Identification of CNS Injury-Related microRNAs as Novel Toll-Like Receptor 7/8 Signaling Activators by Small RNA Sequencing

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)

Intrathecal heat shock protein 60 mediates neurodegeneration and demyelination in the CNS through a TLR4- and MyD88-dependent pathway

Background Toll-like receptors (TLR) constitute a highly conserved class of receptors through which the innate immune system responds to both pathogen- and host-derived factors. Although TLRs are involved in a wide range of central nervous system (CNS) disorders including neurodegenerative diseases, the molecular events leading from CNS injury to activation of these innate immune receptors remain elusive. The stress protein heat shock protein 60 (HSP60) released from injured cells is considered an endogenous danger signal of the immune system. In this context, the main objective of the present study was to investigate the impact of extracellular HSP60 on the brain in vivo. Results We show here that HSP60 injected intrathecally causes neuronal and oligodendrocyte injury in the CNS in vivo through TLR4-dependent signaling. Intrathecal HSP60 results in neuronal cell death, axonal injury, loss of oligodendrocytes, and demyelination in the cerebral cortex of wild-type mice. In contrast both mice lacking TLR4 and the TLR adaptor molecule MyD88 are protected against deleterious effects induced by HSP60. In contrast to the exogenous TLR4 ligand, lipopolysaccharide, intrathecal HSP60 does not induce such a considerable inflammatory response in the brain. In the CNS, endogenous HSP60 is predominantly expressed in neurons and released during brain injury, since the cerebrospinal fluid (CSF) from animals of a mouse stroke model contains elevated levels of this stress protein compared to the CSF of sham- operated mice. Conclusions Our data show a direct toxic effect of HSP60 towards neurons and oligodendrocytes in the CNS. The fact that these harmful effects involve TLR4 and MyD88 confirms a molecular pathway mediated by the release of endogenous TLR ligands from injured CNS cells common to many forms of brain diseases that bi-directionally links CNS injury and activation of the innate immune system to neurodegeneration and demyelination in vivo

MicroRNA-100-5p and microRNA-298-5p released from apoptotic cortical neurons are endogenous Toll-like receptor 7/8 ligands that contribute to neurodegeneration

Background: MicroRNA (miRNA) expression in the brain is altered in neurodegenerative diseases. Recent studies demonstrated that selected miRNAs conventionally regulating gene expression at the post-transcriptional level can act extracellularly as signaling molecules. The identity of miRNA species serving as membrane receptor ligands involved in neuronal apoptosis in the central nervous system (CNS), as well as the miRNAs' sequence and structure required for this mode of action remained largely unresolved. Methods. Using a microarray-based screening approach we analyzed apoptotic cortical neurons of C56BL/6 mice and their supernatant with respect to alterations in miRNA expression/presence. HEK-Blue Toll-like receptor (TLR) 7/8 reporter cells, primary microglia and macrophages derived from human and mouse were employed to test the potential of the identified miRNAs released from apoptotic neurons to serve as signaling molecules for the RNA-sensing receptors. Biophysical and bioinformatical approaches, as well as immunoassays and sequential microscopy were used to analyze the interaction between candidate miRNA and TLR. Immunocytochemical and -histochemical analyses of murine CNS cultures and adult mice intrathecally injected with miRNAs, respectively, were performed to evaluate the impact of miRNA-induced TLR activation on neuronal survival and microglial activation. Results: We identified a specific pattern of miRNAs released from apoptotic cortical neurons that activate TLR7 and/or TLR8, depending on sequence and species. Exposure of microglia and macrophages to certain miRNA classes released from apoptotic neurons resulted in the sequence-specific production of distinct cytokines/chemokines and increased phagocytic activity. Out of those miRNAs miR-100-5p and miR-298-5p, which have consistently been linked to neurodegenerative diseases, entered microglia, located to their endosomes, and directly bound to human TLR8. The miRNA-TLR interaction required novel sequence features, but no specific structure formation of mature miRNA. As a consequence of miR-100-5p- and miR-298-5p-induced TLR activation, cortical neurons underwent cell-autonomous apoptosis. Presence of miR-100-5p and miR-298-5p in cerebrospinal fluid led to neurodegeneration and microglial accumulation in the murine cerebral cortex through TLR7 signaling. Conclusion: Our data demonstrate that specific miRNAs are released from apoptotic cortical neurons, serve as endogenous TLR7/8 ligands, and thereby trigger further neuronal apoptosis in the CNS. Our findings underline the recently discovered role of miRNAs as extracellular signaling molecules, particularly in the context of neurodegeneration

The impact of single and pairwise Toll-like receptor activation on neuroinflammation and neurodegeneration

Background 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. Methods 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. Results 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. Conclusions 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

Human endogenous retrovirus mediates neurodegeneration through murine Toll-like receptor 7 and Toll-like receptor 8 in humans.

