Multiscale networks in multiple sclerosis.

Complex diseases such as Multiple Sclerosis (MS) cover a wide range of biological scales, from genes and proteins to cells and tissues, up to the full organism. In fact, any phenotype for an organism is dictated by the interplay among these scales. We conducted a multilayer network analysis and deep phenotyping with multi-omics data (genomics, phosphoproteomics and cytomics), brain and retinal imaging, and clinical data, obtained from a multicenter prospective cohort of 328 patients and 90 healthy controls. Multilayer networks were constructed using mutual information for topological analysis, and Boolean simulations were constructed using Pearson correlation to identified paths within and among all layers. The path more commonly found from the Boolean simulations connects protein MK03, with total T cells, the thickness of the retinal nerve fiber layer (RNFL), and the walking speed. This path contains nodes involved in protein phosphorylation, glial cell differentiation, and regulation of stress-activated MAPK cascade, among others. Specific paths identified were subsequently analyzed by flow cytometry at the single-cell level. Combinations of several proteins (GSK3AB, HSBP1 or RS6) and immune cells (Th17, Th1 non-classic, CD8, CD8 Treg, CD56 neg, and B memory) were part of the paths explaining the clinical phenotype. The advantage of the path identified from the Boolean simulations is that it connects information about these known biological pathways with the layers at higher scales (retina damage and disability). Overall, the identified paths provide a means to connect the molecular aspects of MS with the overall phenotype.

Serum neurofilament light chain concentration predicts disease worsening in multiple sclerosis.

BACKGROUND: Serum neurofilament light (sNfL) chain is a promising biomarker reflecting neuro-axonal injury in multiple sclerosis (MS). However, the ability of sNfL to predict outcomes in real-world MS cohorts requires further validation. OBJECTIVE: The aim of the study is to investigate the associations of sNfL concentration, magnetic resonance imaging (MRI) and retinal optical coherence tomography (OCT) markers with disease worsening in a longitudinal European multicentre MS cohort. METHODS: MS patients (n = 309) were prospectively enrolled at four centres and re-examined after 2 years (n = 226). NfL concentration was measured by single molecule array assay in serum. The patients' phenotypes were thoroughly characterized with clinical examination, retinal OCT and MRI brain scans. The primary outcome was disease worsening at median 2-year follow-up. RESULTS: Patients with high sNfL concentrations (⩾8 pg/mL) at baseline had increased risk of disease worsening at median 2-year follow-up (odds ratio (95% confidence interval) = 2.8 (1.5-5.3), p = 0.001). We found no significant associations of MRI or OCT measures at baseline with risk of disease worsening. CONCLUSION: Serum NfL concentration was the only factor associated with disease worsening, indicating that sNfL is a useful biomarker in MS that might be relevant in a clinical setting.

Mesenchymal stem cells instruct a beneficial phenotype in reactive astrocytes.

Transplanted mesenchymal stromal/stem cells (MSC) ameliorate the clinical course of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), reducing inflammation and demyelination. These effects are mediated by instructive cross-talk between MSC and immune and neural cells. Astroglial reaction to injury is a prominent feature of both EAE and MS. Astrocytes constitute a relevant target to control disease onset and progression and, based on their potential to acquire stem cell properties in situ, to foster recovery in the post-acute phase of pathology. We have assessed how MSC impact astrocytes in vitro and ex vivo in EAE. Expression of astroglial factors implicated in EAE pathogenesis was quantified by real-time PCR in astrocytes co-cultured with MSC or isolated from EAE cerebral cortex; astrocyte morphology and expression of activation markers were analyzed by confocal microscopy. The acquisition of neural stem cell properties by astrocytes was evaluated by neurosphere assay. Our study shows that MSC prevented astrogliosis, reduced mRNA expression of inflammatory cytokines that sustain immune cell infiltration in EAE, as well as protein expression of endothelin-1, an astrocyte-derived factor that inhibits remyelination and contributes to neurodegeneration and disease progression in MS. Moreover, our data reveal that MSC promoted the acquisition of progenitor traits by astrocytes. These data indicate that MSC attenuate detrimental features of reactive astroglia and, based on the reacquisition of stem cell properties, also suggest that astrocytes may be empowered in their protective and reparative actions by MSC.

