Central and peripheral myeloid-derived suppressor cell-like cells are closely related to the clinical severity of multiple sclerosis.

Multiple sclerosis (MS) is a highly heterogeneous demyelinating disease of the central nervous system (CNS) that needs for reliable biomarkers to foresee disease severity. Recently, myeloid-derived suppressor cells (MDSCs) have emerged as an immune cell population with an important role in MS. The monocytic-MDSCs (M-MDSCs) share the phenotype with Ly-6Chi-cells in the MS animal model, experimental autoimmune encephalomyelitis (EAE), and have been retrospectively related to the severity of the clinical course in the EAE. However, no data are available about the presence of M-MDSCs in the CNS of MS patients or its relation with the future disease aggressiveness. In this work, we show for the first time cells exhibiting all the bona-fide phenotypical markers of M-MDSCs associated with MS lesions, whose abundance in these areas appears to be directly correlated with longer disease duration in primary progressive MS patients. Moreover, we show that blood immunosuppressive Ly-6Chi-cells are strongly related to the future severity of EAE disease course. We found that a higher abundance of Ly-6Chi-cells at the onset of the EAE clinical course is associated with a milder disease course and less tissue damage. In parallel, we determined that the abundance of M-MDSCs in blood samples from untreated MS patients at their first relapse is inversely correlated with the Expanded Disability Status Scale (EDSS) at baseline and after a 1-year follow-up. In summary, our data point to M-MDSC load as a factor to be considered for future studies focused on the prediction of disease severity in EAE and MS.

Cellular and Molecular Processes Are Differently Influenced in Primary Neural Cells by Slight Changes in the Physicochemical Properties of Multicore Magnetic Nanoparticles.

Herein, we use two exemplary superparamagnetic iron oxide multicore nanoparticles (SPIONs) to illustrate the significant influence of slightly different physicochemical properties on the cellular and molecular processes that define SPION interplay with primary neural cells. Particularly, we have designed two different SPION structures, NFA (i.e., a denser multicore structure accompanied by a slightly less negative surface charge and a higher magnetic response) and NFD (i.e., a larger surface area and more negatively charged), and identified specific biological responses dependent on SPION type, concentration, exposure time, and magnetic actuation. Interestingly, NFA SPIONs display a higher cell uptake, likely driven by their less negative surface and smaller protein corona, more significantly impacting cell viability and complexity. The tight contact of both SPIONs with neural cell membranes results in the significant augmentation of phosphatidylcholine, phosphatidylserine, and sphingomyelin and the reduction of free fatty acids and triacylglycerides for both SPIONs. Nonetheless, NFD induces greater effects on lipids, especially under magnetic actuation, likely indicating a preferential membranal location and/or a tighter interaction with membrane lipids than NFA, in agreement with their lower cell uptake. From a functional perspective, these lipid changes correlate with an increase in plasma membrane fluidity, again larger for more negatively charged nanoparticles (NFD). Finally, the mRNA expression of iron-related genes such as Ireb-2 and Fth-1 remains unaltered, while TfR-1 is only detected in SPION-treated cells. Taken together, these results demonstrate the substantial impact that minor physicochemical differences of nanomaterials may exert in the specific targeting of cellular and molecular processes. A denser multicore structure generated by autoclave-based production is accompanied by a slight difference in surface charge and magnetic properties that become decisive for the biological impact of these SPIONs. Their capacity to markedly modify the lipidic cell content makes them attractive as lipid-targetable nanomedicines.

TLR4 pathway impairs synaptic number and cerebrovascular functions through astrocyte activation following traumatic brain injury.