Endogene retrovirale Elemente machen etwa 8 % des menschlichen Genoms aus jedoch ist über deren Funktion und biologische Bedeutung nur sehr wenig bekannt. Einige Hinweise deuten eine direkte Beteiligung einiger HERVs an neurodegenera- tiven Erkrankungen wie Amyotrophic Lateral Sclerosis (ALS) oder Multiple Skle- rose (MS) an. In dieser Arbeit konnte erstmalig gezeigt werden, dass die HERV-K(HML-2) env RNA das TLR7/8 Bindungsmotif (GUUGUGU) enthält und durch die Bindung an humanen TLR8 sowie murinen Tlr7, welche in Neuronen exprimiert werden, Neurodegeneration induziert. Diese Neurotoxizität wird dabei zellautonom über die Bindung an Tlr7/TLR8 in Neuronen erzeugt und in Anwesenheit von Mikrog- liazellen noch verstärkt. Die Induktion der Neurotoxizität erfolgt in Neuronen über das Adapterprotein SARM1 und nicht wie in Mikroglia über MyD88. Der durch HERV-K verursachte neuronale Schaden wurde sowohl in vitro als auch in vivo in Abhängigkeit des Vorhandenseins der Tlr7 und TLR8 nachgewiesen. Die erhöhte Expression von HERV-K env RNA in der CSF von Alzheimer Patient*innen lässt eine Beteiligung von HERV-K am Verlauf dieser Krankheit vermuten. Aus diesen Ergebnisse wurde ein Modell entwickelt, dass die Beteiligung an HERV-K an neu- rodegenerativen Erkrankungen wie der Alzheimer Krankheit zeigt. HERV-K env RNA beinhaltet das gut untersuchte Erkennungsmotif für TLR7 - GUUGUGU. Daher wurde anfänglich das Potential dieser RNA Tlr7 in der Maus und TLR im Menschen zu binden und zu aktivieren genauer untersucht. HERV-K aktiviert murine Mikroglia und induziert Neurotoxizität in primären Neuronenkul- turen in Abhängigkeit von Tlr7. Die zellautonome Neurodegeneration konnte unter Verwendung der neuronalen Zelllinien N1E-115 und SH-SY5Y bestätigt werden. Ein Zellverlust nach Stimulation mit HERV-K war auch hier zu beobachten. Der HERV-K induzierte neuronale Zelltod ist zeit- und dosisabhängig. In humanen Neuronen wird die HERV-K Neurotoxizität über den TLR8 vermittelt, wie Versuche an der THP-1- und TLR8 überexprimierende HEK-Reporterzelllinie zeigten. Humaner TLR7 wie auch der murine Tlr8 zeigten keine Aktivierung nach HERV-K Stimulation. MST-Messungen zeigen eine direkte HERV-K RNA Bin- dung an TLR8. HERV-K scheint somit ein möglicher neuer endogener Ligand für mTlr7 und hTLR8 zu sein. 1 Weiter konnte in dieser Arbeit gezeigt werden, dass die neurotoxischen Effekte auch in vivo durch HERV-K über Tlr7 und TLR8 induziert werden. Intratheka- le Injektionen des HERV-K führten zu signifikanten Absterben von Neuronen im zerebralen Kortex, was durch die Vermittlung der Kontrolloligoribonukleotide aus HERV-K env (HERV-neg.) und einem scrambled Oligo (#4) ohne GUUGUGU- Motif nicht zu beobachten war. Die Notwendigkeit der Expression von Tlr7 oder TLR8 in vivo konnte mit Hilfe der in-utero Elektroporation bestätigt werden. Hier- zu wurden mTlr7 und hTLR8 in das Gehirn 14,5 Tage alter Tlr7-/- Mausembryos eingebracht und diesen dann 19 Tage nach der Geburt per intrathekaler Injektion HERV-K verabreicht. Die immunhistochemische Analyse der Hirnschnitte zeigte deutliche neuronale Schädigungen und Zelltod in den mit Tlr7 und TLR8 trans- fizierten Tieren auf. Im Gegensatz dazu waren die neuronalen Zellen in den Tlr7- und TLR8-defizienten Tieren vor dem Zelltod geschützt, was die Abhängigkeit der HERV-K induzierten Neurotoxizität von Tlr7 und TLR8 in vivo beweist. Die vorherige Verabreichung eines spezifischen HERV-K LNA-Inhibitors konnte den neurotoxischen Effekte des HERV-K sowohl in vitro wie auch in vivo vorbeu- gen und Neurodegeneration verhindern. Die Expression von HERV-K(HML-2) ist größtenteils durch epigenetische Mechanismen unterdrückt, wird in manchen Ge- weben wie dem Gehirn aber ubiquitär exprimiert (Flockerzi et al., 2008; Mayer et al., 2018). Durch Schädigungen oder Erkrankungen kann diese Inhibierung je- doch aufgehoben werden und somit die Bildung von Proviren ermöglichen. So konnte in der humanen Neuronenzelllinie SH-SY5Y eine HERV-K Expression de- tektiert und auch durch induzierten Zelltod eine Sezernierung von HERV-K in das Medium nachgewiesen werden. Die isolierten sezernierten Partikel konnten eben- falls eine Tlr7-abhängige Neurodegeneration in vitro in primären Neuronen indu- zieren. Zur Überprüfung einer möglichen Beteiligung an einer neurodegenerativen Erkrankung wurden die CSFs von Kontrol-, FTD- und AD- Patient*innen auf eine mögliche Expression von HERV-K(HML-2) untersucht. Hierbei konnte in über 50 % der