Interleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. γδ T cells are an important innate source of IL-17. Both IL-17+ γδ T cells and microglia, the major ...resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear.
In this study, we investigated the crosstalk between γδ T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ γδ T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays.
We report here that IL-17+ γδ T cells but not naïve γδ T cells induce a dose- and time-dependent decrease of neuronal viability in vitro. While direct stimulation of γδ T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of γδ T cells through IL-1β and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both γδ T cells and microglia, but did not require the expression of TLRs by γδ T cells. Similarly to cytokine-primed IL-17+ γδ T cells, IL-17+ γδ T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ γδ T cells required a direct cell-cell contact between T cells and neurons.
Taken together, these results point to a crucial role for microglia activated through TLRs in polarization of γδ T cells towards neurotoxic IL-17+ γδ T cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs (miRNAs) are non-coding RNAs originally involved in RNA silencing and post-transcriptional regulation of gene expression. We have shown in previous work that the miRNA let-7b can act as a ...signalling molecule for Toll-like receptor 7, thereby initiating innate immune pathways and apoptosis in the central nervous system. Here, we investigated whether different members of the miRNA family let-7, abundantly expressed in the brain, are released into the human cerebrospinal fluid (CSF) and whether quantitative differences in let-7 copies exist in neurodegenerative diseases. RNA isolated from CSF of patients with Alzheimer´s disease (AD) and from control patients with frontotemporal lobe dementia (FTLD), major depressive episode (MDE) without clinical or neurobiological signs of AD, and healthy individuals, was reverse transcribed with primers against nine let-7 family members, and miRNAs were quantified and analyzed comparatively by quantitative PCR. let-7 miRNAs were present in CSF from patients with AD, FTLD, MDE, and healthy controls. However, the amount of individual let-7 miRNAs in the CSF varied substantially. CSF from AD patients contained higher amounts of let-7b and let-7e compared to healthy controls, while no differences were observed regarding the other let-7 miRNAs. No increase in let-7b and let-7e was detected in CSF from FTLD patients, while in CSF from MDE patients, let-7b and let-7e copy levels were elevated. In CSF from AD patients, let-7b and let-7e were associated with extracellular vesicles. let-7 family members present in the CSF mediated neurotoxicity in vitro, albeit to a variable extent. Taken together, neurotoxic let-7 miRNAs are differentially and specifically released in AD, but also in MDE patients. Thus, these miRNAs may mirror common neuropathological paths and by this serve to unscramble mechanisms of different neurodegenerative diseases.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The invasiveness of malignant gliomas is one of the major obstacles in glioma therapy and the reason for the poor survival of patients. Glioma cells infiltrate into the brain parenchyma and thereby ...escape surgical resection. Glioma associated microglia/macrophages support glioma infiltration into the brain parenchyma by increased expression and activation of extracellular matrix degrading proteases such as matrix metalloprotease (MMP) 2, MMP9 and membrane‐type 1 MMP. In this work we demonstrate that, MMP9 is predominantly expressed by glioma associated microglia/macrophages in mouse and human glioma tissue but not by the glioma cells. Supernatant from glioma cells induced the expression of MMP9 in cultured microglial cells. Using mice deficient for different Toll‐like receptors we identified Toll‐like receptor 2/6 as the signaling pathway for the glioma induced upregulation of microglial MMP9. Also in an experimental mouse glioma model, Toll‐like receptor 2 deficiency attenuated the upregulation of microglial MMP9. Moreover, glioma supernatant triggered an upregulation of Toll‐like receptor 2 expression in microglia. Both, the upregulation of MMP9 and Toll‐like receptor 2 were attenuated by the antibiotic minocycline and a p38 mitogen‐activated protein kinase antagonist in vitro. Minocycline also extended the survival rate of glioma bearing mice when given to the drinking water. Thus glioma cells change the phenotype of glioma associated microglia/macrophages in a complex fashion using Toll‐like receptor 2 as an important signaling pathway and minocycline further proved to be a potential candidate for adjuvant glioma therapy.
What's New?
Gliomas invade the brain and march inexorably onward, rebuffing all attempts against them. The glioma's arsenal includes proteins that degrade the extracellular matrix, allowing the cancer to burrow into the parenchyma and evade the surgeon's knife. These authors demonstrate how the tumor cells press the surrounding microglia into their service: glioma cells up regulate Toll‐like receptor 2 in microglia, which then spurs the local microglia into producing matrix‐metalloprotease 9. Riding to the rescue, however, is the antibiotic minocycline, which hampered the production of both TLR2 and MMP‐9 as well as extending the life of mice burdened with gliomas, making it an attractive candidate for glioma therapy.
