Supplementary Materials for · Fig. S1. WIHN– wounds have higher numbers of cells with nuclear...

advances.sciencemag.org/cgi/content/full/6/12/eaay3704/DC1

Supplementary Materials for

Phagocytosis of Wnt inhibitor SFRP4 by late wound macrophages drives chronic

Wnt activity for fibrotic skin healing

Denise Gay*, Giulia Ghinatti, Christian F. Guerrero-Juarez, Rubén A. Ferrer, Federica Ferri, Chae Ho Lim, Shohei Murakami, Nathalie Gault, Vilma Barroca, Isabelle Rombeau, Philippe Mauffrey, Lamya Irbah, Elsa Treffeisen,

Sandra Franz, Alexandre Boissonnas, Christophe Combadière, Mayumi Ito, Maksim V. Plikus, Paul-Henri Romeo*

*Corresponding author. Email: [email protected] (D.G.); [email protected] (P.-H.R.)

Published 20 March 2020, Sci. Adv. 6, eaay3704 (2020)

DOI: 10.1126/sciadv.aay3704

The PDF file includes:

Fig. S1. WIHN– wounds have higher numbers of cells with nuclear β-Cat. Fig. S2. Whole wound dermis and myofibroblast comparisons in WIHN+ and WIHN– wounds. Fig. S3. Comparisons of macrophage/DC populations in WIHN+ and WIHN– wounds. Fig. S4. WD16 wound examined for macrophage marker expression. Fig. S5. Lower macrophage numbers in the late wound dermis correlate with reduced wound collagen. Fig. S6. SFRP4 is located within lower wound dermis ECM and macrophages. Fig. S7. Hemin abrogates phagocytosis in cultured macrophages but does not alter wound healing, wound architecture, or wound macrophage numbers in vivo. Fig. S8. EDA-FN, Tlr4, and integrin studies. Fig. S9. Comparisons of WT, EDA-FN–/–, and EDA-FN+/– wounds. Fig. S10. SFRP4 is phagocytized and degraded by CD68+ macrophages in newly wounded HS skin dermis. Fig. S11. Examination of ECM components and macrophages/DCs in human normal and HS skin. Fig. S12. A proposed model for macrophage phagocytosis contribution to fibrosis. Table S1. Gene signatures of myofibroblast groups I to III. Table S2. STRING analyses of WIHN+ and WIHN– macrophage clusters. Table S3. List of the top 200 genes shared by macrophage subsets from integrated macrophage/DC analysis. Legend for movie S1

Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/6/12/eaay3704/DC1)

Movie S1 (.avi format). Localization of external SFRP4 and SFRP4 vesicles within MerTK+ macrophage.

Fig. S1. WIHN– wounds have higher numbers of cells with nuclear β-Cat. See Fig. 1E. -

Catenin (-Cat, red) in WD22 WIHN– (left) and WIHN

+ (right) wounds. Nuclei are green.

Dotted squares represent insets below. Arrows point to examples of nuclear -Cat. Scale bar 200

m. Results from 3 independent experiments.

Fig. S2. Whole wound dermis and myofibroblast comparisons in WIHN+ and WIHN

–

wounds. A. Color-blocked T-SNE plots of whole WIHN+ and WIHN

– wound dermis at WD18.

Clusters are color-coded and defined in adjacent legend. B. and I. T-SNE plots of whole wounds

with color-blocked regions selected for analysis (blue). C. and J. show t-SNE plots of WIHN+

(C) or WIHN– (I) myofibroblasts and adjacent color-blocked plots show Group I-III identities. D.

Clustertree analysis (Seurat) of myofibroblast clusters reveals three groups. E. and K. Heatmaps

show top 10 genes within each WIHN+ (C) or WIHN

– (F) cluster. F. and L. Feature plots show

selected genes to define WIHN+ (F) or WIHN

– (K) myofibroblast groups I-III. G. Dotplots

reveal 3 major groups within myofibroblast clusters. H. Violin plots show dermal papilla (DP)

signature in cluster 3 (Group II).

Fig. S3. Comparisons of macrophage/DC populations in WIHN+ and WIHN

– wounds. A.

and E. T-SNE plots of whole WIHN+ (A) and WIHN

– (E) wounds with color-blocked regions

selected for analysis (blue). B. and F. T-SNE plots of subclustered macs/DCs and color-coded

plots (right) assigning group identity. C. and G. Immgen Wplots comparing WIHN+ or WIHN

–

gene lists respectively to Immgen databases of immune, stromal, stem cell gene signatures.

Dotted rectangles define Mac and DC regions. D. and H. Featureplots show selected genes for

WIHN+ (D) or WIHN

– (H) Mac and DC groups. I. Venn diagram shows gene relatedness

between WIHN+ and WIHN

– Mac DEGs. J. Dotplots compare M1 (top) and M2 (bottom)

marker expression in WIHN+ and WIHN

– macrophage subsets from integrated analysis. Chil4

and Clec7a were not found. K. Violin plots show various genes in Mac/DC integrated analysis

(see Fig. 2).

