Objective:
Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive ...microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS).
Methods:
Electron and fluorescence microscopy and flow cytometry were used to detect myeloid MVs in the cerebrospinal fluid (CSF) of healthy controls, MS patients, and rodents affected by experimental autoimmune encephalomyelitis (EAE), the animal model of MS.
Results:
Myeloid MVs were detected in CSF of healthy controls. In relapsing and remitting EAE mice, the concentration of myeloid MVs in the CSF was significantly increased and closely associated with disease course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome from 2 independent cohorts revealed higher levels of myeloid MVs than in 13 age‐matched controls, indicating a clinical value of MVs as a companion tool to capture disease activity. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo at the site of administration; mice impaired in MV shedding were protected from EAE, suggesting a pathogenic role for MVs in the disease. Finally, FTY720, the first approved oral MS drug, significantly reduced the amount of MVs in the CSF of EAE‐treated mice.
Interpretation:
These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation. ANN NEUROL 2012;72:610–624
Significance A major obstacle to the eradication of HIV-1 by combination antiretroviral therapy (cART) is the formation of cellular reservoirs in CD4 ⺠T lymphocytes (carrying latently integrated ...provirus) and tissue macrophages. Infected macrophages assemble new virions in subcellular vacuoles known as virus-containing compartments (VCC), hiding them from the immune system and, in part, from antiretroviral agents. Here we report that extracellular ATP is capable of inducing the rapid release of virions accumulated in VCC via interaction with the P2X7 receptor and without inducing cell death, whereas the antidepressant agent Imipramine blocks the release. Thus, our study identifies two âdruggableâ targets affecting the release of stored virions from infected human macrophages that could bear relevance for purging HIV-1 reservoirs in individuals receiving cART.
HIV type 1 (HIV-1) infects CD4 ⺠T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as âTrojan horsesâ carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.
Microvesicles released by activated macrophages, and detected in biological fluids, may constitute a valuable biomarker to identify myeloid cell activation phenotype during disease.
MVs are known ...vehicles of horizontal communication among cells, currently under scrutiny as powerful biomarkers in several pathological processes. The potential advantage of MVs relies on the assumption that their content reflects processes ongoing in pathologically relevant cell types. We have described that MVs of myeloid origin in the CSF are a marker of microglia/macrophage activation. Myeloid cells have different activation types, resulting in diverse functional phenotypes. Knowledge on the activation type of myeloid cells during disease would be of paramount importance for the understanding of ongoing pathogenic processes. We show here that macrophages activated in vitro in different ways all release increased amounts of MVs compared with NS cells. Moreover, we show that macrophage‐derived MVs contain a repertoire of mRNAs that is not the result of casual sampling from the parental cells, as it is characterized by distinct mRNA enrichments and species. Nevertheless, mRNA content of MVs clearly allows identification in vivo of the activated phenotype of the cell of origin, indicating carryover of functional macrophage traits. We propose that detection of mRNAs in myeloid MVs permits identification of myeloid cell activation type during disease, allowing for further stratification of pathological processes.
The cross-talk between T cells and astrocytes occurring under physiological and, even more, neuroinflammatory conditions may profoundly impact the generation of adaptive immune responses in the ...nervous tissue. In this study, we used a standardized in vitro co-culture assay to investigate the immunomodulatory properties of astrocytes differing for age, sex, and species. Mouse neonatal astrocytes enhanced T cell vitality but suppressed T lymphocyte proliferation in response to mitogenic stimuli or myelin antigens, regardless of the Th1, Th2 or Th17 T cell phenotype. Studies comparing glia cells from adult and neonatal animals showed that adult astrocytes were more efficient in inhibiting T lymphocyte activation than neonatal astrocytes, regardless of their sex. Differently from primary cultures, mouse and human astrocytes derived from reprogrammed fibroblasts did not interfere with T cell proliferation. Overall, we describe a standardized astrocyte-T cell interaction in vitro assay and demonstrate that primary astrocytes and iAstrocytes may differ in modulating T cell function.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Microvesicles are a recently described way of cell communication that has been implicated in a number of biological processes, including neuroinflammation. Widely investigated as biomarkers in ...oncology and neurological disorders, little is known of the role of microvesicles in the pathogenesis of diseases such as multiple sclerosis (MS). Several evidences suggest that pro-inflammatory microglia and infiltrating macrophages release microvesicles that spread inflammatory signals and alter neuronal functions. We review here available information on microvesicles, with a special focus on microglia and macrophage microvesicles, in the pathogenesis of MS, and as potential biomarkers and therapeutic targets.
