redefining the heterogeneity of peripheral nerve cells in ......supplementary information for...
TRANSCRIPT
Supplementary Information for
Redefining the heterogeneity of peripheral nerve cells in health and
autoimmunity
Jolien Wolbert1,*
, Xiaolin Li1,*
, Michael Heming1,*
, Anne K. Mausberg2, Dagmar Akkermann
3, Clara
Frydrychowicz3, Robert Fledrich
4, Linda Groeneweg
5, Christian Schulz
6, Mark Stettner
2, Noelia
Alonso Gonzalez5, Heinz Wiendl
1, Ruth Stassart
3,$, and Gerd Meyer zu Hörste
1,$
1) Department of Neurology with Institute of Translational Neurology, University Hospital Münster, Münster, Germany.
2) Department of Neurology, University Hospital Essen, University Duisburg Essen, Essen, Germany.
3) Department of Neuropathology, University Hospital Leipzig, Leipzig, Germany.
4) Institute of Anatomy, Leipzig University, Leipzig, Germany
5) Institute of Immunology, Westfälische Wilhelms University, Münster, Germany
6) Medizinische Klinik und Poliklinik I, Klinikum der Universität München, Ludwig-Maximilians Universität, München,
Germany
* These authors contributed equally
$ These authors co-supervised the study
Correspondence to: Gerd Meyer zu Hörste, MD
Email: [email protected]
This PDF file includes:
Supplementary Methods
Figures & legends S1 to S15
Table legends S1 to S14
Supplementary references
Other supplementary materials for this manuscript include the following:
Tables S1 to S14
www.pnas.org/cgi/doi/10.1073/pnas.1912139117
Supplementary Methods
Supplementary Methods
Animals
C57BL/6J mice were originally purchased from the Jackson Laboratory and subsequently maintained at
the animal facility of the Medical Faculty of the Westfälische Wilhelms University Münster. Female
C57BL/6J mice were sacrificed at the age of 8-12 weeks. Icam1tm1Jcgr1
NOD mice (named ICAM-1-/-
NOD
mice for simplicity) were previously described (4) and maintained under specific pathogen free conditions
in the animal facility of the University Hospital in Essen. Mice were analyzed for clinical signs of
neuropathy as described (5). Female ICAM-1-/-
NOD mice were sacrificed at the age of 36 weeks and did
not show clinical symptoms of neuropathy. Parts of sciatic nerves of selected donors were processed for
cyro-sectioning and then stained for Hematoxylin and Eosin. Donor mice were without obvious immune
cell infiltrates (Fig. S7A). Young female, prediabetic NOD/ShiLtJ mice at an age of 8-10 weeks were used
as controls (NOD control) and obtained from Charles River laboratories. Lewis rats were originally
purchased from Harlan Laboratory and subsequently maintained at the animal facility of the University
Hospital Essen. Rats were housed under specific pathogen free conditions. Female rats were sacrificed at
the age of 10 weeks. CX3CR1-GFP mice have been described (6), were initially obtained from the
Jackson laboratories and maintained at the Institute of Immunology of the Westfälische Wilhelms-
University Münster. hGFAP-GFP mice (7) (Jax strain #007669) and PDGFRɑ-EGFP mice (8) (strain
#003257) have been described and were purchased from The Jackson Laboratory. Flt3Cre mice (9) and
mice expressing membrane-targeted tandem dimer Tomato (mT) prior to and membrane-targeted green
fluorescent protein (mG) after Cre-mediated excision from the Rosa26 locus (mT/mG mice) (10) and their
intercross (Flt3Cre-mT/mG mice) (11–13) have been described previously. Flt3Cre-mT/mG mice were
maintained at the Animal Facility of the Ludwig Maximilians Universität, München, Germany and
processed in Münster immediately after transport. Sciatic nerves were processed into single cell
suspension for flow cytometry or preserved for immunohistochemistry.
Cell extraction and purification
Sciatic nerves and the brachial nerve plexus were dissected from intracardially PBS perfused, female
C57BL/6 mice (8-12 weeks), NOD/ShiLtJ mice (8-10 weeks), Icam1tm1Jcgr1
NOD mice (36 weeks) and male
Flt3Cre-mT/mG mice (10 weeks) and transferred into ice cold Hank’s buffered salt solution (HBSS)
supplemented with 10mM HEPES (Gibco). Nerve fractions were finely chopped without epineurium
removal and teased as described(14). Nerve tissue was then digested using three different protocols to
test the optimal enzyme combination for collection of viable PNS cells.
Protocol #1 was adapted from a previous study (5) and nerves were incubated in collagenase/dispase
(Roche, 0.5 mg/ml) for 1 h at 37˚C. Protocol #2 was modified from a previous study (15). Digestion mix
contained Trypsin (Gibco, 0.25%): Collagenase II (Worthington, 1.62 U/μl): Hyaluronidase (Worthington,
1%) in a ratio of 1:1:0.04 respectively. 1 μl Pronase (Roche, 1%) was added per 50 μl of mix. Nerves were
digested for 20 minutes at 37°C with a 10 seconds vortex after 10 minutes. Protocol #3 was previously
described (16) and involved cold protease activity. In short, nerves incubated in 10 mg/ml Native Bacillus
Lichenformis protease (Creative Enzymes NATE0633) with 125 U/ml DNAse (Sigma) for 7 minutes at 6°C
Supplementary Methods
while shaking. Tissue was transferred to a C-tube and the gentleMACS brain_03 program was completed
twice (gentleMACS Dissociator, Miltenyi Biotec). Incubation at 6°C was repeated for 8 min and all
subsequent steps were carried out at 4°C. Digestion was in all protocols terminated by the addition of 10
ml IMDM (Gibco) with 10% FCS and the cell suspension was filtered through a 70-μm cell strainer
(Falcon). Myelin was depleted using anti-myelin beads (Miltenyi Biotec) according to the manufacturer’s
protocol. Single cells were subsequently sorted (BD FACSAria III, BD FACSDiva v8.0.1 Software) for
intact viable cells using three viability markers: Zombi NIR APC Cy7, Calcein-AM FITC, and DAPI
(Biolegend) (Fig. S1B). Cell extraction from rat sciatic nerve was performed as previously described (17).
Briefly, sciatic nerves were homogenized in DMEM with 5% FCS using a scalpel and incubated with 1 mg
collagenase/dispase (Roche, Mannheim, Germany) and 100 µg DNase I (Roche) at 37° C for 45 minutes
each. Cells were washed twice with DMEM containing 5% FCS, resuspended in cold medium, and passed
through a 70µm cell strainer. Cells were centrifuged on a 30%/70% percoll gradient (GE healthcare,
Freiburg, Germany) at 1,000g for 30 minutes. Nerve mononuclear cells were collected from the interphase
and washed in culture media.
Generation of single cell libraries and sequencing
Single cell suspensions were loaded onto the Chromium Single Cell Controller using the Chromium Single
Cell 3' Library & Gel Bead Kit v2 (both from 10X Genomics) chemistry following the manufacturer’s
instructions. Sample processing and library preparation was performed according to manufacturer
instructions using AMPure XP beads (Beckman Coulter). Sequencing was either carried out on a local
Illumina Nextseq 500 using the High-Out 75 cycle kit with a 26-8-0-57 read setup or commercially
(Microanaly, China) on a NovaSeq 6000 using the 300 cycle kit with paired end 150 read setup. All the
samples were sequenced with a sequencing depth >50,000 reads per cell. Average sequencing depth
was 82,321 ± 12,332 SEM reads/cell (Table S1).
Preprocessing of sequencing data
Processing of sequencing data was performed with the cellranger pipeline v3.0.2 (10x Genomics) and
according to the manufacturer’s instructions. Raw bcl files were de-multiplexed using the cellranger
mkfastq pipeline. Subsequent reads alignments and transcript counting was done individually for each
sample using the cellranger count pipeline with standard parameters. The cellranger aggr pipeline was
employed, to generate a single cell-barcode matrix containing all the mice samples without normalization.
