Analysis of the immune response to sciatic nerve injury identifies efferocytosis as a key mechanism of nerve debridement

  1. Ashley L Kalinski
  2. Choya Yoon
  3. Lucas D Huffman
  4. Patrick C Duncker
  5. Rafi Kohen
  6. Ryan Passino
  7. Hannah Hafner
  8. Craig Johnson
  9. Riki Kawaguchi
  10. Kevin S Carbajal
  11. Juan Sebastian Jara
  12. Edmund Hollis
  13. Daniel H Geschwind
  14. Benjamin M Segal
  15. Roman J Giger  Is a corresponding author
  1. Department of Cell and Developmental Biology, University of Michigan Medical School, United States
  2. Neuroscience Graduate Program, University of Michigan Medical School, United States
  3. Department of Neurology, University of Michigan Medical School, United States
  4. Program in Neurogenetics, Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, United States
  5. Burke Neurological Institute, United States
  6. The Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, United States
  7. Department of Neurology, The Ohio State University Wexner Medical Center, United States
  8. The Neurological Institute, The Ohio State University, United States
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Immune cell profiles in the injured murine sciatic nerve.

(A) Anatomy of lumbar spinal cord and DRGs connected to the sciatic nerve. The location of the crush site within the nerve trunk and the tissue segment collected for flow cytometry (red bracket) are …

Figure 1—figure supplement 1
Gating scheme for flow cytometry.

Cells were first gated with forward scatter (FSC-A) and side scatter (SSC-A) to exclude debris. Cells were then gated with forward scatter height (FSC-H) and FSC-A to find single cells and to …

Figure 1—figure supplement 2
Immune cell profiles in the sciatic nerve proximal to the injury site.

(A) Anatomy of lumbar spinal cord and DRGs connected to the sciatic nerve. The location of the crush site within the nerve trunk and the tissue segment collected for flow cytometry (red bracket) are …

Figure 2 with 1 supplement
Immune cell profiles in axotomized DRGs.

(A) Representative images of L4 DRG cross sections from naïve mice, d1, d3, and d7 post-SNC. Macrophages were stained with anti-Iba1 and anti-F4/80. Neurons were stained with anti-NFH. Scale bar, 50 …

Figure 2—figure supplement 1
T cells in naive and axotomized DRGs.

Representative images of L4 DRG cross sections from naïve mice, d1, d3, and d7 post-SNC. T cells were labeled with anti-CD3 (red), neurons with anti-NF200 (white). DAPI (blue) was used for nuclear …

Figure 3 with 1 supplement
Sciatic nerve injury triggers massive accumulation of hematogenous leukocytes in the injured nerve but not axotomized DRGs.

(A) Parbiosis complex of a wildtype (WT) and a tdTomato (tdTom) mouse. Mice were surgically paired at postnatal day 56. The timeline of the experiment is shown. (B) Flow cytometric analysis of …

Figure 3—figure supplement 1
Flow cytometry gating scheme to assess chimerism of parabiotic mice.

(A) Cells were first gated with forward scatter (FSC-A) and side scatter (SSC-A) to exclude debris. Cells were then gated with forward scatter height (FSC-H) and FSC-A to find single cells and to …

Figure 4 with 1 supplement
Stable upregulation of immune function associated gene co-expression networks in axotomized DRGs.

Analysis of bulk RNAseq data from naïve and axotomized DRGs. DRGs were harvested from sham operated mice, d1, d3, and d7 post-SNC. (A) Network analysis of whole transcriptomes from naive and …

Figure 4—figure supplement 1
Module – trait relationships in axotomized DRGs and analysis of the turquoise module.

(A) Heatmap of the correlation of WGCNA modules with indicated experimental conditions (sham operated mice and following nerve injury). The values in each cell are Pearson's correlation co-efficient …

Figure 5 with 6 supplements
The cellular landscape of injured peripheral nerve.