Fig. S4. WD16 wound examined for macrophage marker expression. F4/80 (green), MERTK

(red) and CD64 (pink) localization in a healed wound. Rectangle defines magnified inset below.

Scale bar 500 m. Representative of 4 independent experiments.

Fig. S5. Lower macrophage numbers in the late wound dermis correlate with reduced

wound collagen. A. Cytometric dot plots of wound dermal cells. Wound dermal cells

preselected for size and granularity (SSC/FSC), viability (DAPI) and CD45 expression. Plots

show comparisons of F4/80+MERTK

+ cells in WIHN

+ and WIHN

– wounds. B. WIHN

– wounds

have increased levels of collagen protein compared to WIHN+ wounds. Representative WT

WIHN–(top), WIHN

+ (middle) and Cx3cr1

–/– (bottom) WD18 frozen sections stained with Sirius

Red for collagen content. Bar graph (right) shows relative Sirius Red intensity. See Methods for

quantification. Data are expressed as means ±s.e.m. * P value is 0.05, ** P value is 0.01.

Fig. S6. SFRP4 is located within lower wound dermis ECM and macrophages. A. Frozen

sections of W15 WIHN+

(left) and WIHN– (right) stained for extracellular SFRP4 (white). Scale

bar 50 m. Representative of results from 5 experiments. B. Representative frozen section of

W15 C57Bl/6J WIHN– wound stained for MerTK (purple) and SFRP4 (intracellular and

extracellular, green). Dotted line denotes epidermal-dermal interface. Dotted rectangle shows

location of magnified region (right). Scale bar 50 m. Representative of results from 5

experiments. C. Upper panels: WIHN– dermis stained for SFRP4 (white) and CD64 (purple)

within field of macrophages in lower dermis. Lower panels: Localization of SFRP4 within

dermal CD64+ macrophages. Dotted rectangle defines magnified region (right panel). Scale bars

50m and 25m respectively.

Fig. S7. Hemin abrogates phagocytosis in cultured macrophages but does not alter wound

healing, wound architecture, or wound macrophage numbers in vivo. A. Pre-incubation with

hemin reduces the ability of macrophages to phagocytize fibronectin-coupled micro beads.

BMDM-derived IL4-polarized macrophages cultured with hemin and fibronectin-coupled

fluorescent microbeads, were stained for FN1 (see Methods for details). FITC+ beads bound to or

phagocytized by cells were selected according to FITC-positivity and then analyzed for anti-FN1

binding. Beads detected by anti-FN1 antibody were considered extracellular. Top:

Representative dot plots of gated cells (left), gated FITC+ beads (center) and FN1 on beads

(right). Bottom: Line graph showing % beads with reduced FN1 representing % phagocytosis.

Results from 2 independent experiments. B. Whole mount comparisons of vehicle- and hemin-

treated WD14 wounds. Scale bar 2 mm. Representative examples from 20 wounds. C. WD18

wound epidermis stained for LEF1 expression. Dotted rectangle shows area of magnification

with hair buds (below). Scale bar 1 mm. D., E. Frozen sections stained for CD31 D., CD45 E.

Scale bar 200 m. C. – E. Representative of 6 wounds. F. Frozen sections stained for CD64.

Scale bar 200 m. Rectangles denote magnified regions. Dot plot compares numbers of wound

macrophages per mm2. N=4 for each group. G. Frozen sections stained for COL1a1. Scale bar

500 m. Lower panels show magnified regions of collagen staining. Scale bar 50 m. Bar graph

compares relative fluorescences (CTCF) for collagen staining in lower wound dermis. N=3 for

each group.

Fig. S8. EDA-FN, Tlr4, and integrin studies. A. Western blots compare plasma FN1 (FNpl),

adult cellular FN1 (FNa or EDA-LO) and newborn FN1 (FNnb or EDA-HI) for relative

quantification of total FN (left panel) and EDA-FN (right panel). Molecular weight markers left

lanes. Results are representative of 2 independent experiments. See Fig. 6A. B. IL-4-polarized

BMDM-derived macrophage phagocytizing SFRP4-bound EDA-FN beads. Cytofluorographic

histogram shows EDA-FN (pink) and SFRP4 (tan) content on EDA-FN-coupled beads

preincubated with purified SFRP4. Right: LAMP1+ vesicles (left), co-localized with

SFRP4:EDA-FN beads (right) in concanamycin A-treated BM-derived IL-4-polarized

macrophages in vitro. White arrows denote locations of LAMP1+ vesicles. Scale bar 5 m.

