Synergistic Targeting of Innate Receptors TLR7 and NOD2 for Therapeutic Intervention in Multiple Sclerosis.

Regulation of neuroinflammation is critical for maintaining central nervous system (CNS) homeostasis and holds therapeutic promise in autoimmune diseases such as multiple sclerosis (MS). Previous studies have highlighted the significance of selective innate signaling in triggering anti-inflammatory mechanisms, which play a protective role in an MS-like disease, experimental autoimmune encephalomyelitis (EAE). However, the individual intra-CNS administration of specific innate receptor ligands or agonists, such as for toll-like receptor 7 (TLR7) and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2), failed to elicit the desired anti-inflammatory response in EAE. In this study, we investigated the potential synergistic effect of targeting both TLR7 and NOD2 simultaneously to prevent EAE progression. Our findings demonstrate that simultaneous intrathecal administration of NOD2- and TLR7-agonists led to synergistic induction of Type I IFN (IFN I) and effectively suppressed EAE in an IFN I-dependent manner. Suppression of EAE was correlated with a significant decrease in the infiltration of monocytes, granulocytes, and natural killer cells, reduced demyelination, and downregulation of IL-1ß, CCL2, and IFNγ gene expression in the spinal cord. These results underscore the therapeutic promise of concurrently targeting the TLR7 and NOD2 pathways in alleviating neuroinflammation associated with MS, paving the way for novel and more efficacious treatment strategies.

Myelin basic protein mRNA levels affect myelin sheath dimensions, architecture, plasticity, and density of resident glial cells.

Myelin basic protein (Mbp) is essential for both elaboration and maintenance of CNS myelin, and its reduced accumulation results in hypomyelination. How different Mbp mRNA levels affect myelin dimensions across the lifespan and how resident glial cells may respond to such changes are unknown. Here, to investigate these questions, we used enhancer-edited mouse lines that accumulate Mbp mRNA levels ranging from 8% to 160% of wild type. In young mice, reduced Mbp mRNA levels resulted in corresponding decreases in Mbp protein accumulation and myelin sheath thickness, confirming the previously demonstrated rate-limiting role of Mbp transcription in the control of initial myelin synthesis. However, despite maintaining lower line specific Mbp mRNA levels into old age, both Mbp protein levels and myelin thickness improved or fully normalized at rates defined by the relative Mbp mRNA level. Sheath length, in contrast, was affected only when mRNA levels were very low, demonstrating that sheath thickness and length are not equally coupled to Mbp mRNA level. Striking abnormalities in sheath structure also emerged with reduced mRNA levels. Unexpectedly, an increase in the density of all glial cell types arose in response to reduced Mbp mRNA levels. This investigation extends understanding of the role Mbp plays in myelin sheath elaboration, architecture, and plasticity across the mouse lifespan and illuminates a novel axis of glial cell crosstalk.

Targeting Signaling Pathway Downstream of RIG-I/MAVS in the CNS Stimulates Production of Endogenous Type I IFN and Suppresses EAE.

Type I interferons (IFN), including IFNß, play a protective role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Type I IFNs are induced by the stimulation of innate signaling, including via cytoplasmic RIG-I-like receptors. In the present study, we investigated the potential effect of a chimeric protein containing the key domain of RIG-I signaling in the production of CNS endogenous IFNß and asked whether this would exert a therapeutic effect against EAE. We intrathecally administered an adeno-associated virus vector (AAV) encoding a fusion protein comprising RIG-I 2CARD domains (C) and the first 200 amino acids of mitochondrial antiviral-signaling protein (MAVS) (M) (AAV-CM). In vivo imaging in IFNß/luciferase reporter mice revealed that a single intrathecal injection of AAV-CM resulted in dose-dependent and sustained IFNß expression within the CNS. IFNß expression was significantly increased for 7 days. Immunofluorescent staining in IFNß-YFP reporter mice revealed extraparenchymal CD45+ cells, choroid plexus, and astrocytes as sources of IFNß. Moreover, intrathecal administration of AAV-CM at the onset of EAE induced the suppression of EAE, which was IFN-I-dependent. These findings suggest that accessing the signaling pathway downstream of RIG-I represents a promising therapeutic strategy for inflammatory CNS diseases, such as MS.

SARS-CoV-2 and Immunity: Natural Infection Compared with Vaccination.