The normalization of each library was subsequently performed in Seurat (see below). The cellranger
computations were carried at the High Performance Computing Facility of the Westfälische Wilhems
University Münster. The pre-quality control (QC) total cell number was 28,550 with an average of 5,350 ±
50 SEM cells per mouse sample type (Table S1).
Clustering and differential expression analysis
Subsequent analysis steps were carried out with the R-package Seurat v3.0.0 (18) using R v3.6.0 as
recommended by the Seurat tutorials. Briefly, cells were filtered to exclude cell doublets and low-quality
cells with few genes or high mitochondrial counts. Specifically, cells with <200 genes / cell (C57BL/6J
Supplementary Methods
mice, rat, ICAM-1-/-
NOD and NOD control mice) or >2300 genes / cell (C57BL/6J mice, ICAM-1-/-
NOD and
NOD control mice), >3000 genes / cell (rat) and cells with >8% (C57BL/6J mice, ICAM-1-/-
NOD and NOD
control mice) or >10% (rat) mitochondrial genes were filtered out. After QC the total remaining cell number
used for further analysis was 5,400 (C57BL/6J mice), 12,500 (rat), 5,250 (ICAM-1-/-
NOD mice) and 5,400
(NOD control mice) (Table S1). In order to account for differences in the total number of molecules per
cell, the UMI data were normalized using a recently described approach with regularized negative
binomial regression(19). Dimensionality reduction was done by Principal Component analysis (PCA).
Statistically significant Principal Components (PCs) were identified by a combination of a JackStraw
significance test and an elbow plot. Dimensionality reduction was done by Uniform Manifold
Approximation and Projection (UMAP) with default parameters. Clusters were identified using the
“FindNeighbors“ and “FindClusters” function in Seurat. To annotate the clusters, genes differentially
expressed in a one vs. all cluster comparison were queried for known functions in a literature search and
plotted in feature plots.
Identifying differentially expressed genes between different conditions
In order to determine differentially expressed genes between ICAM-1-/-
NOD and NOD control mice, we
performed alignment using Harmony, a newly described alignment method that projects cells into a shared
embedding to cluster cell types across multiple experiments and conditions (20). Further downstream
analysis was conducted with the resulting harmony embeddings using UMAP, “FindNeighbors” and
“FindClusters” function in Seurat. We split each cluster into ICAM-1-/-
NOD and NOD control mice data and
used the “FindMarker” function to determine differentially expressed genes between the groups. Volcano
plots were generated with the R package EnhancedVolcano. Differentially expressed (DE) genes
identified by Seurat were used as input genes. The threshold for p values was set at 0.001 and for the
average log fold change at 0.5.
Identifying cellular interactions
Molecular interactions between the cells were identified by the recently developed CellPhoneDB (21).
Normalized and filtered scRNA-seq data with the clusters previously identified by Seurat were used for
CellPhoneDB analysis. Since the current CellPhoneDB release only accepts human ensembl IDs as input,
murine ensembl IDs were converted to human ensembl IDs using biomaRt(22). In total, 1,381 (9.82%) of
the murine ensembl IDs could not be matched by the ensembl database as suitable orthologues and were
discarded. As recommended, statistical iterations were set at 1000 and genes expressed by less than
10% of cells in the cluster were removed. The interactions are based on the CellPhoneDB repository (21).
Statistical significance of the cellular interactions were calculated as described (21). Briefly, the cell
clusters were randomly permuted and the mean of the average receptor and ligand expression of each
cluster was calculated. The p value for a given receptor-ligand complex was determined by calculating the
proportion of the means which are as or more extreme than the actual mean. Significant interactions
between clusters were visualized in a heatmap and clustered with complete linkage and Euclidean
distance measure using the R package pheatmap. Network visualization was performed with Cytoscape
v.3.7.1 using the previously identified significant interactions between the clusters. The network layout was
Supplementary Methods
set to compound spring embedder. To improve the comparability between different datasets, we
calculated the relative cluster size (cell count in cluster / total cell count in dataset) and the proportional
number of interactions (number of cell-cell interactions / total number of interactions in dataset). The width
and transparency of connecting arrows encoded the proportional number of interactions and the
directionality of ligand/receptor interaction. Node size encodes the relative cluster size. Network analysis
such as determination of betweenness centrality was performed with the integrated NetworkAnalzyer.
Gene set enrichment analysis
We used the Enrichr tool (23) to perform gene set enrichment analysis with the top markers of the
respective clusters identified by Seurat. The following reference datasets, which are integrated in Enrichr,
were employed: TF Perturbations Followed by Expression, Transcription Factor PPI, Enrichr Submission
TF Gene Cooccurrence Enrichment Analysis, WikiPathways 2019 Mouse, KEGG 2019 Mouse, Reactome
2016 and Panther 2016. Enrichment computation was conducted by Enrichr as described (23, 24). Briefly,
Fisher exact text was used to compute enrichment for input gene lists to determine a mean rank with
standard deviation from the expected rank. A z-score for deviation from this expected rank was calculated
by using a reference table of expected ranks with variances.
Comparison with published datasets
We compared DE genes in ICAM-1-/-
NOD vs. NOD control mice in specific clusters with a published
dataset of DE genes in EAE vs. control mice (2). To improve comparability, DE genes in our dataset were
identified using MAST (25) instead of Wilcoxon rank sum test with a lower average log fold change of
0.095. All DE genes with an adjusted p-value greater than 0.05 were removed. The top up- and
downregulated DE genes of the Falcao dataset (2) (cutoff gene expression >|4|) were compared with our
top DE genes using Venn Diagrams (R package VennDiagram (26)). The ‘Interferome’ database (3) was
then searched for the 30 intersecting genes.
Immunohistochemistry on mouse sciatic nerve
For histology-based methods, sciatic nerves were dissected from intracardially PBS perfused CD57BL/6
mice, CX3CR1-GFP mice and Flt3Cre-mT/mG mice and fixed in 4% PFA (Paraformaldehyde, Merck) for
24 hours. For fixed frozen slides (FF), tissue was dehydrated in a series of 15% and 30% sucrose and
embedded into OCT (Tissue-Tek). Snap freezing of the tissue blocks was done using dry ice. Cross
sections were cut (10 μm, Cryostar NX50, Thermo Scientific) and slides were stored at -80°C until staining
with fluorescent antibodies.
For immunocytochemistry, cytospin samples were prepared from peripheral nerve cells and autologous
bone marrow of intra-cardial PBS perfused C57BL/6 mice. PNS cells were isolated with protocol #2 as
previously described. Bone marrow was isolated from the femur by flushing the bone with PBS. Cells were
immediately filtered through a 70 μm cell strainer (Falcon), followed by ammonium chloride–based
erythrocyte lysis (BD Biosciences). Cyto-centrifugation was done with ±150’000 bone marrow cells or
±2’000 PNS cells. The aliquots were centrifuged at 100 xg for 5 minutes (Rotofix 32A, Hettich). Cytospin
slides were fixed in methanol for 5 minutes and air dried before staining.
Supplementary Methods
For antibody staining, tissue cross sections or cytospins were permeabilized with 0.1% TritonX-100
(Sigma) in PBS, and stained with the following primary antibodies: Cd68 (1:50, rat, Biocarta), F4/80
(1:500, rat, Serotec), Cxcl4 (1:100, rabbit, Thermofisher), Cd169 (1:100, gift from Dr. Antonio Castrillo),
Cd11b (1:200, rat, BD Biosciences) and SIGNR1 (1:50, A. hamster, Invitrogen) and incubated overnight at
4°C. Cells were washed with PBS and then incubated with Alexa Fluor (AF) conjugated antibodies (AF594
anti-rabbit, AF488 anti-rabbit, AF488 anti-rat, all 1:1000, Invitrogen) in blocking reagent (Roche), for 45
minutes at room temperature. The F4/80 signal was amplified by streptavidin-HRP (1:100, 45 min, RT),
tyramid (1:100, 15 min, RT) and streptavidin-594 (1:100, 45 min, RT). Slides were mounted in
Fluoromount G with DAPI (Invitrogen). Images were taken using a three laser fluorescent microscope
(Biorevo BZ-900 microscope with BZII Viewer software, Keyence) and processed in ImageJ.