Representative of 6 independent experiments. C. Tlr4 is downregulated in the dermis by WD12

and negligible in myofibroblasts and macrophage/DCs by WD18. C.i. Comparison of Tlr4

expression in gene arrays of WD10 and WD12 wounds C.ii. Left: Violinplots of myofibroblast

clusters for Tlr4 expression. Right: Dotplots comparing Tlr4 expression in WIHN+ (+) and

WIHN– (–) myofibroblasts in integrated analysis. Note the level range. C.iii. Violinplots of

macrophage/DC clusters for Tlr4 expression. D. Co-localization of 5-integrin and SFRP4 on

MERTK+ macrophages in lower dermis. Scale bar 100 m. Box defines region of magnification

(right). Scale bar 50 m. Representative of 6 experiments. E. Co-localization of SFRP4 (white,

left), -integrin (green, middle) in lower dermis. Co-localization of SFRP4 (white, left), -

integrin (green, middle) in lower dermis. Pink arrows point to SFRP4:integrin overlap in upper

and lower panels. Dotted lines represent macrophage boundaries. Scale bars 10 m. ** P value is

0.01. Representative of 6 (Upper) and 3 independent experiments (Lower). F. Inhibition of EDA-

FN phagocytosis by Anti-1-integrin. Bar graph compares percent internalized (inaccessible to

EDA-FN antibody) EDA-FN-beads in cultured BMDM-derived IL4-polarized macrophages

pretreated with isotype control (white) or anti-1-integrin (black) antibody. N=4. See Methods

for details.

Fig. S9. Comparisons of WT, EDA-FN–/–

, and EDA-FN+/–

wounds. A. Confocal comparisons

of WD16 WT (top), EDA-FN+/–

(middle) and EDA-FN–/–

(lower) frozen sections for EDA-FN

(green, left) or Sirius Red staining (right). Scale bar 500 m. B. Confocal comparisons of WD16

WT (left) and EDA-FN–/–

(right) lower wound dermis stained for total FN1 (red) and SFRP4

(green). Insets (below) defined by boxes. Scale bar 50 m. Representative of 4 independent

experiments. C. Left: MERTK (white) in WT (top), EDA-FN+/–

(middle) and EDA-FN–/–

WD16

wounds. Scale bar 500 m. Right: Bar graph shows relative numbers of MERTK+ macrophages

per mm2 dermis in WD16 WT (white), EDA-FN

+/– (green) and EDA-FN

–/– (black) wounds.

Representative of 3 independent experiments. D. Bar graph comparing percent of WT (white)

and EDA-FN+/–

(green) BM-derived IL-4-polarized macrophages with phagocytized EDA-FN

beads. See Methods for details. Combined results from 3 independent experiments. E. Confocal

comparisons of MERTK (pink) and LAMP1 (red) co-localization in WT and EDA+/–

wounds.

Blue is DAPI. Images correspond to magnified images in Fig. 6F to show that LAMP1

expression is primarily observed within MERTK+ macrophages compared to other cells. Scale

bar is 200 m.

Fig. S10. SFRP4 is phagocytized and degraded by CD68+ macrophages in newly wounded

HS skin dermis. A. Schematic model of Hidradenitis suppurativa skin: Follicular rupture

(gray), immune cell infiltrate (aqua blue, region i.), prototypic wound (light blue, region ii.),

healed fibrotic region (yellow) B. Fibrotic (left panels) or infiltrate/wound region (right panels)

from HS patient for collagen (aqua, left), EDA-FN (red, middle), SFRP4 (white, right).

Epidermal:dermal (left panels) and immune infiltrate:dermal (right panels) boundaries defined by

dotted line. Squares (far right panel) show immune infiltrate (region i) and wound areas (region

ii). Scale bar 1mm. See Fig. S12A for comparisons of normal skin. C. Left: CD68+ macrophages

(purple) in immune infiltrate (region i, upper panel) and adjacent wound (region ii, lower panel).

See Fig. S12C for macrophage markers in normal skin. Right: SMA+ (red) myofibroblasts in

wound (region ii). Scale bar 15 m. D. Co-localization of SFRP4 (white) and CD68 (purple) in

region i. Scale bar is 40 m. Stacked bar graph shows numbers per 200m2 macrophages in

normal and HS skin with SFRP4 vesicles (black, % shown above bar). E. Co-localization of

SFRP4 (green) with EDA-FN (upper) or LAMP1 (lower) in region ii. Macrophage borders

defined by dotted lines. Mauve strip in lower panel is a collagen fiber. Scale bar 5m. F. Nuclear

-Cat (purple) in regions i and ii. Dotted rectangles define magnified insets. Infiltrate:wound

boundary is defined by thick dotted line. Blue strands correspond to collagen fibers. Arrows

point to representative -Cat+ nuclei (purple). Scale bar is 200 m. Stacked bar graph compares

number -Cat+ cells (black, % shown above bar) in normal and HS skin. G. Magnified view

shows localization of nuclear -Cat (purple) and CD68 (green) in region ii. Scale bar 15 m. For

Fig. S12B - G, results of 6 independent experiments. H. qRT-PCR comparing normal (white)

and HS (black) dermis for expression of AXIN2, SFRP4, FN1, TGFB1, COL1A1. See Fig. 1 for

details. Data are expressed as means ±s.e.m. * P value is 0.05, ** P value is 0.01, **** P value is

0.001, ****** P value is 0.0001 (10e-4).

