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The chaperone protein Unc-93 homolog B1 (UNC93B1) regulates internalization, trafficking, and stabilization of nucleic acid-sensing Toll-like receptors (TLR) in peripheral immune cells. We sought to determine UNC93B1 expression and its functional relevance in inflammatory and injurious processes in the central nervous system (CNS). We found that UNC93B1 is expressed in various CNS cells including microglia, astrocytes, oligodendrocytes, and neurons, as assessed by PCR, immunocyto-/histochemistry, and flow cytometry. UNC93B1 expression in the murine brain increased during development. Exposure to the microRNA let-7b, a recently discovered endogenous TLR7 activator, but also to TLR3 and TLR4 agonists, led to increased UNC93B1 expression in microglia and neurons. Microglial activation by extracellular let-7b required functional UNC93B1, as assessed by TNF ELISA. Neuronal injury induced by extracellular let-7b was dependent on UNC93B1, as UNC93B1-deficient neurons were unaffected by the microRNA's neurotoxicity in vitro. Intrathecal application of let-7b triggered neurodegeneration in wild-type mice, whereas mice deficient for UNC93B1 were protected against injurious effects on neurons and axons. In summary, our data demonstrate broad UNC93B1 expression in the murine brain and establish this chaperone as a modulator of neuroinflammation and neuronal injury triggered by extracellular microRNA and subsequent induction of TLR signaling.

Additional file 4. miR-672-5p, let-7g-5p, miR-100-5p, and miR-298-5p induce TNF-�� release from microglia in a time- and dose-dependent fashion. (a) C57BL/6 (wild-type, WT) microglia were incubated with 5 ��g/ml of miRNA, as indicated, for indicated durations. (b) WT microglia were incubated with various doses of indicated miRNAs for 24 h followed by TNF-�� ELISA. In all experiments (a, b), the TLR7 agonist loxoribine (1 mM) and the TLR4 agonist LPS (100 ng/ml) served as positive control. Unstimulated condition served as negative control. Data are represented as mean �� SD, n = 3.

Additional file 8. let-7g-5p and miR-672-5p induce neuronal injury in vitro. (a, b) Co-cultures of C57BL/6 (wild-type, WT) microglia and neurons were incubated with 5 ��g/ml of indicated miRNAs for 5 d. Mutant control oligonucleotide and unstimulated cells were used as negative control. Cells were subsequently immunostained with NeuN antibody and stained with TUNEL assay and DAPI. Quantification of NeuN (depicted as relative neuronal viability, left)- and TUNEL (right)-positive cells in co-cultures. Data are expressed as mean �� SD, n = 4. *P

Additional file 3. Gene Ontology (GO) enrichment analysis of biological processes for miRNAs that are enriched in supernatant of apoptotic cortical neurons. Color refers to significance (FDR, false discovery rate), while size indicates the corresponding number of associated genes. S/N, supernatant.

Additional file 11. Intrathecal pre-treatment with LPS enhances miR-298-5p- and miR-100-5p-induced neurodegeneration and microglial accumulation. 1 ��g LPS was injected intrathecally into C57BL/6 mice. After 16 h, mice were injected intrathecally with 10 ��g of miR-298-5p, miR-100-5p, or control oligoribonucleotide (LPS alone, n = 4; LPS + control oligo, n = 4; LPS + miR-298-5p, n = 4; LPS + miR-100-5p, n = 4). Naive mice and mice solely injected with control oligoribonucleotide, miR-100-5p, or miR-298-5p were included in this experimental set-up (naive, n = 4; control oligo, n = 4; miR-298-5p, n = 4; miR-100-5p, n = 4). After 3 d, brain sections were immunostained with NeuN or Iba1 antibody, and with DAPI. Representative images of brain sections labeled with NeuN antibody (a), Iba1 antibody (c), and DAPI (a, c) are shown. Scale bar, 50 ��m; inserts, scale bar, 10 ��m. NeuN+ (b) and Iba1+ (d) cells in the cerebral cortex were quantified. Data are shown as mean �� SD. P values for relevant groups as determined using the Student���s t-test are shown in (b) and (d). n.s., not significant.

Additional file 5. Low dose response of miR-100-5p- and miR-298-5p-treated microglia and HEK293 hTLR7/8 reporter cells. (a) Images of C57BL/6 microglia incubated with 5 ��g/ml of miR-100-5p or miR-298-5p, or PBS (control) for 4 h. Subsequently, cells were fixed and immunolabeled with Iba1 antibody, while nuclei were visualized with DAPI. Compared to control, miRNA-treated microglia displayed an amoeboid morphology, indicating an activated state. Scale bar, 30 ��m. (b) Microglia were incubated with indicated doses of miR-100-5p or miR-298-5p for 24 h. Loxoribine (1 mM) and LPS (100 ng/ml) served as positive control. Unstimulated condition served as negative control. Subsequently, supernatants were analyzed by TNF-�� ELISA. Data are represented as mean �� SD, n = 3. (c) HEK-Blue cells co-expressing human TLR7 (left, center) or human TLR8 (right), and an NF-��B/AP1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were incubated with various doses of miR-100-5p or miR-298-5p, as indicated, for 24 h. Loxoribine (1 mM), R848 (100 ng/ml), or TNF-�� (100 ng/ml) served as positive control. Unstimulated HEK-Blue TLR-expressing cells and HEK-Blue Null1 or Null1-k cells served as negative control. Data are expressed as fold change of optical density of the SEAP protein normalized to unstimulated control. Data are represented as mean �� SD, n = 3. *P