Exploring Alzheimer's disease mouse brain through X-ray phase contrast tomography: From the cell to the organ.

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder associated with aberrant production of beta-amyloid (Aß) peptide depositing in brain as amyloid plaques. While animal models allow investigation of disease progression and therapeutic efficacy, technology to fully dissect the pathological mechanisms of this complex disease at cellular and vascular levels is lacking. X-ray phase contrast tomography (XPCT) is an advanced non-destructive 3D multi-scale direct imaging from the cell through to the whole brain, with exceptional spatial and contrast resolution. We exploit XPCT to simultaneously analyse disease-relevant vascular and neuronal networks in AD mouse brain, without sectioning and staining. The findings clearly show the different typologies and internal structures of Aß plaques, together with their interaction with patho/physiological cellular and neuro-vascular microenvironment. XPCT enables for the first time a detailed visualization of amyloid-angiopathy at capillary level, which is impossible to achieve with other approaches. XPCT emerges as added-value technology to explore AD mouse brain as a whole, preserving tissue chemistry and structure, enabling the comparison of physiological vs. pathological states at the level of crucial disease targets. In-vivo translation will permit to monitor emerging therapeutic approaches and possibly shed new light on pathological mechanisms of neurodegenerative diseases.

Detrimental and protective action of microglial extracellular vesicles on myelin lesions: astrocyte involvement in remyelination failure.

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.

IFN-γ orchestrates mesenchymal stem cell plasticity through the signal transducer and activator of transcription 1 and 3 and mammalian target of rapamycin pathways.

BACKGROUND: Mesenchymal stem cells (MSCs) display a therapeutic plasticity because of their ability to modulate immunity, foster tissue repair, and differentiate into mesodermal cells. IFN-γ has been described to differently affect human mesenchymal stem cell (hMSC) and mouse mesenchymal stem cell (mMSC) immunomodulation and differentiation, depending on the inflammatory milieu. OBJECTIVE: We aimed at dissecting the relevant intracellular pathways through which IFN-γ affects MSC plasticity and the consequence of their manipulation on MSC functions. METHODS: Modification of relevant IFN-γ-dependent pathways in mMSCs was carried out in vitro through gene silencing or chemical inhibition of key components. Functional outcomes were assessed by means of Western blotting, real-time PCR, differentiation, and proliferation assays on MSCs. The effect on T cells was addressed by T-cell proliferation assays; the effect of mammalian target of rapamycin (mTOR) manipulation in MSCs was studied in vivo in a mouse model of delayed-type hypersensitivity assay. To address whether similar mechanisms are involved also in hMSCs on IFN-γ stimulation, the effect of chemical inhibition on the same intracellular pathways was assessed by means of Western blotting, and the final outcome on immunomodulatory properties was evaluated based on real-time PCR and T-cell proliferation. RESULTS: We revealed that in mMSCs IFN-γ-induced immunoregulation is mediated by early phosphorylation of signal transducer and activator of transcription (STAT) 1 and STAT3, which is significantly enhanced by an extracellular signal-regulated kinase 1/2-dependent mTOR inhibition, thereby promoting pSTAT1 nuclear translocation. Accordingly, after intracellular mTOR inhibition, MSCs augmented their ability to inhibit T-cell proliferation and control delayed-type hypersensitivity in vivo. Similarly, on mTOR blockade, hMSCs also enhanced their immunoregulatory features. A sustained exposure to IFN-γ led to inhibition of STAT3 activity, which in mMSCs resulted in an impaired proliferation and differentiation. CONCLUSION: These results provide new insights about MSC intracellular pathways affected by IFN-γ, demonstrating that pharmacologic or genetic manipulation of MSCs can enhance their immunomodulatory functions, which could be translated into novel therapeutic approaches.

Dysregulation of regulatory CD56(bright) NK cells/T cells interactions in multiple sclerosis.