Fig. S11. Examination of ECM components and macrophages/DCs in human normal and

HS skin. Expression and localization of SFRP4 and EDA-FN in Normal A. and HS B. Skin. A.

Human adult skin examined for colocalization of EDA-FN (red) and SFRP4 (white). Scale bar

100 m. B. Co-localization of EDA-FN (red) and SFRP4 (green) in HS skin dermis. Asterisks

show regions of overlap on ECM strands. Dotted lines show positions of CD68+ macrophages.

Scale bar 50 m. C. Examination of Normal Human Skin for Macrophage Markers. Left and

Upper panels: Human skin examined for colocalization of macrophage markers CD68 (purple),

CD11b (white), CD209 (green). Square shows inset area. Scale bar 500 m. Lower: Human skin

frozen section examined for macrophage markers CD68 (purple) and CD206 (green).

Representative results from 3 independent experiments.

Fig. S12. A proposed model for macrophage phagocytosis contribution to fibrosis. In a

WIHN+ wound, which exhibits a more regenerative response (Left), WNT2 drives myofibroblast

Wnt activation and subsequent production of Wnt inhibitor SFRP4 resulting in Wnt

downmodulation (tan background) and hair neogenesis. In a fibrotic wound (Right), EDA-FN-

bound SFRP4 is phagocytized by macrophages in an integrin-dependent manner, thus reducing

the local concentration of this inhibitor and promoting prolonged Wnt activity (blue

background). Because macrophages as well as myofibroblasts are a source of wound EDA-FN, it

remains unknown whether myofibroblast-secreted SFRP4 can be directly loaded onto EDA-FN

on the macrophage surface for more efficient phagocytosis.

Table S1. Gene signatures of myofibroblast groups I to III.

GROUP I SIGNATURE

Extracellular matrix degradation*

Ctsk, Fbn2, Mmp2, Mmp23, Timp2

Extracellular matrix organization*

Bgn, Col1a1, Col1a2, Col3a1, Col5a2, Col8a1, Col14a1, Ctsk, Fbn2, Fmod, Lox, Mfap2, Mfap4, Mmp2, Mfap4, Sparc, Timp2, Lox, Eln

Other secreted* Aebp1, Postn, Il33, Mdk, Itgbl1, Cpz, Smoc2, Ogn, Wisp2, C1qtnf3, Apod, Gpx3, Igf1, Cpxm2, Gas6, Serpinf1, Pdgfrl

Others Itm2a, Tmed3, Mtch1, Il1ra1, Nov, Ddah1

GROUP II SIGNATURE Proliferation, migration

Proliferation* Anapc15, Arpp19, Banf1, Birc5, Bub3, Capg, Ccna2, Ccnb1, Ccnb2, Cdc20, Cdca8, Cdk1, Cenpa, Cenpf, Cenpm, Cks1b, Gins2, H2afv, H2afx, H2afz, H3f3b, Hist1h2ap, Hsp90aa1, Lmnb1, Nudc, Pmf1, Ppp2ca, Psmb7, Psmd14, Rad21, Rangap1, Smc2, Smc4, Spc24, Spc25, Tmpo, Tubb2b, Tubb4b, Tubb5, Tubb6, Ywhae, Mki-67

Others† Ackr3, Anxa2, S100a10, Ly6c1, Ly6a, Emilin2, Gas7, Cd55, Creb5, S100a4, Heg1, Sbsn, Arpc1b, Ackr3, Anxa3, Ugdh, Stmn1, Cd44, Wnt2

GROUP III SIGNATURE

Smooth muscle contraction*

Acta2, Anxa1, Cald1, Calm1, Gucy1a1, Itga1, Myh11, Myl12a, Myl6, Myl9, Mylk, Tpm1, Tpm2, Tpm4, Vcl, Des

Laminin interactions*

Col18a1, Col4a1, Col4a2, Itga1, Itgb1

Collagen fibrils* Cd151, Col18a1, Col4a1, Col4a2

RHO GTPases ActivateROCKs*

Cfl1, Myh11, Myl6, Myl9, Ppp1r12a

Others Tagln, Rgs5, Notch3, Fabp4, Pdgfrb

Signatures were derived from comparisons of DEG lists of WIHN+ and WIHN

– myofibroblast

subsets. Signatures are common to each group within both WIHN+ and WIHN

– phenotypes.

† Proliferation genes are shared between Group II WIHN+ c3 and WIHN

– c3 whereas all Group

II clusters (WIHN+ c2, c3) and WIHN

– c3) share other genes.

* Based on DAVID Ontology and Reactome analyses.