Additional file 9. miR-298-5p and miR-100-5p enter neurons and co-localize to their endosomal compartment and TLR7. (a) Enriched C57BL/6 cortical neurons were incubated with 40 ��g/ml pHrodo Red Dextran serving as endosomal marker for 20 min. Subsequently, neurons were exposed to 5 ��g/ml of Alexa488-labeled miR-298-5p or Alexa488-labeled miR-100-5p, and fixed after 4 h. Scale bar, 10 ��m. (b) Neurons exposed to the fluorescence-tagged miRNAs, as described above, were fixed and immunolabeled with TLR7 antibody. Scale bar, 20 ��m. (a, b) Cells were analyzed by confocal microscopy with sequential analysis. Representative images of neurons incubated with the indicated fluorescent miRNAs (488 nm, green) and pHrodo Red Dextran or TLR7 (552 nm, red) are shown (left panel). Diagrams depict fluorescence intensities of the marked ROI in neurons for the sequential analysis used (pHrodo Red Dextran/TLR7: red line; fluorescent miRNA: green line, right panel).

Additional file 6. Extracellular miR-100-5p and miR-298-5p increase microglial phagocytic activity. Microglia from C57BL/6 mice were exposed to 5 ��g/ml of the indicated miRNAs, LPS (100 ng/ml), or solvent control (PBS). After 2 h, microglia were incubated with red fluorescent beads of 1 ��m size for 1 h. Subsequently, cells were labeled with Iba1 antibody, and nuclei were visualized with DAPI (Scale bar, 30 ��m, left). Bead-linked red fluorescence within Iba1-positive image areas was quantified using FiJi software. Fluorescence intensity (FI) was expressed in arbitrary units (a.u., right). Data are represented as mean �� SD. n = 3. *P

Additional file 7. Microscale thermophoresis measurement of let-7g-5p and specificity control. Binding affinity measurements of the purified polyhistidine-tagged human TLR8 protein and (a) let-7g-5p and (b) miR-298-5p using microscale thermophoresis (MST). (b) The hTLR8 protein fragment was incubated with the control peptide before start of the measurement. (a, b) TLR8-let-7g-5p and TLR8-miR-298-5p interaction was monitored by titrating oligonucleotides from 500 ��M to 30 nM against 50 nM RED-tris-NTA-labeled hTLR8-protein measured with the NanoTemper Monolith NT.115 MST device. Kd values were calculated from dose response curves, which were obtained from titration experiments (n = 4). Data are expressed as mean �� SD.

Additional file 1: Primary cultures of microglia, astrocytes, and neurons. Microglia, astrocytes, and neurons were isolated from C57BL/6 mice as described in the Methods section. Phase contrast images display the respective cell type, as indicated, after 3 d in vitro. Scale bar, 10 μm.

Toll-like receptors (TLRs) belong to pattern recognition receptors, which respond to danger signals such as pathogen-associated molecular patterns or damage-associated molecular patterns. Upon TLR activation in microglia, the major immune cells in the brain, distinct signaling cascades trigger the production of inflammatory molecules, being a critical feature in neuroinflammation and neurodegenerative processes. Recently, individual microRNAs (miRNAs) were shown to act as endogenous TLR ligands. Here, we conducted systematic screening for miRNAs as potential TLR7/8 ligands by small RNA sequencing of apoptotic neurons and their corresponding supernatants. Several miRNA species were identified in both supernatants and injured neurons, and 83.3% of the media-enriched miRNAs activated murine and/or human TLR7/8 expressed in HEK293-derived TLR reporter cells. Among the detected extracellular miRNAs, distinct miRNAs such as miR-340-3p and miR-132-5p induced cytokine and chemokine release from microglia and triggered neurotoxicity in vitro. Taken together, our systematic study establishes miRNAs released from injured neurons as new TLR7/8 activators, which contribute to inflammatory and neurodegenerative responses in the central nervous system (CNS).

Additional file 4: Neither TLR5 deficiency nor exposure to flagellin does affect glioma growth ex vivo. (a) mCherry GL261 glioma cells were inoculated into organotypic brain slices (OBS) derived from P14-P16 C57BL/6 (n = 2; number of inoculated tumors: 27) and P14-P16 Tlr5−/− mice (n = 3; number of inoculated tumors: 21) and cultured for 4 d. Subsequently, OBS were fixed, stained with Hoechst, and scanned by confocal microscopy, followed by 3D surface reconstruction of gliomas and measurement of tumor volumes. (b) OBS derived from P14-P16 C57BL/6 mice were inoculated with mCherry glioma GL261 cells and stimulated with FLA (100 ng/ml, n = 3; number of analyzed tumors: 12). Tumor volume was compared to unstimulated control (n = 2; number of analyzed tumors: 16). Results are represented as mean ± SEM. Data were analyzed by Student’s t-test. n.s., not significant.