Recent evidence has shown that CD56(bright) NK cells, a subset of NK cells abundant in lymph nodes, may have an immunoregulatory function. In multiple sclerosis (MS), expansion of CD56(bright) NK cells has been associated to successful response to different treatments and to remission of disease during pregnancy; how whether they exert immunoregulation in physiologic conditions and whether this is impaired in MS is not known. We dissected the immunoregulatory role of CD56(bright) NK cells function in healthy subjects (HS) and compared it with that of untreated MS subjects or patients with clinically isolated syndrome suggestive of MS (CIS). We found that CD56(bright) NK cells from HS acquire, upon inflammatory cues, the capability of suppressing autologous CD4+T cell proliferation through direct cytotoxicity requiring engagement of natural cytotoxicity receptors (NCRs) and secretion of granzyme B. CD56(bright) NK cells from patients with MS/CIS did not differ in frequency and share a similar phenotype but displayed a significantly lower ability to inhibit autologous T cell proliferation. This impairment was not related to deficient expression of NCRs or granzyme B by CD56(bright) NK cells, but to increased HLA-E expression on T cells from MS/CIS subjects, which could enhance the inhibitory effect mediated by NKG2A that is homogeneously expressed on CD56(bright) NK cells. The defect in controlling autologous T cells by CD56(bright) NK cells in MS/CIS might contribute to the excess of autoimmune response that is associated to disease development.

NG2, a common denominator for neuroinflammation, blood-brain barrier alteration, and oligodendrocyte precursor response in EAE, plays a role in dendritic cell activation.

In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs) and is an early marker of pericyte activation in pathological conditions. NG2 could, therefore, play a role in experimental autoimmune encephalomyelitis (EAE), a disease associated with increased blood-brain barrier (BBB) permeability, inflammatory infiltrates, and CNS damage. We induced EAE in NG2 knock-out (NG2KO) mice and used laser confocal microscopy immunofluorescence and morphometry to dissect the effect of NG2 KO on CNS pathology. NG2KO mice developed milder EAE than their wild-type (WT) counterparts, with less intense neuropathology associated with a significant improvement in BBB stability. In contrast to WT mice, OPC numbers did not change in NG2KO mice during EAE. Through FACS and confocal microscopy, we found that NG2 was also expressed by immune cells, including T cells, macrophages, and dendritic cells (DCs). Assessment of recall T cell responses to the encephalitogen by proliferation assays and ELISA showed that, while WT and NG2KO T cells proliferated equally to the encephalitogenic peptide MOG35-55, NG2KO T cells were skewed towards a Th2-type response. Because DCs could be responsible for this effect, we assessed their expression of IL-12 by PCR and intracellular FACS. IL-12-expressing CD11c+ cells were significantly decreased in MOG35-55-primed NG2KO lymph node cells. Importantly, in WT mice, the proportion of IL-12-expressing cells was significantly lower in CD11c+ NG2- cells than in CD11c+ NG2+ cells. To assess the relevance of NG2 at immune system and CNS levels, we induced EAE in bone-marrow chimeric mice, generated with WT recipients of NG2KO bone-marrow cells and vice versa. Regardless of their original phenotype, mice receiving NG2KO bone marrow developed milder EAE than those receiving WT bone marrow. Our data suggest that NG2 plays a role in EAE not only at CNS/BBB level, but also at immune response level, impacting on DC activation and thereby their stimulation of reactive T cells, through controlling IL-12 expression.

Fumarates modulate microglia activation through a novel HCAR2 signaling pathway and rescue synaptic dysregulation in inflamed CNS.