Table S2. STRING Analysis of WIHN+ Macrophage cluster 0.

Biological Process (GO)

pathway description False discovery rate

GO:0070887 cellular response to chemical stimulus 1.65e-17 GO:0006952 defense response 2.07e-17 GO:0048518 positive regulation of biological process 2.10e-15

GO:0042221 response to chemical 2.10e-15 GO:0006950 response to stress 2.10e-15 Molecular Function (GO)


GO:0005515 protein binding 9.77e-14 GO:0005488 binding 3.87e-11 GO:0042802 Identical protein binding 1.93e-08 GO:0019899 enzyme binding 2.78e-06 GO:0033218 Amide binding

Cellular Components (GO)


GO:0005576 Extracellular region 7.18e-11 GO:0005615 Extracellular space 3.14e-10

GO:0005764 lysosome 3.20e-10

GO:0044421 Extracellular region part 9.20e-10

GO:0031982 vesicle 1.24e-08

KEGG Pathways


mmu04142 Lysosome 8.91e-08

mmu04210 Apoptosis 1.52e-06

mmu05133 Pertussis 3.03e-05

mmu04978 Mineral absorption 3.03e-05

mmu05150 Staphylococcus aureus infection 3.52e-05

Reactome Pathways


MMU-168256 Immune System 2.76e-14

MMU-168249 Innate Immune System 2.16e-16

MMU-6798695 Neutrophil degranulation 5.31e-15

MMU-109582 Hemostasis 9.56e-07

MMU-2132295 MHC class II antigen presentation 4.26e-06

number of nodes: 133 number of edges: 725 average node degree: 10.9 avg. local clustering coefficient: 0.492 expected number of edges: 162 PPI enrichment p-value: < 1.0e-16

Table S2 continued. STRING Analysis of WIHN+ Macrophage cluster 1.



GO:0051707 response to other organism 2.50e-06 GO:0006955 immune response 2.50e-06 GO:0006952 defense response 3.58e-05 GO:0002376 immune system process 3.99e-05 GO:0098542 defense response to other organism 7.16e-05 Molecular Function (GO)


GO:0019864 IgG binding 0.0290 GO:0019763 immunoglobulin receptor activity 0.0290 GO:0005515 protein binding 0.0290 GO:0042802 identical protein binding 0.0423 Cellular Components (GO)


GO:0009986 cell surface 0.0190 KEGG Pathways


mmu04380 Osteoclast differentiation 0.0010

mmu05152 Tuberculosis 0.0238

mmu05140 Leishmaniasis 0.0238

mmu04612 Antigen processing and presentation 0.0238

Reactome Pathways





MMU-2172127 DAP12 interactions 9.68e-05

MMU-391160 Signal regulatory protein family interactions 0.00015

number of nodes: 51 number of edges: 153 average node degree: 6 avg. local clustering coefficient: 0.563 expected number of edges: 18 PPI enrichment p-value: < 1.0e-16

Table S2 continued. STRING Analysis of WIHN– Macrophage cluster 0.



GO:0006955 immune response 1.07e-19 GO:0002376 immune system process 4.92e-19 GO:0006952 defense response 1.21e-18

GO:0050896 response to stimulus 4.59e-13 GO:0006909 phagocytosis 7.24e-13 Molecular Function (GO)


GO:0005515 protein binding 3.03e-09 GO:0005488 binding 1.50e-06 GO:0019864 IgG binding 6.66e-05 GO:0019763 immunoglobulin receptor activity 6.66e-05 GO:0004888 transmembrane signaling receptor activity 0.00021



GO:0044425 membrane part 1.92e-10 GO:0009986 cell surface 1.92e-10

GO:0005886 plasma membrane 7.41e-10

GO:0031224 intrinsic component of membrane 1.08e-09

GO:0031982 vesicle 2.16e-09

KEGG Pathways


mmu05150 Staphylococcus aureus infection 1.80e-10

mmu04145 Phagosome 1.78e-09

mmu05140 Leishmaniasis 1.55e-08

mmu04380 Osteoclast differentiation 1.32e-07

mmu05152 Tuberculosis 1.98e-07

Reactome Pathways





MMU-418594 G alpha (i) signalling events 7.81e-06



Table S2 continued. STRING Analysis of WIHN– Macrophage cluster 2.