AD-Patient*innen eine HERV-K(HML-2) Expression nachgewiesen werden. Dieses Ergebnis zeigt, dass eine Beteiligung von HERV-K an der Pathologie der Alzheimer Krankheiten wahrscheinlich ist.Endogenous retroviral elements account for about 8 % of the human genome, yet very little is known about their function and biological significance. Some evidence suggests a direct involvement of certain human endogenous retroviruses (HERVs) in neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) or Mul- tiple Sclerosis (MS). In this study, it was demonstrated for the first time that HERV-K(HML-2) env RNA contains the TLR7/8 binding motif (GUUGUGU) and induces neurodege- neration by binding to human TLR8 and murine Tlr7, which are expressed also in neurons. This neurotoxicity is generated in a cell-autonomous manner through binding to Tlr7/TLR8 in neurons and is further amplified in the presence of mi- croglial cells. The induction of neurotoxicity occurs in neurons via the adapter protein SARM1 and not, as in microglia, via MyD88. The neuronal damage cau- sed by HERV-K was demonstrated both in vitro and in vivo, depending on the presence of Tlr7 and TLR8. The increased expression of HERV-K env RNA in the cerebrospinal fluid (CSF) of Alzheimer’s patients suggests a role of HERV-K in the progression of this disease. Based on these results, a model was developed that illustrates the involvement of HERV-K in neurodegenerative diseases such as Alzheimer’s disease. HERV-K env RNA contains the well-studied recognition motif for TLR7 - GUU- GUGU. Therefore, the potential of this RNA to bind and activate Tlr7 in mice and TLR in humans was initially investigated in more detail. HERV-K activates murine microglia and induces neurotoxicity in primary neuron cultures depending on Tlr7. The cell-autonomous neurodegeneration was confirmed using the neuro- nal cell lines N1E-115 and SH-SY5Y. Cell loss after stimulation with HERV-K was also observed here. The HERV-K-induced neuronal cell death is time- and dose-dependent. In human neurons, the HERV-K neurotoxicity is mediated via TLR8, as experi- ments with the THP-1 and TLR8 overexpressing HEK reporter cell line showed. Human TLR7 as well as murine Tlr8 showed no activation after HERV-K stimu- lation. MST measurements indicate a direct binding of HERV-K RNA to TLR8. Thus, HERV-K appears to be a potential new endogenous ligand for mTlr7 and hTLR8. Furthermore, this study showed that the neurotoxic effects are also indu- 3 ced in vivo by HERV-K via Tlr7 and TLR8. Intrathecal injections of HERV-K led to significant neuronal death in the cerebral cortex, which was not observed with the control oligoribonucleotides from HERV-K env (HERV-neg.) and a scrambled oligo (#4) without the GUUGUGU motif. The necessity of Tlr7 or TLR8 expres- sion in vivo was confirmed by using in-utero electroporation. For this, mTlr7 and hTLR8 were introduced into the brains of 14.5-day-old Tlr7-/- mouse embryos, and HERV-K was administered intrathecally 19 days postnatal. The immunohistoche- mical analysis of brain sections showed significant neuronal damage and cell death in the animals transfected with Tlr7 and TLR8. In contrast, the neuronal cells in the Tlr7- and TLR8-deficient animals were protected from cell death, demonstra- ting the dependence of HERV-K-induced neurotoxicity on Tlr7 and TLR8 in vivo. The prior administration of a specific HERV-K LNA inhibitor was able to prevent the neurotoxic effects of HERV-K both in vitro and in vivo. The expression of HERV-K(HML-2) is largely suppressed by epigenetic mecha- nisms but is ubiquitously expressed in some tissues such as the brain. (Flockerzi et al., 2008; Mayer et al., 2018). However, this strong regulation can be rever- sed due to damage or diseases, thus enabling the formation of proviruses. In the human neuronal cell line SH-SY5Y, HERV-K expression was detected, and the se- cretion of HERV-K into the medium was demonstrated through induced cell death. The isolated secreted retroviral particles were also able to induce Tlr7-dependent neurodegeneration in primary neurons in vitro. To investigate a possible HERV- K involvement in a neurodegenerative disease, the cerebrospinal fluids (CSFs) of control, FTD, and AD patients were examined for possible expression of HERV- K(HML-2). In over 50 % of the AD patients, HERV-K(HML-2) expression was detected. This result indicates that the involvement of HERV-K in the pathology of Alzheimer’s disease is most likely