(A) Singe-cell transcriptome of injured mouse sciatic nerve at d3 post-SNC, n = 3 biological replicates. Unsupervised Seurat-based clustering identifies 24 cell clusters. Cell type identity for each …

Figure 5—source data 1

List of top 100 cluster enriched genes for all cell clusters identified in the 3d post-SNC nerve.

First column (P_val), probability of getting the ‘elevated’ expression values in these cells under the null hypothesis that all cells have the same expression of the gene. Second column (avg_logFC), average log2 Fold-Change between cells in this cluster relative to cells in all other clusters. Third column (pct.1), percent of the cluster's cells which express the gene. Fourth column (pct.2), percent of non-cluster cells which express the gene. Fifths column (p_val_adj), p_val adjusted so that 5% of the list is expected to have false positives. Links to STRING REACTOME pathway analysis for each cell cluster are included on the last page.

https://cdn.elifesciences.org/articles/60223/elife-60223-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Cell-cluster-specific expression of ECM components and molecules that regulate axon growth in the injured sciatic nerve.

(A) Dotplot analysis of extracellular matrix molecules prominently expressed in the d3 post-SNC nerve. (B) Dotplot analysis of gene products implicated in axon growth, guidance, and regeneration in …

Figure 5—figure supplement 2
Single-cell gene expression in mesenchymal cell clusters in injured sciatic nerve.

Heatmap of top genes enriched in perineural mesenchymal cells (pMES), endoneurial mesenchymal cells (eMES), differentiating mesenchymal cells (dMES), and epineural fibroblasts (Fb) in the d3 …

Figure 5—figure supplement 3
Single-cell gene expression in Schwann cell clusters in injured sciatic nerve.

Heatmap of top genes enriched in repair Schwann cell clusters (SC1, SC2, and SC3) in the d3 post-SNC nerve. Expression levels are calibrated to median gene expression. STRING REACTOME pathways are …

Figure 5—figure supplement 4
Single-cell gene expression in endothelial cell clusters in injured sciatic nerve.

Heatmap of top genes enriched in endothelial cell clusters (EC1, EC2, and EC3) in the d3 post-SNC nerve. Expression levels are calibrated to median gene expression. STRING KEGG Pathway analysis and …

Figure 5—figure supplement 5
Single-cell gene expression in pericyte cell clusters in injured sciatic nerve.

Heatmap of top genes enriched in pericyte clusters (PC1 and PC2) in the d3 post-SNC nerve. Expression levels are calibrated to median gene expression. STRING REACTOME Pathways are listed. FDRs …

Figure 5—figure supplement 6
Single-cell gene expression of immune modulatory molecules in the injured PNS tissue.

Dotplot analysis of gene products with immune modulatory function in the d3 post-SNC nerve. Expression levels, normalized to average gene expression (color coded). For each cell cluster, the …

Figure 6 with 6 supplements
Macrophage subpopulation in the injured nerve are functionally distinct and localize to specific sites.

(A–E) Feature plots of Adgre1 (F4/80), Aif1 (Iba1), Cd68 (Scavenger receptor class D), Cx3cr1 (Fractalkine receptor), and CD209a (DC-SIGN) expression in the d3 post-SNC nerve. (F) scRNAseq dot plot …

Figure 6—figure supplement 1
Single-cell gene expression in myeloid cell clusters of injured sciatic nerve.

Heatmap of top genes enriched in monocytes (Mo), macrophage clusters 1–5 (Mac1-5), MoDC, and cDC in the d3 post-SNC nerve. Expression levels are calibrated to median gene expression. STRING KEGG …

Figure 6—figure supplement 2
Identification of blood-borne, stem-like myeloid cells in the injured sciatic nerve.

(A) Feature plots of Stmn1 (Stathmin-1) and (B) Top2a (DNA topoisomerase II alpha) highlight proliferating cells, including Mac5 cells and a small group of myeloid cells located between clusters …

Figure 6—figure supplement 3
Monocyte-to-macrophage differentiation based on single-cell expression modeling.