GO:0006950 response to stress 2.75e-28 GO:0050896 response to stimulus 5.00e-27 GO:0006952 defense response 1.26e-23 GO:0042221 response to chemical 6.30e-23 GO:0009605 response to external stimulus 6.96e-22 Molecular Function (GO)


GO:0005515 protein binding 2.35e-19 GO:0005488 binding 1.08e-17 GO:0042802 identical protein binding 3.23e-10 GO:0019899 Enzyme binding 5.03e-09 GO:0046983 protein dimerization activity 4.39e-07



GO:0005829 cytosol 5.90e-12 GO:0005623 cell 5.90e-12

GO:0044464 Cell part 7.07e-12

GO:0043231 intracellular membrane-bounded organelle 1.30e-11

GO:0043227 membrane-bounded organelle 2.07e-11

KEGG Pathways


mmu04380 Osteoclast differentiation 7.88e-10

mmu05418 Fluid shear stress and atherosclerosis 2.47e-08

mmu05152 Tuberculosis 2.47e-08

mmu04145 Phagosome 2.47e-08

mmu05140 Leishmaniasis 3.57e-08

Reactome Pathways






MMU-2262752 Cellular responses to stress 9.30e-09


Table S3. List of the top 200 genes shared by macrophage subsets

from integrated macrophage/DC analysis. Shared Genes 10X_wihn1_avg_logFC 10X_PBMC_avg_logFC Adjusted p_value

