Spatial transcriptomics and neurofilament light chain reveal changes in lesion patterns in murine autoimmune neuroinflammation.

OBJECTIVE: Ongoing neuroaxonal damage is a major contributor to disease progression and long-term disability in multiple sclerosis. However, spatio-temporal distribution and pathophysiological mechanisms of neuroaxonal damage during acute relapses and later chronic disease stages remain poorly understood. METHODS: Here, we applied immunohistochemistry, single-molecule array, spatial transcriptomics, and microglia/axon co-cultures to gain insight into spatio-temporal neuroaxonal damage in experimental autoimmune encephalomyelitis (EAE). RESULTS: Association of spinal cord white matter lesions and blood-based neurofilament light (sNfL) levels revealed a distinct, stage-dependent anatomical pattern of neuroaxonal damage: in chronic EAE, sNfL levels were predominately associated with anterolateral lumbar lesions, whereas in early EAE sNfL showed no correlation with lesions in any anatomical location. Furthermore, neuroaxonal damage in late EAE was largely confined to white matter lesions but showed a widespread distribution in early EAE. Following this pattern of neuroaxonal damage, spatial transcriptomics revealed a widespread cyto- and chemokine response at early disease stages, whereas late EAE was characterized by a prominent glial cell accumulation in white matter lesions. These findings were corroborated by immunohistochemistry and microglia/axon co-cultures, which further revealed a strong association between CNS myeloid cell activation and neuroaxonal damage both in vivo and in vitro. INTERPRETATION: Our findings indicate that CNS myeloid cells may play a crucial role in driving neuroaxonal damage in EAE. Moreover, neuroaxonal damage can progress in a stage-dependent centripetal manner, transitioning from normal-appearing white matter to focal white matter lesions. These insights may contribute to a better understanding of neurodegeneration and elevated sNfL levels observed in multiple sclerosis patients at different disease stages.

Activation of non-classical NMDA receptors by glycine impairs barrier function of brain endothelial cells.

Blood-brain barrier (BBB) integrity is necessary to maintain homeostasis of the central nervous system (CNS). NMDA receptor (NMDAR) function and expression have been implicated in BBB integrity. However, as evidenced in neuroinflammatory conditions, BBB disruption contributes to immune cell infiltration and propagation of inflammatory pathways. Currently, our understanding of the pathophysiological role of NMDAR signaling on endothelial cells remains incomplete. Thus, we investigated NMDAR function on primary mouse brain microvascular endothelial cells (MBMECs). We detected glycine-responsive NMDAR channels, composed of functional GluN1, GluN2A and GluN3A subunits. Importantly, application of glycine alone, but not glutamate, was sufficient to induce NMDAR-mediated currents and an increase in intracellular Ca2+ concentrations. Functionally, glycine-mediated NMDAR activation leads to loss of BBB integrity and changes in actin distribution. Treatment of oocytes that express NMDARs composed of different subunits, with GluN1 and GluN3A binding site inhibitors, resulted in abrogation of NMDAR signaling as measured by two-electrode voltage clamp (TEVC). This effect was only detected in the presence of the GluN2A subunits, suggesting the latter as prerequisite for pharmacological modulation of NMDARs on brain endothelial cells. Taken together, our findings argue for a novel role of glycine as NMDAR ligand on endothelial cells shaping BBB integrity.

Pro-inflammatory T helper 17 directly harms oligodendrocytes in neuroinflammation.

T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes, and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate, which induced cell stress and changes in biosynthesis of cholesterol and lipids, as revealed by single-cell RNA-sequencing analysis. Finally, exposure to glutamate decreased myelination, whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities in MS.

The frequency of follicular T helper cells differs in acute and chronic neuroinflammation.

