www.sciencemag.org/content/363/6425/eaat7554/suppl/DC1

Supplementary Material for

Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation

Marta Joana Costa Jordão, Roman Sankowski, Stefanie M. Brendecke, Sagar, Giuseppe

Locatelli, Yi-Heng Tai, Tuan Leng Tay, Eva Schramm, Stephan Armbruster, Nora Hagemeyer, Olaf Groß, Dominic Mai, Özgün Çiçek, Thorsten Falk, Martin

Kerschensteiner, Dominic Grün, Marco Prinz*

*Corresponding author. Email: marco.prinz@uniklinik-freiburg.de

Published 25 January 2019, Science 363, eaat7554 (2017) DOI: 10.1126/science.aat7554

This PDF file includes:

Figs. S1 to S8 Tables S1 to S3

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Fig. S1.

Fig. S1: Single-cell profiling of hematopoietic cells during inflammation as revealed by

scRNA-seq

(a) t-SNE representation of 3,461 individual hematopoietic cells from different CNS

compartments of C57BL/6 mice under homeostasis and neuroinflammation as measured by

scRNA-seq and RaceID3 clustering. (b) Distribution of cells (in %) from each phase contributing

to the different clusters from the leptomeninges, perivascular space and parenchyma and choroid

plexus. Subpopulations are displayed from homeostasis to inflammation (left to right).

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Fig. S2.

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Fig. S2: Molecular census of tissue-resident macrophages during inflammation

(a) t-SNE representation of 618 individual myeloid cells from different CNS compartments

under homeostasis as measured by scRNA-seq. Identification of the cell populations was based

on the expression of core signature genes for different myeloid populations. MG: microglia,

CAM: CNS-associated macrophages, MCs: monocyte-derived cells, Granul: granulocytes,

Lympho: lymphocytes. (b) Clustering-based definition of cell populations found in the naïve

CNS. (c) CAM subsets during homeostasis identified by unbiased clustering. (d) Number of

cells (in %) from each compartment contributing to the different clusters. (e) mRNA expression

levels of enriched genes in CAMs compared to other myeloid cells. Whisker plots with mean ±

s.e.m. of expression value in a logarithmic scale are exhibited. Data are representative of 6 mice

pooled from three independent experiments. (f) Most differentially regulated genes in hCAM1 in

comparison to hCAM3. Data are presented as log2 fold changes and are representative of 6 mice

from two independent experiments. (g, h, i, j) mRNA expression levels of enriched genes in

mMΦ (g), pvMΦ (h), cpMΦ (i), and microglial (j) subsets. Whisker plots with mean ± s.e.m. of

expression value are depicted. Data are representative of 16 mice pooled from three independent

experiments. (k) Quantification of differentially expressed genes between mMΦ, pvMΦ, and

cpMΦ during health and disease with a p-value < 0.05 present in one CAM population compared

to the other two populations. (l) Transcriptomic profile of daMG1 subset. Arrows depict the

upregulation or stable expression of the specified genes. (m) Left: Representative flow

cytometric gating strategy for resident LYVE-1-expressing pvMΦ

(CD45hiCD11b+tdTomato+LYVE-1+) from Cx3cr1CreERT2:R26tdTomato mice at peak of the disease.

Right: Quantification of the mean fluorescence intensity (MFI) for LYVE-1 at naïve stage and at

peak of the disease. Data are representative of 4 mice from two independent experiments. A two-

tailed Mann–Whitney U test revealed a significant difference between the groups. (n)

Representative immunofluorescence of naïve Cx3cr1CreERT2:R26tdTomato mice showing the

expression of P2RY12 and absence of MD-1 in microglial cells. Pictures are representative of 3

mice from two independent experiments. Asterisks indicate resident tdTomato+ microglia. Scale

bars: 500 µm (overview) and 10 µm (inset). (o) Representative immunofluorescence pictures of

the lesion site of the spinal cord from Cx3cr1CreERT2:R26tdTomato mice at peak of the disease.

Squares highlight the different tomato+ daMG subsets as presented in Fig. 3g. Pictures are

representative of 4 mice from two independent experiments. Scale bars: 50 µm.

