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

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\u27s 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

Naïve γδ T cells express TLRs but do not secrete IL-17 in response to TLR stimulation.

<p><b>(A, B)</b> γδ T cells were isolated from C57BL/6J mice and stained at their cell surface (TLR1, TLR2, TLR4) or intracellularly (TLR7, TLR9, MyD88) with antibodies directed against the indicated TLR (solid line) and the respective isotype control (dotted line). Data are displayed as delta (Δ) mean fluorescence intensity (MFI) of the specific antibody in relation to the isotype control ± SEM from 3 to 5 individual experiments. <b>(C, D)</b> γδ T cells were isolated from C57BL/6J mice and stimulated with the TLR ligands Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), imiquimod (10 μg/ml), CpG (1 μM), or IL-1β and IL-23 (10 ng/ml each). Unstimulated cells served as a negative control. After 3 days, cells were re-stimulated and analyzed by flow cytometry for intracellular IFN-γ and IL-17 expression. Results in <b>(D)</b> are shown as mean ± SEM of 3 experiments, ANOVA with Dunnett´s multiple comparison post test of each ligand <i>vs</i>. unstimulated control. <b>(E)</b> γδ T cells were stimulated as described in <b>(C)</b>, but supernatants were collected directly after 3 days and analyzed by IL-17 ELISA. Results are shown as mean ± SEM of 3 individual experiments, ANOVA with Dunnett´s multiple comparison post test of each ligand <i>vs</i>. unstimulated control, (D) <i>p</i> = 0.3262, <i>p</i> = 0.0355, (E) <i>p</i> = 0.4647.</p

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 from microglia stimulated via TLRs induce activation patterns alike to IL-17+ γδ T cells dependent on MyD88 expressed in microglia and in γδ T cells.

<p><b>(A)</b> Wild-type (WT) or MyD88KO microglia were stimulated for 24 h with the TLR ligands Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), imiquimod (10 μg/ml) or CpG (1 μM). Microglia-conditioned supernatants were transferred to freshly isolated naïve WT, TLR2KO, TLR7KO, or MyD88KO γδ T cells. After 3 days, supernatants were collected and analyzed regarding IL-17 by ELISA. Mean ± SEM of 3–4 individual experiments. <b>(B, C)</b> WT microglia were stimulated as in <b>(A)</b> and microglia-conditioned supernatants were either used for cytokine analysis or transferred to freshly isolated naïve γδ T cells. Also, naïve γδ T cells were directly stimulated with TLR ligands as indicated or IL-1β, IL-23, anti-CD3, and anti-CD28. After 3 days, supernatants were analyzed by bead based multiplex assay or ELISA for indicated cytokines. Mean ± SEM of 3 individual experiments. Amounts of IL-1β and IL-18 were analyzed by ELISA for n = 6 individual experiments. <b>(D)</b> Naïve γδ T cells were directly analyzed. <i>In vitro</i> polarized IL-17+ γδ T cells were harvested after 3 days in culture with IL-1β, IL-23, anti-CD3, and anti-CD28, re-stimulated with PMA/ionomycin, and analyzed by flow cytometry for intracellular granzyme B expression. Representative FACS plots of n = 3 individual experiments are shown. <b>(E, F)</b> WT microglia were stimulated as in <b>(A)</b> and microglia-conditioned supernatants were preincubated with 10 μg/ml anti-IL-1β, anti-IL-23 or respective isotype controls before transfer to naïve γδ T cells. After 2 days, γδ T cells were collected and analyzed by flow cytometry regarding CD3, CD25, CD69, and CD62L expression <b>(E)</b>. After 3 days supernatants were analyzed by ELISA regarding IL-17 secretion <b>(F)</b>. Each condition was performed in duplicate and averaged. Mean ± SEM of 4 individual experiments. <b>(A, B, C, E, F)</b> ANOVA followed by Bonferroni multiple comparison post test, (A) TLR2KO <i>p</i> = 0.3340, TLR7KO <i>p</i> = 0.0989, (B) IL-6 <i>p</i> = 0.0705, IL-23 <i>p</i> = 0.5709, IL-1β <i>p</i> = 0.0011, IL-18 <i>p</i> = 0.7380, (C) IL-13 i = 0.0148, IL-17 <i>p</i><0.0001, IL-22 <i>p</i><0.0001, IFN-γ <i>p</i> = 0.0001, granzyme B <i>p</i> = 0.4176, (E) <i>p</i><0.0001.</p

Microglia Induce Neurotoxic IL-17+ γδ T Cells Dependent on TLR2, TLR4, and TLR9 Activation

<div><p>Background</p><p>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.</p><p>Objective</p><p>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.</p><p>Results</p><p>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 <i>in vitro</i>. 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 <i>in vitro</i>. 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.</p><p>Conclusion</p><p>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.</p></div

Supernatants from microglia stimulated via TLR2, TLR4, or TLR9 induce neurotoxic γδ T cells.

<p><b>(A)</b> Microglia were stimulated with Pam3CysSK4 (100 ng/ml) LPS (100 ng/ml) or CpG (1 μM) for 24 h. Unstimulated cells served as a control. Microglia-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. After 3 days, γδ T cells or microglia-conditioned supernatant only were supplemented with cortical neurons for additional 5 days. Neuronal cultures without γδ T cells in the presence of Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), CpG (1 μM) or imiquimod (10 μg/ml) alone, served as controls. Subsequently, cultures were immunostained with antibodies against CD3 (γδ T cells, green), NeuN, and neurofilament (neurons, red), magnification 100x, scale bar 50 μm. In <b>(C)</b> 1*10⁵ microglia were added to neuronal cultures concurrent with γδ T cells. <b>(D)</b> BMDMs were stimulated with Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml) or CpG (1 μM) for 24 h. Unstimulated cells served as a control. BMDM-conditioned supernatants were transferred to freshly isolated naïve γδ T cells. After 3 days, γδ T cells or microglia-conditioned supernatant only were supplemented with cortical neurons for additional 5 days. Neuronal cultures without γδ T cells in the presence of Pam3CysSK4 (100 ng/ml), LPS (100 ng/ml), CpG (1 μM) or imiquimod (10 μg/ml) alone served as controls. Subsequently, cultures were immunostained as in <b>(A)</b>. <b>(B, C, D)</b> NeuN-positive cells were quantified and expressed as relative neuronal viability. Each condition was performed in duplicate and averaged. Mean ± SEM of 3–5 individual experiments with ANOVA followed by Bonferroni multiple comparison post test, (B) <i>p</i><0.0001, (C) <i>p</i><0.0001, (D) <i>p</i><0.0001.</p