Beyond the major role of T cells in the pathogenesis of the autoimmune neuroinflammatory disorder multiple sclerosis (MS), recent studies have highlighted the impact of B cells on pathogenic inflammatory processes. Follicular T helper cells (Tfh) are essential for the promotion of B cell-driven immune responses. However, their role in MS and its murine model, experimental autoimmune encephalomyelitis (EAE), is poorly investigated. A first step to achieving a better understanding of the contribution of Tfh cells to the disease is the consideration of Tfh cell localization in relation to genetic background and EAE induction method. Here, we investigated the Tfh cell distribution during disease progression in disease relevant organs in three different EAE models. An increase of Tfh frequency in the central nervous system (CNS) was observed during peak of C57BL/6 J EAE, paralleling chronic disease activity, whereas in relapsing-remitting SJL EAE mice Tfh cell frequencies were increased during remission. Furthermore, transferred Tfh-skewed cells polarized in vitro induced mild clinical symptoms in B6.Rag1-/- mice. We identified significantly higher levels of Tfh cells in the dura mater than in the CNS both in C57BL/6 and in SJL/J mice. Overall, our study emphasizes diverse, non-static roles of Tfh cells during autoimmune neuroinflammation.

CNS-localized myeloid cells capture living invading T cells during neuroinflammation.

To study the role of myeloid cells in the central nervous system (CNS) in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), we used intravital microscopy, assessing local cellular interactions in vivo in EAE animals and ex vivo in organotypic hippocampal slice cultures. We discovered that myeloid cells actively engulf invading living Th17 lymphocytes, a process mediated by expression of activation-dependent lectin and its T cell-binding partner, N-acetyl-D-glucosamine (GlcNAc). Stable engulfment resulted in the death of the engulfed cells, and, remarkably, enhancement of GlcNAc exposure on T cells in the CNS ameliorated clinical EAE symptoms. These findings demonstrate the ability of myeloid cells to directly react to pathogenic T cell infiltration by engulfing living T cells. Amelioration of EAE via GlcNAc treatment suggests a novel first-defense pathway of myeloid cells as an initial response to CNS invasion and demonstrates that T cell engulfment by myeloid cells can be therapeutically exploited in vivo.

ß1-Integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells.

Although the impact of Th17 cells on autoimmunity is undisputable, their pathogenic effector mechanism is still enigmatic. We discovered soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex proteins in Th17 cells that enable a vesicular glutamate release pathway that induces local intracytoplasmic calcium release and subsequent damage in neurons. This pathway is glutamine dependent and triggered by binding of ß1-integrin to vascular cell adhesion molecule 1 (VCAM-1) on neurons in the inflammatory context. Glutamate secretion could be blocked by inhibiting either glutaminase or KV1.3 channels, which are known to be linked to integrin expression and highly expressed on stimulated T cells. Although KV1.3 is not expressed in CNS tissue, intrathecal administration of a KV1.3 channel blocker or a glutaminase inhibitor ameliorated disability in experimental neuroinflammation. In humans, T cells from patients with multiple sclerosis secreted higher levels of glutamate, and cerebrospinal fluid glutamine levels were increased. Altogether, our findings demonstrate that ß1-integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells upon direct cell-cell contact between Th17 cells and neurons.

Neuronal ICAM-5 Plays a Neuroprotective Role in Progressive Neurodegeneration.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) leading to CNS inflammation and neurodegeneration. Current anti-inflammatory drugs have only limited efficacy on progressive neurodegenerative processes underlining the need to understand immune-mediated neuronal injury. Cell adhesion molecules play an important role for immune cell migration over the blood-brain barrier whereas their role in mediating potentially harmful contacts between invading immune cells and neurons is incompletely understood. Here, we assess the role of the CNS-specific neuronal adhesion molecule ICAM-5 using experimental autoimmune encephalomyelitis (EAE), an animal model of MS. ICAM-5 knockout mice show a more severe EAE disease course in the chronic phase indicating a neuroprotective function of ICAM-5 in progressive neurodegeneration. In agreement with the predominant CNS-specific function of ICAM-5, lymphocyte function-associated antigen 1 (LFA-1)/ICAM-1 contact between antigen-presenting cells and T helper (Th)17 cells in EAE is not affected by ICAM-5. Strikingly, intrathecal application of the shed soluble form, sICAM-5, ameliorates EAE disease symptoms and thus might serve locally as an endogenous neuronal defense mechanism which is activated upon neuroinflammation in the CNS. In humans, cerebrospinal fluid from patients suffering from progressive forms of MS shows decreased sICAM-5 levels, suggesting a lack of this endogenous protective pathway in these patient groups. Overall, our study points toward a novel role of ICAM-5 in CNS autoinflammation in progressive EAE/MS.

