Augmentation of a neuroprotective myeloid state by hematopoietic cell transplantation

Abstract

Multiple sclerosis (MS) is an autoimmune disease associated with inflammatory demyelination in the central nervous system (CNS). Autologous hematopoietic cell transplantation (HCT) is under investigation as a promising therapy for treatment-refractory MS. Here we identify a reactive myeloid state in chronic experimental autoimmune encephalitis (EAE) mice and MS patients that is surprisingly associated with neuroprotection and immune suppression. HCT in EAE mice leads to an enhancement of this myeloid state, as well as clinical improvement, reduction of demyelinated lesions, suppression of cytotoxic T cells, and amelioration of reactive astrogliosis reflected in reduced expression of EAE-associated gene signatures in oligodendrocytes and astrocytes. Further enhancement of myeloid cell incorporation into the CNS following a modified HCT protocol results in an even more consistent therapeutic effect corroborated by additional amplification of HCT-induced transcriptional changes, underlining myeloid-derived beneficial effects in the chronic phase of EAE. Replacement or manipulation of CNS myeloid cells thus represents an intriguing therapeutic direction for inflammatory demyelinating disease.

Extended Data Figure 1

(a) Absolute number of nuclei per library. (b) Number of features and counts per library. (c) Annotated UMAP plot of 68,593 nuclei integrated from all 8 libraries. Clustering based on the first 20 principal components and a resolution of 0.6. (d) Dotplot demonstrating annotation and canonical marker genes of clusters. (e) UMAPs of individual libraries. (f) Upset plot showing significant DEGs (padj < 0.05) for main cell types between the Control and EAE condition. The top 15 intersections are illustrated.

Extended Data Figure 2

(a) GSEA-derived top 20 significantly enriched pathways (control vs EAE) of the Gene Ontology biological process term for the myeloid and astrocyte clusters. (b)+(c) Representative genes of selected enriched pathways

Extended Data Figure 3

(a) GSEA-derived top 20 significantly enriched pathways (control vs EAE) of the Gene Ontology biological process term for OLC clusters. (b) Representative genes of selected enriched pathways. (c) Immunofluorescent stain for Olig2 and complement component 4 (C4) in the spinal cord white matter. White arrows demonstrate spatial association between C4 and OLC. Scale bar is 20µm.

Extended Data Figure 4

(a) Absolute number of nuclei per original dataset. (b) Number of features and counts original dataset. (c) UMAP plots of 132,425 nuclei integrated from the original datasets. Clustering based on the first 20 principal components and a resolution of 0.6. Lesion-dependent conditions of the original datasets are shown (A_, Absinta; J_, Jäkel; S_, Schirmer; A, active; A/CA, acute/chronic active; CA, chronic active; CI, chronic inactive; remyel, remyelinating; wm, white matter), which were combined into 4 groups: control, active/chronic active (A/CA), chronic inactive (CI), and MS white matter (WM). (d) Dotplot demonstrating annotation and canonical marker genes of clusters. (e) Relative distribution of main cluster groups between different original datasets and control/MS condition. (f) Upset plot showing significant DEGs (padj < 0.05) for main cell types between the Control and MS condition. The top 10 intersections are illustrated.

Extended Data Figure 5

(a) Representative images of GFP+ chimerism in brain myeloid cells. (b) Representative immunofluorescent images of ramified myeloid cells in the brain. Brightness/contrast was adjusted individually per image for morphological assessment despite differing Iba1 intensity between conditions. Scale bar is 20µm. (c) Morphological analysis of ramified myeloid cells of the cortex in different conditions. Bars represent the group mean. Each dot represents one cell, dot colors represent different animals. Mann–Whitney U test; ns: p>0.05, *: p≤0.05, **: p≤0.01, ***: p≤0.001, ****: p≤0.0001.

Extended Data Figure 6

(a) Venn diagram showing significant DEGs (padj <0.05) of the whole NucSeq dataset between different conditions. (b)-(d) Selected genes of certain EAE-enriched pathways in the context of BMT and BMT+PLX for myeloid cells (b), astrocytes (c), and OLC (d).