Pseudo time trajectory analysis of cell differentiation in the d3 post-SNC nerve (A–D) Predicted cell differentiation in the myeloid cell compartment. Slingshot analysis was used to predict how Mo …

Figure 6—figure supplement 4
Mo/Mac maturation in axotomized DRGs, assessed by Ly6C surface expression.

Flow cytometric analysis of Mo/Mac in naive and axotomized DRGs. The surface distribution of Ly6C on Mo/Mac (CD45+, CD11b+, Ly6G-, CD11c-) is shown. (A) The percentile of Ly6C cells and (B) the …

Figure 6—figure supplement 5
Evidence for distinct immune compartments in the injured sciatic nerve.

(A) Longitudinal sections of naïve and injured (3d and 7d) sciatic nerves from Arg1-YFP reporter mice. The injury site is underlined with a dotted line. Proximal is to the left. Distribution of …

Figure 6—figure supplement 6
Expression of gene products that regulate cholesterol transport and metabolism in macrophages of injured and naive nerve.

Feature plots of gene products implicated in cholesterol transport and lowering of cellular cholesterol levels. (A) Abca1/CERP (ATP-binding cassette subfamily A1). (B) Abcg1 (ATP-binding cassette …

Figure 7 with 3 supplements
Macrophages ‘eat’ dying leukocytes in the injured nerve.

(A) Cartoon of phagocyte with actin rich phagocytic cup eating a tdTom+apoptotic cell (AC). ‘Eat me’ signals displayed on the surface of AC allow direct or indirect recognition via engulfment …

Figure 7—figure supplement 1
Expression of gene products implicated in myelin binding and phagocytosis.

(A–F) Feature plots and (G–L) Violin plots of Axl (TAM receptor tyrosine kinase AXL), Mertk (TAM receptor tyrosine kinase MER), Lrp1 (Low-density lipoprotein receptor-related protein 1), Pirb

Figure 7—figure supplement 2
Expression analysis of bridging molecules and engulfment receptors in macrophages of naive nerve and injured nerve.

(A) Dotplot showing expression of engulfment receptors in in naïve Mac in comparison to Mo and Mac1-5 in injured nerve. (B). Dotplot showing expression of ‘don’t eat me’ (Cd47 and Sirpa) signals, as …

Figure 7—figure supplement 3
Contribution of MoDC to efferocytosis in the injured sciatic nerve.

(A) Flow cytometric analysis of 3d sciatic nerve from non-parabiotic (tdTom single) mice. Dot plot shows that 96.8% of MoDC (CD11b+, CD11c+, Ly6G-) are tdTom+(B) Flow cytometric analysis of 3d …

Figure 8 with 1 supplement
GM-CSF is required for conditioning lesion induced dorsal column axon regeneration.

(A) Schematic showing conditioning lesion to the sciatic nerve (1) followed by dorsal column lesion (2) and tracer injection in the nerve (3). Experimental time line of conditioning lesion (CL), …

Figure 8—figure supplement 1
Locus and PCR genotyping of Csf2-/- mice.