H2-Eb1 7.10E-41 1.14E-85 2.28E-85

Cd74 9.91E-41 4.97E-84 9.94E-84

H2-Aa 1.12E-41 6.54E-84 1.31E-83

H2-Ab1 1.23E-40 3.87E-83 7.74E-83

Gsr 1.66E-21 1.28E-76 2.56E-76

Hp 3.95E-32 2.71E-71 5.42E-71

Chil3 1.01E-14 4.77E-68 9.54E-68

Ifitm6 4.38E-24 4.61E-66 9.21E-66

Ifitm3 5.41E-26 1.44E-60 2.87E-60

Mgst1 5.85E-13 7.31E-58 1.46E-57

Plac8 1.43E-16 9.11E-58 1.82E-57

Lyz2 4.18E-21 1.99E-53 3.98E-53

Msrb1 3.30E-16 3.85E-52 7.69E-52

Cebpb 7.99E-26 5.65E-49 1.13E-48

H2-DMb1 1.69E-17 6.88E-49 1.38E-48

Srgn 8.82E-05 3.00E-43 6.01E-43

Smpdl3a 1.12E-06 2.25E-42 4.51E-42

Tyrobp 4.58E-15 8.41E-38 1.68E-37

Clec4d 1.78E-05 1.92E-37 3.84E-37

Lst1 3.34E-21 1.23E-33 2.45E-33

H2-DMa 1.26E-15 5.82E-33 1.16E-32

Prdx5 0.000462163 1.80E-32 3.60E-32

Tpd52 0.000134607 4.47E-31 8.94E-31

Sirpb1c 1.76E-09 2.53E-30 5.07E-30

Ifitm2 7.13E-12 2.11E-28 4.23E-28

F13a1 1.68E-07 3.13E-28 6.26E-28

Thbs1 4.98E-09 3.41E-28 6.83E-28

Vcan 3.59E-06 2.25E-27 4.51E-27

Gda 0.003915725 8.42E-26 1.68E-25

Klra2 3.51E-07 1.22E-25 2.45E-25

Cd9 5.26E-05 3.41E-25 6.83E-25

Ptms 2.00E-08 5.45E-25 1.09E-24

Ppia 1.95E-09 1.28E-24 2.56E-24

Sell 1.86E-06 1.41E-24 2.81E-24

Mgl2 1.14E-07 3.58E-24 7.16E-24

H3f3b 0.000215483 7.67E-24 1.53E-23

Clec4e 3.84E-05 5.57E-23 1.11E-22

Rps2 6.25E-05 9.77E-23 1.95E-22

Msr1 0.0402741 2.07E-22 4.13E-22

Atpif1 1.04E-06 3.17E-22 6.34E-22

Cybb 0.031858798 9.08E-22 1.82E-21

Tubb5 2.47E-09 5.21E-21 1.04E-20

Naaa 0.000146142 2.61E-20 5.21E-20

Ms4a6c 5.56E-15 4.35E-20 8.70E-20

Ly6c2 5.36E-15 1.32E-19 2.64E-19

Tmem176a 4.44E-07 6.21E-19 1.24E-18

Mrpl33 0.042612448 9.56E-19 1.91E-18

Emilin2 0.045143913 1.38E-18 2.76E-18

Mrc1 0.000868268 1.83E-18 3.66E-18

Slpi 0.008069491 2.46E-18 4.92E-18

Cd44 0.903061932 6.81E-18 1.36E-17

Ly6e 1.60E-08 9.98E-18 2.00E-17

Fcer1g 4.73E-10 2.18E-17 4.35E-17

Syngr2 4.38E-09 3.10E-17 6.20E-17

WIHN+ WIHN–

Shared Genes WIHN+_avg_logFC WIHN–_avg_logFC Adjusted p_value

Itgal 0.007981844 4.59E-17 9.19E-17

Npc2 0.008215559 4.84E-17 9.69E-17

Lyn 0.088804962 9.38E-17 1.88E-16

Cd53 3.45E-08 1.17E-16 2.34E-16

Cd209a 1.70E-05 1.83E-16 3.66E-16

Clec10a 0.000146472 1.91E-16 3.83E-16

Rpl12 0.000895915 2.75E-16 5.50E-16

Gm9733 2.84E-13 2.96E-16 5.92E-16

Cxcl16 0.000588186 3.71E-16 7.43E-16

Metrnl 0.000817791 8.54E-16 1.71E-15

Cbfa2t3 0.000285109 2.12E-15 4.24E-15

Tmsb10 2.41E-06 2.45E-15 4.90E-15

Gm5150 8.57E-05 6.55E-15 1.31E-14

Ms4a6b 2.58E-09 1.13E-14 2.26E-14

Slamf7 0.001193356 1.57E-14 3.14E-14

Ifi204 0.000187919 2.57E-14 5.15E-14

Ciita 0.006179068 3.91E-14 7.81E-14

Ptma 0.00014858 4.38E-14 8.75E-14

Aldh2 0.083928539 5.24E-14 1.05E-13

Btg1 0.003638714 9.26E-14 1.85E-13

Atp5g2 0.000645689 9.50E-14 1.90E-13

Clec4b1 0.00198232 1.18E-13 2.35E-13

Tgfbi 0.001804621 1.19E-13 2.38E-13

Sem1 8.82E-06 1.69E-13 3.39E-13

Klrd1 0.000153569 1.98E-13 3.96E-13

Olfm1 0.000598856 2.19E-13 4.38E-13

Sat1 0.15727175 4.32E-13 8.65E-13

Rgs10 0.00085646 6.50E-13 1.30E-12

Ccnd1 0.000313449 7.31E-13 1.46E-12

Samhd1 0.040918648 7.81E-13 1.56E-12

Tmem176b 1.69E-06 1.28E-12 2.55E-12

H2-DMb2 0.000314961 1.38E-12 2.76E-12

Klrb1b 0.001596468 2.09E-12 4.19E-12

Fcgrt 0.003157395 2.75E-12 5.50E-12

Basp1 0.000858485 2.95E-12 5.90E-12

C1qb 4.89E-06 3.31E-12 6.61E-12

Sec61g 0.003025887 4.10E-12 8.21E-12

Pkm 0.000427018 4.15E-12 8.30E-12

Hspa8 1.05E-05 4.82E-12 9.64E-12

Cd209d 0.00020023 4.96E-12 9.92E-12

Eef1g 1.51E-06 6.40E-12 1.28E-11

Cst3 3.71E-06 6.48E-12 1.30E-11

Bcl2a1d 7.50E-05 6.86E-12 1.37E-11

Rap1b 0.315743048 8.25E-12 1.65E-11

Nfil3 0.011557565 1.11E-11 2.21E-11

Fxyd5 1.45E-05 1.24E-11 2.48E-11

Nr4a1 0.044208677 1.29E-11 2.58E-11

Plp2 0.002036566 2.74E-11 5.48E-11

Fam49b 0.002570119 3.01E-11 6.01E-11

Gm14005 0.000216029 3.17E-11 6.34E-11

Tmsb4x 3.39E-11 1.32E-09 6.78E-11

Batf3 3.62E-05 3.43E-11 6.86E-11

C1qa 0.000350061 6.85E-11 1.37E-10

Sdf2l1 3.41E-06 7.41E-11 1.48E-10