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Fig. S3.

Fig. S3: Expression of CAMs and microglial core signature cells across all immune cells as

revealed by scRNA-seq

(a) t-SNE and whisker plot showing the expression of Fcrls across all myeloid cells in different

CNS compartments. Left: The green dotted line in the t-SNE plot encircles cells belonging to

CAM subsets, whereas the red dotted line depicts the microglial subsets. Below: whisker plots

with mean ± s.e.m. of expression value are depicted as logarithmic scale. Data are representative

of 16 mice from two independent experiments. (b) t-SNE plots showing the expression of Mrc1,

Cbr2, Stab1, and Pf4 during homeostasis and neuroinflammation in all CNS hematopoietic cells.

The green dotted line encircles cells belonging to CAM subsets. (c) t-SNE plots showing the

expression of microglia-enriched genes during homeostasis and neuroinflammation. The red

dotted line depicts the microglia subsets.

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Fig. S4.

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Fig. S4: Gene signature of individual circulating monocytes during health and disease

(a) t-SNE representation of 384 individual monocytes measured by scRNA-seq and RaceID3

clustering. Yellow dots highlight the analyzed cells from homeostasis and different stages of

EAE. The different monocytic populations are highlighted in the plot. (b) t-SNE plot depicting

the expression level of Ly6c2, Nr4a1, and Ccr2 in all circulating monocytes from the blood that

underwent scRNA-seq. (c) mRNA expression levels of enriched genes in monocytes. Whisker

plots are displayed as mean ± s.e.m. of expression value in a logarithmic scale. Data are

representative of four mice from two independent experiments. (d, e) Expression levels of

enriched genes in different monocyte-derived populations from the leptomeninges (mMCs) (d),

and perivascular space and parenchyma (pMCs) (e). Data are depicted as whisker plots where

mean ± s.e.m. of expression value in a logarithmic scale is presented. Data are representative of

16 mice pooled from three independent experiments. (f) Most differentially regulated genes

between damMΦ1 and mMC4. Data are presented as log2 fold changes. Data are representative

of 16 mice from three independent experiments. (g) mRNA expression levels of enriched genes

in cDCs and CD209+ MCs from the different CNS compartments. Whisker plots are displayed as

mean ± s.e.m of expression value in a logarithmic scale. Data are representative of 16 mice from

three independent experiments. (h) t-SNE plots showing the mRNA expression of Ly75 during

homeostasis and neuroinflammation in all CNS hematopoietic cells. The dotted line delineates

the cells belonging to DCs subsets. (i) Representative immunofluorescence image of CD205

(encoded by Ly75) in all CNS compartments. Scale bars: 50 µm (overview) and 10 µm (insert).

The dashed line delimits the barrier between the meninges (Men) and parenchyma (PC), or

delimits the vessel lumen and perivascular space (PV). A representative picture from 4 mice

from two independent experiments is shown. (j) Quantification of the tdTomato-expressing

microglia (MG) and CD209+ and MerTK+ MCs as revealed by flow cytometric analysis from

Cx3cr1CreERT2:R26tdTomato mice at peak of the disease. Data are depicted as mean ± s.e.m. and are

representative of 5 mice from two independent experiments. (k) Representative flow cytometric

gating strategy for MerTK+ MCs (MerTK+CD64+Ly6C+CD44+) in the CNS that can also express

CD209a. Histograms show the comparison between MerTK+ MCs and CD209+ MCs for the

expression level of CD44, CD11c, and MHC-II. Gating strategy is representative of 5 mice from

two independent experiments.

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Fig. S5.

Fig. S5: Gene ontology enrichment (GO) analysis across the myeloid subsets at different

CNS compartments.

Selected GO pathways associated to innate and adaptive immunity were selected and depicted

across the different subsets found in the perivascular space (PV) and parenchyma,

leptomeninges, and choroid plexus.

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Fig. S6.

Fig. S6: Expression of previously reported myeloid phenotypic markers in all the immune

cells analyzed during both homeostasis and disease.

Expression of Arg1, linked to an anti-inflammatory monocytic profile (70) was highly expressed

in MCs. CD44 (32) could be confirmed as being highly expressed by peripheral immune cells.

