Neuroinflammation and neurodegeneration may represent two poles of brain pathology. Brain myeloid cells, particularly microglia, play key roles in these conditions. We employed single-cell mass cytometry (CyTOF) to compare myeloid cell populations in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, the R6/2 model of Huntington’s disease (HD) and the mutant superoxide dismutase 1 (mSOD1) model of amyotrophic lateral sclerosis (ALS). We identified three myeloid cell populations exclusive to the CNS and present in each disease model. Blood-derived monocytes comprised five populations and migrated to the brain in EAE, but not in HD and ALS models. Single-cell analysis resolved differences in signaling and cytokine production within similar myeloid populations in EAE compared to HD and ALS models. Moreover, these analyses highlighted α5 integrin on myeloid cells as a potential therapeutic target for neuroinflammation. Together, these findings illustrate how neuropathology may differ between inflammatory and degenerative brain disease.
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We thank O. Butovsky (Harvard University) for the generous gift of 4D4 and FCRLS antibody. We thank A. Trejo and A. Jager for mass cytometry quality control and maintenance, and S. Douglas and V. Giangarra for technical assistance. This work was supported by grants to G.P.N.: U19 AI057229, 1U19AI100627, Department of Defense (CDMRP), Northrop-Grumman Corporation, R01CA184968, 1R33CA183654-01, R33CA183692, 1R01GM10983601, 1R21CA183660, 1R01NS08953304, OPP1113682, 5UH2AR067676, 1R01CA19665701, R01HL120724. G.P.N. is supported by the Rachford & Carlotta A. Harris Endowed Chair. B.A was supported by a three-year postdoctoral fellowship from the Canadian Institute for Health Research (201102MFE-246400-166230).
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 Composite minimum spanning tree (MST) of CNS samples reveals distinct CD45+ immune cell populations.
Composite MST represents X-shift clusters constructed by combining CNS samples from all the disease conditions and their biological replicates (n = 37). All the samples in this experiment were barcoded and analyzed together on CyTOF (Methods). All CD45pos immune cells are circled in the center panel of this figure. Lymphocytes are depicted to the left, with clusters of CD3, CD4, and B220 populations circled in individual panels. CD11b myeloid cells are shown to the right, with clusters of Ly6G, Ly6C and CD11c populations circled in individual panels. The color code shows the expression level (zero to high) of each expression marker in each panel.
Composite MSTs (n = 37) of CNS samples demonstrate CD86 and CD39 expression on populations A, B, C. a) The color code shows the expression level of CD86. b) The color code shows the expression level of CD39.
Supplementary Figure 3 Comparisons of the expression of several markers in three CNS-resident myeloid populations.
a) Similarities. Populations A, B, and C expressed different levels of CD88, MHC class I (H-2), TAM receptor tyrosine kinases Mer (MerTK), and the newly introduced microglia markers 4D4 and FCRLS. This panel represents data from Peak EAE from 10 independent experiments. b) Variations. Differential expression of a number of markers were detected in three CNS-resident myeloid populations. Populations B and C expressed different levels of CD80, TAM receptor Axl, T-cell immunoglobulin mucin protein 4 (TIM4), CD274 (PD-L1), CD195 (CCR5), CD194 (CCR4), and low levels of CD206 and TREM2. Population A lacked the expression of all these markers. This panel represents data from Peak EAE from 10 independent experiments.
MSTs from each disease condition (n = 5) demonstrates the expression level of a) CD80 and CD274 (PD-L1); b) CD194 (CCR4) and CD195 (CCR5); and c) TAM receptor Axl and T-cell immunoglobulin mucin protein 4 (TIM4).
Composite MSTs of CNS samples (n = 37) and MSTs from each disease condition (n = 5) demonstrate that FCRLS and 4D4 (new antibodies that mark microglia) are only expressed on CNS-resident myeloid populations A, B, and C. a) The color code shows the expression level of FCRLS. b) The color code show the expression level of 4D4.
a) Expression of YFP. In healthy conditional Cx3cr1CreER Rosa26-YFP mice, populations A and B (the only two populations that exist in the healthy condition) were manually gated and the expression of YFP was confirmed. The gating strategy is described in Fig. 2b. Cells were stained with a similar CyTOF panel as the rest of the experiment. GFP conjugated antibody was used to detect YFP. Histogram from a representative experiment (n = 5). b) MSTs from healthy condition wild-type mice (n = 5) suggests that populations A and B are the two prominent CNS-resident myeloid populations. Population C was barely detectable in the healthy CNS.
a) Expression of CD11c in population C under different disease conditions. MSTs from different disease conditions (n = 5) demonstrates that CD11c is only expressed in population C among the three CNS-resident myeloid populations. CD11c expression in population C is only detected in the active phase of the EAE disease (presymptomatic, onset and peak). This marker is not expressed in population C in end-stage HD, chronic EAE, or recovered EAE. b) Expansion rate of each CD11b CNS-resident myeloid population under each disease condition. Representative biaxial dot plot showing the fraction of Ki-67+ proliferating cells within each CNS-resident myeloid population under each disease condition (n = 5).