Recently, the protective and/or pathological role of virus-specific T cells in SARS-CoV-2 infection has been the focus of many studies. We investigated the anti-spike IgG levels and SARS-CoV-2-specific T cells in 125 donors (90 vaccinated with four different vaccine platforms, 16 individuals with a previous natural infection, and 19 not vaccinated donors who did not report previous SARS-CoV-2 infections). Our data show that anti-spike IgG titers were similar between naturally infected subjects and those vaccinated with adenoviral vector vaccines. Of note, all immunized donors produced memory CD4+ and/or CD8+ T cells. A sustained polyfunctionality of SARS-CoV-2-specific T cells in all immunized donors was also demonstrated. Altogether, our data suggest that the natural infection produces an overall response like that induced by vaccination. Therefore, this detailed immunological evaluation may be relevant for other vaccine efforts especially for the monitoring of novel vaccines effective against emerging virus variants.

An Experimental Model of Neuromyelitis Optica Spectrum Disorder-Optic Neuritis: Insights Into Disease Mechanisms.

Background: Optic neuritis (ON) is a common inflammatory optic neuropathy, which often occurs in neuromyelitis optica spectrum disease (NMOSD). An experimental model of NMOSD-ON may provide insight into disease mechanisms. Objective: To examine the pathogenicity of autoantibodies targeting the astrocyte water channel aquaporin-4 [aquaporin-4 (AQP4)-immunoglobulin G (AQP4-IgG)] in the optic nerve. Materials and Methods: Purified IgG from an AQP4-IgG-positive NMOSD-ON patient was together with human complement (C) given to wild-type (WT) and type I interferon (IFN) receptor-deficient mice (IFNAR1-KO) as two consecutive intrathecal injections into cerebrospinal fluid via cisterna magna. The optic nerves were isolated, embedded in paraffin, cut for histological examination, and scored semi-quantitatively in a blinded fashion. In addition, optic nerves were processed to determine selected gene expression by quantitative real-time PCR. Results: Intrathecal injection of AQP4-IgG+C induced astrocyte pathology in the optic nerve with loss of staining for AQP4 and glial fibrillary acidic protein (GFAP), deposition of C, and demyelination, as well as upregulation of gene expression for interferon regulatory factor-7 (IRF7) and CXCL10. Such pathology was not seen in IFNAR1-KO mice nor in control mice. Conclusion: We describe induction of ON in an animal model for NMOSD and show a requirement for type I IFN signaling in the disease process.

Central Nervous System-Endogenous TLR7 and TLR9 Induce Different Immune Responses and Effects on Experimental Autoimmune Encephalomyelitis.

Innate receptors, including Toll like receptors (TLRs), are implicated in pathogenesis of CNS inflammatory diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). TLR response to pathogens or endogenous signals includes production of immunoregulatory mediators. One of these, interferon (IFN)ß, a Type I IFN, plays a protective role in MS and EAE. We have previously shown that intrathecal administration of selected TLR ligands induced IFNß and infiltration of blood-derived myeloid cells into the central nervous system (CNS), and suppressed EAE in mice. We have now extended these studies to evaluate a potential therapeutic role for CNS-endogenous TLR7 and TLR9. Intrathecal application of Imiquimod (TLR7 ligand) or CpG oligonucleotide (TLR9 ligand) into CNS of otherwise unmanipulated mice induced IFNß expression, with greater magnitude in response to CpG. CD45+ cells in the meninges were identified as source of IFNß. Intrathecal CpG induced infiltration of monocytes, neutrophils, CD4+ T cells and NK cells whereas Imiquimod did not recruit blood-derived CD45+ cells. CpG, but not Imiquimod, had a beneficial effect on EAE, when given at time of disease onset. This therapeutic effect of CpG on EAE was not seen in mice lacking the Type I IFN receptor. In mice with EAE treated with CpG, the proportion of monocytes was significantly increased in the CNS. Infiltrating cells were predominantly localized to spinal cord meninges and demyelination was significantly reduced compared to non-treated mice with EAE. Our findings show that TLR7 and TLR9 signaling induce distinct inflammatory responses in the CNS with different outcome in EAE and point to recruitment of blood-derived cells and IFNß induction as possible mechanistic links between TLR9 stimulation and amelioration of EAE. The protective role of TLR9 signaling in the CNS may have application in treatment of diseases such as MS.

Mitochondria-A target for attenuation of astrocyte pathology.