Extended Data Figure 7

(a) UMAP of the immune cell cluster split by condition. (b) Microglia marker genes are shown for cluster M.2 and M.5 for different conditions. (c) Immunostaining of the white matter in a Control spinal cord demonstrating a CD206 positive border-associated macrophage. Scale bar is 30µm. (d) Expression of additional DAM (disease-associated microglia) genes associated with subcluster M.7. (e) Co-expression of Spp1 and Itgax in cluster M.7 is demonstrated. Dots are ordered towards the front based on expression. (f) Venn diagram showing significant DEGs (padj <0.05) of the myeloid subcluster between different conditions. (g) UMAP of the immune cell cluster of the human dataset, representing 7822 cells. BC, B cells. DC, dendritic cells. M, myeloid. NP, neutrophils. TC/NK, T cells/natural killer cells. (h) Canonical marker genes of immune cells. (i) Distribution of immune cell clusters between control and MS conditions. (j) UMAPs split by control and MS conditions. (k) UMAPs split by original dataset. (l) Marker genes of clusters M.3 (top row), M.5 (bottom row, left), and M.6 (bottom row, right).

Extended Data Figure 8

(a) Immunofluorescent stain for Gpnmb in the spinal cord white matter. Scale bar is 20µm. (b) Immunofluorescence images of the grey/white matter border in the spinal cord. Neuronal nuclei show bright Histone deacetylase 9 (Hdac9) signal. Light blue arrows indicate colocalization of myeloid cells and Hdac9 positive nuclei. Scale bar is 30µm.

Figure 1:

Myeloid expansion and transcriptional activation are features of chronic EAE and MS (a) Experimental design and timeline. Representative image of the lumbothoracic section of the spinal cord used for NucSeq. Contour plots show representative gating for flow-cytometric isolation of NeuN-negative nuclei. Pictogram shows the number of pooled animals per NucSeq library. BU-BMT, busulfan conditioned bone marrow transplantation. EAE, experimental autoimmune encephalomyelitis. NucSeq, single nucleus RNA sequencing. (b) Representative image of a lesion in the dorsal column of an EAE animal featuring immune infiltration and demyelination. Scale bar 100µm. (c) Annotation and canonical marker genes of main clusters. COP, committed oligodendrocyte precursor. MFOD, myelin forming oligodendrocytes. MOD, mature oligodendrocytes. OPC, oligodendrocyte precursor cells. (d) Uniform manifold approximation and projection (UMAP) of 68,593 nuclei integrated from all 8 libraries with annotation of main cell clusters. (e) Distribution of cell populations between Control and EAE animals. (f) Venn diagram of differentially expressed genes (DEG) of myeloid, astrocyte (Astro), and oligodendrocyte (Oligo) lineage between Control and EAE condition with an adjusted p value (padj) of <0.05. (g) Disease scores were calculated via the VISION pipeline using gene signatures based on common or cell type-specific DEGs. Score expression changes are shown between Control and EAE groups. (h) Forty-three significant DEGs shared with genes of a multiple sclerosis genome wide association study (GWAS) are demonstrated with their expression profile over different cell populations. Expression values (average log2 fold-change) are normalized by columns. If no significant DEG was present for a cell type, the value 0 was applied. (i) UMAP of 132,425 nuclei integrated from three human NucSeq datasets with annotation of main cell cluster groups. (j) Relative proportion of the myeloid and lymphocyte cell clusters. Dots represent different original datasets. A/CA, active / chronic active lesion; CI, inactive lesion; WM, white matter. (k) Expression of the EAE Myeloid Score in the myeloid cell population of the human NucSeq dataset. Expression between Control and MS groups differs significantly (p < 2.2e-16, Mann–Whitney U test).

Figure 2:

BMT changes the density and morphology of CNS myeloid cells in chronic EAE (a) Representative images of lumbar spinal cord sections demonstrating Iba1+ myeloid cell distribution as well as engraftment of donor derived GFP+ cells. Brightness/contrast was adjusted individually per image for illustrative reasons. (b) Chimerism of donor derived GFP+ cells within the myeloid cluster (CNS tissue based on Iba1+ immunofluorescence; peripheral blood (PB) based on CD45+CD11b+ flow cytometry) for different tissues and compared between the EAE-BMT and EAE-BMT+PLX conditions. Replicates for brain and PB: n=7 and n=9 for EAE-BMT and EAE-BMT+PLX, respectively. Mean values based on a total number of 136 brain regions of interest (ROIs). Spinal cords do not include tissue used for NucSeq, number of replicates: n=3 and n=4 for EAE-BMT and EAE-BMT+PLX, respectively, with mean values based on a total of 35 sections. Mann–Whitney U test; ns: p>0.05, *: p≤0.05, **: p≤0.01, ***: p≤0.001. (c) Pearson correlation coefficient (r) and scatter plot demonstrate the correlation between brain and spinal cord GFP+ chimerism. Regression line based on linear model. (d) A gene signature for CDMCs was based on previously published 1296 DEGs between CDMCs and endogenous microglia and a score was calculated with the VISION pipeline. Expression in the myeloid cluster is demonstrated. (e) The fractions of nuclei in the myeloid cluster with a CDMC score expression of > - 0.05 are shown. Bars represent the group mean. (f) Quantification of ramified Iba1+ cell density. Dots represent the average of multiple measurements per animal. Bars represent the mean of all animals per condition. A total number of 78 spinal cord ROIs and 221 brain ROIs were assessed. t-test; ns: p>0.05, *: p≤0.05, **: p≤0.01, ***: p≤0.001, ****: p≤0.0001. (g) Representative immunofluorescent images of ramified myeloid cells in the spinal cord. Brightness/contrast was adjusted individually per image for morphological assessment due to differing Iba1 intensity between conditions. Scale bar is 20µm. (h) Morphological analysis of ramified myeloid cells of the spinal cord in different conditions. Bars represent the group mean. Each dot represents one cell, dot colors represent different animals. Mann–Whitney U test; ns: p>0.05, *: p≤0.05, **: p≤0.01, ***: p≤0.001, ****: p≤0.0001.

Figure 3:

BMT and microglia replacement enhance the myeloid transcriptional response and improve clinical outcome (a) Mean clinical score ± standard deviation over an observation period of 90 days. Thirty animals derived from two independent cohorts were included (EAE n=9, EAE-BMT n=8, EAE-BMT+PLX n=9, EAE-PLX n=4). Two animals showed EAE related mortality before completion of the observation period (EAE (day 68) and EAE-BMT (day 66)). One animal of the EAE-BMT+PLX group was sacrificed on day 76 due to non-EAE related reasons (with clinical score of 1.5) and was excluded from the analysis after this timepoint. (b) Boxplots show the distribution of clinical scores for the indicated timepoints. Mann–Whitney U test; ns: p>0.05, *: p≤0.05, **: p≤0.01. (c) Fractions of nuclei of main cell types in different conditions. Bars represent condition means, dots individual libraries. (d) Expression changes of EAE-scores for main cell types between different libraries. Black crossbars represent the median. (e) Clinical score of EAE animals used for different NucSeq libraries. (f) Upset plots show significant DEGs (padj < 0.05) for main cell types between the EAE and EAE-BMT or EAE-BMT+PLX groups. The top 15 intersections are illustrated.

Figure 4:

BMT modulates the myeloid and lymphoid response to EAE (a) Subcluster analysis was performed on the immune cell clusters of the integrated main dataset. The dot plot demonstrates the resulting subclusters with an expression profile of canonical marker genes and CDMC Score. BC, B cells. DC, dendritic cells. M, myeloid. NK, natural killer cells. TC, T cells. (b) UMAP plot of 10,314 nuclei of the immune cluster. (c) Bar chart showing the shifted distribution of myeloid cell subclusters between different conditions. (d) Immunofluorescent stain for CD206 (Mrc1) in the spinal cord of an EAE-BMT animal. Scale bar is 30µm. (e) Violin plots of selected genes associated with subcluster M.4 and the interferon pathway. Black crossbars represent the median. (f) Immunofluorescent stain for Stat1 in the spinal cord white matter of an EAE animal. Red arrows indicate nuclear and paranuclear Stat1 signal colocalizing with Iba1. Scale bar is 20µm. (g) Violin plots of selected DAM (disease-associated microglia) genes associated with subcluster M.7. Black crossbars represent the median. (h) Representative immunofluorescent image of Osteopontin (Spp1) positive myeloid cells with strong Iba1 signal forming a cluster adjacent to an inflammatory lesion (asterisk) in the spinal cord of an EAE animal. Rectangle indicates magnified area with cell cluster. Scale bar is 30µm. (i) Scatterplots show the relationship of significant DEGs (padj < 0.05 for both conditions) of disease-associated (Control vs EAE) and treatment-associated (EAE vs EAE-BMT and EAE-BMT+PLX, respectively) conditions of the myeloid cluster. Numbers represent the DEGs per quadrant. Differences between quadrants have been tested with the Pearson’s Chi-squared test with Yates’ continuity correction: p = 2.3e-10 (EAE-BMT), p < 2.2e-16 (EAE-BMT+PLX). (j) Representative immunofluorescent images of hemi spinal cords stained for Iqgap1 including EAE-BMT animals with low and high GFP+ chimerism. (k) Immunofluorescent stain for Iqgap1 in the spinal cord white matter. Scale bar is 20µm. (l) Marker gene expression of different T cell populations. (m) UMAP of the T cell subcluster. (n) Distribution of T cell populations between different conditions.