BACKGROUND AND PURPOSE: Activation of astrocytes contributes to synaptic remodelling, tissue repair and neuronal survival following traumatic brain injury (TBI). The mechanisms by which these cells interact to resident/infiltrated inflammatory cells to rewire neuronal networks and repair brain functions remain poorly understood. Here, we explored how TLR4-induced astrocyte activation modified synapses and cerebrovascular integrity following TBI. EXPERIMENTAL APPROACH: To determine how functional astrocyte alterations induced by activation of TLR4 pathway in inflammatory cells regulate synapses and neurovascular integrity after TBI, we used pharmacology, genetic approaches, live calcium imaging, immunofluorescence, flow cytometry, blood-brain barrier (BBB) integrity assessment and molecular and behavioural methods. KEY RESULTS: Shortly after a TBI, there is a recruitment of excitable and reactive astrocytes mediated by TLR4 pathway activation with detrimental effects on post-synaptic density-95 (PSD-95)/vesicular glutamate transporter 1 (VGLUT1) synaptic puncta, BBB integrity and neurological outcome. Pharmacological blockage of the TLR4 pathway with resatorvid (TAK-242) partially reversed many of the observed effects. Synapses and BBB recovery after resatorvid administration were not observed in IP3 R2-/- mice, indicating that effects of TLR4 inhibition depend on the subsequent astrocyte activation. In addition, TBI increased the astrocytic-protein thrombospondin-1 necessary to induce a synaptic recovery in a sub-acute phase. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that TLR4-mediated signalling, most probably through microglia and/or infiltrated monocyte-astrocyte communication, plays a crucial role in the TBI pathophysiology and that its inhibition prevents synaptic loss and BBB damage accelerating tissue recovery/repair, which might represent a therapeutic potential in CNS injuries and disorders.

Tissue plasminogen activator worsens experimental autoimmune encephalomyelitis by complementary actions on lymphoid and myeloid cell responses.

BACKGROUND: Tissue plasminogen activator (tPA) is a serine protease involved in fibrinolysis. It is released by endothelial cells, but also expressed by neurons and glial cells in the central nervous system (CNS). Interestingly, this enzyme also contributes to pathological processes in the CNS such as neuroinflammation by activating microglia and increasing blood-brain barrier permeability. Nevertheless, its role in the control of adaptive and innate immune response remains poorly understood. METHODS: tPA effects on myeloid and lymphoid cell response were studied in vivo in the mouse model of multiple sclerosis experimental autoimmune encephalomyelitis and in vitro in splenocytes. RESULTS: tPA-/- animals exhibited less severe experimental autoimmune encephalomyelitis than their wild-type counterparts. This was accompanied by a reduction in both lymphoid and myeloid cell populations in the spinal cord parenchyma. In parallel, tPA increased T cell activation and proliferation, as well as cytokine production by a protease-dependent mechanism and via plasmin generation. In addition, tPA directly raised the expression of MHC-II and the co-stimulatory molecules CD80 and CD86 at the surface of dendritic cells and macrophages by a direct action dependent of the activation of epidermal growth factor receptor. CONCLUSIONS: Our study provides new insights into the mechanisms responsible for the harmful functions of tPA in multiple sclerosis and its animal models: tPA promotes the proliferation and activation of both lymphoid and myeloid populations by distinct, though complementary, mechanisms.

Expression of Early Growth Response Gene-2 and Regulated Cytokines Correlates with Recovery from Guillain-Barré Syndrome.

Guillain-Barré syndrome (GBS) is an immune-mediated peripheral neuropathy. The goal of this research was the identification of biomarkers associated with recovery from GBS. In this study, we compared the transcriptome of PBMCs from a GBS patient and her healthy twin to discover possible correlates of disease progression and recovery. The study was then extended using GBS and spinal cord injury unrelated patients with similar medications and healthy individuals. The early growth response gene-2 (EGR2) was upregulated in GBS patients during disease recovery. The results provided evidence for the implication of EGR2 in GBS and suggested a role for EGR2 in the regulation of IL-17, IL-22, IL-28A, and TNF-ß cytokines in GBS patients. These results identified biomarkers associated with GBS recovery and suggested that EGR2 overexpression has a pivotal role in the downregulation of cytokines implicated in the pathophysiology of this acute neuropathy.

Myeloid cell distribution and activity in multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease in which an exacerbated immune response provokes oligodendrocyte loss and demyelination, the hallmarks of this neurological disease. The destruction of myelin due to the uncontrolled activity of the invading immune cells leads to the formation of MS plaques. Among the different leukocytes that participate in the immune response associated with MS, the role of myeloid cells has been analyzed extensively (i.e. macrophages, dendritic cells -DCs- and neutrophils). Hence, in this review we will summarize what is known about the distribution, expression and markers available to study myeloid cells, and their histopathology, not only in a standard animal model of MS (autoimmune experimental encephalomyelitis -EAE) but also in MS tissue. In this review, we will not only refer to mature myeloid cells but also to the undifferentiated and almost unexplored myeloid-derived suppressor cells (MDSCs). The active role of MDSCs in the prompt resolution of an immune episode is gaining importance, yet is still the subject of some debate. Finally, the similarities and differences between MS and EAE are discussed, particularly in terms of myeloid cell phenotype, activity and the markers used.