Astrocyte pathology is a feature of neuromyelitis optica spectrum disorder (NMOSD) pathology. Recently mitochondrial dysfunction and metabolic changes were suggested to play a role in NMOSD. To elucidate the role of mitochondrial dysfunction, astrocyte pathology was induced in C57BL/6 J female mice by intracerebral injection of aquaporin-4-immunoglobulin G from an NMOSD patient, together with complement. Etomoxir has been shown to cause mitochondrial dysfunction. Etomoxir was delivered to the central nervous system and resulted in decreased astrocyte pathology. The ameliorating effect was associated with increases in different acylcarnitines and amino acids. This suggests that mitochondria may be a therapeutic target in NMOSD.

Innate Signaling in the CNS Prevents Demyelination in a Focal EAE Model.

The pathological hallmark of multiple sclerosis (MS) is the formation of multifocal demyelinating lesions in the central nervous system (CNS). Stimulation of innate receptors has been shown to suppress experimental autoimmune encephalomyelitis (EAE), an MS-like disease in mice. Specifically, targeting Toll-like receptor 9 (TLR9) and NOD-like receptor 2 (NOD2) significantly reduced disease severity. In the present work we have developed a novel focal EAE model to further study the effect of innate signaling on demyelinating pathology. Focal lesions were induced by stereotactic needle insertion into the corpus callosum (CC) of mice previously immunized for EAE. This resulted in focal pathology characterized by infiltration and demyelination in the CC. We find that intrathecal delivery of MIS416, a TLR9 and NOD2 bispecific innate ligand, into the cerebrospinal fluid reduced focal lesions in the CC. This was associated with upregulation of type I and II interferons, interleukin-10, arginase-1, CCL-2 and CXCL-10. Analysis of draining cervical lymph nodes showed upregulation of type II interferons and interleukin 10. Moreover, intrathecal MIS416 altered the composition of early CNS infiltrates, increasing proportions of myeloid and NK cells and reducing T cells at the lesion site. This study contributes to an increased understanding of how innate immune responses can play a protective role, which in turn may lead to additional therapeutic strategies for the prevention and treatment of demyelinating pathologies.

The protective effect of Angiotensin AT2-receptor stimulation in Neuromyelitis optica spectrum disorder is independent of astrocyte-derived BDNF.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an antibody-mediated autoimmune inflammatory disease of the central nervous system (CNS), resulting in primary astrocytopathy. We have previously shown that Angiotensin AT2-receptor (AT2R) stimulation with the specific agonist Compound 21 (C21) attenuated NMOSD-like pathology. Recent studies have proposed that the mechanism behind protective effects of AT2R includes induction of brain derived neurotrophic factor (BDNF). Astrocytes are a major cellular source of BDNF. In this study we used mice with conditional BDNF deficiency in astrocytes (GfapF) to examine the involvement of astrocyte-derived BDNF in NMOSD-like pathology and in mediating the protective effect of AT2R stimulation. METHODS: Anti-aquaporin-4 IgG (AQP4-IgG) from an NMOSD patient and human complement (C) were co-injected intrastriatally to GfapF and wildtype littermate BDNFfl/fl mice (WT), together with either C21 or vehicle at day 0, followed by intrathecal injection of C21 or vehicle at day 2 and tissue collection at day 4. RESULTS: Intracerebral/intrathecal injection of C21, alone or in combination with AQP4-IgG + C, induced BDNF expression in WT mice. Injection of AQP4-IgG + C induced NMOSD-like pathology, including loss of AQP4 and GFAP. There was no difference in the severity of pathological changes between GfapF and WT mice. C21 treatment significantly and equally ameliorated NMOSD-like pathology in both WT and GfapF mice. CONCLUSION: Our findings indicate that astrocyte-derived BDNF neither reduces the severity of NMOSD-like pathology nor is it necessary for the protective effect of AT2R stimulation in NMOSD-like pathology.

Microglia-Secreted Factors Enhance Dopaminergic Differentiation of Tissue- and iPSC-Derived Human Neural Stem Cells.

Microglia have recently been established as key regulators of brain development. However, their role in neuronal subtype specification remains largely unknown. Using three different co-culture setups, we show that microglia-secreted factors enhance dopaminergic differentiation of somatic and induced pluripotent stem cell-derived human neural stem cells (NSCs). The effect was consistent across different NSC and microglial cell lines and was independent of prior microglial activation, although restricted to microglia of embryonic origin. We provide evidence that the effect is mediated through reduced cell proliferation and decreased apoptosis and necrosis orchestrated in a sequential manner during the differentiation process. tumor necrosis factor alpha, interleukin-1ß, and insulinlike growth factor 1 are identified as key mediators of the effect and shown to directly increase dopaminergic differentiation of human NSCs. These findings demonstrate a positive effect of microglia on dopaminergic neurogenesis and may provide new insights into inductive and protective factors that can stimulate in vitro derivation of dopaminergic neurons.