Immunohistochemistry of human sural nerve biopsies
Human samples were selected according to the histological findings in sural nerve biopsies. Sural nerve
biopsies with no major pathological findings (with respect to inflammation, axonal and myelin pathology,
vascular pathology) were selected. Samples were anonymized and processed in a blinded manner.
Selected patients were of mixed age, between >30 years and <70 years of age (inclusion criteria) and did
not suffer from a severe neurological disorder at time-point of biopsy (exclusion criteria). In total five sural
nerve samples from five independent patients were analyzed. No other criteria besides the described
characteristics were applied. The study received ethical approval by the ethic board of the University Clinic
Leipzig, Germany. The staining was performed by the fully automated immunostainer Benchmark XT
(Roche, Basel, Switzerland). Staining protocol included deparaffinization and counterstain with
hematoxylin and blue colouring reagent according to manufacturers’ instructions. No pretreatment was
performed for MBP and SMA/ACTA2. Pretreatment with cell conditioning 1 (CC1, Roche, Basel,
Switzerland) was performed for all other antibodies. The following antibodies were used: LCA (CD45)
(leukocyte common antigen, monoclonal mouse, autostainer Dako, #IS751,), CD68 (monoclonal mouse,
1:100, Dako, #M0876), CD8 (monoclonal mouse, 1:50, Dako, #M7103), CD4 (monoclonal mouse, 1:100,
Dako, #M7310), CD34 (monoclonal mouse, 1:100, Dako, #M7165), SMA/ACTA2 (monoclonal mouse,
1:300, Dako, #M0851), SOX10 (polyclonal rabbit, 1:40, Cell Marque, #383A-76), MBP (monoclonal rabbit,
1:100, Cell Marque, #295A-16).
RNA in situ hybridization
RNA in situ hybridization (ISH) was performed on fixed frozen (FF), fresh frozen and paraffin embedded
sections of sciatic nerves from intra-cardial PBS perfused C57BL/6 mice, hGFAP-GFP mice and
PDGFRɑ-EGFP mice. Three different ISH kits were used according to manufacturer’s protocol. The
ViewRNA ISH Tissue Assay Kit (1-plex) from Thermo Fisher was used on fixed frozen tissue, to test the
probes Mm-Mbp, Mm-Apod, Mm-Smoc2, Mm-Sfrp4, and Mm-Pf4 and compared to Mm-Gapdh (positive
control) and Ba-DapB (negative control). Briefly, sections were dehydrated in a series of 50%-70%-90%
ethanol each for 10 min and baked in a dry oven at 60°C for 1 h. Protease QF (1:100) was applied to the
slides and incubated for 12,5 min at room temperature (RT), followed by two PBS washes. Probes were
diluted 1:40, hybridized for 3 hours at 40°C, 0% CO2 (CO2 cell culture incubator MCO-17A1, Sanyo) and
Supplementary Methods
slides were washed three times with wash buffer. Amplification steps were performed by incubating with
PreAmp1 QF (1:100, 25 min, 40°C), Amp1 QF (1:100, 15 min, 40°C) and Label Probe-AP (1:1000, 15
min, 40°C) with wash buffer washes of 3x2 min in between steps. Slides then incubated in AP-Enhancer
Solution for 5 min at RT and FastRed for 30 min at 40°C (ViewRNA Chromogenic Signal Amplification Kit,
1-plex, Thermo Fisher).
The ThermoFisher ViewRNA Cell Assay Kit (multiplex) was used, in combination with the first steps of the
previously mentioned Tissue Assay kit, to detect Mm-Apod, Mm-Smoc2, Mm-Ngfr, Mm-S100b, Mm-
Sox10, Mm-Sfrp4 and Mm-Pi16 and compared to Mm-Gapdh (positive control) and Ba-DapB (negative
control) in different single and co-stain settings. For the multiplex staining on sciatic mouse nerves, the
hybridization steps were performed with the ViewRNA Tissue Assay kit as previously described.
For the multiplex amplification steps, the ViewRNA ISH Cell Assay kit (multiplex) from Thermo Fisher was
used. Briefly, steps were performed by incubating with PreAmplifier Mix (1:100, 60 min, 40°C), Amplifier
Mix (1:100, 60 min, 40°C) and Label ProbeMix (1:100, 60 min, 40°C) with wash buffer washes of 3x2 min
in between steps. Slides then incubated in DAPI (1:100, 10 min, RT) followed by a PBS wash. Slides
processed with the Thermo Fisher ViewRNA ISH kits were mounted in Fluoromount G (Invitrogen).
The BaseScopeTM
Detection Reagent Kit – RED from ACD Biotech, was used to test the following probes:
Mm-Smoc2 and Mm-Sfrp4, co-stained with an antibody for Mbp, and compared to Mm-Ppib (positive
control) and Ba-DapB (negative control). The kit was used according to manufacturer’s instructions on
paraffin embedded samples. In short, paraffin embedded nerves were cut to 5 µm sections, dried
overnight at RT, baked one hour at 60 °C and subsequently de-paraffinized in a xylol/ethanol series.
Target retrieval was performed in RNAScope target retrieval buffer for 15 min at about 99 °C, then
washed in water and ethanol shortly. Protease Plus was applied on the samples and incubated for 30 min
at 40 °C. Target and control probes were applied to the sections, slides were hybridized for 2 h at 40°C
and washed twice with wash buffer for 2 min. Amplification steps were as follows, incubation with AMP1
(30 min, 40°C), AMP2 (15 min, 40°C), AMP3 (30 min, 40°C), AMP4 (15 min, 40°C), AMP 5 (30 min, RT)
and AMP 6 (15 min, RT) with wash buffer washes of 2x2 min in between steps. Signal was visualised by
incubation with the FastRED substrate (10 min at RT).
For myelin counterstaining the samples were washed twice in PBS after RNAScope incubation and
blocked with goat serum. Samples were incubated overnight at 4°C with Mbp antibody (1:200, rabbit,
CellMarque) and with its corresponding cyanine dye (1:1000, Dianova) for 1hr at RT and mounted in Aqua
Polymount. All images were obtained with an Axio Observer Z1 (Zeiss) and processed in AxioVision and
ImageJ.
Flow cytometry of leukocytes
Flow cytometry analysis was performed on isolated PNS cells, using protocol #2 as previously described.
After myelin removal, cells were stained for extracellular antibodies (25 min, RT). As a control, autologous
brain, spleen and bone marrow were isolated and processed into single cell suspension as previously
described(5). In short, the brain was first mechanically digested followed by enzymatic digestion with
CollagenaseD (2.5 mg/ml) and DNaseI (0.05 mg/ml). The digested brain, bone marrow and spleen were
mashed through a 70 μm cell strainer (Falcon) followed by ammonium chloride–based erythrocyte lysis
Supplementary Methods
(BD Biosciences). The following viability dye and murine antibodies were used: zombi NIR APC-Cy7;
CD45 BV421; CD11b BV510; CD68 PE Cy7; B220 PE; CD3 FITC; CD3 BV510; CD4 APC; CD8 Pacific
Blue; NK1.1 PE Cy7; NKG2AB6 PE; NKp46 FITC; F4/80 APC; CD14 FITC; Ly6C Percp Cy5.5; CD317
Pacific Blue; CCR9 PE Cy7; CD11c AF700; MHCII PE. Samples were measured on the Gallios (10
Colors, 3 lasers, Beckman Coulter) with Kaluza for Gallios software and analysed with FlowJo_V10.