The synthetic retinoid Am80 delays recovery in a model of multiple sclerosis by modulating myeloid-derived suppressor cell fate and viability.

Relapsing-remitting multiple sclerosis (RR-MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). It is characterized by relapsing phases with ongoing neurological affectation that are followed by a remitting period in which inflammatory events are controlled and the patients partially recover. Experimental Autoimmune Encephalomyelitis (EAE) is the animal model most often used to study the inflammatory component of MS. Several cell types are involved in controlling the immune response in EAE and immature myeloid-derived suppressor cells (MDSCs) have emerged as important actors in the immunomodulation that occurs in EAE due to their ability to suppress inflammatory responses by inducing T cell apoptosis. In this study, we assessed whether MDSC differentiation may have consequences on the clinical course of EAE by treating mice around the peak of the clinical course EAE with the MDSC-differentiating agent Am80, an analogue of retinoid acid. Am80 administration abrogates the immunomodulation that occurs in EAE mice through different MDSC-related mechanisms: i) induction of MDSC apoptosis; ii) polarization of MDSCs to mature subsets of myeloid cells (dendritic cells/macrophages/neutrophils); and iii) altering their immunosuppressor phenotype. Consequently, T cell density increases and their viability is promoted, delaying the animal's recovery. Therefore, our data point to MDSC behaviour as a crucial factor in facilitating the transition from the relapsing to the remission phase in EAE, which should be considered for future immune-related therapies for MS.

Myeloid-derived suppressor cells limit the inflammation by promoting T lymphocyte apoptosis in the spinal cord of a murine model of multiple sclerosis.

Multiple Sclerosis (MS) is a demyelinating/inflammatory disease of the central nervous system. Relapsing-remitting MS is characterized by a relapsing phase with clinical symptoms and the production of inflammatory cell infiltrates, and a period of remission during which patients recover partially. Myeloid-derived suppressor cells (MDSCs) are immature cells capable of suppressing the inflammatory response through Arginase-I (Arg-I) activity, among other mechanisms. Here, we have identified Arg-I(+) -MDSCs in the spinal cord during experimental autoimmune encephalomyelitis (EAE), cells that were largely restricted to the demyelinating plaque and that always exhibited the characteristic MDSC surface markers Arg-I/CD11b/Gr-1/M-CSF1R. The presence and density of Arg-I(+) -cells, and the proportion of apoptotic but not proliferative T cells, were correlated with the EAE time course: peaked in parallel with the clinical score, decreased significantly during the remitting phase and completely disappeared during the chronic phase. Spinal cord-isolated MDSCs of EAE animals augmented the cell death when co-cultured with stimulated control splenic CD3 T cells. These data point to an important role for MDSCs in limiting inflammatory damage in MS, favoring the relative recovery in the remitting phase of the disease. Thus, the MDSC population should be considered as a potential therapeutic target to accelerate the recovery of MS patients.

IFN-gamma-induced TNF-alpha expression is regulated by interferon regulatory factors 1 and 8 in mouse macrophages.