Neuronal damage in autoimmune neuroinflammation is the correlate for long-term disability in multiple sclerosis (MS) patients. Here, we investigated the role of immune cells in neuronal damage ...processes in animal models of MS by monitoring experimental autoimmune encephalomyelitis (EAE) by using two-photon microscopy of living anaesthetized mice. In the brainstem, we detected sustained interaction between immune and neuronal cells, particularly during disease peak. Direct interaction of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 and neuronal cells in demyelinating lesions was associated with extensive axonal damage. By combining confocal, electron, and intravital microscopy, we showed that these contacts remarkably resembled immune synapses or kinapses, albeit with the absence of potential T cell receptor engagement. Th17 cells induced severe, localized, and partially reversible fluctuation in neuronal intracellular Ca
2+ concentration as an early sign of neuronal damage. These results highlight the central role of the Th17 cell effector phenotype for neuronal dysfunction in chronic neuroinflammation.
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► Th17 cells establish immune-neuronal synapses and induce neuronal cell death in vitro ► Th17 cells directly contact neurons irrespective of their CNS-antigen specificity ► Th17 cell-neuronal interaction leads to severe neuronal dysfunction ► Th17 cell-mediated Ca
2+ elevation is partially reversible by blocking excitotoxicity
Toll-like receptors (TLRs) enable innate immune cells to respond to pathogen- and host-derived molecules. The central nervous system (CNS) exhibits most of the TLRs identified with predominant ...expression in microglia, the major immune cells of the brain. Although individual TLRs have been shown to contribute to CNS disorders, the consequences of multiple activated TLRs on the brain are unclear. We therefore systematically investigated and compared the impact of sole and pairwise TLR activation on CNS inflammation and injury.
Selected TLRs expressed in microglia and neurons were stimulated with their specific TLR ligands in varying combinations. Cell cultures were then analyzed by immunocytochemistry, FlowCytomix, and ELISA. To determine neuronal injury and neuroinflammation in vivo, C57BL/6J mice were injected intrathecally with TLR agonists. Subsequently, brain sections were analyzed by quantitative real-time PCR and immunohistochemistry.
Simultaneous stimulation of TLR4 plus TLR2, TLR4 plus TLR9, and TLR2 plus TLR9 in microglia by their respective specific ligands results in an increased inflammatory response compared to activation of the respective single TLR in vitro. In contrast, additional activation of TLR7 suppresses the inflammatory response mediated by the respective ligands for TLR2, TLR4, or TLR9 up to 24 h, indicating that specific combinations of activated TLRs individually modulate the inflammatory response. Accordingly, the composition of the inflammatory response pattern generated by microglia varies depending on the identity and combination of the activated TLRs engaged. Likewise, neuronal injury occurs in response to activation of only selected TLRs and TLR combinations in vitro. Activation of TLR2, TLR4, TLR7, and TLR9 in the brain by intrathecal injection of the respective TLR ligand into C57BL/6J mice leads to specific expression patterns of distinct TLR mRNAs in the brain and causes influx of leukocytes and inflammatory mediators into the cerebrospinal fluid to a variable extent. Also, the intensity of the inflammatory response and neurodegenerative effects differs according to the respective activated TLR and TLR combinations used in vivo.
Sole and pairwise activation of TLRs modifies the pattern and extent of inflammation and neurodegeneration in the CNS, thereby enabling innate immunity to take account of the CNS diseases' diversity.
Microglia express Toll-like receptors (TLRs) that sense pathogen- and host-derived factors, including single-stranded RNA. In the brain, let-7 microRNA (miRNA) family members are abundantly ...expressed, and some have recently been shown to serve as TLR7 ligands. We investigated whether let-7 miRNA family members differentially control microglia biology in health and disease. We found that a subset of let-7 miRNA family members function as signaling molecules to induce microglial release of inflammatory cytokines, modulate antigen presentation, and attenuate cell migration in a TLR7-dependent manner. The capability of the let-7 miRNAs to control microglial function is sequence specific, mapping to a let-7 UUGU motif. In human and murine glioblastoma/glioma, let-7 miRNAs are differentially expressed and reduce murine GL261 glioma growth in the same sequence-specific fashion through microglial TLR7. Taken together, these data establish let-7 miRNAs as key TLR7 signaling activators that serve to regulate the diverse functions of microglia in health and glioma.
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•let-7 miRNAs act as Toll-like receptor (TLR) 7 signaling molecules in glioma•Extracellular let-7 miRNAs differentially modulate microglial biology•The sequence motif UUGU is responsible for let-7-induced microglial activation•Select let-7 miRNAs attenuate glioma growth by directly activating microglial TLR7
Buonfiglioli et al. elucidate the role of let-7 miRNAs acting as Toll-like receptor (TLR) ligands in the brain. Select let-7 miRNAs function as signaling molecules to modulate diverse microglial functions and glioma growth through TLR7. These data establish let-7 miRNAs as TLR7 signaling activators of microglia in health and glioma.