Supplementary Figure 8 Expression of intracellular cytokine levels in the CNS-resident myeloid population by disease condition.
a) Distribution plots (Violin plots) shows the expression levels of indicated intracellular cytokines, grouped by disease condition and in each CNS-resident myeloid population. The gating strategy for each population is described in Fig. 2b. Plots were created in Mathematica. This plot is a representative from three independent experiments. b) Box-and-whisker charts showing the same distribution of intracellular cytokine levels in individual cells, grouped by cluster and disease state. The distribution plots (Violin plots from above) have been converted to box-and-whisker charts, which allow for quantitative evaluation of the median and quantiles of cytokine expression for the individual cells. Center line is median; boxes extend to 25th and 75th quantile; whiskers extend to 1.5x the interquartile range. This chart is a representative from three independent experiments.
Supplementary Figure 9 Single-cell analysis of cytokine production in CNS-resident myeloid populations in early time points of HD.
a) Frequency of populations A, B, and C based on manual gating (Fig. 2b) confirms that populations A, B, and C are present in early time points of HD. Center line is average; boxes extend to 25th and 75th percentile; whiskers extend to 5th and 95th percentiles. This graph presents data from four independent experiments. b) Single-cell analysis of cytokine production by different CNS-resident myeloid populations in early time points of HD. X-shift analysis of the co-expression of cytokines in CNS-resident myeloid populations suggests that each population contains heterogeneous subsets depending on each disease conditions. Percentages of single-cells expressing zero, one or two cytokines are represented in a stacked bar graph. This graph presents data from four independent experiments.
Supplementary Figure 10 Single-cell analysis of cytokine production in CNS-resident myeloid populations in mSOD1 mice, the transgenic model of amyotrophic lateral sclerosis (ALS).
a) Frequency of populations A, B, and C based on manual gating (Fig. 2b) confirms that populations A, B, and C are present in ALS. Center line is average; boxes extend to 25th and 75th percentile; whiskers extend to 5th and 95th percentiles. mSOD1 onset represents six independent experiments, mSOD1 disease end stage represents five independent experiments. b) Single-cell analysis of cytokine production by different CNS-resident myeloid populations in ALS. X-shift analysis of the co-expression of cytokines in CNS-resident myeloid populations reveals that each population contains heterogeneous subsets depending on each disease conditions. Percentages of single-cells expressing zero, one or two cytokines are represented in a stacked bar graph. This graph presents data from four independent experiments.
Supplementary Figure 11 Variation in expression of several markers in five peripheral monocyte populations.
Differential expression of a number of markers were detected in peripheral monocyte populations. Populations D and E compared to the other three populations have a higher expression of phagocytic receptors like TAM receptor tyrosine kinases (Mer, Axl), costimulatory molecules (CD80, CD86), receptors involved in purinergic signaling (CD38, CD39), and TREM2 as well as CD206. This panel represents data from Peak EAE from 10 independent experiments.
a) Distribution plots (Violin plots) shows the expression levels of indicated intracellular cytokines grouped by disease condition and in each peripheral monocyte population. The gating strategy for each populations is described in Fig. 4b. Plots were created in Mathematica. This plot is a representative from three independent experiments. b) Box-and-whisker charts showing the same distribution of intracellular cytokine levels in individual cells, grouped by cluster and disease state. The distribution plots (Violin plots from above) have been converted to box-and-whisker charts, which allow quantitative evaluation of the median and quantiles of cytokine expression for the individual cells. Center line is median; boxes extend to 25th and 75th quantile, whiskers extend to 1.5x the interquartile range. This chart is a representative from three independent experiments.
a) Expression of YFP in peripheral blood cells. The lack of YFP expression in peripheral blood cells of conditional Cx3cr1CreER Rosa26-YFP mice was confirmed by CyTOF. Both peripheral blood cells and CNS were stained with a similar CyTOF panel. Histogram is a representative experiment (n = 5). b) Gating strategy on CyTOF samples for singlets and live/dead. Samples are labeled with iridium nucleic acid (intercalator 191/193Ir DNA intercalator, DVS Sciences/Fluidigm, Markham, ON). For each event, many features are recorded, including signal duration (called Event Length) and iridium intensity. Single events have lower iridium intensity (since they have less DNA) and lower Event Length values compared to aggregates. These characteristics enable gating of single cells (singlets). Live cells are identified by the lack of cleaved poly-(ADP)-ribose polymerase (c-PARP) binding as previously reported60.
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Ajami, B., Samusik, N., Wieghofer, P. et al. Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models. Nat Neurosci 21, 541–551 (2018). https://doi.org/10.1038/s41593-018-0100-x
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