Immune cells play an important role in the pathogenesis of MS. However, our knowledge of the diversity of immune cell types and states in health and disease has been limited by the restrictions of traditional oligo-marker immunological approaches. Single-cell RNA sequencing (scRNA-seq) provides new means for discovery and characterization of immune cells by measuring expression levels of thousands of genes simultaneously at the single cell level.
Here, we investigated the immune cell composition of cerebrospinal fluid (CSF), as the most accessible immune compartment in contact with the central nervous system, using scRNA-seq in MS and non-MS neuroinflammatory conditions (ONID).
Fresh CSF samples were collected at the time of diagnostic lumbar puncture from 8 untreated RRMS and 7 ONID patients. In addition, to examine the context of the reconstituted CSF after ocrelizumab treatment, we sampled 6 progressive MS patients on ocrelizumab for >1 year, 5 months after their last dose. A replication set of 3 untreated RRMS and 3 ONID are also collected.
After removing doublets, RBCs, and low-quality cells, 59,288 cells were remained for analysis. Data were integrated using canonical correlation analysis. Over-clustering using ‘Seurat’ and iterative merging of clusters with more similar gene expression using ‘SCCAF’ resulted in 13 major clusters: 1 αβ-T cell cluster (consisting of 24 subclusters), 4 other T (NK, NKT, γδ-T), 5 myeloid (microglia-like monocytic, myeloid DC, granulocytes), B cells, plasmablasts and plasmacytoid DC.
Consistent with earlier smaller studies, we observed subsets of myeloid cells with microglia-like features (3% frequency). Using our reference brain-derived microglia scRNA-seq dataset (221,126 cells), we have characterized the microglial subtypes to which the CSF cells most resemble. Interestingly, these are part of the 4 myeloid subtypes which percentage were reduced in newly diagnosed RRMS relative to ONID. In addition, 3 CD4+ and 1 CD8+ memory T cell subsets were increased in RRMS. If replicated, these intriguing differences could guide our understanding of earliest stages of MS vs. other elements of the differential.
B cell component was largely reconstituted in the ocrelizumab group, and the patients had a CD4+ and CD8+ T cell pattern largely similar to ONID (vs RRMS). However, myeloid cell percentages were increased even more in this group. Further studies are needed to resolve whether these changes are due to progression vs. age vs. ocrelizumab.
Our data offer a new level of resolution in cell population shifts and suggest that measuring certain combinations of subsets could be useful clinically. In addition to these results, we will present on gene-level expression differences and a first draft of a CSF molecular network map across the 3 conditions.
Results of B-cell depleting therapies have underscored important antibody-independent functions of B cells in multiple sclerosis (MS) pathogenesis. Bruton’s Tyrosine Kinase (BTK) is a key down-stream signaling molecule of the B cell receptor, and BTK inhibitors (BTKi) have been used for treating various B-cell malignancies. BTKi are now being pursued as a ‘next generation’ non-depleting approach to B-cell targeting in MS. However, the contributions of BTK to MS-relevant functional B-cell responses, and the impact of BTKi on such responses, remain largely unknown.
We would like to assess the impact of BTK inhibition on MS implicated B-cell functions and explore underlying mechanisms.
We applied a series of functional assays and RNA sequencing to human B cells, isolated from peripheral blood of healthy individuals or MS patients, as well as from human tonsils, to study influence of BTKi on B-cell survival, activation, proliferation, antibody production, antigen presenting functions, cytokine production and metabolism.
As expected, BTKi strongly decreased B-cell activation with minimal effects on B cell survival. BTKi also significantly limited the induction of co-stimulatory molecules (CD80, CD86) expression on activated B cells, which in turn resulted in a decreased capacity of the B cells to support both polyclonal as well as antigen-specific T-cell activation. Interestingly, BTKi treatment preferentially reduced pro-inflammatory B-cell cytokine (GM-CSF, TNFα and IL-6) secretion with only marginal influence on B-cell IL-10 production, resulting in a decreased capacity of the B cells to promote myeloid cell pro-inflammatory responses. Unbiased transcriptomic analysis of either vehicle- or BTKi-treated B cells suggested that BTKi could limit metabolism-related pathways, and subsequent seahorse analyzer experiments confirmed that BTKi decreased B-cell mitochondrial respiration and glycolysis. Metabolic manipulation further revealed that the level of mitochondrial respiration could control the balance between pro- and anti-inflammatory B cell responses.
BTKi preferentially limits pro-inflammatory B-cell responses that are implicated in MS pathophysiology. Our study also reveals a fundamental role for metabolism in regulating B-cell functions, and points to a novel therapeutic strategy targeting the balance between pro- and anti-inflammatory responses of B cells through modulation of their energy-utilization pathways.
Dysfunctional RNA binding proteins (RBPs), including heterogeneous nuclear ribonucleoprotein A1 (A1), are key pathological features of numerous neurologic diseases. Recently, evidence suggests that dysfunctional RBP biology, including mislocalization of the RBP from its homeostatic nuclear location to the cytoplasm, stress granule (SG) formation, and changes in RNA metabolism, are associated with neurodegeneration and worsened disease in multiple sclerosis (MS) and its models, such as experimental autoimmune encephalomyelitis (EAE).