Type I interferon-activated microglia are critical for neuromyelitis optica pathology.

Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system (CNS) most frequently mediated by serum autoantibodies against the water channel aquaporin 4, expressed on CNS astrocytes, resulting in primary astrocytopathy. There is no cure for NMO, and treatment with Type I interferon (IFNI)-IFNß is ineffective or even detrimental. We have previously shown that both NMO lesions and associated microglial activation were reduced in mice lacking the receptor for IFNß. However, the role of microglia in NMO is not well understood. In this study, we clarify the pathomechanism for IFNI dependence of and the role of microglia in experimental NMO. Transcriptome analysis showed a strong IFNI footprint in affected CNS tissue as well as in microglial subpopulations. Treatment with IFNß led to exacerbated pathology and further microglial activation as evidenced by expansion of a CD11c+ subset of microglia. Importantly, depletion of microglia led to suppression of pathology and decrease of IFNI signature genes. Our data show a pro-pathologic role for IFNI-activated microglia in NMO and open new perspectives for microglia-targeted therapies.

Protective roles for myeloid cells in neuroinflammation.

Myeloid cells represent the major cellular component of innate immune responses. Myeloid cells include monocytes and macrophages, granulocytes (neutrophils, basophils and eosinophils) and dendritic cells (DC). The role of myeloid cells has been broadly described both in physiological and in pathological conditions. All tissues or organs are equipped with resident myeloid cells, such as parenchymal microglia in the brain, which contribute to maintaining homeostasis. Moreover, in case of infection or tissue damage, other myeloid cells such as monocytes or granulocytes (especially neutrophils) can be recruited from the circulation, at first to promote inflammation and later to participate in repair and regeneration. This review aims to address the regulatory roles of myeloid cells in inflammatory diseases of the central nervous system (CNS), with a particular focus on recent work showing induction of suppressive function via stimulation of innate signalling in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE).

Absence of miRNA-146a Differentially Alters Microglia Function and Proteome.

Background: MiR-146a is an important regulator of innate inflammatory responses and is also implicated in cell death and survival. Methods: By sorting CNS resident cells, microglia were the main cellular source of miR-146a. Therefore, we investigated microglia function and phenotype in miR-146a knock-out (KO) mice, analyzed the proteome of KO and wild-type (WT) microglia by LC-MS/MS, and examined miR-146a expression in different brain lesions of patients with multiple sclerosis (MS). Results: When stimulated with LPS or myelin in vitro, microglia from KO mice expressed higher levels of IL-1ß, TNF, IL-6, IL-10, CCL3, and CCL2 compared to WT. Stimulation increased migration and phagocytosis of WT but not KO microglia. CD11c+ microglia were induced by cuprizone (CPZ) in the WT mice but less in the KO. The proteome of ex vivo microglia was not different in miR-146a KO compared to WT mice, but CPZ treatment induced differential and reduced protein responses in the KO: GOT1, COX5b, CRYL1, and cystatin-C were specifically changed in KO microglia. We explored discriminative features of microglia proteomes: sparse Partial Least Squares-Discriminant Analysis showed the best discrimination when control and CPZ-treated conditions were compared. Cluster of ten proteins separated WT and miR-146a KO microglia after CPZ: among them were sensomes allowing to perceive the environment, Atp1a3 that belongs to the signature of CD11c+ microglia, and proteins related to inflammatory responses (S100A9, Ppm1g). Finally, we examined the expression of miR-146a and its validated target genes in different brain lesions of MS patients. MiR-146 was upregulated in all lesion types, and the highest expression was in active lesions. Nineteen of 88 validated target genes were significantly changed in active lesions, while none were changed in NAWM. Conclusion: Our data indicated that microglia is the major source of miR-146a in the CNS. The absence of miR-146a differentially affected microglia function and proteome, and miR-146a may play an important role in gene regulation of active MS lesions.

Protective Microglial Subset in Development, Aging, and Disease: Lessons From Transcriptomic Studies.

