The microglial triggering receptor expressed on myeloid cells 2 (TREM2) signals via the activatory membrane adaptor molecule TYROBP. Genetic variants or mutations of TREM2 or TYROBP have been linked ...to inflammatory neurodegenerative diseases associated with aging. The typical aging process goes along with microglial changes and mild neuronal loss, but the exact contribution of TREM2 is still unclear. Aged TREM2 knock‐out mice showed decreased age‐related neuronal loss in the substantia nigra and the hippocampus. Transcriptomic analysis of the brains of 24 months old TREM2 knock‐out mice revealed 211 differentially expressed genes mostly downregulated and associated with complement activation and oxidative stress response pathways. Consistently, 24 months old TREM2 knock‐out mice showed lower transcription of microglial (Aif1 and Tmem119), oxidative stress markers (Inos, Cyba, and Cybb) and complement components (C1qa, C1qb, C1qc, C3, C4b, Itgam, and Itgb2), decreased microglial numbers and expression of the microglial activation marker Cd68, as well as accumulation of oxidized lipids. Cultured microglia of TREM2 knock‐out mice showed reduced phagocytosis and oxidative burst. Thus, microglial TREM2 contributes to age‐related microglial changes, phagocytic oxidative burst, and loss of neurons with possible detrimental effects during physiological aging.
Main Points
TREM2 mutations are linked to neurodegenerative diseases, but its exact role in aging is still unclear.
Microglial TREM2 triggers inflammatory reaction, phagocytic oxidative burst and loss of neurons during physiological aging.
Microglial cells can be derived directly from the dissociated brain tissue by sorting procedures, from postnatal glial cultures by mechanic isolation or from pluripotent stem cells by ...differentiation. The detailed molecular phenotype of microglia from different sources is still unclear. Here, we performed a whole transcriptome analysis of flow cytometry‐sorted microglia, primary postnatal cultured microglia, embryonic stem cell derived microglia (ESdM), and other cell types. Microglia and ESdM, both cultured in serum‐free medium, were closely related to sorted microglia and showed a unique transcriptome profile, clearly distinct to other myeloid cell types, T cells, astrocytes, and neurons. ESdM and primary cultured microglia showed strong overlap in their transcriptome. Only 143 genes were differentially expressed between both cell types, mainly derived from immune‐related genes with a higher activation status of proinflammatory and immune defense genes in primary microglia compared to ESdM. Flow cytometry analysis of cell surface markers CD54, CD74, and CD274 selected from the microarray confirmed the close phenotypic relation between ESdM and primary cultured microglia. Thus, assessment of genome‐wide transcriptional regulation demonstrates that microglial cells are unique and clearly distinct from other macrophage cell types.
The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on myeloid‐derived cell types. The extracellular immunoglobulin‐like domain of TREM2 binds anionic ligands ...including Apolipoprotein E and Amyloid‐β. The transmembrane domain interacts with its adaptor protein DAP12/TYROBP that is responsible for propagation of downstream signaling upon ligand interaction. Several sequence variants of TREM2 have been linked to different neurodegenerative diseases including Alzheimer's disease. Here, we generated HEK 293 Flp‐In cell lines stably expressing human TREM2 and DAP12 using a bicistronic construct with a T2A linker sequence allowing initial expression of both proteins in stoichiometric amounts. Cell biological and biochemical analyses revealed transport of TREM2 to the cell surface, and canonical sequential proteolytic processing and shedding of TREM2 (sTREM2). The functionality of this cell system was demonstrated by detection of phosphorylated spleen tyrosine kinase (SYK) upon stimulation of TREM2 with the anionic membrane lipid phosphatidylserine or anti‐TREM2 antibodies. Using this cell model, we demonstrated impaired signaling of disease associated TREM2 variants. We also identified a monoclonal antibody against the stalk region of TREM2 with agonistic activity. Activation of TREM2‐DAP12 signaling with the monoclonal antibody and the partial loss of function of disease associated variants were recapitulated in induced pluripotent stem cell derived microglia. Thus, this reporter cell model represents a suitable experimental system to investigate signaling of TREM2 variants, and for the identification of ligands and compounds that modulate TREM2‐DAP12 signaling.
MAIN POINTS
Disease associated variants impair the signaling activity of TREM2 by distinct mechanisms.