Myeloid cells, such as macrophages and microglia, play a crucial role in neuroinflammation and have been recently identified as a novel therapeutic target, especially for chronic forms. The general ...aim would be to change the phenotype of myeloid cells from pro- to anti-inflammatory, favoring their tissue-trophic and regenerative functions. Myeloid cells, however, display a number of functional phenotypes, not immediately identifiable as pro- or anti-inflammatory, and associated to ambiguous markers.
We employed in vitro assays to study macrophage polarization/differentiation in the presence of classical polarizing stimuli such as IFNγ (pro-inflammatory) and IL4 (anti-inflammatory). We induced neuroinflammation in mice by immunization with a myelin antigen and treated diseased mice with intracisternal delivery of an IL4-expressing lentiviral vector. We analyzed clinical, pathological, and immunological outcomes with a focus on myeloid cells.
We found that IL6, usually considered a pro-inflammatory cytokine, was released in vitro by macrophages treated with the anti-inflammatory cytokine IL4. We show the existence of macrophages expressing IL6 along with classical anti-inflammatory markers such as CD206 and demonstrate that these cells are immunosuppressive in vitro. In neuroinflamed mice, we show that IL4 delivery in the central nervous system (CNS) is associated with clinical and pathological protection from disease, associated with increased IL6 expression in infiltrating macrophages.
IL6 is known to mediate both pro- and anti-inflammatory effects, having two distinct ways to induce cell-signaling: either through the membrane bound receptor (anti-inflammatory) or through trans-signaling (pro-inflammatory). We show here that IL6-expressing macrophages are associated to protection from neuroinflammation, suggesting that IL6 anti-inflammatory properties prevail in the CNS, and calling for a general reconsideration of IL6 in macrophage polarization.
CD4(+)Foxp3(+) regulatory T cells (Tregs) have been considered crucial in controlling immune system homeostasis, and their derangement is often associated to autoimmunity. Tregs identification is, ...however, difficult because most markers, including CD25 and Foxp3, are shared by recently activated T cells. We show in this paper that CD4(+)Foxp3(+) T cells are generated in peripheral lymphoid organs on immunization and readily accumulate in the target organ of an autoimmune reaction, together with classical inflammatory cells, constituting up to 50% of infiltrating CD4(+) T cells. Most CD4(+)Foxp3(+) T cells are, however, CD25(-) and express proinflammatory cytokines such as IL-17 and IFN-γ, questioning their suppressive nature. Moreover, in vitro CD4(+) T lymphocytes from naive and autoimmune mice, stimulated to differentiate into Th1, Th2, Th17, and induced Tregs, display early mixed expression of lineage-specific markers. These results clearly point to an unprecedented plasticity of naive CD4(+) T cells, that integrating inflammatory signals may change their fate from the initial lineage commitment to a different functional phenotype.
The role of myeloid cells in the pathogenesis of MS is determined by the polarization they acquire after activation, and mediated by release of extracellular vesicles (MVs). We assessed the effects ...of treatments for MS on activation and polarization of myeloid cells. MVs levels and markers of polarization of myeloid cells have been assessed at baseline and up to 6 months after the start of a MS treatment. Patients had higher levels of MVs than controls, and these increased significantly over 6 months under natalizumab. Interferon β-1a significantly decreased M1 pro-inflammatory marker IL1β and upregulated Trem2, a receptor important for debris clearance; both interferon β-1a and fingolimod decreased pro-inflammatory marker IL6. Current treatments for MS significantly modulate myeloid cells activity.
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