Additional file 3: Flagellin does not induce microglial injury or cell loss. C57BL/6 microglia were incubated with different flagellin (FLA) doses, as indicated, for 72 h. Unstimulated cells served as negative control. Subsequently, cells were stained with IB4 to mark microglia, TUNEL assay to mark apoptotic cells, and DAPI. (a) Representative image of microglia exposed to 5 μg/ml FLA. Scale bar, 50 μm. (b) IB4- and (c) TUNEL-positive cells were quantified. Results were expressed as absolute number of IB4-positive or TUNEL-positive cells per field (c). Data are expressed as mean ± SEM. Results were analyzed by Kruskal–Wallis test followed by Dunn’s post hoc test. n.s., not significant.

Additional file 2: Protein concentrations of cytokines/chemokines released from wild-type and Tlr5−/− microglia. Multiplex immunoassay was used to detect cytokines/chemokines, as indicated, in supernatants of cultured neonatal microglia from C57BL/6 (wild-type, WT) and Tlr5−/− mice in response to 100 ng/ml flagellin (FLA) after 24 h. Unstimulated cells served as negative control, while LPS (100 ng/ml) was used as positive control (n = 3). Data are expressed in pg/ml ± SD. n.d., not detectable.

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 2, matrix-metalloprotease 9 and membrane-type 1 matrix metalloprotease. In this work we demonstrate that, matrix-metalloprotease 9 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 matrix-metalloprotease 9 in cultured microglial cells. Using mice deficient for different Toll-like receptors we identified Toll-like receptor 2/6 as the signalling pathway for the glioma induced upregulation of microglial matrix-metalloprotease 9. Also in an experimental mouse glioma model, Toll-like receptor 2 deficiency attenuated the upregulation of microglial matrix-metalloprotease 9. Moreover, glioma supernatant triggered an upregulation of Toll-like receptor 2 expression in microglia. Both, the upregulation of matrix-metalloprotease 9 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 signalling pathway and minocycline further proved to be a potential candidate for adjuvant glioma therapy.

Der primäre Vorteil einer inflammatorischen Reaktion im Zentralen Nervensystem (ZNS) ist der Schutz vor eindringenden Pathogenen und Neoplasmen sowie die Beseitigung zellulären Abfalls. Der Nachteil eines solchen inflammatorischen Prozesses im ZNS ist die Möglichkeit einer beschleunigten Neurodegeneration. Wir haben die durch Pathogene induzierte Aktivierung des angeborenen Immunsystems und die hierdurch ausgelöste Inflammation im ZNS untersucht und die in diesem Zusammenhang existierende Literatur analysiert. Der Hauptfokus lag auf der Rolle des Toll-like Rezeptor (TLR) 4 bei der Schädigung von Oligodendrozyten und Neuronen. Darüberhinaus wurde die Rolle des Toll-like Rezeptor 2 bei der Schädigung und dem Zelltod von Mikroglia und Neuronen beleuchtet. Im letzten Teil der Arbeit wurde die Rolle endogener Liganden von Rezeptoren der angeborenen Immunität bei der Schädigung des ZNS untersucht. Mit Hilfe des Schlaganfallmodells der Maus wurde eine kritische Rolle für TLR2 bei der nicht-infektiösen Schädigung des ZNS nachgewiesen. Der in diesem Kontext verantwortliche Ligand des TLR2 wurde bisher nicht identifiziert. Hingegen wurde Heat shock protein 60 (HSP60), ein ubiquitär exprimiertes Chaperon, als endogener Ligand für TLR4 in Mikroglia identifiziert. Die Bindung an diesen Rezeptor und die anschliessende Aktivierung der Zelle führt zu neuronaler Schädigung. In der vorliegenden Arbeit wurden die oben aufgeführten Resultate diskutiert und zusammengefasst., The predictable advantage of an inflammatory response in the central nervous system (CNS) is protection from pathogens and neoplasms and clearing of non- viable cellular remains. However, an unanticipated disadvantage of inflammation may be the possibility of accelerated neurodegeneration. We have investigated and reviewed the field of pathogen-induced activation of the innate immune system and subsequent inflammation in the CNS. A major focus has been on the role of Toll-like receptor (TLR) 4 in oligodendrocyte and neuronal injury. Furthermore, the role of Toll-like receptor 2 in neuronal and microglial cell death has been elucidated. Finally, the role of endogenous ligands of innate immune receptors in CNS injury has been investigated. In the context of stroke, TLR2 plays a critical role in non-infectious CNS injury. Whereas in this context the endogenous ligand of TLR2 remains unknown, heat shock protein 60 (HSP60), a ubiquitously expressed chaperone molecule, has been identified as an endogenous ligand of TLR4 in microglia inducing neuronal injury. In this work the above-named results have been discussed and summarized.