Dimethyl fumarate (DMF), recently approved as an oral immunomodulatory treatment for relapsing-remitting multiple sclerosis (MS), metabolizes to monomethyl fumarate (MMF) which crosses the blood-brain barrier and has demonstrated neuroprotective effects in experimental studies. We postulated that MMF exerts neuroprotective effects through modulation of microglia activation, a critical component of the neuroinflammatory cascade that occurs in neurodegenerative diseases such as MS. To ascertain our hypothesis and define the mechanistic pathways involved in the modulating effect of fumarates, we used real-time PCR and biochemical assays to assess changes in the molecular and functional phenotype of microglia, quantitative Western blotting to monitor activation of postulated pathway components, and ex vivo whole-cell patch clamp recording of excitatory post-synaptic currents in corticostriatal slices from mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, to study synaptic transmission. We show that exposure to MMF switches the molecular and functional phenotype of activated microglia from classically activated, pro-inflammatory type to alternatively activated, neuroprotective one, through activation of the hydroxycarboxylic acid receptor 2 (HCAR2). We validate a downstream pathway mediated through the AMPK-Sirt1 axis resulting in deacetylation, and thereby inhibition, of NF-κB and, consequently, of secretion of pro-inflammatory molecules. We demonstrate through ex vivo monitoring of spontaneous glutamate-mediated excitatory post-synaptic currents of single neurons in corticostriatal slices from EAE mice that the neuroprotective effect of DMF was exerted on neurons at pre-synaptic terminals by modulating glutamate release. By exposing control slices to untreated and MMF-treated activated microglia, we confirm the modulating effect of MMF on microglia function and, thereby, its indirect neuroprotective effect at post-synaptic level. These findings, whereby DMF-induced activation of a new HCAR2-dependent pathway on microglia leads to the modulation of neuroinflammation and restores synaptic alterations occurring in EAE, represent a possible novel mechanism of action for DMF in MS.

Can we switch microglia's phenotype to foster neuroprotection? Focus on multiple sclerosis.

Microglia cells, the resident innate immune cells in the brain, are highly active, extending and retracting highly motile processes through which they continuously survey their microenvironment for 'danger signals' and interact dynamically with surrounding cells. Upon sensing changes in their central nervous system microenvironment, microglia become activated, undergoing morphological and functional changes. Microglia activation is not an 'all-or-none' process, but rather a continuum depending on encountered stimuli, which is expressed through a spectrum of molecular and functional phenotypes ranging from so-called 'classically activated', with a highly pro-inflammatory profile, to 'alternatively activated' associated with a beneficial, less inflammatory, neuroprotective profile. Microglia activation has been demonstrated in most neurological diseases of diverse aetiology and has been implicated as a contributor to neurodegeneration. The possibility to promote microglia's neuroprotective phenotype has therefore become a therapeutic goal. We have focused our discussion on the role of microglia in multiple sclerosis, a prototype of inflammatory, demyelinating, neurodegenerative disease, and on the effect of currently approved or on-trial anti-inflammatory therapeutic strategies that might mediate neuroprotection at least in part through their effect on microglia by modifying their behaviour via a switch of their functional phenotype from a detrimental to a protective one. In addition to pharmaceutical approaches, such as treatment with glatiramer acetate, interferon-ß, fingolimod or dimethyl fumarate, we address the alternative therapeutic approach of treatment with mesenchymal stem cells and their potential role in neuroprotection through their 'calming' effect on microglia.

Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status.

Deoxycytidine kinase (dCK) and 5' deoxynucleotidase (NT5C2) are involved in metabolism of cladribine (2CdA), the immunomodulatory drug for multiple sclerosis; by mediating phosphorylation (activation) or phosphorolysis (deactivation) of 2CdA, respectively, these enzymes promote or prevent its accumulation in the cell, which leads to cell death. In particular, lymphocytes which present with a high intracellular dCK/NT5C2 ratio are more sensitive to 2CdA than other immune cells. We aim at determining if the expression of these enzymes and/or their activity differ in specific progenitor and mature immune cells and are influenced by cellular activation and/or exposure to 2CdA. Flow cytometry analysis showed no difference in dCK/NT5C2 ratio in progenitor and mature immune cells. 2CdA induced apoptosis in stimulated T and B cells and unstimulated B cells. dCK expression was enhanced by 2CdA at mRNA and protein levels in activated T cells and mRNA level in activated B cells. dCK activity, measured through an in-house luminescence release enzyme assay was higher in activated T and B cells, and such an increase was abrogated in activated B cells, but not T cells, upon exposure to 2CdA. These results reveal an important relationship between dCK activity and the effect of 2CdA on B and T cells, according to their activation status. Further study is warranted to evaluate whether dCK activity could, in the future, be a suitable predictive biomarker of lymphocyte response to 2CdA.