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scRNA-seq
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MC
EC2
lymph
●
●
0 50K 100K 150K 200K 250K
FSC-A
0
-103
103
104
105
Calc
ein
AM
0 50K 100K 150K 200K 250K
FSC-A
0
50K
100K
150K
200K
250K
0-103
103
104
105
DAPI
0
103
104
105
Zom
bie
NIR
SSC-
A
Figure S1: Optimizing the peripheral nerve cell extraction protocol. (A) Peripheral nerve cells were isolated from the combined sciatic nerve and brachial plexus of naive adult C57BL/6 mice after intracardial PBS perfusion . Three different protocols were tested for enzymatic digestion: #1 collagenase/dispase (Roche, 0.5 mg/ml), 1 h at 37 ̊ C; #2 Trypsin (Gibco, 0.25%): Collagenase II (Worthington, 1.62 U/μl): Hyaluronidase (Worthington, 1%) ratio 1:1:0.04 + 1 μl Pronase (Roche, 1%)/50 μl of mix, 20 min at 37°C; #3 Native Bacillus Lichenformis protease (Creative Enzymes NATE0633, 10 mg/ml) + DNAse (Sigma, 125 U/ml), 7 min at 6°C, 2x gentleMACS brain_03 program (gentleMACS Dissociator, Miltenyi Biotec), 8 min at 6°C. The proportion of viable cells (grey part) of the plot against non-viable cells (white part) after each protocol is depicted in a stacked bar plot. Viable cells were defined as Calcein-AM+Zombie-NIR-DAPI- cells. (B) Gating strategy for flow cytometry-based viable cell sorting. (C) Experimental scheme of the five step cell extraction protocol: 1) intra-cardial PBS perfusion and isolation of sciatic nerve and brachial plexus, 2) peripheral nerve dissection and mechanical dissoci-ation, 3) four enzyme digestion, 4) magnetic bead-based myelin debris removal, 5) flow sorting for viable cells. This figure was modified from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic License (D) After multi-step purification of peripheral nerve cells, single cell (sc) transcriptomes were generated from n = 36 naive adult female C57BL/6 mice in three biological replicates (each replicate n = 12). The biological replicates are highlighted: red = batch #1, blue = batch #2, green = batch #3
A
lymph
MC
MP
TC BC EC2
mySC
fibronm
SCvSM
CEC
1PC
Dhtkd1MmeDnmt1SetxSlc25a46GarsYarsAtp7aPtrh2Cntnap1Gnb4Cox6a1Dnajb2Inf2Mfn2Dctn1Med25LmnaLrsam1Kif5aRetreg1Drp2Egr2Pmp22MpzPrxSco2Abhd12Rab7Hint1Atp1a1Coa7Fgd4HarsSpg11LitafSgpl1Aifm1Bscl2Pdk3Mpv17Trim2Sbf2Ndrg1GanArhgef10Fig4Fbln5Sigmar1Sbf1Mcm3apMorc2aDync1h1Dnm2VcpPlekhg5Hoxd10Mtmr2WarsPrps1Ighmbp2Sptlc1Hspb1KarsBag3Hspb8Prps1l3Dctn2Trpv4Kif1bNagluMarsAarsmt−Atp6Hk1Surf1
−3
−2
−1
0
1
2
3 Figure S2: Many hereditary neuropathy genes show highest expression in non-glia cells. (A) Genes known to cause hereditary neuropathies were retrieved from a public database (www.molgen.ua.ac.be/CMTMutations) and plotted in the clusters we identified in peripheral nerve cells of C57BL/6 mice. The average gene expression is color-coded. mySC: myelinating Schwann cells, nmSC: non-myelinating Schwann cells, fibro: fibroblasts, vSMC: vascular smooth muscle cells, PC: pericytes, EC1: endothelial cells cluster 1, EC2: endothe-lial cells cluster 2, lymph: lymphatic vessel endothelial cells, BC: B cells, TC: T cells and natural killer cells, MC: myeloid lineage cells, MP: macrophages.
●●●●
●●●●
●●●
●●●●●
●●
●●●●●
●●●●●●●●
●●●●●●
●●●●●
●●●●
MPMCBCTC
lymphPC
vSMCEC2EC1fibro
nmSCmySC
Btg2 Fth1 Mt2 Mt1Soc
s3 Jun
Fos
Apoe
Cryab Ptn
Mbp
−1012
Average Expression
Percent Expressed●
●●
406080
100
●●●●●●●●●●●
●●●
●●
●●●
●●●●●
●●●●●
●●
●●●
●●
MPMCBCTC
lymphPC
vSMCEC2EC1fibro
nmSCmySC
Sox9Mmp2
Ccl11
Ebf1 Osr2Ceb
pdSpry
2Tcf4
Lama2
Hspg2
Myoc
ApodMatn
2
0
1
2
Average Expression
Percent Expressed●
●●●
204060
80
A
B
UMAP1
UM
AP2
Ngfr
1.0
0.0
2.0Cspg4
0.75
0.0
1.5
Pdgfrb
0.8
0.0
1.6gene score
2.0
0.0
4.0
C
Ngfr Sox10 S100b0
20
40
60
80
100
% o
f all A
pod+ a
ndAp
od+ S
moc
2+ cel
ls
D
Figure S3: Localization of novel transcripts in non-myelinating Schwann cells and fibroblasts. (A-B) Dotplots of selected mySC marker genes (A) and nmSC marker genes (B) grouped by cluster. The average gene expression level per cluster is color coded and circle size represents the percentage of cells expressing the gene. Threshold was set to a minimum of 10% of cells expressing the gene. (C) Feature plots were generated to show expression of Ngfr, Cspg4 and Pdgfrb individually and as gene score combined. Plots are corresponding to Fig. 1A. Magnifications are zoomed in on the pericyte (PC) cluster. (D) This graph shows a quantification of the RNA ISH stainings performed in Fig. 2B and Fig. S4-6. The percentage of cells that co-stained for Ngfr, Sox10 or S100b was calculated within cells that expressed Apod alone or Apod together with Smoc2. Data are depicted as mean ± SEM, n=12.
DAPI ApodNgfr Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
Ngfr DAPIApod Smoc2
*
Figure S4: co-staining of the nmSC markers Apod and Smoc2 with NgfrFresh-frozen sections of sciatic nerves of naive adult C57BL/6 mice were stained for Apod, Smoc2 together with the Schwann cell marker Ngfr by RNA ISH as described in the methods. This figure corresponds to Fig. 2B. Please note that each dot repre-sents a single RNA molecule. White dotted line shows the epineurium border of the sciatic nerve. Nuclei were stained with DAPI. Scale bars 20 μm (left) and 10 μm (magnification). Arrows indicate co-staining of all markers, asterisks indicate co-stain of a new marker with a known lineage marker and arrowheads indicate individual staining.
DAPI ApodS100b Smoc2
S100b DAPIApod
Smoc2
*
*
*
S100b DAPIApod
S100b DAPIApod
S100b DAPIApod
S100b DAPIApod
S100b DAPIApod
Smoc2S100b DAPIApod
Smoc2S100b DAPIApod
Smoc2S100b DAPIApod
Smoc2S100b DAPIApod
*
*
*
*
Figure S5: co-staining of the nmSC markers Apod and Smoc2 with S100bFresh-frozen sections of sciatic nerves of naive adult C57BL/6 mice were stained for Apod, Smoc2 together with the Schwann cell marker S100b by RNA ISH as described in the methods. This figure corresponds to Fig. 2B. Please note that each dot represents a single RNA molecule. White dotted line shows the epineurium border of the sciatic nerve. Nuclei were stained with DAPI. Scale bars 20 μm (left) and 10 μm (magnification). Arrows indicate co-staining of all markers, asterisks indicate co-stain of a new marker with a known lineage marker and arrowheads indicate individual staining.
DAPI ApodSox10 Smoc2
Sox10 DAPIApod
Smoc2
*
*
Sox10 DAPIApod
Sox10 DAPIApod
Sox10 DAPIApod
Sox10 DAPIApod
Sox10 DAPIApod
Sox10 DAPIApod
Sox10 DAPIApod
Sox10 DAPIApod
Sox10 DAPIApod
Smoc2
Smoc2
*
**
*
*
*
*
*
*
*
Figure S6: co-staining of the nmSC markers Apod and Smoc2 with Sox10Fresh-frozen sections of sciatic nerves of naive adult C57BL/6 mice were stained for Apod, Smoc2 together with the Schwann cell marker Sox10 by RNA ISH as described in the methods. This figure corresponds to Fig. 2B. Please note that each dot repre-sents a single RNA molecule. White dotted line shows the epineurium border of the sciatic nerve. Nuclei were stained with DAPI. Scale bars 20 μm (left) and 10 μm (magnification). Arrows indicate co-staining of all markers, asterisks indicate co-stain of a new marker with a known lineage marker and arrowheads indicate individual staining.