The Role of ERK Signaling in Experimental Autoimmune Encephalomyelitis.

Extracellular signal-regulated kinase (ERK) signaling plays a crucial role in regulating immune cell function and has been implicated in autoimmune disorders. To date, all commercially available inhibitors of ERK target upstream components, such as mitogen-activated protein (MAP) kinase/ERK kinase (MEKs), but not ERK itself. Here, we directly inhibit nuclear ERK translocation by a novel pharmacological approach (Glu-Pro-Glu (EPE) peptide), leading to an increase in cytosolic ERK phosphorylation during T helper (Th)17 cell differentiation. This was accompanied by diminished secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine influencing the encephalitogenicity of Th17 cells. Neither the production of the cytokine interleukin (IL)-17 nor the proliferation rate of T cells was affected by the EPE peptide. The in vivo effects of ERK inhibition were challenged in two independent variants of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Overall, ERK inhibition had only a very minor impact on the clinical disease course of EAE. This indicates that while ERK translocation might promote encephalitogenicity in T cells in vitro by facilitating GM-CSF production, this effect is overcome in more complex in vivo animal models of central nervous system (CNS) autoimmunity.

Protein kinase CK2 governs the molecular decision between encephalitogenic TH17 cell and Treg cell development.

T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance. Thus, the balance between Treg cells and TH17 cells determines the severity of a TH17 cell-driven disease and therefore is a promising target for treating autoimmune diseases. However, the molecular mechanisms controlling this balance are still unclear. Here, we report that pharmacological inhibition as well as genetic ablation of the protein kinase CK2 (CK2) ameliorates experimental autoimmune encephalomyelitis (EAE) severity and relapse incidence. Furthermore, CK2 inhibition or genetic ablation prevents TH17 cell development and promotes the generation of Treg cells. Molecularly, inhibition of CK2 leads to reduced STAT3 phosphorylation and strongly attenuated expression of the IL-23 receptor, IL-17, and GM-CSF. Thus, these results identify CK2 as a nodal point in TH17 cell development and suggest this kinase as a potential therapeutic target to treat TH17 cell-driven autoimmune responses.

Increase of Alternatively Activated Antigen Presenting Cells in Active Experimental Autoimmune Encephalomyelitis.

The importance of CD11c+ antigen-presenting cells (APCs) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) is well accepted and the gate keeper function of perivascular CD11c+ APCs has been demonstrated. CD11c can be expressed by APCs from external sources or by central nervous system (CNS) resident APCs such as microglia. Yet, changes in the gene expression pattern of CNS CD11c+ APCs during disease are still unclear and differentially expressed genes might play a decisive role in EAE progression. Due to their low numbers in the diseased brain and due to the absence of considerable numbers in the healthy CNS, analysis of CNS CD11c+ cells is technically difficult. To ask whether the CD11c+ APC population contributes to remission of EAE disease, we used Illumina deep mRNA sequencing (RNA-Seq) and quantitative real time polymerase chain reaction (qRT-PCR) analyses to identify the transcriptome of CD11c+ APCs during disease course. We identified a battery of genes that were significantly regulated during the exacerbation of the disease compared to remission and relapse. Three of these genes, Arginase-1, Chi3l3 and Ms4a8a, showed a higher expression at the exacerbation than at later time points during the disease, both in SJL/J and in C57BL/6 mice, and could be attributed to alternatively activated APCs. Expression of Arginase-1, Chi3l3 and Ms4a8a genes was linked to the disease phase of EAE rather than to disease score. Expression of these genes suggested that APCs resembling alternatively activated macrophages are involved during the first wave of neuroinflammation and can be directly associated with the disease progress.