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

Expression of Toll-Like Receptors in the Developing Brain

Toll-like receptors (TLR) are key players of the innate and adaptive immune response in vertebrates. The original protein Toll in Drosophila melanogaster regulates both host defense and morphogenesis during development. Making use of real-time PCR, in situ hybridization, and immunohistochemistry we systematically examined the expression of TLR1–9 and the intracellular adaptor molecules MyD88 and TRIF during development of the mouse brain. Expression of TLR7 and TLR9 in the brain was strongly regulated during different embryonic, postnatal, and adult stages. In contrast, expression of TLR1–6, TLR8, MyD88, and TRIF mRNA displayed no significant changes in the different phases of brain development. Neurons of various brain regions including the neocortex and the hippocampus were identified as the main cell type expressing both TLR7 and TLR9 in the developing brain. Taken together, our data reveal specific expression patterns of distinct TLRs in the developing mouse brain and lay the foundation for further investigation of the pathophysiological significance of these receptors for developmental processes in the central nervous system of vertebrates

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

Antigenpräsentation und Aktivierung von T-Zellen in der Leber

Die Ätiologie und Pathogenese autoimmuner Lebererkrankungen sind nur unvollständig verstanden. Bei der primär sklerosierenden Cholangitis bzw. bei der autoimmunen Hepatitis sind Cholangiozyten der größeren Gallengänge und Hepatozyten die Zielzellen der Autoimmunreaktion in der Leber. Mausmodelle sind zur Analyse initialer pathophysiologischer Prozesse notwendig und tragen zum besseren Verständnis der immunologischen Vorgänge in der Leber bei. Mit Hilfe transgener Mauslinien, die das Modellantigen Ovalbumin gewebespezifisch in den Cholangiozyten (ASBT-OVA) oder in den Hepatozyten (TF-OVA) exprimieren, sowie adoptiven Transfers antigenspezifischer CD4+ und CD8+ T-Zellen wurden Untersuchungen zur Antigenpräsentation, T-Zell-Aktivierung und Toleranzinduktion in der Leber durchgeführt. Die Expression von Ovalbumin in Cholangiozyten resultierte in einer Aktivierung der CD8+ T-Zellen in der Leber und den Lymphknoten. Im Gegensatz dazu ignorierten naive CD4+ T-Zellen das Antigen und wurden nicht aktiviert. Die Expression von Ovalbumin in Hepatozyten resultierte in einer vollständigen Aktivierung der CD8+ T-Zellen zu Effektorzellen über Kreuzpräsentation durch professionelle antigenpräsentierende Zellen (APZ) in der Leber. Diese Aktivierung war transient und selbst-limitiert. Die Induktion von CD4+Foxp3+ regulatorischen T-Zellen trug entscheidend zur Limitierung der induzierten Autoimmunität und Kontrolle der Expansion von antigenspezifischen CD8+ T-Zellen bei. Naive CD4+ T-Zellen benötigten die Aktivierung durch APZ in einem anderen Organ, bevor sie in die Leber relokalisierten und wiesen keinen Effektorphänotyp auf. Beide Modelle repräsentieren nicht die chronische Eigenschaft humaner autoimmuner Lebererkrankungen, ermöglichen jedoch Untersuchungen zum besseren Verständnis der Rolle verschiedener T-Zell-Populationen in der Pathogenese autoimmuner Lebererkrankungen sowie der Antigenpräsentation und Aktivierung von T-Zellen durch hepatisches Antigen.Aetiology and pathogenesis of autoimmune liver diseases are still incompletely understood. Cholangiocytes of the larger bile ducts and hepatocytes are the target structures of autoimmune reactions in the liver in primary sclerosing cholangitis and autoimmune hepatitis, respectively. Mouse models are necessary to analyse initial pathophysiological processes and contribute to a better understanding of immunological processes in the liver. With the help of transgenic mouse strains, in which the model antigen ovalbumin is expressed specifically in the cholangiocytes (ASBT-OVA) or in hepatocytes (TF-OVA), as well as the adoptive transfer of antigen specific CD4+ and CD8+ T cells, antigen presentation, T cell activation and tolerance induction in the liver, were analyzed. Expression of ovalbumin in cholangiocytes resulted in activation of CD8+ T cells in the liver and lymph nodes. In contrary, naïve antigen specific CD4+ T cells ignored the antigen expressed by cholangiocytes and were not activated. Expression of ovalbumin in hepatocytes resulted in complete activation of CD8+ T cells to become effector cells by crosspresentation depending on professional antigen presenting cells (APCs) in the liver. This activation was transient and self-limiting. Induction of CD4+Foxp3+ regulatory T cells played a crucial role in limiting autoimmunity and controlling the expansion of antigen specific CD8+ T cells in the liver. By contrast, naïve CD4+ T cells required activation by professional APCs in a different organ before relocating to the liver and did not display an effector phenotype. Both models do not represent the chronic characteristics of human autoimmune liver diseases, but help to gain a better understanding regarding the role of specific T cell populations in the pathogenesis of autoimmune liver diseases, as well as regarding antigen presentation and activation of T cells by hepatic antigen

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

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

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

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