Trem2, previously seen as being upregulated in specific monocyte populations infiltrating the

CNS during EAE (57), was observed as being highly expressed by microglia in the CNS

parenchyma while low expression was observed for monocyte populations within the

leptomeninges and CP. Bst2, seen to be expressed by activated microglia (57), was shown to be

highly expressed in pvMΦ. Dotted lines delimit the cells belonging to a specific immune cell

population. MCs: monocyte-derived cells, Per. immune cells: peripheral immune cells, daMG:

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disease-associated microglia, pvMΦ: perivascular macrophages, cpMΦ: choroid plexus

macrophages

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Fig. S7.

Fig. S7: Dynamics of resident macrophages and infiltrating monocytes during the

progression of EAE

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(a) Recombination of Ly6Chi and Ly6Clo monocytes 3 days and 14 and 18 weeks after TAM as

shown in the percentage of tdTomato-expressing cells. Data are representative of 5 mice from

two independent experiments. Two-way ANOVA followed by Sidak’s multiple comparisons test

revealed significant differences between the groups. (b) Recombination of pvMΦ, mMΦ and

microglia at 14 weeks of age (8 weeks after TAM) shown in the percentage of tdTomato-

expressing cells. Data are presented as mean ± s.e.m. of at least 4 mice from two independent

experiments. Kruskal-Wallis followed by Dunn’s multiple comparisons test revealed significant

differences between the groups (c) Recombination of CAMs, microglia, and Ly6Chi and Ly6Clo

monocytes at the peak phase of EAE of Cre+ mice not induced with tamoxifen. Data are

representative of 3 mice from two independent experiments. (d) IBA-1 immunofluorescence of

myeloid cells in the spinal cords of naïve or EAE-diseased Cx3cr1CreERT2:R26tdTomato animals at

indicated time points. CNS endogenous tissue macrophages are characterized by IBA-

1+tdTomato+ immunoreactivity whereas infiltrating monocyte-derived cells are IBA-

1+tdTomato−. Scale bar: 500 µm (overviews) and 50 µm (insets). Immunofluorescence pictures

are representative of 4 mice from two representative experiments. (e) Representative

immunofluorescence images of proliferating resident macrophages (tdTomato+Ki67+) in

different CNS compartments. Scale bar: 10 µm. Representative pictures of 6 mice from two

independent experiments are shown. (f) Representative Imaris 3D reconstructions from confocal

pictures of resident MΦ (tdTomato+, red) and infiltrating monocyte-derived cells (IBA-

1+tomato−, green) found at the leptomeninges and perivascular space from the spinal cord during

the different phases of EAE. Pictures are representative of 10 mice from four independent

experiments. Scale bars = 2 µm. N.P.: not present. (g) Morphology as shown in volume (µm3) of

the resident MΦ and infiltrating monocytes at different phases of EAE. Data are presented as

mean ± s.e.m. and are representative of 6-10 mice from three independent experiments. At least

15 cells per mouse were evaluated. Two-way ANOVA followed by Sidak’s multiple

comparisons test revealed significant differences between the groups. (h) Scheme of the

Cx3cr1CreERT2:R26Confetti mouse model used for the analysis of microglia expansion during EAE.

Tamoxifen treatment of Cx3cr1CreERT2:R26Confetti mice induces a stochastic recombination of

microglia and CAMs which allows us to study the potential clonal expansion of these cells

during EAE.

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Fig. S8.

Fig. S8: Expression of antigen-presentation related genes on a single-cell level

(a, b) t-SNE plot showing the expression of core signature genes associated with antigen-

presentation capacity (APC) in all myeloid cells from all compartments of naïve mice (a), and in

all myeloid cells from all CNS compartments during homeostasis and inflammation (b). The

population that expresses the highest levels of the APC-associated genes is highlighted. hCAM2:

CNS-associated macrophages subset 2, hCAM3: CNS-associated macrophages subset 2.

daCAM: disease-associated CNS-associated macrophages, MCs: monocyte-derived cells,

daMG3: disease-associated microglia population 3. (c) Histological quantification of the number

of CD4+ cells found at the meninges (Men), perivascular space (PV) and parenchyma (PC)

during the progression of EAE. Data are representative of 7 mice from three independent

experiment. Data are presented as mean ± s.e.m. (d) t-SNE plots showing the expression level of

Ccr2 across all myeloid cells found at the meninges, and perivascular space and parenchyma,

and choroid plexus during EAE. Ccr2 expression was highest in monocyte-derived cells (MCs).