We have previously described that IFN-gamma induces cyclooxygenase 2 and inducible NO synthase expression by a mechanism that involved endogenously produced TNF-alpha. In this study, we report that TNF-alpha production is induced by IFN-gamma treatment in the murine macrophage cell line RAW 264.7. TNF-alpha mRNA levels are increased in cells treated with IFN-gamma in a time-dependent manner and IFN-gamma also increased human TNF-alpha promoter-dependent transcription. Two regions in the TNF-alpha promoter seem to be responsible for the IFN-gamma response: a distal region between -1311 and -615 bp of the human TNF-alpha promoter, and a proximal region located between -95 and -36 bp upstream of the transcriptional start. In contrast, IFN-gamma stimulation induces the expression of the transcription factors IRF-1 and IRF-8. Overexpression of these transcription factors produces an increase in the transcriptional activity of the human TNF-alpha promoter. There is a correlation between the regions of the TNF-alpha promoter responsible of the transcriptional activation elicited by IRF-1 and IRF-8 and those required for IFN-gamma response. In addition, IRF-1 and IRF-8 are recruited to the TNF-alpha promoter in IFN-gamma-treated RAW 264.7 cells, as demonstrated by chromatin immunoprecipitation assays. Moreover, overexpression of IRF-1 and IRF-8 induces TNF-alpha production in unstimulated RAW 264.7 macrophages, comparable to the production of TNF-alpha elicited by IFN-gamma stimulation, and silencing of IRF-1 and/or IRF-8 with specific small interfering RNAs, decreases IFN-gamma-elicited TNF-alpha production. In summary, IFN-gamma treatment induces TNF-alpha expression at transcriptional level requiring the coordinate action of IRF-1 and IRF-8.

Requirement of tumor necrosis factor alpha and nuclear factor-kappaB in the induction by IFN-gamma of inducible nitric oxide synthase in macrophages.

IFN-gamma induces NO production, inducible NO synthase (iNOS) protein, and promoter expression in mouse macrophage cells. Mutation of IFN regulatory factor 1 responsive element, gamma-activated site, as well as NF-kappaB elements in the murine iNOS promoter strongly reduced IFN-gamma-induced iNOS transcriptional activity. The role of NF-kappaB activation in iNOS induction by IFN-gamma was corroborated by overexpression of the NF-kappaB inhibitory protein IkappaBalpha, which inhibited iNOS promoter activity induced by IFN-gamma. In addition, IFN-gamma treatment induced p65 binding to the iNOS promoter by chromatin immunoprecipitation assay and NF-kappaB binding to DNA by EMSA, although with a delayed kinetics, suggesting an indirect autocrine role for another cytokine produced in response to IFN-gamma. It is interesting that we found that IFN-gamma induced TNF-alpha secretion, and the induction of iNOS expression by IFN-gamma was abolished in primary peritoneal macrophages from TNF-alpha-deficient (TNF-alpha-/-) mice or in RAW 264.7 cells treated with anti-TNF-alpha neutralizing antibodies. Moreover, exogenous addition of recombinant mouse TNF-alpha restored iNOS expression induced by IFN-gamma in TNF-alpha-/- mice. It is intriguing that NF-kappaB binding to DNA in response to IFN-gamma treatment was absent in TNF-alpha-/- mice. Taken together, our data suggest that the TNF-alpha produced in response to IFN-gamma is required for iNOS induction by activating NF-kappaB transcription factor.

Involvement of TNF and NF-kappa B in the transcriptional control of cyclooxygenase-2 expression by IFN-gamma in macrophages.

IFN-gamma induces cyclooxygenase (COX)-2 expression and PG production in mouse macrophage cells. IFN-gamma activates COX-2 promoter-driven transcription. Deletion of the IFN sequence regulatory element (ISRE) I -1541/-1522 and ISRE II -1215/-1206 sites of the mouse COX-2 promoter minimally decrease this IFN-gamma induction. In contrast, deletion of the -965/-150 region from the COX-2 promoter abrogated IFN-gamma induction. In this region a NF-kappaB site has been described and mutation of this site impairs the induction of the full COX-2 promoter by IFN-gamma. Moreover, IFN-gamma induction of the COX-2 promoter was also strongly reduced by transfection of plasmid encoding the NF-kappaB inhibitor, IkappaBalpha. Interestingly, IFN-gamma induction of the COX-2 and PGE(2) synthesis was absent in macrophages from TNF(-/-) mice, and neutralizing anti-TNF Abs inhibited COX-2 promoter induction by IFN-gamma in RAW 264.7 macrophages. Moreover, NF-kappaB activity was induced late after stimulation with IFN-gamma correlating with the effect of autocrine TNF, and this NF-kappaB activation was absent in macrophages from TNF(-/-) mice. Taken together our results suggest a model in which IFN-gamma-induced TNF activates NF-kappaB, which is required for full COX-2 expression.