Pi16 DAPISfrp4DAPI Sfrp4 Pi16
DAPI ApodSmoc2 Vim
DAPI PdgfraGFP Smoc2 Apod
*
*
DAPI Apod Smoc2 Vim
DAPI PdgfraGFP Smoc2 Apod
DAPI PdgfraGFP DAPISmoc2
Apod
DAPISmoc2
Apod DAPIVim
A
C
B
Pi16 DAPISfrp4
Pi16 DAPISfrp4
Pi16 DAPISfrp4
Pi16 DAPISfrp4
Pi16 DAPISfrp4
Pi16 DAPISfrp4
Pi16 DAPISfrp4
Pi16 DAPISfrp4
Figure S7: RNA ISH staining of multiple fibro markers(A) Fresh frozen sections from C57BL/6 mice, as in Fig. 2, were stained for Apod and Smoc2 and the fibroblast marker Vim (encoding Vimentin) by ISH. (B) Fresh frozen sections from PDGFRa-GFP mice, as in Fig. 2, were stained for Apod and Smoc2 by ISH. Scale bars 50 μm (left), 20 μm (right), and 10 μm (magnification). Asterisk indicates co-stain of Apod and Smoc2, arrowhead indicates single stain of fibroblast markers Vim and PdgfraGFP. (C) Fresh-frozen sections of sciatic nerves of naive adult C57BL/6 mice, as in Fig. 2, were stained for Sfrp4 with the fibro marker Pi16 by RNA ISH as described in the methods. This figure corres-ponds to Fig. 2D. Please note that each dot represents a single RNA molecule. The GFP protein signal is more homogeneously distributed. White dotted line shows the epineurium border of the sciatic nerve. Nuclei were stained with DAPI. Scale bars 20 μm (left) and 10 μm (magnification). Arrows indicate co-staining of the two markers and arrowheads indicate indivi-dual staining.
PdgfraGFP Pi16 DAPIDAPI PdgfraGFP Pi16
Sfrp4 DAPIDAPI PdgfraGFP Sfrp4B
PdgfraGFP Pi16 DAPI
PdgfraGFP Pi16 DAPI
PdgfraGFP Pi16 DAPI
PdgfraGFP Pi16 DAPI
PdgfraGFP Pi16 DAPI
PdgfraGFP
Sfrp4 DAPIPdgfraGFP
Sfrp4 DAPIPdgfraGFP
Sfrp4 DAPIPdgfraGFP
Sfrp4 DAPIPdgfraGFP
Sfrp4 DAPIPdgfraGFP
A
Figure S8: Sciatic nerve of a PDGFRαGFP reporter mouse stained with the fibro markers Pi16 and Sfrp4PFA Fixed-frozen sections of sciatic nerves of naive adult PDGFRαGFP mice were stained for Pi16 (A) and Sfrp4 (B) by RNA ISH as described in the methods. This figure corresponds to Fig. 2D. Please note that each dot represents a single RNA molecule. The GFP protein signal is more homogene-ously distributed. White dotted line shows the epineurium border of the sciatic nerve. Nuclei were stained with DAPI. Scale bars 20 μm (left) and 10 μm (magnification). Arrows indicate co-staining of the two markers and arrowheads indicate individual staining.
A SC (SOX10)B mySC (MBP)C
fibro + EC (CD34)D vSMC + PC (ACTA2)E
MP (CD68)G TC (CD8)H
Leuko (CD45)F
TC (CD4)I
Figure S9: Confirmation of some cell populations in human sural nerves.Representative histological stainings of human sural nerve biopsies of a control patient (out of n=5 patients) without signs of peripheral nerve pathology are shown. (A) Semithin cross section of a representative sural nerve. cale bar 50µm. (B-I) Immunohistochemistry (IHC) stainings of control sural nerve for SOX10 (B), MBP (C), CD34 (D), smooth muscle actin (ACTA2) (E), pan leukocyte marker CD45 (F), CD68 (G), CD8 (H) and CD4 (I). Scale bars 50µm. SC: Schwann cells, mySC: myelinating Schwann cells, fibro: fibroblasts, EC: endothelial cells, vSMC: vascular smooth muscle cells, PC: pericytes, Leuko: leukocytes, MP: macrophages, TC: T cells
A
B
100
101
102
103
104
105
106
100
101
102
103
104
105
106
1,03% 0,21%
3,30%10
010
110
210
310
410
510
6
100
101
102
103
104
105
106
GPVI
CD
41
live/
dead
CD45
Bone marrow Sciatic nerve
UMAP1
UM
AP2
Ms4a7
0.0
2.0Ccl4
0.0
5.02.5
0.0
0.60.3
Cx3cr1Ccl17
0.0
4.02.0
C1qb
0.0
4.02.0
Rt1-Bb
0.0
4.02.0
C
8,14%
DAPI
Cxcl4F4/80
DAPI
Cxcl4F4/80
DAPI
Cxcl4F4/80
Figure S10: Cxcl4-expressing cells in peripheral nerves are not megakaryocytes. (A) Cytospins were generated from bone marrow and sciatic nerve from a female C57BL/6 mouse. Slides were stained for F4/80, Cxcl4 and DAPI by immunocytochemistry. Arrow indicates co-stain, arrow-heads indicate single stain of Cxcl4. (B) PNS cells were purified from two female C57BL/6 mice, pooled, and analyzed by flow cytometry. The viable CD45+ cell population (left), was stained for CD41 and GPVI (right) to determine the percentage of megakaryocytes. One repre-sentative dotplot out of two independent experiments is shown. (C) Feature plots of selected myeloid genes correspon-ding to Fig. 3E (rat dataset).
Cx3cr1GFP Cd68Cxcl4 DAPIDAPI Cx3cr1GFP
Cxcl4 Cd68
Cx3cr1GFP Cd68Cxcl4 DAPI
Cx3cr1GFP Cd68Cxcl4 DAPI
Cx3cr1GFP Cd68Cxcl4 DAPI
Cx3cr1GFP Cd68Cxcl4 DAPI
Cx3cr1GFP Cd68Cxcl4 DAPI
Figure S11: Sciatic nerve of a CX3CR1GFP reporter mouse stained with Cxcl4 and Cd68 antibodies PFA Fixed-frozen sections of sciatic nerves from CX3CR1-GFP reporter mice were stained for Cxcl4, Cd68 and DAPI using immunohistochemistry. This figure corresponds to Fig. 3H. Scale bar 50 μm (left), 20 μm (magnification). Please note that the samples were stained on protein level, so the signal is homogeneously distributed. Arrows indicate co-stain of two markers and arrowheads indicate individual staining.