Human endogenous retrovirus HERV-K(HML-2) RNA causes neurodegeneration through Toll-like receptors

Full author list omitted for brevity. For the full list of authors, see article.Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs, such as HERV-K(HML-2), are reported to be involved in neurological disorders, little is known about their biological function. We report that RNA from an HERV-K(HML-2) envelope gene region binds to and activates human Toll-like receptor (TLR) 8, as well as murine Tlr7, expressed in neurons and microglia, thereby causing neurodegeneration. HERV-K(HML-2) RNA introduced into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimer's disease (AD), resulted in neurodegeneration and microglia accumulation. Tlr7-deficient mice were protected against neurodegenerative effects but were resensitized toward HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Transcriptome data sets of human AD brain samples revealed a distinct correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from individuals with AD compared with controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for species-specific TLRs 7/8 and imply a functional contribution of human endogenous retroviral transcripts to neurodegenerative processes, such as AD

IL-17+ γδ T cells are neurotoxic <i>in vitro</i>.

<p><b>(A-E)</b> γδ T cells were isolated from C57BL/6J mice and cultured with IL-1β (10 ng/ml), IL-23 (10 ng/ml), anti-CD3 (1μg/ml) and anti-CD28 (10μg/ml) to induce IL-17+ γδ T cells. After 3 days, polarized IL-17+ γδ T cells with their culture media or supernatants only were co-cultured with cortical neurons for 72 h. Neuronal cultures without the addition of γδ T cells served as a control. To evaluate neuronal viability cultures were subsequently immunostained with antibodies against neuronal nuclei (NeuN), neurofilament (NF) to mark neurons (both in red), CD3 to mark γδ T cells (green) and DAPI (blue). Representative images are shown for co-culture with 1x10⁵ polarized γδ T cells after 72 h, in <b>(A)</b> magnification 20x, scalebar 100μm, in <b>(D)</b> magnification 100x, scalebar 50μm, white arrowheads indicate apoptotic nuclei. In <b>(B, C)</b> γδ T cells were isolated, polarized, and co-cultured as described above at indicated concentrations for 72 h or 1x10⁵ polarized γδ T cells were co-cultured for up to 4 days. (<b>E)</b> Quantification of DAPI+ nuclei displaying apoptotic hallmarks. <b>(F, G)</b> Primary cortical neurons were incubated with recombinant IL-17 for 72 h at indicated concentrations or with 50 ng/ml for indicated time points. Neurons treated with imiquimod (10 μg/ml) or LPS (100 ng/ml) served as a positive and negative control, respectively. Cultures were then stained with NeuN Ab and DAPI. Each condition was performed in duplicate and averaged. NeuN-positive cells were quantified and expressed as relative neuronal viability. Mean ± SEM of 3–5 individual experiments, ANOVA with Dunnett´s multiple comparison post test of each time point/condition <i>vs</i>. control, (B) <i>p</i><0.0001, (C) <i>p</i> = 0.0032, (E) <i>p</i> = 0.0151, (F) <i>p</i> = 0.7851, (G) <i>p</i> = 0.0064.</p