(A) Schematic of Csf2 gene locus of WT and germline Csf2-/- mice. Primer sets were designed both inside (set 2) and outside (set 2) of the deleted region. (B) Agarose gel shows PCR genotyping …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
AntibodyNeurofilament heavy chain
(chicken polyclonal)
Aves LabNFH1:750
AntibodyAnti-chicken Cy3
(donkey polyclonal)
Jackson Immunoresearch703-165-1551:200
AntibodyIba1
(rabbit polyclonal)
Wako Chemicals019–197411:500
AntibodyF4/80
(Rat IgG2b monoclonal)
Thermo Fisher Scientificma1-911241:500 - 1:1000
AntibodyCD68
(rabbit polyclonal)
Abcamab1252121:500
AntibodyGFAP
(rabbit polyclonal)
DAKOZ033429-21:500
AntibodySCG10
(rabbit polyclonal)
Novus BiologicalsNBP1494611:500 - 1:1000
AntibodyCTB
(goat polyclonal)
List Biological Laboratories7031:10,000
AntibodyCD11b
(rabbit monoclonal)
Abcamab1333571:200-1:1000
AntibodyERK1/2
(rabbit polyclonal)
Cell Signaling91021:5000
AntibodyAnti-rabbit HRP
(donkey polyclonal)
EMD MilliporeAP182P1:2000-1:10000
AntibodyCD16/32
(Rat-IgG2a monoclonal)
BD Pharmingen5531411 µg / 1 million cells / 25 µL
AntibodyCD11b-PE-Cy7
(Rat-IgG2b monoclonal)
Thermo Fisher Scientific25-0112-821:200
AntibodyIsotype Control-PE-Cy7
(Rat-IgG2b monoclonal)
Thermo Fisher Scientific25-4031-821:100
AntibodyCD45-e450
(Rat-IgG2b monoclonal)
Thermo Fisher Scientific48-0451-821:100
AntibodyIsotype Control-e450
(Rat-IgG2b monoclonal)
Thermo Fisher Scientific48-4031-821:100
AntibodyCD45.1-e450
(Mouse-IgG2a monoclonal)
Biolegend1107211:100
AntibodyIsotype Control-e450
(Mouse-IgG2a monoclonal)
Biolegend4002351:100
AntibodyCD45.2-APC
(Mouse-IgG2a monoclonal)
Biolegend1098131:100
AntibodyIsotype Control-APC
(Mouse-IgG2a monoclonal)
Biolegend4002211:100
AntibodyLy6G-APC-Cy7
(Rat-IgG2a monoclonal)
BD Biosciences5606001:100
AntibodyIsotype Control-APC-Cy7
(Rat-IgG2a monoclonal)
BD Biosciences5527701:100
AntibodyCD11c-PerCP-Cy5.5
(ArmHam-IgG monoclonal)
Thermo Fisher Scientific45-0114-821:100
AntibodyIsotype Control-PerCP-Cy5.5
(ArmHam-IgG monoclonal)
Thermo Fisher Scientific45-4888-801:100
AntibodyLy6C-FITC
(Rat-IgM monoclonal)
BD Biosciences5610851:100
AntibodyIsotype Control-FITC
(Rat-IgM monoclonal)
BD Biosciences5539421:100
AntibodyIba1 (goat polyclonal)Novus BiologicalsNB100-10281:200
AntibodyAnti-goat Alexa Fluor
488 (donkey polyclonal)
Jackson Immunoresearch705-545-1471:200
Chemical CompoundTOPRO pan-nuclear stainThermo Fisher ScientificT3605
Chemical CompoundFixable Viability DyeThermo Fisher Scientific650866141:500
Chemical CompoundProteinase KNew England BiolabsP8107S
Chemical Compound10 mM dNTP mixPromegaC1141
Chemical Compound5X Green GoTaq BufferPromegaM791A
Chemical CompoundGoTaq DNA polymerasePromegaM3005
Chemical CompoundBuprenorphinePar PharmaceuticalNDC12496-0757-1
Chemical CompoundKetaminePar PharmaceuticalNDC42023-115-10
Chemical CompoundXylazineAkornNDC59399-110-20
Chemical CompoundIsofluraneMcKesson Corporation667940172
Chemical CompoundRhodamine-conjugated
dextran MW 3,000