Infection, ischemia, trauma, and neoplasia elicit a similar inflammatory response in the CNS characterized by activation of microglia, the resident CNS monocyte. The molecular events leading from CNS ...injury to the activation of innate immunity is not well understood. We show here that the intracellular chaperone heat shock protein 60 (HSP60) serves as a signal of CNS injury by activating microglia through a toll-like receptor 4 (TLR4)-dependent and myeloid differentiation factor 88 (MyD88)-dependent pathway. HSP60 is released from CNS cells undergoing necrotic or apoptotic cell death and specifically binds to microglia. HSP60-induced synthesis of neurotoxic nitric oxide by microglia is dependent on TLR4. HSP60 induces extensive axonal loss and neuronal death in CNS cultures from wild-type but not TLR4 or MyD88 loss-of-function mutant mice. This is the first evidence of an endogenous molecular pathway common to many forms of neuronal injury that bidirectionally links CNS inflammation with neurodegeneration.
Accumulation and infiltration of microglia/brain macrophages around and into glioma tissue promote tumor invasion and expansion. One tumor-promoting mechanism of microglia/brain macrophages is ...upregulation of membrane type 1 matrix metalloprotease (MT1-MMP), which promotes the degradation of extracellular matrix. MT1-MMP upregulation is induced by soluble factors released by glioma cells activating microglial Toll-like receptor 2 (TLR2).
Versican identified by proteomics was silenced in glioma cells by short interference RNA and short hairpin RNA approaches and studied in vitro and after injection into mouse brains or organotypic brain slices.
The splice variants V0/V1 of the endogenous TLR2 ligand versican are highly expressed in mouse and human glioma tissue. Versican-silenced gliomas induced less MT1-MMP expression in microglia both in vitro and in vivo, which resulted in smaller tumors and longer survival rates as compared with controls. Recombinant versican V1 induced significantly higher levels of MT1-MMP in wild-type microglia compared with untreated and treated TLR2 knockout microglial cells. Using glioma-injected organotypic brain slices, we found that the impact of versican signaling on glioma growth depended on the presence of microglia. Moreover, we found that TLR2 expression is upregulated in glioma-associated microglia but not in astrocytes. Additionally, an established TLR2 neutralizing antibody reduced glioma-induced microglial MT1-MMP expression as well as glioma growth ex vivo.
Our results show that versican released from glioma promotes tumor expansion through glioma-associated microglial/macrophage TLR2 signaling and subsequent expression of MT1-MMP. This signaling cascade might be a novel target for glioma therapies.
•The central TLR/IL-1R signaling adaptor MyD88 modulates neocortical structure.•MyD88-deficiency affects neuronal branching.•MyD88 expression modulates mouse CNS development.•Specific and natural ...behaviors are changed in MyD88-deficient mice.
While the original protein Toll in Drosophila melanogaster regulates both host defense and morphogenesis, the role of its ortholog Toll-like receptors (TLRs), the interleukin 1 receptor (IL-1R) family, and the associated signaling pathways in mammalian brain development and structure is poorly understood. Because the adaptor protein myeloid differentiation primary response protein 88 (MyD88) is essential for downstream signaling of most TLRs and IL-1R, we systematically investigated the effect of MyD88 deficiency on murine brain structure during development and on behavior. In neonatal Myd88−/− mice, neocortical thickness was reduced, while density of cortical neurons was increased. In contrast, microglia, astrocyte, oligodendrocyte, and proliferating cell numbers were unchanged in these mice compared to wild-type mice. In adult Myd88−/− mice, neocortical thickness was unaltered, but neuronal density in neocortex and hippocampus was increased. Neuron arborization was less pronounced in adult Myd88−/− mice compared to wild-type animals. In addition, numbers of microglia and proliferating cells were increased in the neocortex and subventricular zone, respectively, with unaltered astrocyte and oligodendrocyte numbers, and myelinization was enhanced in the adult Myd88−/− neocortex. These morphologic changes in the brain of adult Myd88−/− mice were accompanied by specific behavioral traits, such as decreased locomotor activity, increased anxiety-like behavior, but normal day/light activity, satisfactory learning, short- and long-term spatial memory, potential cognitive inflexibility, and increased hanging and locomotor behavior within their home cage. Taken together, MyD88 deficiency results in morphologic and cellular changes in the mouse brain, as well as in altered natural and specific behaviors. Our data indicate a pathophysiological significance of MyD88 for mammalian CNS development, structure, and function.
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