A
B
m-tdTomatom-GFP
% w
ithin
CD
11b+ C
D68
+
brain PNS BM SPC 0
25
50
75
10075.7%liv
e/de
adC
D11
b
CD68 m-tdTomato
m-G
FP
0.86% 66.0%
34.0%
C
●●●
●●
●●
●●
●●●
●●●
●●●●●
●●●
●●●
●●●
●●
●●
MPMCBCTC
lymphPC
vSMCEC2EC1fibro
nmSCmySC
Tgfbr1 Hexb Frcls Sparc Tmem119 Pry12
Average Expression
−1012
Percent Expressed ●●●
255075
D
Yolk sac hematopoiesis (<E10)
Microglia&
Some tissue macrophages
Yolk sacstem cell
M
EMP
YSC
Erythro-myeloidprogenitors
Definitive hematopoiesis (>E10)
TC BC
HSC
MPP
LPMP
GRM MK
Myeloid progenitor
Lymphoid progenitor
Multi potentprogenitor
Hematopietic stem cellFlt3- or Flt3+
MacrophagesGranulocytes
MegakaryocytesT cells
B cells
m-GFPm-tdTomato
m-GFPm-tdTomato
Figure S12: Nerve-associated macrophages are heterogeneous and transcriptionally different from microglia. (A) Simplified schematic overview of hematopoiesis, that takes place until embryonic day E8.0, and definitive hematopoiesis, starting from embryonic day E12.5. Colors of schematic cells indicate their expression of membrane-targeted tdTomato (mT; red) and membrane-targeted GFP (mG; green) in Flt3Cre-mT/mG mice. (B) Representative gating of pooled leukocytes extracted from the PNS of three Flt3Cre-mT/mG mice and stained against Cd11b and Cd68. (C) Three male adult Flt3Cre-mTmG mice were intracardially perfused with PBS. The proportion of viable Cd11b+Cd68+ cells expressing mT and mG in brain, peripheral nerve cells (PNS), bone marrow (BM), and spleen (SPC) was quantified by flow cytometry as in B. No CD45 antibodies were intravenously injected (1). Bar graph shows the proportion of cells in the m-GFP+ and m-tdTomato+ gates. One out of two expe-riments is shown. (C) Fresh frozen sections of sciatic nerves of a male Flt3Cre-mT/mG mouse were stained for DAPI. Scale bar 50 μm (left), 20 μm (right), arrowheads indicate single stain. (D) Dotplot of the expression of microglia markers in the cell clusters derived from naive C57BL/6 mice is shown. The circle size correlates with the percentage of cells expressing the gene and the average gene expression level per cluster is color-coded. Threshold was set to a minimum of 1% of cells in the cluster expres-sing the gene.
A
B
UMAP1
UM
AP2
0.0
2.01.0
H2-DMb2
0.0
2.01.0
Ms4a1
0.0
2.01.0
Ighd
0.0
6.03.0
Ighg1
0.0
2.01.0
Cx3cr1
0.0
2.01.0
Vps37b
0.0
2.01.0
Cxcr6
0.0
2.01.0
Cd8a
0.0
2.01.0
Klrc1 Xcl1
0.0
3.01.5
Adgre1
0.0
1.0Pf4
0.0
2.0Cd86
0.0
1.6
0.8
0.0
1.00.5
Flt3 Siglech
0.0
2.01.0
C Pmp22
2.0
0.0
4.0
1.50.0
3.0
2.5
0.0
5.0
2.0
0.0
4.0
Gsn Mpz
2.0
0.0
4.0
1.50.0
3.0
1.25
0.0
2.5Sostdc1
1.00.0
2.0
NO
D
ICAM
-1-/-N
OD
2.0
0.0
4.0
2.5
0.0
5.0
Ccl5Psmb8
0.8
0.0
1.6
1.0
0.0
2.0
B2m
1.5
0.0
3.0
1.50.0
3.0
H2-K1
1.0
0.0
2.0
1.25
0.0
2.5
NO
D
ICAM
-1-/-N
OD
UMAP1
UM
AP2
Figure S13: Pre-inflammatory stage of sciatic nerve and gene expression of ICAM-1-/-NOD mice. (A) Longitudinal cryo-section of the sciatic nerve of pre-clinical female ICAM-1-/-NOD mice stained with hematoxylin and eosin. One represen-tative out of 10 mice is shown. Scale bars represent 200μm and 50μm. (B) Feature plots of selected leukocyte markers corresponding to Fig. 4A. Insets show higher magnification of smaller clusters of interest. Intensity of red indicates expression level. (C) Genes differentially expressed (DE) in selected cell clusters between NOD control (top rows) and ICAM-1-/- NOD (bottom rows) derived samples were calculated. Selected DE genes are shown in feature plots corres-ponding to Fig. 4A
NF1
ISRE
STAT
ICSBP
IRF
IRF7
STAT1
IRF8
IRF1
STAT3
NFKB
IRF3
NFKAPPAB65
NFKAPPAB50
NFKAPPAB
SIRT6
-1500bp -1000bp -500bp 0bp 500bp
Rtp4H2-D1
Ifi27Calm2
Iigp1Psmb10
ApodPlp1
Shisa5Bst2
TapbpTap2Tap1
Oasl2Psmb8H2-K1H2-Q4
Irf1Hsp90b1
Hspa5Gas7B2m
Gbp7Igtp
Irgm2Irgm1
Ifi35H2-T23
Stat1Gbp3
Figure S14: Transcriptional response to autoimmunity is conserved between oligos and mySC and mimicking an IFN-responseDifferentially expressed genes (up- or down-regulated) were identified in the mySC cluster in ICAM-1-/-NOD vs. NOD control mice. Published DE genes of oligos in EAE vs. control CNS were obtained (2). DE gene lists from both sources were tested for overlap (compared with Venn diagram in Fig. 4G). Shared DE genes were submitted to the Interferome database (3) and the plot generated by the database in ‘transcription factor (TF) analysis’ was downloaded. Binding sites of the indicated TF are depicted as colored boxes relative to the transcriptional start site (0 bp) of the indicated genes.
CD
74_MIF
APP_CD
74SELL_SELPLGG
AS6_AXLSELL_C
D34
CXC
L12_CXC
R4
SPP1_CD
44C
CL11_C
CR
2C
XCL12_A C
KR3
GR
N_TN
FRSF1A
CC
L11_DPP4
CXC
L12_DPP4
PDG
FA_PDG
FRA
a7b1 complex_LAM
C1
a6b4 complex_IG
F1a6b1 com
plex_LAMC
1C
CL2_C
CR
2C
CL7_C
CR
2C
CL24_C
CR
2C
CL8_C
CR
2C
CL7_C
CR
5TN
F_LTBRC
CL8_C
CR
5PTPR
C_M
RC
1TSLPR
_CR
LF2PSAP_G
PR37L1
JAM2_JAM
3FG
FR1_N
CAM
1JAG
1_NO
TCH
2TSLPR
_TSLPC
D48_C
D244
CC
L5_CC
R5
LTB_L TBRD
LL4_NO
TCH
2C
CR
5_CC
L4SEM
A4D_C
D72
LILRB4_LAIR
1EN
TPD1_AD
OR
A2AIN
SL6_NO
TCH
1D
LL4_NO
TCH
1PTPR
C_C
D22
SELL_POD
XLTG
FBR3_TG
FB1PR
OS1_AXL
aVb5 complex_FN
1H
BEGF_C
D44
PTN_PTPR
SC
D8 receptor_LC
KIG
F1R_IG
F1JAG
1_NO
TCH
3PTH
LH_PTH
1RJAG
1_NO
TCH
1TN
F_TNFR
SF1AFAS_TN
FIL34_C
SF1RD
LL4_NO
TCH
3C
SF1_CSF1R
C3_C
3AR1