Targeting the stalk region of TREM2 with bivalent antibodies activates TREM2 signaling.
Main Points
Disease associated variants impair the signaling activity of TREM2 by distinct mechanisms.
Targeting the stalk region of TREM2 with bivalent antibodies activates TREM2 signaling.
Age‐related macular degeneration (AMD) is a major cause of blindness in the elderly population. Its pathophysiology is linked to reactive oxygen species (ROS) and activation of the complement system. ...Sialic acid polymers prevent ROS production of human mononuclear phagocytes via the inhibitory sialic acid‐binding immunoglobulin‐like lectin‐11 (SIGLEC11) receptor. Here, we show that low‐dose intravitreal injection of low molecular weight polysialic acid with average degree of polymerization 20 (polySia avDP20) in humanized transgenic mice expressing SIGLEC11 on mononuclear phagocytes reduced their reactivity and vascular leakage induced by laser coagulation. Furthermore, polySia avDP20 prevented deposition of the membrane attack complex in both SIGLEC11 transgenic and wild‐type animals. In vitro, polySia avDP20 showed two independent, but synergistic effects on the innate immune system. First, polySia avDP20 prevented tumor necrosis factor‐α, vascular endothelial growth factor A, and superoxide production by SIGLEC11‐positive phagocytes. Second, polySia avDP20 directly interfered with complement activation. Our data provide evidence that polySia avDP20 ameliorates laser‐induced damage in the retina and thus is a promising candidate to prevent AMD‐related inflammation and angiogenesis.
Synopsis
Low molecular weight polysialic acid (polySia avDP20) ameliorates laser‐induced inflammation and choroidal neovascularization in a humanized mouse model of AMD‐like laser‐induced retinal injury.
Low‐dose polySia avDP20 reduces reactivity of mononuclear phagocytes and vascular leakage induced by laser coagulation in humanized transgenic mice expressing sialic acid‐binding immunoglobulin‐like lectin‐11 (SIGLEC11).
PolySia avDP20 prevents deposition of the membrane attack complex in both SIGLEC11 transgenic and wild‐type mouse retinal pigment epithelium/choroids.
PolySia avDP20 prevents tumor necrosis factor‐α, vascular endothelial growth factor A, and superoxide production of cultured SIGLEC11‐positive phagocytes.
PolySia avDP20 directly interferes with complement activation in vitro.
Low molecular weight polysialic acid (polySia avDP20) ameliorates laser‐induced inflammation and choroidal neovascularization in a humanized mouse model of AMD‐like laser‐induced retinal injury.
The complement system has been implicated in the removal of dysfunctional synapses and neurites during development and in disease processes in the mouse, but it is unclear how far the mouse data can ...be transferred to humans. Here, we co‐cultured macrophages derived from human THP1 monocytes and neurons derived from human induced pluripotent stem cells, to study the role of the complement system in a human model. Components of the complement system were expressed by the human macrophages and human neuronal culture, while receptors of the complement cascade were expressed by human macrophages as shown via gene transcript analysis and flow cytometry. We mimicked pathological conditions leading to an altered glycocalyx by treatment of human neurons with sialidases. Desialylated human neurites were opsonized by the complement component C1q. Furthermore, human neurites with an intact sialic acid cap remained untouched, while desialylated human neurites were removed and ingested by human macrophages. While blockage of the complement receptor 1 (CD35) had no effect, blockage of CD11b as part of the complement receptor 3 (CR3) reversed the effect on macrophage phagocytosis of desialylated human neurites. Data demonstrate that in the human system sialylation of the neuronal glycocalyx serves as an inhibitory flag for complement binding and CR3‐mediated phagocytosis by macrophages. GLIA 2016;64:35–47
Main points
Desialylation on neurites resulted in complement C1q opsonization and removal in a human neuron‐macrophage co‐culture system.
Desialylated human neurites were removed and ingested by human macrophages via complement receptor 3‐mediated phagocytosis.