We hypothesized that correcting dysfunctional RBP biology with the nuclear export inhibitor, KPT-276, would restore A1 function by decreasing its nucleocytoplasmic mislocalization and reduce SG formation and neurodegeneration in spinal cord neurons from EAE animals as compared to vehicle controls.
C57BL/6 female mice were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) to induce EAE. Upon reaching a clinical score of 2.5, mice were treated with KPT-276 (75 mg/kg, oral gavage) or vehicle control every other day for a total of six treatments. Two days following the final treatment, spinal cords were harvested and analyzed using immunofluorescence for A1 localization and SG formation in neurons as well as markers of neurodegeneration.
EAE mice treated with KPT-276 showed a decrease in clinical score severity as compared to vehicle control EAE mice. Furthermore, mice treated with KPT-276 showed a significant reduction in the number of neurons with A1 mislocalization (*p=0.0351), the amount of A1 in the cytoplasm (*p=0.0341), and SG formation (*p=0.0208) as compared to vehicle mice. In the same areas of KPT-276 treated mice where there was a reduction in these features of dysfunctional RBP biology, there was a significant increase in the number of neurons indicative of increased neuronal survival (*p=0.0373).
These experiments suggest that modulating dysfunctional RBP biology through KPT-276 restores RBP function and is a potential therapeutic, which increases neuronal survival and decreases neurodegeneration and clinical disease severity.
Emerging evidence from both human multiple sclerosis (MS) tissue and rodent experimental autoimmune encephalomyelitis (EAE) models has demonstrated that subsets of oligodendrocyte lineage cells are capable of expressing antigen presenting and processing molecules, including MHC class I and MHC class II molecules, in an inflammatory environment. Antigen presentation by inflammatory oligodendroglia (iOPCs/iOLs) to CD4 and CD8 T cells may result in cytotoxic death of oligodendroglia and perpetuation of the inflammatory response. However, the dynamics of MHC class I and MHC class II expression by oligodendrocyte lineage cells in an inflammatory environment are unknown.
To better define these dynamic changes in phenotype, we developed two new mouse reporter lines by targeting Beta-2-microglobulin (B2m) and CD74 invariant chain (Ii) genes, which are upregulated by oligodendroglia in human MS and mouse EAE. Beta-2-microglobulin (B2m) is a component of both classical and non-classical MHC class I molecules and CD74 invariant chain (Ii) is involved in occupancy of the peptide binding groove of MHC class II molecules in the endoplasmic reticulum and formation of stable MHC class II-antigen complexes.
We generated reporter lines by Crispr/Cas9-mediated insertion of a P2A-TdTomato-WPRE-pA sequence, replacing the stop codon of B2m (for class I reporter) and CD74 (for class II reporter).
Both B2m-TdT and CD74-TdT mice demonstrate robust reporter expression in cultured OPCs upon exposure to interferon gamma. In naïve CD74-TdT reporter mice, TdT reporter expression was not detected in the brain or spinal cord parenchyma, but strong expression was observed in peripheral immune cells in the meninges and choroid plexus. B2m-TdT reporter mice exhibited TdT reporter expression in microglia throughout the brain and spinal cord, and some expression in spinal cord white matter oligodendroglia near the meningeal surface. No TdT reporter expression was detected in brain oligodendroglia at baseline. Cortical stab injury in B2m-TdT reporter mice resulted in TdT reporter co-localization with Iba1+ microglia and Mac2+ macrophages within the lesion core and Olig2+CC1+ oligodendrocytes in the corpus callosum ipsilateral and contralateral to the stab injury.
Further investigation of MHC class I and MHC class II expression in oligodendroglia in the setting of inflammatory demyelinating models will help reveal the spatial and temporal dynamics of this phenotypic change in oligodendroglia.
Multiple Sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS) characterized by cortical demyelinating lesions containing activated microglia and phagocytic macrophages. The complex dynamics between microglia and oligodendrocytes during demyelination remain to be established. Bruton’s Tyrosine Kinase (BTK) is a key regulator of microglial phagocytosis; however, it is unclear whether modulation of BTK directly affects immune-mediated demyelination.
To visualize and manipulate in vivo microglia-oligodendrocyte interactions during cortical myelin loss and repair.
We developed a novel in vivo model of immune-mediated cortical demyelination through the application of recombinant antibodies derived from MS patients and human complement (MSrAb+huC’) onto the cortical surface. We characterized cellular interactions in real-time via longitudinal in vivo two-photon microscopy of myelinating oligodendrocytes and microglia in transgenic mice.
We found that MSrAb+huC’ application resulted in robust demyelination that recapitulated MS pathology. Microglia rapidly engulfed myelin sheaths following the application of MSrAb+huC’, and subsequently increased their density, accumulating around heavily affected oligodendrocytes. Oral administration of a brain-penetrant BTK-inhibitor prior to the application of MSrAb+huC’ drastically altered microglia dynamics in the 72 hours post-surgery, notably by diminishing engulfment morphology and density changes. Moreover, BTK-inhibition prevented the loss of oligodendrocytes.
Inhibition of BTK signaling alters microglia-oligodendrocyte interactions and limits complement-dependent antibody-mediated demyelination. These findings provide a novel context to define glial interactions during immune-regulated demyelination and outline a crucial role for microglia in driving myelin loss.