Micro-RNAs Shuttled by Extracellular Vesicles Secreted from Mesenchymal Stem Cells Dampen Astrocyte Pathological Activation and Support Neuroprotection in In-Vitro Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with no effective cure. Astrocytes display a toxic phenotype in ALS and contribute to motoneuron (MN) degeneration. Modulating astrocytes' neurotoxicity can reduce MN death. Our previous studies showed the beneficial effect of mesenchymal stem cell (MSC) administration in SOD1G93A ALS mice, but the mechanisms are still unclear. We postulated that the effects could be mediated by extracellular vesicles (EVs) secreted by MSCs. We investigated, by immunohistochemical, molecular, and in vitro functional analyses, the activity of MSC-derived EVs on the pathological phenotype and neurotoxicity of astrocytes isolated from the spinal cord of symptomatic SOD1G93A mice and human astrocytes (iAstrocytes) differentiated from inducible neural progenitor cells (iNPCs) of ALS patients. In vitro EV exposure rescued mouse and human ALS astrocytes' neurotoxicity towards MNs. EVs significantly dampened the pathological phenotype and neuroinflammation in SOD1G93A astrocytes. In iAstrocytes, exposure to EVs increased the antioxidant factor Nrf2 and reduced reactive oxygen species. We previously found nine miRNAs upregulated in MSC-derived EVs. Here, the transfection of SOD1G93A astrocytes with single miRNA mimics reduced astrocytes' activation and the expression of neuroinflammatory factors. Moreover, miR-466q and miR-467f mimics downregulate Mapk11, while miR-466m-5p and miR-466i-3p mimics promote the nuclear translocation of Nrf2. In iAstrocytes, transfection with miR-29b-3p mimic upregulated NQO1 antioxidant activity and reduced neurotoxicity towards MNs. MSC-derived EVs modulate astrocytes' reactive phenotype and neurotoxicity through anti-inflammatory and antioxidant-shuttled miRNAs, thus representing a therapeutic strategy in ALS.

Microglia-derived CCL2 has a prime role in neocortex neuroinflammation.

BACKGROUND: In myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), several areas of demyelination are detectable in mouse cerebral cortex, where neuroinflammation events are associated with scarce inflammatory infiltrates and blood-brain barrier (BBB) impairment. In this condition, the administration of mesenchymal stem cells (MSCs) controls neuroinflammation, attenuating astrogliosis and promoting the acquisition of stem cell traits by astrocytes. To contribute to the understanding of the mechanisms involved in the pathogenesis of EAE in gray matter and in the reverting effects of MSC treatment, the neocortex of EAE-affected mice was investigated by analyzing the cellular source(s) of chemokine CCL2, a molecule involved in immune cell recruitment and BBB-microvessel leakage. METHODS: The study was carried out by immunohistochemistry (IHC) and dual RNAscope IHC/in situ hybridization methods, using astrocyte, NG2-glia, macrophage/microglia, and microglia elective markers combined with CCL2. RESULTS: The results showed that in EAE-affected mice, hypertrophic microglia are the primary source of CCL2, surround the cortex neurons and the damaged BBB microvessels. In EAE-affected mice treated with MSCs, microgliosis appeared diminished very soon (6 h) after treatment, an observation that was long-lasting (tested after 10 days). This was associated with a reduced CCL2 expression and with apparently preserved/restored BBB features. In conclusion, the hallmark of EAE in the mouse neocortex is a condition of microgliosis characterized by high levels of CCL2 expression. CONCLUSIONS: This finding supports relevant pathogenetic and clinical aspects of the human disease, while the demonstrated early control of neuroinflammation and BBB permeability exerted by treatment with MSCs may have important therapeutic implications.

The Gut-Brain Axis in Multiple Sclerosis. Is Its Dysfunction a Pathological Trigger or a Consequence of the Disease?