Sialic‐acid‐binding immunoglobulin‐like lectin‐h (Siglec‐h) is a recently identified mouse‐specific CD33‐related Siglec that signals via DAP12/TYROBP. Expression of Siglec‐h has been observed on ...plasmacytoid dendritic cells and microglia, but the ligand and the function of Siglec‐h remained elusive. Here, we demonstrate gene transcription and protein expression of Siglec‐h by mouse microglia after interferon‐γ treatment or polarization into a M1‐subtype. Microglial Siglec‐h acted as phagocytosis receptor since targeting of microsphere beads to Siglec‐h triggered their uptake into the microglia. The extracellular domain of Siglec‐h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells. Microglial cells stimulated to express Siglec‐h engulfed intact glioma cells without prior induction of apoptosis and slightly reduced glioma cell number in culture. Phagocytosis of glioma cells by activated microglia was dependent on Siglec‐h and its adapter molecule DAP12. Thus, data show that M1‐polarized microglial cells can engulf glioma cells via a DAP12‐mediated Siglec‐h dependent mechanism.
Sialic acid-binding Ig-like lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-E is a mouse CD33-related Siglec that preferentially binds ...to sialic acid residues of the cellular glycocalyx. Here, we demonstrate gene transcription and protein expression of Siglec-E by cultured mouse microglia. Siglec-E on microglia inhibited phagocytosis of neural debris and prevented the production of superoxide radicals induced by challenge with neural debris. Soluble extracellular Siglec-E receptor protein bound to the neural glycocalyx. Coculture of mouse microglia and neurons demonstrated a neuroprotective effect of microglial Siglec-E that was dependent on neuronal sialic acid residues. Increased neurotoxicity of microglia after knockdown of Siglece mRNA was neutralized by the reactive oxygen species scavenger Trolox. Data suggest that Siglec-E recognizes the intact neuronal glycocalyx and has neuroprotective function by preventing phagocytosis and the associated oxidative burst.
The Eph‐ephrin system plays pivotal roles in cell adhesion and migration. The receptor‐like functions of the ephrin ligands allow the regulation of intracellular processes via reverse signaling. ...γ‐Secretase mediated processing of ephrin‐B has previously been linked to activation of Src, a kinase crucial for focal adhesion and podosome phosphorylation. Here, we analyzed the role of γ‐secretase in the stimulation of reverse ephrin‐B2 signaling in the migration of mouse embryonic stem cell derived microglia. The proteolytic generation of the ephrin‐B2 intracellular domain (ICD) by γ‐secretase stimulates Src and focal adhesion kinase (FAK). Inhibition of γ‐secretase decreased the phosphorylation of Src and FAK, and reduced cell motility. These effects were associated with enlargement of the podosomal surface. Interestingly, expression of ephrin‐B2 ICD could rescue these effects, indicating that this proteolytic fragment mediates the activation of Src and FAK, and thereby regulates podosomal dynamics in microglial cells. Together, these results identify γ‐secretase as well as ephrin‐B2 as regulators of microglial migration.
Main Points
γ‐Secretase cleaves ephrin‐B2 in microglia.
The ephrin‐B2 intracellular domain regulates phosphorylation of Src/FAK, podosomal dynamics, and microglial motility.
γ‐Secretase regulates reverse signalling of ephrin‐B2 in microglia.
A broad spectrum of diseases is characterized by myelin abnormalities and/or oligodendrocyte pathology. In most, if not all, of these diseases, early activation of microglia occurs. Our knowledge ...regarding the factors triggering early microglia activation is, however, incomplete. In this study, we used the cuprizone model to investigate the temporal and causal relationship of oligodendrocyte apoptosis and early microglia activation. Genome-wide gene expression studies revealed the induction of distinct chemokines, among them Cxcl10, Ccl2, and Ccl3 in cuprizone-mediated oligodendrocyte apoptosis. Early microglia activation was unchanged in CCL2- and CCL3-deficient knockouts, but was significantly reduced in CXCL10-deficient mice, resulting in an amelioration of cuprizone toxicity at later time points. Subsequent in vitro experiments revealed that recombinant CXCL10 induced migration and a proinflammatory phenotype in cultured microglia, without affecting their phagocytic activity or proliferation. In situ hybridization analyses suggest that Cxcl10 mRNA is mainly expressed by astrocytes, but also oligodendrocytes, in short-term cuprizone-exposed mice. Our results show that CXCL10 actively participates in the initiation of microglial activation. These findings have implications for the role of CXCL10 as an important mediator during the initiation of neuroinflammatory processes associated with oligodendrocyte pathology.
Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to ...re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear.
As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1β, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed.
Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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