BC−BCBC−TC(CD8)BC−fibroBC−mySCBC−MCBC−EC1BC−vSMC/PCBC−pDCBC−EC2TC(CD8)−(TC)CD4 TC(CD8)−nmSCTC(CD8)−BCTC(CD8)−TC(CD8)TC(CD8)−fibroTC(CD8)−mySCTC(CD8)−MCTC(CD8)−EC1TC(CD8)−vSMC/PCTC(CD8)−pDCTC(CD8)−EC2fibro−TC(CD4)fibro−nmSCfibro−BCfibro−(TC)CD8fibro−fibrofibro−mySCfibro−MCfibro−EC1fibro−vSMC/PCfibro−pDCfibro−EC2mySC−TC(CD4)mySC−nmSCmySC−BCmySC−TC(CD8)mySC−fibromySC−mySCmySC−MCmySC−EC1mySC−vSMC/PCmySC−pDCmySC−EC2MC−TC(CD4)MC−nmSCMC−BCMC−TC(CD8)MC−fibroMC−mySCMC−MCMC−EC1MC−vSMC/PCMC−pDCMC−EC2EC1−TC(CD4)EC1−nmSCEC1−BCEC1−TC(CD8)EC1−fibroEC1−mySCEC1−MCEC1−EC1EC1−vSMC/PCEC1−pDCEC1−EC2vSMC/PC−TC(CD4)vSMC/PC−nmSCvSMC/PC−BCvSMC/PC−TC(CD8)vSMC/PC−fibrovSMC/PC−mySCvSMC/PC−MCvSMC/PC−EC1vSMC/PC−vSMC/PCvSMC/PC−pDCvSMC/PC−EC2pDC−TC(CD4)pDC−nmSCpDC−BCpDC−TC(CD8)pDC−fibropDC−mySCpDC−MCpDC−EC1pDC−vSMC/PCpDC−pDCpDC−EC2EC2−TC(CD4)EC2−nmSCEC2−BCEC2−TC(CD8)EC2−fibroEC2−mySCEC2−MCEC2−EC1EC2−vSMC/PCEC2−pDCEC2−EC2
0
1
2
3
4
5
6BA
TC(CD8)
MC
EC2
pDC
vSMC/PC
mySC
EC1
fibro
TC (CD4)
nmSC
BC
NOD control
C ICAM-1-/-NOD
mySC
MC
fibro
BC
vSMC/PC
EC1
pDC
TC(CD8)
TC (CD4)
nmSC
EC2
D
CD
74_MIF
APP_CD
74C
XCL12_C
XCR
3C
CL11_C
CR
2C
CL7_C
CR
5C
CL7_C
CR
2C
XCL12_C
XCR
4EG
FR_TG
FB1TG
FBR3_TG
FB1C
CL2_C
CR
2SELL_SELPLGTG
Fbeta receptor1_TGFB1
SELL_CD
34PSAP_G
PR37L1
IFNG
_Type II IFNR
a4b1 comple x_FN
1a4b7 com
plex_FN1
DLL4_N
OTC
H2
a6b4 compl ex_IG
F1a6b1 com
pl ex_LAMC
1PD
GFA_PD
GFR
Aa7b1 com
plex_LAMC
1aVb1 com
plex_VTNaVb1 com
plex_FN1
CD
86_CTLA4
PLAUR
_a4b1 comple x
CXC
L16_CXC
R6
CXC
L9_CXC
R3
CXC
L10_CXC
R3
CC
L8_CC
R5
CC
L8_CC
R2
PECAM
1_CD
38JAM
2_a4b1 complex
TNFR
SF1A_ FASLGG
RN
_TNFR
SF1BPTN
_PTPRS
JAM2_JAM
3a1b1 com
plex_CO
L18A1EFN
A1_EPHA5
a1b1 complex_C
OL27A1
a1b1 complex_C
OL16A1
a1b1 complex_C
OL5A2
a1b1 complex_C
OL5A3
a1b1 complex_C
OL1A1
a1b1 complex_C
OL4A2
a1b1 complex_C
OL4A1
a1b1 comple x_C
OL6A3
a1b1 complex_C
OL1A2
a1b1 complex_C
OL3A1
RAR
RES2_C
MKLR
1TH
Y1_aXb2 complex
ICAM
1_aXb2 complex
ALCAM
_CD
6C
D8 receptor_LC
KC
CL5_C
CR
5aLb2 com
plex_ICAM
1IC
AM1_ITG
ALC
CR
5_CC
L4PTPR
C_C
D22
CD
28_CD
86SEM
A4D_C
D72
LILRB4_LAIR
1aVb5 com
plex_FN1
a11b1 complex_C
OL6A3
a11b1 compl ex_C
OL1A2
a11b1 complex_FN
1a11b1 com
plex_CO
L8A1a11b1 com
plex_CO
L6A1a11b1 com
ple x_CO
L15A1FG
F2_FGFR
1a11b1 com
plex_CO
L6A2a11b1 com
pl ex_CO
L18A1a11b1 com
plex_CO
L5A2a11b1 com
plex_CO
L4A2a11b1 com
plex_CO
L4A1a11b1 com
pl ex_CO
L3A1a11b1 com
plex_CO
L5A3a11b1 com
pl ex_CO
L1A1C
XCL12_D
PP4C
CL11_D
PP4N
RP2_SEM
A3CN
RP2_VEG
FALR
P1_MD
KSELL_PO
DXL
TNF_TN
FRSF1A
CXC
L9_DPP4
CXC
L10_DPP4
TGFbeta receptor1_TG
FB3C
D44_H
GF
EFNA4_EPH
A2SPP1_C
D44
a4b1 comple x_SPP1
aLb2 compl ex_F11R
aLb2 complex_IC
AM2
FASLG_FAS
CLEC
2D_FAM
3CVC
AM1_a4b1 com
plexSEM
A4D_PLXN
B1IL34_C
SF1RH
BEGF_C
D44
a4b7 comple x_VC
AM1
CC
L5_CC
R1
CC
L18_CC
R1
CC
L15_CC
R1
CC
L8_CC
R1
C3_C
3AR1
PDG
FB_PDG
FRB
IGF1R
_IGF1
PDG
FB_PDG
FRA
ANG
PT2_TEKN
RP1_VEG
FAD
LL4_NO
TCH
3a1b1 com
pl ex_CO
L17A1EFN
A1_EPHA2
TNFSF12_TN
FRSF12A
FGFR
1_NC
AM1
EFNA5_EPH
A5a11b1 com
pl ex_CO
L27A1a11b1 com
pl ex_CO
L16A1a11b1 com
plex_CO
L17A1EFN
A5_EPHA2
LRP1_PD
GFB
RSPO
1_LGR
4PD
GFR
complex_PD
GFB
CC
L7_CC
R1
CSF1_C
SF1RO
SMR
_OSM
F AS_TNF
LIFR_O
SMEFN
A4_EPHA5
FCER
2_aXb2 complex
SEMA7A_a1b1 com
ple xC
D96_PVR
HBEG
F_EGFR
TNF_TN
FRSF1B
TGFBR
3_TGFB3
a11b1 complex_C
OL14A1
a11b1 complex_C
OL5A1
a1b1 compl ex_C
OL8A1
FLT1_VEGFA
a1b1 compl ex_C
OL14A1
a1b1 comple x_C
OL5A1
ENTPD
1_ADO
RA2B
a1b1 comple x_C
OL15A1
a1b1 complex_C
OL6A2
a1b1 complex_C
OL6A1
GAS6_AXL
CXC
L12_ACKR
3PR
OS1_AXL
GR
N_TN
FRSF1A
TC(CD4)−TC(CD4)TC(CD4)−nmSCTC(CD4)−BCTC(CD4)−TC(CD)8TC(CD4)−fibroTC(CD4)−mySCTC(CD4)−MCTC(CD4)−EC1TC(CD4)−vSMC/PCTC(CD4)−pDCTC(CD4)−EC2nmSC−TC(CD4)nmSC−nmSCnmSC−BCnmSC−TC(CD8)nmSC−fibronmSC−mySCnmSC−MCnmSC−EC1nmSC−vSMC/PCnmSC−pDCnmSC−EC2BC−TC(CD4)BC−nmSCBC−BCBC−TC(CD8)BC−fibroBC−mySCBC−MCBC−EC1BC−vSMC/PCBC−pDCBC−EC2TC(CD8)−TC(CD4)TC(CD8)−nmSCTC(CD8)−BCTC(CD8)−TC(CD8)TC(CD8)−fibroTC(CD8)−mySCTC(CD8)−MCTC(CD8)−EC1TC(CD8)−vSMC/PCTC(CD8)−pDCTC(CD8)−EC2fibro−TC(CD4)fibro−nmSCfibro−BCfibro−TC(CD8)fibro−fibrofibro−mySCfibro−MCfibro−EC1fibro−vSMC/PCfibro−pDCfibro−EC2mySC−TC(CD4)mySC−nmSCmySC−BCmySC−TC(CD8)mySC−fibromySC−mySCmySC−MCmySC−EC1mySC−vSMC/PCmySC−pDCmySC−EC2MC−TC(CD4)MC−nmSCMC−BCMC−TC(CD8)MC−fibroMC−mySCMC−MCMC−EC1MC−vMC/PCMC−pDCMC−EC2EC1−TC(CD4)EC1−nmSCEC1−BCEC1−TC(CD8)EC1−fibroEC1−mySCEC1−MCEC1−EC1EC1−vSMC/PCEC1−pDCEC1−EC2vSMC/PC−TC(CD4)vSMC/PC−nmSCvSMC/PC−BCvSMC/PC−TC(CD8)vSMC/PC−fibrovSMC/PC−mySCvSMC/PC−MCvSMC/PC−EC1vSMC/PC−vSMC/PCvSMC/PC−pDCvSMC/PC−EC2pDC−TC(CD4)pDC−nmSCpDC−BCpDC−TC(CD8)pDC−fibropDC−mySCpDC−MCpDC−EC1pDC−vSMC/PCpDC−pDCpDC−EC2EC2−TC(CD4)EC2−nmSCEC2−BCEC2−TC(CD8)EC2−fibroEC2−mySCEC2−MCEC2−EC1EC2−vSMC/PCEC2−pDCEC2−EC2
0
1
2
3
4
5
6
Figure S15: Comparative cell-cell interactomes in healthy and pre-neuropathic mice. (A, C) Cellular interaction networks predicted from genes expressed in the peripheral nervous system of NOD control (A) and ICAM-1-/-NOD mice. Nodes (circles) repre-sent cell clusters and node size correlates with the relative cell count. Significant cell-cell interacti-ons were predicted by CellP-honeDB and represent edges (arrows) in the network. The width and transparency of the edges correlate with the amount of interactions; arrows indicate the directionality of ligand/receptor interactions. Layout was set to compound spring embedder. (B, D) Heatmaps depicting the color-coded negative natural logarithm of the p-value (Methods) of the signifi-cant cell-cell interactions of NOD control (B) and ICAM-1-/-NOD (D) samples. Only cell-cell interactions are shown that show at least one significant interaction. mySC: myelinating Schwann cells, nmSC: non-myelinating Schwann cells, fibro: fibroblasts, vSMC/PC: vascu-lar smooth muscle cells and pericy-tes, EC1: endothelial cells cluster 1, EC2: endothelial cells cluster2, BC: B cells, CD4: CD4 T helper cells, CD8: cytotoxic CD8 T cells and natural killer cells, MC: myeloid lineage cells, pDC: plasmacytoid dendritic cells.