A large and expending body of evidence indicates that the gut-brain axis likely plays a crucial role in neurological diseases, including multiple sclerosis (MS). As a whole, the gut-brain axis can be considered as a bi-directional multi-crosstalk pathway that governs the interaction between the gut microbiota and the organism. Perturbation in the commensal microbial population, referred to as dysbiosis, is frequently associated with an increased intestinal permeability, or "leaky gut", which allows the entrance of exogeneous molecules, in particular bacterial products and metabolites, that can disrupt tissue homeostasis and induce inflammation, promoting both local and systemic immune responses. An altered gut microbiota could therefore have significant repercussions not only on immune responses in the gut but also in distal effector immune sites such as the CNS. Indeed, the dysregulation of this bi-directional communication as a consequence of dysbiosis has been implicated as playing a possible role in the pathogenesis of neurological diseases. In multiple sclerosis (MS), the gut-brain axis is increasingly being considered as playing a crucial role in its pathogenesis, with a major focus on specific gut microbiota alterations associated with the disease. In both MS and its purported murine model, experimental autoimmune encephalomyelitis (EAE), gastrointestinal symptoms and/or an altered gut microbiota have been reported together with increased intestinal permeability. In both EAE and MS, specific components of the microbiota have been shown to modulate both effector and regulatory T-cell responses and therefore disease progression, and EAE experiments with germ-free and specific pathogen-free mice transferred with microbiota associated or not with disease have clearly demonstrated the possible role of the microbiota in disease pathogenesis and/or progression. Here, we review the evidence that can point to two possible consequences of the gut-brain axis dysfunction in MS and EAE: 1. A pro-inflammatory intestinal environment and "leaky" gut induced by dysbiosis could lead to an altered communication with the CNS through the cholinergic afferent fibers, thereby contributing to CNS inflammation and disease pathogenesis; and 2. Neuroinflammation affecting efferent cholinergic transmission could result in intestinal inflammation as disease progresses.

Hydroxycarboxylic Acid Receptor 2, a Pleiotropically Linked Receptor for the Multiple Sclerosis Drug, Monomethyl Fumarate. Possible Implications for the Inflammatory Response.

Monomethyl fumarate (MMF), metabolite of dimethyl fumarate (DMF), an immunosuppressive drug approved for the treatment of multiple sclerosis (MS), is a potent agonist for hydroxycarboxylic acid receptor 2 (HCAR2), eliciting signals that dampen cell activation or lead to inflammation such as the skin flushing reaction that is one of the main side effects of the treatment, together with gastrointestinal inflammation. Our aim is to further understand the molecular basis underlying these differential effects of the drug. We have used wild-type and HCAR2 knock-out mice to investigate, in vitro and ex vivo under steady-state and pathological conditions, the HCAR2-mediated signaling pathways activated by MMF in dendritic cells (DC), which promote differentiation of T cells, and in intestinal epithelial cells (IEC) where activation of a pro-inflammatory pathway, such as the cyclooxygenase-2 pathway involved in skin flushing, could underlie gastrointestinal side effects of the drug. To understand how DMF treatment might impact on gut inflammation induced by experimental autoimmune encephalomyelitis (EAE), the animal model for MS, we have used 3D X-ray phase contrast tomography and flow cytometry to monitor possible intestinal alterations at morphological and immunological levels, respectively. We show that HCAR2 is a pleiotropically linked receptor for MMF, mediating activation of different pathways leading to different outcomes in different cell types, depending on experimental in-vitro and in-vivo conditions. In the small intestine of EAE-affected mice, DMF treatment affected migration of tolerogenic DC from lamina propria to mesenteric lymph nodes, and/or reverted their profile to pro-inflammatory, probably as a result of reduced expression of aldehyde dehydrogenase and transforming growth factor beta as well as the inflammatory environment. Nevertheless, DMF treatment did not amplify the morphological alterations induced by EAE. On the basis of our further understanding of MMF signaling through HCAR2, we suggest that the pleiotropic signaling of fumarate via HCAR2 should be addressed for its pharmaceutical relevance in devising new lead compounds with reduced inflammatory side effects.

Bone Marrow Transfer in Relapsing-Remitting EAE Ameliorates Disease at First Remission, with No Synergistic Effect upon Co-Transplantation with Mesenchymal Stem Cells.