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Table. S1.

Compartments

PV & Parenchyma Leptomeninges Choroid plexus Blood

Naive 326 105 187 241

Preclinical 332 207 191

Onset 383 459 153 143

Peak 283 281 170

Total cells 1324 1052 701 384

Table S1: Number of analyzed cells that underwent scRNA-seq per compartment and

EAE phase. Examined cells were taken from the perivascular space (PV) and parenchyma,

leptomeninges, choroid plexus, and blood.

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Table. S2.

Hematopoietic cell population

Upregulated genes

Homeostatic subsets Disease-associate subsets

Microglia

C1: Sparc, Hexb, Ctss, Csf1r C7: Sparc, Ctss, Ly86, C1qc

C2: Sparc, Ctss, Hexb, Csf1r C8: Sparc, Ctss, Ly86, C1qc

C6: Ctss, Sparc, Apoe, C1qc

CAMs

C13: Apoe, C1qc, Mrc1, Sepp1 C14: Apoe, Cd74, H2-Eb1, H2-Aa

C15: Cd74, Apoe, H2-Aa, H2-Eb1

C27: Cd74, Apoe, H2-Aa, H2-Eb1

C26: Cd74, H2-Eb1, H2-Aa, Apoe

Monocytes

C5: Lyz1/Lyz2, Chil3/Chil4, Ly6c2, Plac8 C18: Arg1, Lyz1/Lyz2, Fn1, Cd74

C25: Lyz1/Lyz2, Hba-a1/Hba-a2, Hbb-bs, CD74

C23: Lyz1/Lyz2, Fn1, Prg4, Arg1

C20: Fn1, Arg1, Lyz1/Lyz2, Thbs1

C19: Lyz1/Lyz2, Ly6c2, Plac8, Hba-a1/Hba-a2

DCs

C21: Ccr7, Fscn1, Ccl5, Ccl22

C11: Cd74, Ifitm1, H2-Ab1, H2-Aa

Granulocytes

C3: S100a9, S100a8, Ngp, Lcn2

C24: S100a9, S100a8, Retnlg, Ifitm1

C4: Irg1, Hdc, Cxcr2, Mxd1

Lymphocytes

C12: Hba-a1/Hba-a2, Lyz1/Lyz2, Pou2f2, Cybb C10: Ttr, Igkc, Enpp2, Ighg2c

C17: Ccl5, Gzma, Ms4a4b, Thy1

C16: Icos, Trbc1/Trbc2, Gzma, Ccl5

C22: Prss34, Ly6c2, Vim, Mpo

Table S2: Top upregulated genes in each hematopoietic subset presented in Fig. S1a.

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Table. S3.

Microglia-enriched genes CAMs-enriched genes MG and CAMs- enriched genes

Gene name

Expression during neuroinflammation

Gene name

Expression during neuroinflammation

Gene name

Expression during neuroinflammation

Sparc Stable Ms4a7 Stable C1qa Stable

Hexb Stable Mrc1 Downregulated C1qb Stable

P2ry12 Downregulated Cbr2 Downregulated C1qc Stable

Tmem119 Downregulated Pf4 Downregulated Csf1 Stable

Siglech Downregulated Cd163 Downregulated Csf1r Stable

Cst3 Downregulated Cx3cr1 Downregulated

Ctsl Upregulated Fcrls Downregulated

Sall1 Downregulated

Gpr34 Downregulated

Olfml3 Stable

Selplg Downregulated

Trem2 Downregulated

Bhlhe41 Stable

Slc2a5 Downregulated

Lgmn Stable

Serpine2 Stable but low expressed

P2ry13 Downregulated

Table S3: Top highly expressed genes in microglia and CAMs populations as revealed by

single-cell RNAseq.