Supplementary Table Legends
Supplementary Table Legends
Table S1: A summary of technical information regarding samples and sequencing.
STDEV: Standard deviation; SEM: Standard error of the mean
Table S2: Marker genes of cluster in naive mice (corresponding to Fig. 1A)
avg_log_FC: log fold change of the average expression between the cluster vs. all remaining clusters;
pct.1: percentage of cells with the gene detected in the cluster; pct.2: percentage of cells with the gene
detected in all remaining clusters; p_val_adj: adjusted p values (based on Bonferroni correction). Average
log FC threshold was set to 0.25.
Table S3: Marker genes of mySC and nmSC in naive mice with lowered threshold
Threshold was lowered to detect panSC markers in mySC and nmSC, genes of interest are highlighted in
yellow. avg_log_FC: log fold change of the average expression between the cluster vs. all remaining
clusters; pct.1: percentage of cells with the gene detected in the cluster; pct.2: percentage of cells with
the gene detected in all remaining clusters; p_val_adj: adjusted p values (based on Bonferroni correction).
Average log FC threshold was set to 0.05.
Table S4: Curated list of transcripts not previously identified in myelinating Schwann cells.
avg_log_FC: log fold change of the average expression between the mySC cluster vs. all remaining
clusters; pct.1: percentage of cells with the gene detected in the mySC cluster; pct.2: percentage of cells
with the gene detected in all remaining clusters; p_val_adj: adjusted p values (based on Bonferroni
correction).Threshold was set on 0.25 avg logFC; PNS-SC/myelination: these genes have been previously
described to be expressed by Schwann cells of the PNS or to be associated with PNS myelination. CNS
glia cells/myelination: these genes have been previously described to be expressed by glial cells of the
CNS or to be associated with CNS myelination. SC: Schwann cell, PNS: peripheral nervous system, CNS:
central nervous system, OL: oligodendrocyte, OPC: oligodendrocyte precursor cell, DRG: dorsal root
ganglion
Table S5: GSEA results of marker genes in the mySC cluster in naive mice
p_val_adj: FDR-adjusted p values
Table S6: GSEA results of marker genes in the nmSC cluster in naive mice
p_val_adj: FDR-adjusted p values
Table S7: GSEA results of marker genes in the fibro cluster in naive mice
p_val_adj: FDR-adjusted p values
Supplementary Table Legends
Table S8: Marker genes of cell clusters in rat samples (corresponding to Fig. 3F)
avg_log_FC: log fold change of the average expression between the cluster vs. all remaining clusters;
pct.1: percentage of cells with the gene detected in the cluster; pct.2: percentage of cells with the gene
detected in all remaining clusters; p_val_adj: adjusted p values (based on Bonferroni correction),
Threshold for average logFC was set to 0.25.
Table S9: Marker genes of cell clusters in ICAM-1-/-
NOD and NOD control mice (corresponding to Fig. 4A)
avg_log_FC: log fold change of the average expression between the cluster vs. all remaining clusters;
pct.1: percentage of cells with the gene detected in the cluster; pct.2: percentage of cells with the gene
detected in all remaining clusters; p_val_adj: adjusted p values (based on Bonferroni correction).
Threshold for average logFC was set at 0.25.
Table S10: Differentially expressed genes in the EC cluster in ICAM-1-/-
NOD vs. NOD control mice
avg_log_FC: log fold change of the average expression between ICAM-1-/-
NOD and NOD control mice
(positive values indicate a higher gene expression in ICAM-1-/-
NOD mice); pct.1: percentage of cells with
the gene detected in ICAM-1-/-
NOD mice; pct.2: percentage of cells with the gene detected in NOD control
mice; p_val_adj: adjusted p values (based on Bonferroni correction). Threshold for average logFC was set
to 0.25.
Table S11: Differentially expressed genes in the nmSC cluster in ICAM-1-/-
NOD vs. NOD control mice
avg_log_FC: log fold change of the average expression between ICAM-1-/-
NOD and NOD control mice
(positive values indicate a higher gene expression in ICAM-1-/-
NOD mice) ; pct.1: percentage of cells with
the gene detected in ICAM-1-/-
NOD mice; pct.2: percentage of cells with the gene detected in NOD control
mice; p_val_adj: adjusted p values (based on Bonferroni correction). Threshold for average logFC was set
at 0.25.
Table S12: Differentially expressed genes in the mySC cluster in ICAM-1-/-
NOD vs. NOD control mice
avg_log_FC: log fold change of the average expression between ICAM-1-/-
NOD and NOD control mice
(positive values indicate a higher gene expression in ICAM-1-/-
NOD mice) ; pct.1: percentage of cells with
the gene detected in ICAM-1-/-
NOD mice; pct.2: percentage of cells with the gene detected in NOD control
mice; p_val_adj: adjusted p values (based on Bonferroni correction). Threshold for average logFC was set
at 0.25.
Supplementary Table Legends
Table S13: Differentially expressed genes in the oligo cluster in naive vs. EAE mice (2), in the mySC
cluster in ICAM-1-/-
NOD vs. NOD control mice and the overlapping genes
Top DE genes (up- and down-regulated) were identified in the mySC cluster in ICAM-1-/-NOD vs. NOD
nerves. Published genes DE in oligo in EAE vs. control CNS (2) were obtained. DE gene lists from both
sources were tested for overlap (Venn diagram, Fig. 4G). Shared DE genes were submitted to the
Interferome database and the plot generated in TF analysis was downloaded. Binding sites of the
indicated TF are depicted as colored boxes relative to the transcriptional start site (0 bp) of the indicated
genes.
Table S14: Network characteristics in NOD control and ICAM-1-/-
NOD mice (corresponding to Fig. S9A
and Fig. S9C).
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