Multiple sclerosis (MS) is a neurological disorder characterized by an autoimmune response, demyelinating plaques and axonal damage. Intense immunosuppression (II) followed by autologous hematopoietic stem cell transplantation has been proposed as a treatment in severe forms of MS. We have used murine relapsing-remitting (RR) experimental autoimmune encephalomyelitis (RR-EAE) to evaluate the transplantation of syngeneic bone marrow cells (BMC) after II, in combination with mesenchymal stem cells (MSCs) as a new therapeutic adjunct capable of improving immune reconstitution. In EAE-affected mice treated with BMC alone, we observed a drastic reduction in the clinical course only during the early RR phase of the disease. There was no difference in the RR-EAE clinical course between mice treated with BMC alone and co-transplanted mice. To analyze the immune reconstitution, we quantified the circulating immune cells in naïve and RR-EAE-affected mice after II, with BMC alone or in combination with MSC. Although II resulted in reduced numbers of circulating immune cells, reconstitution did not differ in co-transplanted mice. During the early phase of the disease, IL-4 was significantly elevated in co-transplanted mice, as compared to those treated with BMC alone. These data suggest that BMC transplantation after II transiently ameliorates the clinical symptoms of RR-EAE, but that co-transplantation with MSC has no synergistic effect.

Role of miRNAs shuttled by mesenchymal stem cell-derived small extracellular vesicles in modulating neuroinflammation.

Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for inflammatory/neurodegenerative diseases, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). One mode of action through which MSCs exert their immunomodulatory effects is release of extracellular vesicles that carry proteins, mRNAs, and microRNAs (miRNAs), which, once transferred, modify the function of target cells. We identified nine miRNAs significantly dysregulated in IFN-γ-primed MSCs, but present at different levels in their derived small extracellular vesicles (s-EV). We show that miR-467f and miR-466q modulate the pro-inflammatory phenotype of activated N9 microglia cells and of primary microglia acutely isolated from late symptomatic SOD1G93A mice, a murine ALS model, by downregulating Tnf and Il1b expression. Further analysis of the mode of action of miR-467f and miR-466q indicated that they dampen the pro-inflammatory phenotype of microglia by modulating p38 MAPK signaling pathway via inhibition of expression of their target genes, Map3k8 and Mk2. Finally, we demonstrated that in vivo administration of s-EV leads to decreased expression of neuroinflammation markers in the spinal cord of EAE-affected mice, albeit without affecting disease course. Overall, our data suggest that MSC-derived exosomes could affect neuroinflammation possibly through specific immunomodulatory miRNAs acting on microglia.

Corrigendum: X-ray Phase Contrast Tomography Serves Preclinical Investigation of Neurodegenerative Diseases.

[This corrects the article DOI: 10.3389/fnins.2020.584161.].

Neuroinflammation induces synaptic scaling through IL-1ß-mediated activation of the transcriptional repressor REST/NRSF.

Neuroinflammation is associated with synapse dysfunction and cognitive decline in patients and animal models. One candidate for translating the inflammatory stress into structural and functional changes in neural networks is the transcriptional repressor RE1-silencing transcription factor (REST) that regulates the expression of a wide cluster of neuron-specific genes during neurogenesis and in mature neurons. To study the cellular and molecular pathways activated under inflammatory conditions mimicking the experimental autoimmune encephalomyelitis (EAE) environment, we analyzed REST activity in neuroblastoma cells and mouse cortical neurons treated with activated T cell or microglia supernatant and distinct pro-inflammatory cytokines. We found that REST is activated by a variety of neuroinflammatory stimuli in both neuroblastoma cells and primary neurons, indicating that a vast transcriptional change is triggered during neuroinflammation. While a dual activation of REST and its dominant-negative splicing isoform REST4 was observed in N2a neuroblastoma cells, primary neurons responded with a pure full-length REST upregulation in the absence of changes in REST4 expression. In both cases, REST upregulation was associated with activation of Wnt signaling and increased nuclear translocation of ß-catenin, a well-known intracellular transduction pathway in neuroinflammation. Among single cytokines, IL-1ß caused a potent and prompt increase in REST transcription and translation in neurons, which promoted a delayed and strong synaptic downscaling specific for excitatory synapses, with decreased frequency and amplitude of spontaneous synaptic currents, decreased density of excitatory synaptic connections, and decreased frequency of action potential-evoked Ca2+ transients. Most important, the IL-1ß effects on excitatory transmission were strictly REST dependent, as conditional deletion of REST completely occluded the effects of IL-1ß activation on synaptic transmission and network excitability. Our results demonstrate that REST upregulation represents a new pathogenic mechanism for the synaptic dysfunctions observed under neuroinflammatory conditions and identify the REST pathway as therapeutic target for EAE and, potentially, for multiple sclerosis.