Microglial heterogeneity has been the topic of much discussion in the scientific community. Elucidation of their plasticity and adaptability to disease states triggered early efforts to characterize microglial subsets. Over time, their phenotypes, and later on their homeostatic signature, were revealed, through the use of increasingly advanced transcriptomic techniques. Recently, an increasing number of these "microglial signatures" have been reported in various homeostatic and disease contexts. Remarkably, many of these states show similar overlapping microglial gene expression patterns, both in homeostasis and in disease or injury. In this review, we integrate information from these studies, and we propose a unique subset, for which we introduce a core signature, based on our own research and reports from the literature. We describe that this subset is found in development and in normal aging as well as in diverse diseases. We discuss the functions of this subset as well as how it is induced.

Innate signaling within the central nervous system recruits protective neutrophils.

There is great interest in understanding how the central nervous system (CNS) communicates with the immune system for recruitment of protective responses. Infiltrating phagocytic monocytes and granulocytes are implicated in neuroinflammation in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). To investigate how CNS endogenous signals can be harnessed to promote anti-inflammatory programs, we have used a particulate Toll-like receptor 9 and nucleotide-oligomerization domain 2 bispecific innate ligand (MIS416), to address whether its phagocytosis within the CNS recruits protective myeloid cells. We find that MIS416 injected intrathecally into the cerebrospinal fluid via the cisterna magna induced a local chemokine response that recruited blood-derived monocytes and neutrophils to the CNS. These cells phagocytosed MIS416. The increase in EAE severity normally seen from time of onset did not occur in mice receiving MIS416. This suppression of disease symptoms was dependent on expression of the type I interferon receptor (IFNAR). Transfer of intrathecal MIS416-induced neutrophils suppressed EAE in recipient mice, while monocytes did not transfer protection. MIS416-induced neutrophils showed increased IL-10 expression that was IFNAR1-driven. In contrast to intrathecal administration, intravenous administration of MIS416 led to monocyte but not neutrophil infiltration to the CNS. We thus identify a CNS-intrinsic and -specific phagocytosis-induced recruitment of anti-inflammatory neutrophils that contribute to CNS homeostasis and may have therapeutic potential.

Angiotensin AT2 receptor-induced interleukin-10 attenuates neuromyelitis optica spectrum disorder-like pathology.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a relapsing inflammatory central nervous system (CNS) disease for which there is no cure. Immunoglobulin G autoantibodies specific for the water channel aquaporin-4 are a serum biomarker, believed to induce complement-dependent astrocyte damage with secondary demyelination. OBJECTIVE: To investigate the effect of angiotensin AT2 receptor (AT2R) stimulation on NMOSD-like pathology and its underlying mechanism. METHODS: NMOSD-like pathology was induced in mice by intracerebral injection of immunoglobulin-G isolated from NMOSD patient serum, with complement. This mouse model produces the characteristic histological features of NMOSD. A specific AT2R agonist, Compound 21 (C21), was given intracerebrally at day 0 and by intrathecal injection at day 2. RESULTS: Loss of aquaporin-4 and glial fibrillary acidic protein was attenuated by treatment with C21. Administration of C21 induced mRNA for interleukin-10 in the brain. NMOSD-like pathology was exacerbated in interleukin-10-deficient mice, suggesting a protective role. C21 treatment did not attenuate NMOSD-like pathology in interleukin-10-deficient mice, indicating that the protective effect of AT2R stimulation was dependent on interleukin-10. CONCLUSION: Our findings identify AT2R as a novel potential therapeutic target for the treatment of NMOSD. Interleukin-10 signaling is an essential part of the protective mechanism counteracting NMOSD pathology.

Diffusion Kurtosis Imaging maps neural damage in the EAE model of multiple sclerosis.

Diffusion kurtosis imaging (DKI) is an imaging modality that yields novel disease biomarkers and in combination with nervous tissue modeling, provides access to microstructural parameters. Recently, DKI and subsequent estimation of microstructural model parameters has been used for assessment of tissue changes in neurodegenerative diseases and associated animal models. In this study, mouse spinal cords from the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) were investigated for the first time using DKI in combination with biophysical modeling to study the relationship between microstructural metrics and degree of animal dysfunction. Thirteen spinal cords were extracted from animals with varied grades of disability and scanned in a high-field MRI scanner along with five control specimen. Diffusion weighted data were acquired together with high resolution T2* images. Diffusion data were fit to estimate diffusion and kurtosis tensors and white matter modeling parameters, which were all used for subsequent statistical analysis using a linear mixed effects model. T2* images were used to delineate focal demyelination/inflammation. Our results reveal a strong relationship between disability and measured microstructural parameters in normal appearing white matter and gray matter. Relationships between disability and mean of the kurtosis tensor, radial kurtosis, radial diffusivity were similar to what has been found in other hypomyelinating MS models, and in patients. However, the changes in biophysical modeling parameters and in particular in extra-axonal axial diffusivity were clearly different from previous studies employing other animal models of MS. In conclusion, our data suggest that DKI and microstructural modeling can provide a unique contrast capable of detecting EAE-specific changes correlating with clinical disability.