Figure 5:

BMT-associated increased myelination is accompanied by partial reversal of the transcriptional response in OLCs (a) Representative fluorescent myelin stain (FluoroMyelin^™) of the lumbar spinal cord. Red arrows indicate demyelinated lesions. (b) Scatterplot demonstrating the correlation between white matter demyelination and clinical score. Regression lines based on linear models; r = Pearson correlation coefficient. (c) Comparison of demyelinated white matter areas between conditions for animals with available spinal cord histology. Dots represent the average of multiple analyzed spinal cord sections, with a median of 4 (range: 2 – 6) sections per anatomical location per animal. Bars represent the mean of all replicates per condition. t-test; ns: p>0.05, *: p≤0.05, **: p≤0.01. (d) Violin plots of selected genes associated with myelin formation and oligodendrocyte differentiation/maturation that are differentially expressed between EAE and EAE-BMT (blue asterisk) or EAE-BMT+PLX (green asterisk) conditions (padj < 0.05). (e) Venn diagrams of DEGs between selected conditions (padj < 0.05) for main OLC subpopulations. (f) Scatterplots show the relationship of significant DEGs (padj < 0.05 for both conditions) of disease-associated (Control vs EAE) and treatment-associated (EAE vs EAE-BMT and EAE-BMT+PLX, respectively) conditions of different OLC subpopulations. Numbers represent the DEGs per quadrant. Differences between quadrants have been tested with the Fisher’s Exact Test: p = 1.4e-08 (EAE-BMT) and p = 4.2e-13 (EAE-BMT+PLX) for OPC. p = 6.9e-13 (EAE-BMT) and p < 2.2e-16 (EAE-BMT+PLX) for MFOD. p = 1.2e-05 (EAE-BMT) and p = 7.5e-06 (EAE-BMT+PLX) for MOD.

Figure 6:

BMT and microglia replacement ameliorate reactive astrogliosis in chronic EAE (a) Immunofluorescent stain for GFAP in the spinal cord. Scale bar is 50µm. (b) Quantification of GFAP+ area in the lumbar spinal cord. Dots represent the average of measurements per animal (median number of spinal cord sections per animal = 2). Bars represent the mean of all animals per condition. t-test; ns: p>0.05, *: p≤0.05, **: p≤0.01. (c) UMAP plot of the astrocyte cluster consisting of 7745 nuclei. (d) Expression of astrocyte subcluster marker genes. (e) Bar chart showing the distribution of astrocyte subclusters between different conditions. (f) Expression of selected genes associated with reactive astrogliosis. All genes shown are differentially expressed between Control and EAE (padj < 0.05). Compared to EAE, Slc1a3 and both Gfap and Slc1a3 were differentially expressed versus EAE-BMT and EAE-BMT+PLX, respectively. (g) Venn diagram of DEGs of the astrocyte lineage (padj < 0.05). (h) Scatterplots show the relationship of significant DEGs (padj < 0.05 for both conditions) of disease-associated (Control vs EAE) and treatment-associated (EAE vs EAE-BMT and EAE-BMT+PLX, respectively) conditions in the astrocyte cluster. Numbers represent the DEGs per quadrant. Differences between quadrants have been tested with the Pearson’s Chi-squared test with Yates’ continuity correction: p < 2.2e-16 (EAE-BMT), p < 2.2e-16 (EAE-BMT+PLX).