Antibiotic treatment significantly enhanced levels of IL-6, TNF-α, IFN-γ, IL-17A, and TGF-ß in EAE-induced male offspring, and IFN-γ, IL-17A, and IL-10 levels in EAE-induced female offspring. There were also sex differences in the onset and severity of EAE disease, and inflammatory cytokines (IL-6, IFN-γ, and IL-17A) between EAE-induced male and female offspring treated with antibiotics. Taken together, this study suggests that antibiotic treatment during pregnancy and lactation in dams might affect the development of the immune system in male and female offspring in adulthood.