(Microruby)
Life TechnologiesD-7162
Chemical CompoundCholera toxin B (CTB)Life TechnologiesC34775
Chemical CompoundPuralube Eye ointmentDechraNDC-17033-211-38
Chemical CompoundN2Gibco17502048
Chemical CompoundN1SigmaN6530
Chemical CompoundLeibovitz-15 (L-15)Gibco21083–027
Chemical CompoundPenicillin/StreptomycinLife Technologies15140–122
Chemical CompoundDMEM Ham’s F-12Gibco10565–018
Chemical CompoundFetal Bovine SerumAtlanta BiologicalsS11550
Chemical CompoundCytosine arabinosideSigma-AldrichC1768
Chemical CompoundCollagenase type 2Worthington
Biochemical
LS004176
Chemical CompoundPBS without Calcium,
Magnesium
Gibco10010023
Chemical CompoundPoly-L-lysine
MW 70,000–150,000
Sigma-AldrichP4707
Chemical CompoundlamininSigma-AldrichL2020
Chemical CompoundParaformaldehydeSigma-Aldrich158127–500G
Chemical CompoundTriton-X100Sigma-AldrichT8787
Chemical CompoundBovine Serum Albumin
(BSA) heat shock fraction V
Fisher ScientificBP1600
Chemical CompoundHoechst 33342InvitrogenH3570
Chemical CompoundTissue-Tek O.C.T. CompoundElectron Microscopy
Sciences
62550–01
Chemical Compoundβ-GlycerophosphateSigma-AldrichG9422-100G
Chemical CompoundSodium Orthovanadate
(Na3VO4)
Sigma-AldrichS6508-10G
Chemical CompoundProtease inhibitor cocktailSigma-AldrichP8340-5ML
Chemical CompoundDC Protein Assay KitBio-Rad5000111
Chemical Compound2x Laemmli sample bufferBio-Rad1610737
Chemical Compoundβ-MercaptoethanolEMD Millipore6010
Chemical CompoundBlotting-grade blockerBio-Rad1706404
Chemical CompoundSuperSignal West
Pico PLUS Chemiluminescent
Substrate
Thermo Fisher
Scientific
34580
Chemical CompoundSuperSignal West
Femto Maximum Sensitivity
Substrate
Thermo Fisher
Scientific
34095
Chemical CompoundWesternSure PREMIUM
Chemi Substrate
Li-Cor Biosciences926–95000
Chemical CompoundFixable Viability Dye eF506Thermo Fisher Scientific65-0866-14
Chemical CompoundTRIzolThermo Fisher Scientific15596026
Chemical CompoundDispaseSigma-AldrichD4693
Chemical CompoundActinomycin DSigma-AldrichA1410
Chemical CompoundPercollSigma-AldrichP4937
Chemical CompoundMACS bufferMiltenyi130-091-376
Chemical CompoundHanks balanced
salt solution
Gibco14025092
Chemical CompoundSucroseFisher ScientificS5-500
OtherPasteur pipetteVWR14673–010
OtherFlat Bottom Cell
Culture Plates, 24-well
Corning3524
OtherSuperfrost Plus
Microscope Slides
FisherScientific12-550-15
OtherZeiss Axio Observer Z1Zeiss491912-0049-000
OtherZeiss Axiocam 503
mono camera
Zeiss426559-0000-000
OtherEC PlnN 10x objectiveZeiss420341-9911-000
OtherMotorized tissue
homogenizer
RPI299200
OtherFisher Scientific
Sonic Dismembrator
Fisher ScientificModel 500
OtherRefrigerated CentrifugeEppendorf5424R
OtherPhotospectrometerMolecular DevicesSpectraMax M5e
OtherPVDF membraneEMD MilliporeIPVH00010
OtherLI-COR C-DigitLi-Cor BiosciencesCDG-001313
Other70 µm cell strainerCorning352350
OtherAmmonium-Chloride-
Potassium (ACK) Lysing
Buffer
GibcoA1049201
OtherClinical CentrifugeBeckman CoulterAllegra 6R
Other40 µm filterBD Falcon352340