Selective localization of IgG from cerebrospinal fluid to brain parenchyma.

BACKGROUND: Encounter of autoantibodies with specific antigens can lead to hypersensitivity reactions and pathology. In multiple sclerosis and neuromyelitis optica spectrum disease (NMOSD), immunoglobulin-G (IgG) deposition has been observed in pathological lesions in the central nervous system. The paradigmatic autoantibodies in NMOSD are specific for the water channel aquaporin-4, localized to astrocytic end-feet at the blood-brain barrier and ependymal cells at the cerebrospinal fluid-brain barrier. We have previously observed that IgG antibodies from NMO patients (NMO-IgG) access brain parenchyma from the cerebrospinal fluid and induce subpial and periventricular NMO-like lesions and blood-brain barrier breakdown, in a complement-dependent manner. OBJECTIVE: To investigate how IgG trafficking from cerebrospinal fluid to brain parenchyma can be influenced by injury. METHODS: IgG from healthy donors was intrathecally injected into the cerebrospinal fluid via cisterna magna at 1, 2, 4, or 7 days after a distal stereotactic sterile needle insertion to the striatum. RESULTS: Antibody deposition, detected by staining for human IgG, peaked 1 day after the intrathecal injection and was selectively seen close to the needle insertion. When NMO-IgG was intrathecally injected, we observed complement-dependent NMO-like pathology (loss of aquaporin-4 and glial fibrillary acidic protein) proximal to the insertion site, with similar kinetics. A fluorescent tracer did not show the same distribution indicating IgG-selective localization. CONCLUSION: These findings suggest that IgG from cerebrospinal fluid localize selectively in brain parenchyma at the site of injury and pathogenic NMO-IgG induce astrocyte pathology at the same location.

Experimental Demyelination and Axonal Loss Are Reduced in MicroRNA-146a Deficient Mice.

Background: The cuprizone (CPZ) model of multiple sclerosis (MS) was used to identify microRNAs (miRNAs) related to in vivo de- and remyelination. We further investigated the role of miR-146a in miR-146a-deficient (KO) mice: this miRNA is differentially expressed in MS lesions and promotes differentiation of oligodendrocyte precursor cells (OPCs) during remyelination, but its role has not been examined during demyelination. Methods: MicroRNAs were examined by Agilent Mouse miRNA Microarray in the corpus callosum during CPZ-induced demyelination and remyelination. Demyelination, axonal loss, changes in number of oligodendrocytes, OPCs, and macrophages/microglia was compared by histology/immunohistochemistry between KO and WT mice. Differential expression of target genes and proteins of miR-146a was analyzed in the transcriptome (4 × 44K Agilent Whole Mouse Genome Microarray) and proteome (liquid chromatography tandem mass spectrometry) of CPZ-induced de- and remyelination in WT mice. Levels of proinflammatory molecules in the corpus callosum were compared in WT versus KO mice by Meso Scale Discovery multiplex protein analysis. Results: miR-146a was increasingly upregulated during CPZ-induced de- and remyelination. The absence of miR-146a in KO mice protected against demyelination, axonal loss, body weight loss, and atrophy of thymus and spleen. The number of CNP+ oligodendrocytes was increased during demyelination in the miR-146a KO mice, while there was a trend of increased number of NG2+ OPCs in the WT mice. miR-146a target genes, SNAP25 and SMAD4, were downregulated in the proteome of demyelinating corpus callosum in WT mice. Higher levels of SNAP25 were measured by ELISA in the corpus callosum of miR-146a KO mice, but there was no difference between KO and WT mice during demyelination. Multiplex protein analysis of the corpus callosum lysate revealed upregulated TNF-RI, TNF-RII, and CCL2 in the WT mice in contrast to KO mice. The number of Mac3+ and Iba1+ macrophages/microglia was reduced in the demyelinating corpus callosum of the KO mice. Conclusion: During demyelination, absence of miR-146a reduced inflammatory responses, demyelination, axonal loss, the number of infiltrating macrophages, and increased the number of myelinating oligodendrocytes. The number of OPCs was slightly higher in the WT mice during remyelination, indicating a complex role of miR-146a during in vivo de- and remyelination.