Supernatants derived from microglia stimulated through TLRs activate naïve γδ T cells and induce expression of IL-17, but not IFN-γ.

<p><b>(A)</b> Microglia were stimulated with the TLR ligands Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), imiquimod (10 μg/ml), or CpG (1 μM) for 24 h. Microglia-conditioned supernatants were transferred to freshly isolated naïve γδ T cells, or γδ T cells were directly stimulated with the TLR ligands. Unstimulated cells served as a control. After 2 days, γδ T cells were collected and analyzed by flow cytometry regarding CD3, CD25, CD69, and CD62L expression. Each condition was performed in duplicate and averaged. Mean ± SEM of 3 to 9 individual experiments. <b>(B, C)</b> Microglia were stimulated for 24 h with TLR ligands as described in <b>(A)</b> for 24 h. Subsequently, either the microglia-conditioned supernatants were transferred to freshly isolated naïve γδ T cells or γδ T cells were co-cultured with both microglia and their supernatant. After 3 days, γδ T cells were harvested, restimulated with PMA/ionomycin, and analyzed by flow cytometry for intracellular IFN-γ and IL-17 expression. <b>(C)</b> Each condition was performed in duplicates and averaged. Mean ± SEM of 4 individual experiments. <b>(D)</b> γδ T cells were cultured with microglia-conditioned supernatant, as described in <b>(A).</b> After indicated time points supernatants were analyzed by ELISA regarding IL-17 production. Each condition was performed in duplicates and averaged. Mean ± SEM of 3 to 7 experiments. <b>(E)</b> Overview of Vγ-chain usage (Vγ1.1, Vγ2, Vγ3 and Vγ5) found on IL-17+ γδ T cells activated by supernatants derived from TLR-stimulated microglia. Mean ± SEM of 3 individual experiments. <b>(F)</b> Bone marrow-derived macrophages (BMDMs) were stimulated for 24 h with various TLR ligands as named in <b>(A)</b>. BMDM-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. γδ T cells were collected after two days and analyzed by flow cytometry regarding CD3, CD25, CD69, and CD62L expression, and supernatants were collected after one, 2 and 3 days, and analyzed regarding the presence of IL-17 by ELISA <b>(G)</b>. Each condition was performed in duplicate and averaged. Mean ± SEM of 4 to 5 individual experiments. <b>(A)</b>, <b>(C)</b> and <b>(F)</b> ANOVA with Dunnett´s multiple comparison post test of each ligand <i>vs</i>. unstimulated control, (A) <i>p</i> = 0.7198, <i>p</i><0.0001, <i>p</i> = 0.9415, <i>p</i><0.0001, <i>p</i> = 0.9707, <i>p</i> = 0.0001, (C) <i>p</i> = 0.0061, <i>p</i> = 0.9883, <i>p</i> = 0.2590, <i>p</i> = 0.1599, (E) <i>p</i><0.0001, <i>p</i> = 0.0004, <i>p</i> = 0.0521. <b>(D)</b> and <b>(G)</b> 2-way ANOVA with Bonferroni post test compared to unstimulated control; <i>p</i>*<0.05, <i>p</i>***<0.001.</p