OtherMyelin Removal BeadsMiltenyi130-096-731
OtherMidiMACS separatorMiltenyi130-042-302
OtherLS ColumnsMiltenyi130-042-401
OtherHemacytometerMillipore SigmaZ359629
OtherChromium Next
GEM Chip G
10X Genomics, IncNC1000127
Other10X Genomic
Chromium Controller
10X Genomics, IncN/A
OtherNovaSeq Illumina 6000IlluminaN/A
OtherCryostatLeica BiosystemsCM3050S
Other70 µm Cell StrainerCorning352350
OtherConfocal MicroscopeNikonC1
OtherConfocal MicroscopeLeica BiosystemsSP8
Genetic Reagent
(Mus musculus C57BL/6)
Csf2-/-Jackson LaboratoriesStock No: 026812PMID:8171324
MGI: J:17978
Genetic Reagent
(Mus musculus C57BL/6)
ROSA26-TdTomJackson LaboratoriesStock No: 007576PMID:17868096
MGI: J:124702
Genetic Reagent
(Mus musculus C57BL/6)
CD45.1Jackson LaboratoriesStock No: 002014PMID:11698303
MGI: J:109863 PMID:11994430
MGI: J:109854 PMID:12004082
MGI: J:109853
Genetic Reagent
(Mus musculus C57BL/6)
Wildtype, WTTaconicsB6NTac
Genetic Reagent
(Mus musculus C57BL/6)
Arg1-eYFPJackson LaboratoriesStock No: 015857PMID:17450126 MGI: J:122735
OligonucleotideCsf2 ForwardIntegrated DNA
Technologies
N/A5’-GTGAAACACAAGTTACCACCTATG-3’
OligonucleotideCsf2 ReverseIntegrated DNA
Technologies
N/A5’-TTTGTCTTCCGCTGTCCAA-3’
OligonucleotideNeomycin ForwardIntegrated DNA
Technologies
N/A5’-CTTGGGTGGAGAGGCTATTC-3’
OligonucleotideNeomycin ReverseIntegrated DNA
Technologies
N/A5’-AGGTGAGATGACAGGAGATC-3’
SoftwareWIS-NeuromathWeizmann Institute
of Science
Version 3.4.8PMID:23055261
SoftwareImage Studio
Software
Li-Cor BiosciencesVersion 5.2.5
SoftwareNovaSeq control
software
IlluminaVersion 1.6
SoftwareReal Time Analysis
(RTA) software
IlluminaVersion 3.4.4
SoftwareCellranger10X Genomics, IncVersion 3.1.0
SoftwareFACSDivaBD BiosciencesVersion 7
SoftwareFlowJoFlowJo LLCVersion 10.6.2
SoftwareSeuratSatija Lab - New York
Genome Center
Version 3.1.2
SoftwareRr-project.orgVersion 3.6.2
SoftwareSlingshotBioconductorVersion 1.4.0
SoftwareRangerComprehensive R
Archive Network
Version 0.12.1
SoftwarePrismGraphPadVersion 7 and 8
SoftwareImarisBitplane
SoftwareLeica Application
Suite (LAS X)
Leica
SoftwareZen Application
Software
ZeissPro 3.8
OtherSomnoSuiteKent ScientificSS-01
OtherPovidone-Iodine
Prep Pad
PDI HealthcareB40600
OtherAlcohol Prep,
Sterile, Md, 2 Ply
Covidien6818
OtherFine Forceps Dumont
#55 Dumoxel
Roboz Surgical InstrumentRS-5063
Other7 mm Reflex Wound ClipsCell Point Scientific203–1000
OtherMicro Friedman RongeurRoboz Surgical InstrumentRS-8306
OtherMcPherson-Vannas
Micro Dissecting Spring
scissors
Roboz Surgical InstrumentRS-5600
OtherCOATED VICRYL
(polyglactin 910) Suture
EthiconJ463G
Otherdumont #7 curved
forceps
Fine Science Tools11271–30
OtherMiltex halsted
mosquito forceps
Integra LifeSciences724
OtherNanofil 10 µL syringeWorld Precision
Instruments
NANOFIL
Other36 g beveled nanofil
needle
World Precision
Instruments
NF36BV-2
OtherNon-absorbable
sutures
Ethicon640G
OtherAbsorbable suturesEthiconJ463G

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