Ckb 1.17E-06 8.09E-11 1.62E-10


Klrk1 0.001433566 9.46E-11 1.89E-10

Tuba1b 0.000242379 1.08E-10 2.16E-10

Ndufa5 0.001248963 1.60E-10 3.19E-10

Emb 0.000643707 1.64E-10 3.28E-10

H2afj 0.000129905 2.33E-10 4.66E-10

Pltp 0.00030981 2.98E-10 5.95E-10

Bcl2a1b 8.57E-06 3.17E-10 6.34E-10

Ywhah 0.000607034 6.73E-10 1.35E-09

Junb 0.000531737 6.75E-10 1.35E-09

Ccr2 0.003946759 7.32E-10 1.46E-09

Gstm1 0.010295156 7.78E-10 1.56E-09

Id2 0.008441733 9.68E-10 1.94E-09

Dusp1 0.000375631 1.05E-09 2.11E-09

Rpl14 2.35E-08 1.10E-09 2.21E-09

Mcemp1 1.82E-05 1.23E-09 2.47E-09

Zfp36l2 0.003942902 1.37E-09 2.75E-09

Gsn 0.001706161 1.58E-09 3.16E-09

Ccl9 0.00075543 2.36E-09 4.72E-09

Rnf149 0.021454135 3.26E-09 6.52E-09

Fcgr1 2.15E-05 4.48E-09 8.96E-09

Ms4a4c 1.09E-07 6.44E-09 1.29E-08

Fos 6.47E-08 6.49E-09 1.30E-08

C1qc 0.000144825 7.32E-09 1.46E-08

Hif1a 0.335072732 8.23E-09 1.65E-08

Ifi27l2a 0.001194042 9.82E-09 1.96E-08

Traf1 0.000290866 1.03E-08 2.06E-08

Ncf2 0.434285584 1.06E-08 2.12E-08

Fgd2 0.00648575 1.22E-08 2.44E-08

Ier2 0.016897254 1.24E-08 2.48E-08

Arhgef6 0.002132283 1.85E-08 3.70E-08

Jaml 0.000986964 2.11E-08 4.22E-08

Cd24a 4.62E-05 2.16E-08 4.31E-08

Naga 0.001147238 2.40E-08 4.80E-08

Arhgdia 2.48E-08 3.72E-05 4.96E-08

Bcl2a1a 0.001194386 3.12E-08 6.23E-08

Anxa4 0.003955057 4.26E-08 8.51E-08

Hmgn2 0.00111197 4.67E-08 9.35E-08

Nupr1 0.00512482 5.42E-08 1.08E-07

Ninj1 0.029237597 5.58E-08 1.12E-07

Commd1 0.00466608 8.50E-08 1.70E-07

Nucks1 0.000158755 9.06E-08 1.81E-07

Sdc4 0.016200662 9.41E-08 1.88E-07

Eva1b 0.00100537 9.76E-08 1.95E-07

Pafah1b3 0.000890499 9.88E-08 1.98E-07

Slamf9 0.000424899 1.09E-07 2.18E-07

Mtss1 0.000714115 1.34E-07 2.68E-07

Psmb9 5.42E-05 1.55E-07 3.10E-07

Tnip3 1.99E-05 1.57E-07 3.14E-07

Ifi211 0.012168028 1.57E-07 3.14E-07

Fyb 0.364871814 2.03E-07 4.07E-07

Axl 0.020733809 2.72E-07 5.44E-07

Mrpl42 0.000182613 2.80E-07 5.61E-07

Mdh1 0.000238232 3.21E-07 6.42E-07

Mpp6 0.00156399 3.21E-07 6.42E-07

Osm 0.010477513 3.50E-07 7.00E-07


Nt5c 0.001175324 3.66E-07 7.32E-07

Stk17b 0.039882472 3.81E-07 7.63E-07

Ndufa12 0.001321524 4.04E-07 8.07E-07

Dctpp1 0.000888182 4.08E-07 8.16E-07

Uqcc2 0.000559165 4.42E-07 8.84E-07

Kdm7a 0.963532551 4.45E-07 8.91E-07

Hfe 0.001771958 4.79E-07 9.58E-07

Tnfaip2 0.002171679 4.85E-07 9.71E-07

Lamp1 6.13E-07 7.20E-05 1.23E-06

Rbms1 0.366747264 6.74E-07 1.35E-06

Rex1bd 0.005863867 7.13E-07 1.43E-06

Mbd3 0.002306159 8.58E-07 1.72E-06

Lamp2 0.091497855 1.07E-06 2.14E-06

H2afv 0.000713305 1.10E-06 2.19E-06

Capza2 0.012366507 1.43E-06 2.87E-06

Trappc5 0.003266906 1.66E-06 3.31E-06

Ass1 0.002893514 1.67E-06 3.35E-06

Tnni2 0.001484672 1.79E-06 3.58E-06

Cd63 0.000130155 2.04E-06 4.08E-06

Aes 0.000456433 2.31E-06 4.62E-06

Ear2 4.93E-05 2.41E-06 4.83E-06

Cd7 0.002095256 2.42E-06 4.84E-06

C1galt1c1 0.000927243 2.78E-06 5.56E-06

Klf13 0.199413995 2.85E-06 5.70E-06

Atf3 0.005528262 2.89E-06 5.78E-06

Fosl2 0.00614697 3.17E-06 6.34E-06

Pf4 0.000270962 3.35E-06 6.70E-06

AW112010 0.127311075 3.56E-06 7.13E-06

Ube2d3 0.071654416 3.57E-06 7.14E-06

Rap1a 0.038395659 4.44E-06 8.88E-06

Trib1 0.351339654 4.45E-06 8.90E-06

Malt1 0.005859063 5.24E-06 1.05E-05

Tmem167 0.260514775 6.34E-06 1.27E-05

Rexo2 8.61E-05 6.98E-06 1.40E-05

Cdkn1a 0.07935423 7.15E-06 1.43E-05

Movie S1. Localization of external SFRP4 and SFRP4 vesicles within MerTK+

macrophage. Stacked confocal reconstruction of central MERTK+ macrophage stained for

EDA-FN (red) and SFRP4 (white). Nuclei are blue. SFRP4 can be seen associated with external

FN fibers (top) as well as within vesicles in macrophage.

Supplementary Materials for · Fig. S1. WIHN– wounds have higher numbers of cells with nuclear...

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Transcript of Supplementary Materials for · Fig. S1. WIHN– wounds have higher numbers of cells with nuclear...